Note: Descriptions are shown in the official language in which they were submitted.
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POSH INTERACTING PROTEINS AND RELATED METHODS
RELATED APPLICATIONS
This application claims the benefit of priority of U.S. Provisional
Application number 60/451,437 filed 3 March 2003; 60/452,284 filed 5 March
2003; 60/456,640 filed 20 Maxch 2003; 60/460,526 filed 3 April 2003;
60/464,285
filed 21 April 2003; 60/469,462 filed 9 May 2003; 60/471,378 filed 15 May
2003;
60/472,327 filed 20 May 2003; 60/474,706 filed 30 May 2003; 60/475,825 filed 3
June 2003; 601479,317 filed 17 June 2003; 60/480,376 filed 19 June 2003;
60/480,215 filed 19 June 2003; 60/493,860 filed 8 August 2003; 60/503,931
filed I6
September 2003; 60/455,760 filed 19 March 2003; 60/460,792 filed 4 April 2003;
601498,634 filed 28 August 2003; and a ,provisional application filed on March
2,
2004, (Attorney Docket No. PROL-P79-024), in the name of Daniel N. Taglicht,
Iris
Alroy, Yuval Reiss, Liora Yaar, Danny Ben-Avraham, Shmuel Tuvia, and Tsvika
Greener entitled "Posh Interacting Proteins and Related Methods"; a PCT
application US03/35712 filed 10 November 2003; and a PCT application filed on
February 5, 2004, (Attorney Docket No. PROL-PWO-039), in the name of Iris
Alroy, Daniel Taglicht, Yuval Reiss, Liora Yaar, and Shmuel Tuvia entitled
"Posh
Associated Kinases and Related Methods". The teachings of the referenced
Applications are incorporated herein by reference in their entirety.
BACKGROUND
Potential dnig target validation involves determining whether a DNA, RNA
or protein molecule is implicated in a disease process and is therefore a
suitable
target for development of new therapeutic drugs. Drug discovery, the process
by
which bioactive compounds are identified and characterized, is a critical step
in the
development of new treatments for human diseases. The landscape of drug
discovery has changed dramatically due to the genomics revolution. DNA and
protein sequences are yielding a host of new dnig targets and an enormous
amount
of associated information.
The identification of genes and proteins involved in various disease states or
lcey biological processes, such as inflammation and immune response, is a
vital part
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of the drug design process. Many diseases and disorders could be treated or
prevented by decreasing the expression of one or more genes involved in the
molecular etiology of the condition if the appropriate molecular target could
be
identif ed and appropriate antagonists developed. For example, cancer, in
which one
or more cellular oncogenes become activated and result in the unchecked
progression of cell cycle processes, could be treated by antagonizing
appropriate cell
cycle control genes. Furthermore many human genetic diseases, such as
Huntington's disease, and certain prion conditions, which are influenced by
both
genetic and epigenetic factors, result from the inappropriate activity of a
polypeptide
as opposed to the complete loss of its function. Accordingly, antagonizing the
aberrant function of such mutant genes would provide a means of treatment.
Additionally, infectious d iseases such as HIV have been successfully treated
with
molecular antagonists targeted to specific essential retroviral proteins such
as HIV
protease or reverse transcriptase. Drug therapy strategies for treating such
diseases
and disorders have frequently employed molecular antagonists which target the
polypeptide product of the disease gene(s). However, the discovery of relevant
gene
or protein targets is often difficult and time consuming.
One area of particular interest is the identification of host genes and
proteins
that are co-opted by viruses during the viral life cycle. The serious and
incurable
nature of many viral diseases, coupled with the high rate of mutations found
in many
viruses, makes the identification of antiviral agents a high priority fox the
improvement of world health. Genes and proteins involved in a viral life cycle
are
also appealing as a subject for investigation because such genes and proteins
will
typically have additional activities in the host cell and may play a role in
other non-
viral disease states.
Other areas of interest include the identification of genes and proteins
involved in cancer, apoptosis and neural disorders (particularly those
associated with
apoptotic neurons, such as Alzheimer's disease).
It would be beneficial to identify proteins involved in one or more of these
processs for use in, among other things, drug screening methods. Additionally,
once
a protein involved in one or more processes of interest has been identified,
it is
possible to identify proteins that associate, directly or indirectly, with the
initially
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identified protein. Knowledge of interactors will provide insight into protein
assemblages and pathways that participate in disease processes, and in many
cases
an interacting protein will have desirable properties for the targeting of
therapeutics.
In some cases, an interacting protein will already be known as a drug target,
but in a
different biological context. Thus, by identifying a suite of proteins that
interact
with an initially identified protein, it is possible to identify novel drug
targets and
new uses for previously known therapeutics.
SUMMARY
This application provides isolated, purified or recombinant complexes
comprising a POSH polypeptide and one or more POSH-associated protein (POSH-
AP). In certain aspects, the POSH-AP comprises a polypeptide selected from the
group consisting of PKA, SNX1, SNX3, ATP6VOC, PTPN12, PPP1CA, GOSR2,
CENTBI, DDEFI, ARFI, ARFS, PACS-1, EPS8L2, HERPUDl, UNC84B,
MSTP028, GOCAP, EIF'353, SRA1, CBL-B, RALA, SIAHI, SMNl, SMN2,
SYNEl, TTC3, VCY2IPI and UBE2N (UBC13). In other aspects, the POSH-AP
comprises a polypeptide selected from the group consisting of ARHV (Chp),
WASFI, HIP55, SPG20, HLA-A, and HLA-B. In further aspects, the POSH-AP
comprises one or more polypeptides set forth in Table 8. In certain
embodiments the
POSH polypeptide is a human POSH polypeptide.
In certain embodiments, this application provides isolated, purified or
recombinant complexes comprising a HERPUDI polypeptides and a ubiquitin
ligase, examples of the ubiquitin ligase include CBL-B, TTC3, and SIAHI .
In certain embodiments, the application provides methods for identifying
agents that modulates an activity of a POSH polypeptide or POSH-AP, comprising
identifying an agent that disrupts a complex of a POSH polypeptide and a POSH-
AP, wherein an agent that disrupts such a complex is an agent that modulates
an
activity of the POSH polypeptide or the POSH-AP.
In yet other embodiments, the application provides methods of identifying an
antiviral agent, comprising identifying a test agent that disrupts a complex
comprising a POSH polypeptide and a POSH-AP and evaluating the effect of the
test agent on either a pro-infective or pro-replicative function of a virus is
an
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antiviral agent, wherein an agent inhibits such a function of a virus is an
antiviral
agent. In certain embodiments the POSH-AP is selected from the group
consisting
of PKA, SNX1, SNX3, PTPN12, GOSR2, CENTBl, ARF1, ARFS, PACS-I,
EPS8L2, HERPUD1, SMN1, SMN2, UNC84B, MSTP028, GOCAP, CBL-B,
SYNE1, UBE2N (UBC13), SIAHl, TTC3, WASF1, HIP55, RALA, and SPG20.
Examples of such viruses include for example, envelope vinises such as the
Human
Immunodeficiency Virus, the West Nile Virus, and the Moloney Murine Leukemia
Virus (MMuLV).
In other embodiments, the application provides methods of identifying an
anti-apoptotic agent, comprising identifying a test agent that disrupts a
complex
comprising a POSH polypeptide and a POSH-AP and evaluating the effect of the
test agent on apoptosis of a cell wherein an agent that decreases apoptosis of
the cell
is an anti-apoptotic agent. In yet other embodiments, the application provides
methods of identifying an anti-cancer agent, comprising identifying a test
agent that
disrupts a complex comprising a POSH polypeptide and a POSH-AP and evaluating
the effect of the test agent on proliferation or survival of a cancer cell,
wherein an
agent that decreases proliferation or survival of a cancer cell is an anti-
cancer agent.
Examples of the POSH-AP include PKA, SNX1, PTPN12, PPPl CA, ARF1, ARFS,
CENTBl, EPS8L2, EIF3S3, CBL-B, R.ALA, SIAHl, TTC3, ATP6VOC, and
VCY2IPI. In certain embodiments, the cancer is a POSH-associated cancer.
In certain aspects, the application provides methods of identifying an agent
that inhibits trafficking of a protein through the secretory pathway,
comprising
identifying a test agent that disrupts a complex comprising a POSH polypeptide
and
a POSH-AP and evaluating the effect of the test agent on the traff eking of a
protein
through the secretory pathway wherein an agent that disrupts localization of
said
POSH-AP is an agent that inhibits traffcking of a protein through the
secretory
pathway. In certain embodiments, the protein is a myristoylated protein. In
yet
other embodiments, the protein is a viral protein. In alternative embodiments,
the
protein is associated with a neurological disorder such as fox example the
amyloid
beta precursor protein.
In yet other embodiments, the application provides methods of identifying an
agent that inhibits the progression of a neurological disorder, comprising
identifying
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a test agent that disrupts a complex comprising a POSH polypeptide and a POSH-
AP evaluating the effect of the test agent on the trafficking of a protein
through the
secretory pathway wherein an agent that disrupts localization of a POSH-AP is
an
agent that inhibits progression of a neurological disorder. In certain aspects
the
POSH-AP is HERPUD1.
In yet other embodiments, this application provides methods of treating a
viral infection in a subject in need thereof, comprising administering an
agent that
inhibits a POSH-AP in an amount sufficient to inhibit the viral infection. The
agent
is one that: inhibits a kinase activity of the POSH-AP; inhibits expression of
the
POSH-AP; inhibits the ubiquitin ligase activity of the POSH-AP; inhibits the
phosphatase activity of the POSH-AP; inhibits the GTPase activity of the POSH-
AP; and inhibits the ubiquitination of the POSH-AP. In certain embodiments,
the
POSH-AP comprises a polypeptide selected from the group consisting of: PKA,
SNXI, SNX3, SMNl, SMN2, PTPN22, GOSR2, CENTBI, ARF1, ARFS, PACS-l,
EPS8L2, HERPUD1, UNC84B, MSTP028, GOCAP, CBL-B, SYNE1, UBE2N
(LTBCI3), SIAHI, TTC3, WASFI, HIP55, IZALA, and SPG20. In certain aspects,
the agent may be an siRNA construct, a small molecule, an antibody, or an
antisense
construct.
In certain embodiments, the agent is an siRNA construct comprising a
nucleic acid sequence that hybridizes to an mRNA encoding the POSH-AP.
Examples include siRNA constructs that inhibit the expression of HERPLTD1 or
MSTP028. Examples of siRNA constructs that inhibit the expression of HERPUD I
include: 5'GGAAGUUCUUCGGAACCUdTdT-3' and
5'- dTdTCCCUUCAAGAAGCCUUGGA-5'. Examples of siRNA constructs that
inhibit the expression of MSTP028 include: 5'-AAGTGCTCACCGACAGTGAAG-
3' and 5'-AAGATACTTATGAGCCTTTCT-3'.
In other aspects, the agents may be a small molecule inhibitor is selected
from
among the following categories: adenosine cyclic monophosphorothioate,
isoquinolinesulfonamide, piperazine, piceatannol, and ellagic acid. In
alternative
embodiments, the agents may be a small molecule inhibitor that inhibits the
Iigase
activity of a POSH polypeptide or inhinbits the ubiquitination of a POSH-AP:
Examples of such small molecules include, for example:
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O
S N O
N CI
N
! ~O \
O . O
0
N \ \ \ ( N+-.O
O ~ ~ \ ' o_
N
S N
> >
o ~ .o
N~ \ / N\
CI O
GI
and
O CI
I ~ N~O \
In certain embodiments, the application provides packaged pharmaceuticals
for treating viral infections, comprising: a pharmaceutical composition
comprising
an inhibitor of a POSH-AP and a pharmaceutically acceptable Garner and
instructions for use.
In certain embodiments, the application provides methods of treating or
preventing a POSH associated cancer in a subject comprising administering an
agent
that inhibits a POSH-AP to a subject in need thereof, wherein said agent
treats or
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prevents cancer. The POSH-AP comprises a polypeptide selected from the group
consisting of: PKA, SNXI, PTPN12, PPPICA, CENTB1, ARFl, ARFS, EPS8L2,
EIF3S3, CBL-B, RALA, SIAH1, TTC3, ATP6VOC, and VCY2IP1.
In yet other aspects, the application provides methods of treating a
S neurological disorder comprising administering an agent to a subject in need
thereof,
wherein said agent either inhibits the Ubiquitin ligase activity of POSH or
inhibits
the ubquitination of a POSH-AP. Examples of the POSH-AP include: PTPN12,
DDEFI, EPS8L2, HERPUDI, GOCAP, CBL-B, SIAHl, SMNl, SMN2, TTC3,
SPG20, SNXl, and ARFI.
Examples of the neurological disorders include Alzheimer's disease,
Parkinson's
disease, Huntington's disease, schizophrenia, Niemann-Pick's disease, and
prion-
associated diseases. In certain aspects, the agent is selected from the group
consisting of: an siRNA construct, a small molecule, an antibody, and an
antisense
construct. Examples of the small molecules include:
O
S~ N O
'N \ ~ ~ ~ N \ ~ CI
wOr
O ~ O
o i
N \ \ \ ~ N+:O
O
N
S N
> >
o ~--~ .o
N~ \ / N\ -
CI O
CI
CA 02517525 2005-08-30
WO 2004/078130 PCT/US2004/006308
and
O CI
O
~ N~O \
In certain aspects, the disclosure provides methods of treating viral
hepatitis
in a subject in need thereof. Such a method may comprise administering an
effective amount of an agent that inhibits POSH or disrupts an interaction
between
POSH and a dynamin, preferably dynamin II. In certain embodiments, the subject
has a viral hepatitis caused by HBV or HCV.
In certain aspects, the disclosure provides methods of inhibiting a
hepatotrophic virus or a method for treating a disease associated with a
hepatrophic
virus, comprising administering an effective amount of an agent, wherein said
agent
inhibits POSH or an interaction between POSH and dynamin. In certain
embodiments, the hepatrophic virus is selected from the group consisting of
HAV,
HBV, HCV, HDV, and HEV. The hepatotrophic virus associated disease may be,
for example, viral hepatitis or hepatocellular carcinoma. An agent for any of
the
above methods may include, for example, a nucleic acid agent that decreases
the
Ievel of POSH in cells of the subject (e.g., an antisense oligonucleotide, an
RNAi
construct, a DNA enzyme, a ribozyme) or small molecule inhibitors of POSH, as
well as antibodies or other binding agents that bind to a surface of POSH or
dynamin that participates in a POSH-dynamin interaction. An agent may be any
of
the following: a small molecule, an antibody, a fragment of an antibody, a
peptidomimetic, and a polypeptide. Examples of small molecules include:
_g_
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STRUCTURE MW CAS number
F 384.2 14567-55-4
F
F ~ O
F O
N
O
O N O
Br O 389.5 414908-38-0
~N
CI
Br
In certain embodiments, the application provides methods for inhibiting an
HBV infection in a subject in need thereof, comprising administering an
effective
amount of a POSH inhibitor, wherein the HBV infection is inhibited in the
subject.
In additional embodiments, the disclosure provides methods for treating an HBV
infection in a patient, comprising administering an effective amount of an
agent that
inhibits POSH or decreases the level of POSH protein or nucleic acid in an
infected
cell. An agent may be, for example, an RNAi construct that inhibits the
expression
of POSH. Optionally the RNAi construct is 20-25 nucleotides in length and
optionally it is selected from any one of SEQ ID NOS: 15, 16, I8, 19, 21, 22,
24,
and 25. The RNAi may be formulated as a liposome. An agent may be a small
molecule inhibitor of POSH ubiquitin ligase activity, as disclosed herein.
Examples
1 S of small molecule inhibitors of POSH include:
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STRUCTURE MW CAS number
F 384.2 14567-55-4
F
F ~ O
F O
N\/
~~O
O N O
Br O 389.5 414908-38-0
~N
CI
Br
In certain aspects, the disclosure provides a method for treating an HBV
infection in a patient, comprising administering an effective amount of an
antisense
oligonucleotide sufficient to bind a nucleic acid molecule, which nucleic acid
molecule encodes a POSH polypeptide.
In certain embodiments, the application provides methods for inhibiting an
HBV infection by administering an effective amount of a compound of the
formula:
F
F
F ~ O
F
N\/
~'O
O N O
In additional embodiments, the application provides methods for treating an
HBV infection by administering an effective amount of a compound of the
formula:
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Br O
~N
CI
Br
In certain aspects, the disclosure provides methods for inhibiting the
maturation of a lentivirus by modulating the activity of a Vpu polypeptide. In
preferred embodiments, maturation of the lentivirus is inhibited by inhibiting
the
transport and/or assembly of viral particles in the TGN and from the TGN to
the
plasma membrane. A preferred lentivinis for application of such a method is
the
human immunodeficiency virus.
In certain aspects, the disclosure provides methods of inhibiting viral
infection comprising administering an agent to a subject in need thereof,
wherein
said agent inhibits the interaction between a POSH polypeptide and Vpu.
In certain aspects, the disclosure provides methods for identifying a target
polypeptide for antiviral therapy, the method comprising: a) selecting a test
polypeptide known to localize or predicted to localize to the trans Golgi
network; b)
inhibiting an activity of the test polypeptide in a cell infected with a viral
construct
under conditions where, but for the inhibition of the activity of the test
polypeptide,
viral particles are released from the cell; and c) determining whether viral
particles
are released from the cell, wherein, if inhibiting the activity of the test
polypeptide in
the cell inhibits the release of viral particles from the cell, the test
polypeptide is a
target polypeptide for antiviral therapy. In a preferred embodiment, the test
polypeptide is Vpu. Vpu activity may be inhibited, for example, by siRNA,
antisense or other nucleic acid based method.
In certain aspects, the disclosure provides isolated, purified or recombinant
complexes comprising a POSH polypeptide and a Vpu polypeptide. The POSH
polypeptide may comprise, for example, a POSH SH3 domain, or a polypeptide at
least 80% identical to such an SH3 domain. An antiviral agent may be selected
based on its ability to disrupt a POSH-Vpu complex.
The practice of the present application will employ, unless otherwise
indicated, conventional techniques of cell biology, cell culture, molecular
biology,
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transgenic biology, microbiology, recombinant D NA, and immunology, which are
within the skill of the art. Such techniques are explained fully in the
literature. See,
for example, Molecular- Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook,
Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA
Clorairag,
Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J.
Gait
ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid
Hybridization (B.
D. Hames & S. J. Higgins eds. 1984); Transcription Atad Translation (B. D.
Hames
& S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.
Liss,
Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A
Practical Guide To Molecular Cloning (1984); the treatise, Methods In
Enzynaology
(Academic Press, Inc., N.Y.); Gene Transfer hectors For Manarraalian Cells (J.
H.
Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods Ira
Enzymology, Vols. 154 and 155 (Wu et al. eds.), lmmunoclaemical Methods Ira
Cell
And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987);
Handbook Of Experimental Immunology, Volumes T-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating tlae Mouse Embryo, (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the application will be apparent from the
following detailed description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows human POSH coding sequence (SEQ ID NO:I).
Figure 2 shows human POSH amino acid sequence (SEQ ID N0:2).
Figure 3 shows human POSH cDNA sequence (SEQ ID N0:3).
Figure 4 shows 5' eDNA fragment of human POSH (public gi:10432611;
SEQ ID N0:4).
Figure 5 shows N terminus protein fragment of hPOSH (public gi:10432612;
SEQ ID NO:S).
Figure 6 shows 3' mRNA fragment of hPOSH (public gi:7959248; SEQ ID
N0:6).
Figure 7 shows C terminus protein fragment of hPOSH (public gi:7959249;
SEO >D N0:7).
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Figure 8 shows human POSH full mRNA, annotated sequence.
Figure 9 shows domain analysis of human POSH.
Figiwe 10 is a diagram of human POSH nucleic acids. The diagram shows
the full-length POSH gene and the position of regions amplified by RT-PCR or
targeted by siRNA used in figure 11.
Figure 11 shows effect of knockdown of POSH mRNA by siRNA duplexes.
HeLa S S-6 c ells w ere t ransfected w ith s iRNA a gainst Lamin A/C ( lanes
1, 2 ) o r
POSH (lanes 3-IO). POSH siRNA was directed against the coding region (153 -
lanes 3, 4; 155 - lanes S, 6) or the 3'UTR (157 - lanes 7, 8; 159 - lanes 9,
10). Cells
were harvested 24 hours post-transfection, RNA extracted, and POSH mRNA levels
compared by RT-PCR of a discrete sequence in the coding region of the POSH
gene
(see figure 10). GAPDH is used an RT-PCR control in each reaction.
Figure 12 shows that POSH affects the release of VLP from cells. A)
Phosphohimages of SDS-PAGE gels of immunoprecipitations of 3~S pulse-chase
labeled Gag proteins are presented for cell and vital lysates from transfected
HeLa
cells that were either untreated or treated with POSH RNAi (50 nM for 48
hours).
The time during the chase period (1, 2, 3, 4, and 5 hours after the pulse) are
presented from left to right for each image.
Figure 13 shows release of VLP from cells at steady state. Hela cells were
transfected with an HIV-encoding plasmid and siRNA. Lanes l, 3 and 4 were
transfected with wild-type HIV-encoding plasmid. Lane 2 was transfected with
an
HIV-encoding plasmid which contains a point mutation in p6 (PTAP to ATAP).
Control siRNA (lamin A/C) was transfected to cells in lanes 1 and 2. siRNA to
Tsg101 was transfected in lane 4 and siRNA to POSH in lane 3.
Figure 14 shows mouse POSH mRNA sequence (public gi:10946921; SEQ
ID NO: 8).
Figure 15 shows mouse POSH Protein sequence (Public gi:10946922; SEQ
ID NO: 9).
Figure 16 s hows D rosophila m elanogaster P OSH m RNA s equence (public
gi:17737480; SEQ ID NO:10).
Figure 17 shows Drosophila melanogaster POSH protein sequence (public
gi:17737481; SEQ ID NO:11).
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Figure 18 shows POSH domain analysis.
Figlxre 19 shows that human POSH has ubiquitin ligase activity.
Figure 20 shows that human POSH co-immunoprecipitates with RAC1.
Figure 21 shows that POSH knockdown results in decreased secretion of
phospholipase D ("PLD")
Figure 22 shows effect of hPOSH on Gag-EGFP intracellular distribution.
Figure 23 shows intracellular distribution of HIV-1 Nef in hPOSH-depleted
cells.
Figure 24 shows intracellular distribution of Src in hPOSH-depleted cells.
Figure 25 shows intracellular distribution of Rapsyn in hPOSH-depleted
cells.
Figure 26 shows that POSH reduction by siRNA abrogates West Nile virus
infectivity.
Figure 27 shows that POSH knockdown decreases the release of extracellular
MMuLV particles.
Figure 28 shows that knock-down of human POSH entraps HIV virus
particles in intracellular vesicles. HIV virus release was analyzed by
electron
microscopy following siRNA and full-length HIV plasmid transfection. Mature
viruses were secreted by cells transfected with HIV plasmid and non-relevant
siRNA
(control, bottom panel). Knockdown of TsgI01 protein resulted in a budding
defect,
the viruses that were released had an immature phenotype (top panel).
Knockdown
of hPOSH levels resulted in accumulation of viruses inside the cell in
intracellular
vesicles (middle panel).
Figure 29A shows siRNA-mediated reduction of MSTP028 expression
inhibits H1V vims-like particle production (Experiment 1).
Figure 29B shows siRNA-mediated reduction of MSTP028 expression
inhibits HIV virus-like particle production (Experiment 2).
Figure 30 shows putative PKA phosphorylation sites in hPOSH. Amino acid
sequence of hPOSH (70 residues per line). Motifs of the low stringency RxxS/T
type are underlined. The high stringency motif R/KRIKxS/T is bordered.
Putative
S/T phosphorylation sites are highlighted in green. Color-coding of domains:
Red -
RING, Blue - SH3, Green - putative Rac-1 Binding Domain.
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Figure 31 shows that phosphorylation of hPOSH regulates binding of GTP-
'loaded Rac-1. Bacterially expressed hPOSH (1 ~,g) (pOSH) or GST (1 ~,g) (NS)
were phosphorylated. Subsequently, GTPyS loaded or unloaded recombinant Rac-1
(0.2 ~.g) was added to hPOSH or GST. Bound racl was isolated as described in
materials and methods and samples separated by SDS-PAGE on a 12% gel and
immunobloted with anti-Rac-1. Input is 0.25 ~,g of Rac-1.
Figure 32 shows domain analysis of various POSH-APs.
Figure 33 shows siRNA-mediated reduction in HERPUDl expression
reduces HIV maturation.
Figure 34A shows that endogenous Herp levels are reduced in H153 cells.
H153 (POSH-RNAi) and H187 (control RNAi) cells were transfected with a plasmid
encoding Flag-ubiquitin. Total cell lysates (A) or Flag-immunoprecipitated
material
(B) were separated on 10% SDS-PAGE and immttnoblotted with anti-Herp
antibodies.
Figure 34B shows that exogenous Herp levels and its ubiquitination are
reduced in POSH-depleted cells. H153 and H187 cells were co-transfected with
Here or control plasmids and a plasmid encoding Flag-ubiquitin (indicated
above the
figure). Total (A) and flag-immunoprecipitated material (B) were separated on
10%
SDS-PAGE and immunoblotted with anti-Herp antibodies.
Figure 35 shows that the compounds CAS number 14567-55-4 and CAS
number 414908-38-0 (lanes 7 and 8) inhibit HBV production.
Figure 36 provides the nucleic acid and amino acid sequences of POSH-APs.
DETAILED DESCRIPTION OF THE APPLICATION
1. Definitions
The term "binding" refers to a direct association between two molecules, due
to, for example, covalent, electrostatic, hydrophobic, ionic and/or hydrogen-
bond
interactions under physiological conditions.
A "chimeric protein" or "fusion protein" is a fusion of a first amino acid
sequence encoding a polypeptide with a second amino acid sequence defining a
domain foreign t o a nd n of s ubstantially h omologous w ith a ny d omain o f
t he ~ first
amino acid sequence. A chimeric protein may present a foreign domain which is
-IS-
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found ( albeit i n a d ifferent p rotein) i n a n o rganism w hick a lso a
xpresses t he first
protein, or it may be an "interspecies", "intergenic", etc. fusion of protein
structures
expressed by different kinds of organisms.
The terms "compound", "test compound" and "molecule" are used herein
interchangeably and are meant to include, but are not limited to, peptides,
nucleic
acids, carbohydrates, small organic molecules, natural product extract
libraries, and
any other molecules (including, but not limited to, chemicals, metals and
organometallic compounds).
The phrase "conservative amino acid substitution" refers to grouping of
amino acids on the basis of certain common properties. A functional way to
define
common properties between individual amino acids is to analyze the normalized
frequencies of amino acid changes between corresponding proteins of homologous
organisms (Schulz, G. E. and R. H. Schirmer., Principles of Protein Structure,
Springer-Verlag). According to such analyses, groups of amino acids may be
defined where amino acids within a group exchange preferentially with each
other,
and therefore resemble each other most in their impact on the overall protein
structure (Schulz, G. E. and R. H. Schirmer, Principles of Protein ,
Structure,
Springer-Verlag). Examples of amino acid groups defined in this manner
include:
(i) a charged group, consisting of Glu and Asp, Lys, Arg and His,
(ii) ~ a positively-charged group, consisting of Lys, Arg and His,
(iii) a negatively-charged group, consisting of Glu and Asp,
(iv) an aromatic group, consisting of Phe, Tyr and Trp,
(v) a nitrogen ring group, consisting of His and Trp,
(vi) a large aliphatic nonpolar group, consisting of Val, Leu and Ile,
(vii) a slightly-polar group, consisting of Met and Cys,
(viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu,
Gln
and Pro,
(ix) an aliphatic group consisting of Val, Leu, Ile, Met and Cys, and
(x) a small hydroxyl group consisting of Ser and Thr.
In addition to the groups presented above, each amino acid residue may form
its own group, and the group formed by an individual amino acid may be
referred to
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simply by the one and/or three letter abbreviation for that amino acid
commonly
used in the art.
A "conserved residue" is an amino acid that is relatively invariant across a
range of similar proteins. Often conserved residues will vary only by being
replaced
with a similar amino acid, as described above for "conservative amino acid
substitution".
The term "domain" as used herein refers to a region of a protein that
comprises a particular structi.~re andlor performs a particular function.
The term "envelope virus" as used herein refers to any virus that uses
cellular
membrane and/or any organelle membrane in the viral release process.
"Homology" or "identity" or "similarity" refers to sequence similarity
between two peptides or between two nucleic acid molecules. Homology and
identity c an each be determined by comparing a position in each sequence
which
may b a a Iigned for p urposes o f c omparison. W hen an equivalent p osition
i n t he
compared sequences is occupied by the same base or amino acid, then the
molecules
are i dentical a t t hat p osition; w hen t he a quivalent s ite o ccupied b y
t he s ame o r a
similar amino acid residue (e.g., similar in steric and/or electronic nature),
then the
molecules can be referred to as homologous (similar) at that position.
Expression as
a percentage of homology/similarity or identity refers to a function of the
number of
identical or similar amino acids at positions shared by the compared
sequences. A
sequence which is "unrelated" or "non-homologous" shares less than 40%
identity,
though preferably less than 25% identity with a sequence of the present
application.
In comparing two sequences, the absence of residues (amino acids or nucleic
acids)
or presence of extra residues also decreases the identity and
homology/similarity.
The term "homology" describes a mathematically based comparison of
sequence similarities which is -used to identify genes or proteins with
similar
functions or motifs. The nucleic acid and protein sequences of the present
application may be used as a "query sequence" to perform a search against
public
databases to, for example, identify other family members, related sequences or
homologs. Such searches can be performed using the NBLAST and XBLAST
programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
BLAST
nucleotide searches can be performed with the NBLAST program, score=100,
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wordlength=12 to obtain nucleotide sequences homologous to nucleic acid
molecules of the application. BLAST protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid sequences
homologous to protein molecules of the application. To obtain gapped
alignments
for comparison purposes, Gapped BLAST can be utilized as described in Altschul
et
al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and
Gapped BLAST programs, the default parameters of the respective programs
(e.g.,
XBLAST and BLAST) can be used. See-http:/lwww.ncbi.nlm.nih.gov.
As used herein, "identity" means the percentage of identical nucleotide or
amino acid residues at corresponding positions in two or more sequences when
the
sequences are aligned to maximize sequence matching, i.e., taking into account
gaps
and insertions. Identity can be readily calculated by known methods, including
but
not limited to those described in (Computational Molecular Biology, Lesk, A.
M.,
ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer
Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds.,
Humane
Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G.,
Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,
J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D.,
SIAM
J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed
to
give the largest match between the sequences tested. Moreover, methods to
determine identity are codified in publicly available computer programs.
Computer
program methods to determine identity between two sequences include, but are
not
limited to, the GCG program package (Devereux, J., et al., Nucleic Acids
Research
12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J.
Molec. Biol. 215: 403-4I0 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-
3402
(1997)). The BLAST X program is publicly available from NCBI and other sources
(BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;
Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well known Smith
Waterman algorithm may also be used to determine identity.
The term "isolated", as used herein with reference to the subject proteins and
protein complexes, refers to a preparation of protein or protein complex that
is
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essentially free from contaminating proteins that normally would be present
with the
protein or complex, e.g., in the cellular milieu in which the protein or
complex is
found endogenously. Thus, an isolated protein complex is isolated from
cellular
components t hat n ormally would " contaminate" o r i nterfere w ith t he s
tudy o f t he
complex in isolation, for instance while screening for modulators thereof. It
is to be
understood, however, that such an "isolated" complex may incorporate other
proteins the modulation of which, by the subj ect protein or protein complex,
is being
investigated.
The term "isolated" as also used herein with respect to nucleic acids, such as
DNA or RNA, refers to molecules in a form which does not occur in nature.
Moreover, an "isolated nucleic acid" is meant to include nucleic acid
fragments
which are not naturally occurring as fragments and would not be found in the
natural
state.
Lentiviruses include primate lentivinzses, e.g., human immunodeficiency
virus types 1 and 2 (HIV-1/HIV-2); simian immunodeficiency virus (SIV) from
Chimpanzee (SIVcpz), Sooty mangabey (SIVsmm), AfnCaIl Green Monkey
(SIVagm), S yke's m onkey (SIVsyk), M andrill ( SIVmnd) and M acaque (SIVmac).
Lentiviruses also include feline lentiviruses, e.g., Feline immunodeficiency
virus
(FIV); Bovine lentiviruses, e.g., Bovine immunodeficiency virus (BIV); Ovine
lentiviruses, e.g., Maedi/Visna virus (MVV) and Caprine arthritis encephalitis
virus
(CAEV); and Equine lentiviruses, e.g., Equine infectious anemia virus (EIAV).
All
lentiviruses express at least two additional regulatory proteins (Tat, Rev) in
addition
to Gag, Pol, and Env proteins. Primate lentiviruses produce other accessory
proteins
including Nef, Vpr, Vpu, Vpx, and Vif. Generally, lentiviruses are the
causative
agents of a variety of disease, including, in addition to immunodeficiency,
neurological degeneration, and arthritis. Nucleotide sequences of the various
lentiviruses can be found in Genbank under the following Accession Nos. (from
J.
M. Coffin, S. H. Hughes, and H. E. Varmus, "Retroviruses" Cold Spring Harbor
Laboratory P ress, 199,7 p 8 04): 1 ) H IV-1: K 03455, M 19921, K 02013, M3843
1,
M38429, K02007 and M17449; 2) HIV-2: M30502, J04542, M30895, J04498,
M15390, M31113 and L07625; 3) SIV:M29975, M30931, M58410, M66437,
L06042, M33262, M19499, M32741, M31345 and L03295; 4) FIV: M25381,
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M36968 and Ul 1820; 5)BIV. M32690; 6) ElAV: M16575, M87581 and U01866;
6) Visna: M10608, M51543, L06906, M60609 and M60610; 7) CAEV: M33677;
and 8) Ovine lentivirus M31646 and M34193. Lentiviral DNA can also be obtained
from the American Type Culture Collection (ATCC). For example, feline
immunodeficiency virus is available under ATCC Designation No. VR-2333 and
VR-3112. Equine infectious anemia virus A is available under ATCC Designation
No. VR-778. Caprine arthritis-encephalitis vims is available under ATCC
Designation No. VR-905. Visna virus is available under ATCC Designation No.
VR-779.
As used herein, the term "nucleic acid" refers to polynucleotides such as
deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
The
term should also be understood to include, as equivalents, analogs of either
RNA or
DNA m ade from nucleotide analogs, and, as applicable to the embodiment b eing
described, single-stranded (such as sense or antisense) and double-stranded
polynucleotides.
The term "maturation" as used herein refers to the production, post-
translational processing, assembly and/or release of proteins that form a
viral
particle. Accrodingly, this includes the processing of viral proteins leading
to the
pinching off of nascent virion from the cell membrane.
A "POSH nucleic acid" is a nucleic acid comprising a sequence as
represented in any of SEQ JD Nos:l, 3, 4, 6, 8, and 10 as well as any of the
variants
described herein.
A "POSH polypeptide" or "POSH protein" is a polypeptide comprising a
sequence as represented in any of SEQ >D Nos: 2, 5, 7, 9 andl 1 as well as any
of the
variations described herein.
A "POSH-associated protein" or "POSH-AP" refers to a protein capable of
interacting with and/or binding to a POSH polypeptide. Generally, the POSH-AP
may interact directly or indirectly with the POSH polypeptide. Preferred POSH-
APs
include those provided in Table 7. Other preferred POSH-APs include those
listed
in Table 8. Examples of these and other POSH-APs are provided throughout.
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The terms peptides, proteins and polypeptides are used interchangeably
herein.
The term "purified protein" refers to a preparation of a protein or proteins
which are preferably isolated from, or otherwise substantially free of, other
proteins
normally associated with the proteins) in a cell or cell lysate. The term
"substantially free of other cellular proteins" (also referred to herein as
"substantially
free of other contaminating proteins") is defined as encompassing individual
preparations of each of the component proteins comprising less than 20% (by
dry
weight) contaminating protein, and preferably comprises less than 5%
contaminating
IO protein. Functional forms of each of the component proteins can be prepared
as
purified preparations by using a cloned gene as described in the attached
examples.
By "purified", it is meant, when referring to component protein preparations
used to
generate a reconstituted protein mixture, that the indicated molecule is
present in the
substantial absence of other biological macromolecules, such as other proteins
(particularly other proteins which may substantially mask, diminish, confuse
or alter
the characteristics of the component proteins either as purified preparations
or in
their function in the subject reconstituted mixture). The teen "purified" as
used
herein preferably means at least 80% by dry weight, more preferably in the
range of
85% by weight, more preferably 95-99% by weight, and most preferably at least
99.8% by weight, of biological macromolecules of the same type present (but
water,
buffers, and other small molecules, especially molecules having a molecular
weight
of less than 5000, can be present). The term "pure" as used herein preferably
has the
same numerical limits as "purified" immediately above.
A "recombinant nucleic acid" is any nucleic acid that has been placed
adjacent to another nucleic acid by recombinant DNA techniques. A "recombined
nucleic acid" also includes any nucleic acid that has been placed next to a
second
nucleic acid by a laboratory genetic technique such as, for example,
tranformation
and integration, transposon hopping or viral insertion. In general, a
recombined
nucleic acid is not naturally located adjacent to the second nucleic acid.
The term "recombinant protein" refers to a protein of the present application
which is produced by recombinant DNA techniques, wherein generally DNA
encoding the expressed protein is inserted into a suitable expression vector
which is
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in turn used to transform a host cell to produce the heterologous protein.
Moreover,
the phrase "derived from", with respect to a recombinant gene encoding the
recombinant protein is meant to include within the meaning of "recombinant
protein" those proteins having an amino acid sequence of a native protein, or
an
amino acid sequence similar thereto which is generated by mutations including
substitutions and deletions of a naturally occurring protein.
A "RING domain" or "Ring Finger" is a zinc-binding domain with a defined
octet of cysteine and histidine residues. Certain RING domains comprise the
consensus sequences as set forth below (amino acid nomenclature is as set
forth in
Table 1): Cys Xaa Xaa Cys Xaalo-zo Cys Xaa His Xaaz_s Cys Xaa Xaa Cys Xaals-so
Cys Xaa Xaa Cys or Cys Xaa Xaa Cys Xaalo _ zo Cys Xaa His Xaa2_s His Xaa Xaa
Cys Xaals-so Cys Xaa Xaa Cys. Certain RING domains are represented as amino
acid sequences that are at least 80% identical to amino acids 12-52 of SEQ ID
NO: 2
and is set forth in SEQ ID No: 26. Preferred RING domains are 85%, 90%, 95%,
98% and, most preferably, 100% identical to the amino acid sequence of SEQ ID
NO: 26. Preferred RING domains of the application bind to various protein
partners
to form a complex that has ubiquitin ligase activity. RING domains preferably
interact with at least one of the following protein types: F box proteins, E2
ubiquitin
conjugating enzymes and cullins.
The term "RNA interference" or "RNAi" refers to any method by which
expression of a gene or gene product is decreased by introducing into a target
cell
one or more double-stranded RNAs which are homologous to the gene of interest
(particularly to the messenger RNA of the gene of interest). RNAi may also be
achieved by introduction of a DNA:RNA hybrid wherein the antisense strand
(relative to the target) is RNA. Either strand may include one or more
modifications
to the base or sugar-phosphate backbone. Any nucleic acid preparation designed
to
achieve an RNA interference effect is referred to herein as an siRNA
construct.
Phosphorothioate is a particularly common modification to the backbone of an
siRNA construct.
"Small molecule" as used herein, is meant to refer to a composition, which
has a molecular weight of less than about 5 kD and most preferably less than
about
2.5 kD. Small molecules can be nucleic acids, peptides, polypeptides,
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peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or
inorganic molecules. Many pharmaceutical companies have extensive libraries of
chemical and/or biological mixtures comprising arrays of small molecules,
often
fungal, bacterial, or algal extracts, which can be screened with any of the
assays of
the application.
An " SH3" o r "Src H oenology 3 " d omain i s a p rotein d omain o f g
enerally
about 60 amino acid r esidues first identified as a conserved sequence in the
non-
catalytic part of several cytoplasmic protein tyrosine kinases (e.g., Src,
Abl, Lck).
SH3 domains mediate assembly of specific protein complexes via binding to
proline-rich peptides. Exemplary SH3 domains are represented by amino acids
137-
192, 199-258, 448-505 and 832-888 of SEQ >D N0:2 and are set forth in SEQ TD
Nos: 27-30. In certain embodiments, an SH3 domain interacts with a consensus
sequence of RXaaXaaPXaaX6P (where X6, as defined in table 1 below, is a
hydrophobic amino acid). Tn certain embodiments, an SH3 domain interacts with
one or more of the following sequences: P(T/S)AP, PFRDY, RPEPTAP,
RQGPTI~EP, I2QGPKEPFR, RPEPTAPEE and I~PLPVAP.
As used herein, the term "specifically hybridizes" refers to the ability of a
nucleic acid ,probe/primer of the application to hybridize to at least 12, 15,
20, 25,
30, 35, 40, 45, 50 or 100 consecutive nucleotides of a POSH sequence, or a
sequence complementary thereto, or naturally occurring mutants thexeof, such
that it
has less than 15%, preferably less than 10%, and more preferably less than 5%
background hybridization to a cellular nucleic acid (e.g., mRNA or genomic
DNA)
other than the POSH gene. A variety of hybridization conditions may be used to
detect specific hybridization, and the stringency is determined primarily by
the wash
stage of the hybridization assay. Generally high temperatures and low salt
concentrations give high stringency, while low temperatures and high salt
concentrations give low stringency. Low stringency hybridization is achieved
by
washing in, for example, about 2.0 x SSC at 50 °C, and high stringency
is acheived
with about 0.2 x SSC at 50 °C. Further descriptions of stringency are
provided
below.
As applied to polypeptides, "substantial sequence identity" means that two
peptide sequences, when optimally aligned, such as by the programs GAP or
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BESTFIT using default gap which share at least 90 percent sequence identity,
preferably at least 95 percent sequence identity, more preferably at least 99
percent
sequence identity or more. Preferably, residue positions which are not
identical
differ by conservative amino acid substitutions. For example, the substitution
of
amino acids having similar chemical properties such as charge or polarity are
not
likely to effect the properties of a protein. Examples include glutamine for
asparagine or glutamic acid for aspartic acid.
As is well known, genes for a particular polypeptide may exist in single or
multiple copies within the genome of an individual. Such duplicate genes may
be
identical or may have certain modifications, including nucleotide
substitutions,
additions or deletions, which all still code for polypeptides having
substantially the
same activity.
A "virion" is a complete viral particle; nucleic acid and capsid (and a lipid
envelope in some viruses. A "viral particle" may be incomplete, as when
produced
by a cell transfected with a defective virus (e.g., an HIV virus-like particle
system).
Table 1: Abbreviations for classes of amino acids
Symbol Category Amino Acids
Represented
X1 Alcohol Ser, Thr
X2 Aliphatic Ile, Leu, Val
Xaa Any Ala, Cys, Asp, Glu,
Phe,
Gly, His, Ile, Lys,
Leu,
Met, Asn, Pro, Gln,
Arg,
Ser, Thr, Val, Trp,
Tyr
X4 Aromatic Phe, His, Trp, Tyr
XS Charged Asp, Glu, His, Lys,
Arg
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X6 Hydrophobic Ala, Cys, Phe, Gly,
His,
Ile, Lys, Leu, Met,
Thr,
Val, Trp, Tyr
X7 Negative Asp, Glu
XS Polar Cys, Asp, Glu, His,
Lys,
Asn, Gln, Arg, Ser,
Thr
X9 Positive His, Lys, Arg
X 10 Small Ala, Cys, Asp, Gly,
Asn,
Pro, Ser, Thr, Val
X11 Tiny Ala, Gly, Ser
X12 Turnlike Ala, Cys, Asp, Glu,
Gly,
His, Lys, Asn, Gln,
Arg,
Ser, Thr
X13 Asparagine-AspartateAsn, Asp
* Abbreviations as adopted from http://smart.embl-
heidelberg.de/SMART_DATA/alignments/consensus/grouping.html.
2. Overview
In certain aspects, the application relates to the discovery of novel
associations between POSH proteins and other proteins (termed POSH-APs), and
related methods and compositions. In certain aspects, the application relates
to
novel associations among certain disease states, POSH nucleic acids and
pxoteins,
and POSH-AP nucleic acids and proteins.
In certain aspects, by identifying proteins associated with POSH, and
particularly human POSH, the present application provides a conceptual link
between the POSH-APs and cellular processes and disorders associated with POSH
APs, and POSH itself. Accordingly, in certain embodiments of the disclosure,
agents that modulate a POSH-AP may now be used to modulate POSH functions
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and disorders associated with POSH function, such as viral disorders, POSH-
associated cancers, and POSH-associated neural disorders. Additionally, test
agents
may be screened for an effect on a POSH-AP and then further tested for an
effect on
a POSH function or a disorder associated with POSH function. Likewise, in
certain
embodiments of the disclosure, agents that modulate POSH may now be used to
modulate POSH-AP functions and disorders associated with POSH-AP function,
including a variety of cancers. Additionally, test agents may be screened for
an
effect on POSH and then further tested for effect on a POSH-AP function or a
disorder associated with POSH-AP function. In further aspects, the application
provides nucleic acid agents (e.g., RNAi probes, antisense nucleic acids),
antibody-
related agents, small molecules and other agents that affect POSH function,
and the
use of same in modulating POSH and/or POSH-AP activity.
POSH intersects with and regulates a wide range of key cellular functions
that may be manipulated by affecting the level of and/or activity of POSH
polypeptides or POSH-AP polypeptides. Many features of POSH, and particularly
human POSH, are described in PCT patent publications WO03/095971A2
(application no. W02002US0036366) and W003/078601A2 (application no.
-.
W02003US0008194) the teachings of which are incorporated by reference herein.
As described in the above-referenced publications, native human POSH is a
large polypeptide containing a RING domain and four SH3 domains. POSH is a
ubiquitin ligase (also termed an "E3" enzyme); the RING domain mediates
ubiquitination of, for example, the POSH polypeptide itself. POSH interacts
with a
large number of proteins and participates in a host of different biological
processes.
As demonstrated in this disclosure, POSH associates with a number of different
proteins in the cell. POSH co-localizes with proteins that are known to be
located in
the trans-Golgi network, implying that POSH participates in the trafficking of
proteins in the secretory system. The term "secretory system" should be
understood
as referring to the membrane compartments and associated proteins and other
molecules that are involved in the movement of proteins from the site of
translation
to a location within a vacuole, a compartment in the secretory pathway itself,
a
lysosome or endosome or to a location at the plasma membrane or outside the
cell.
Commonly cited examples of compartments in the secretory system include the
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endoplasmic reticulum, the Golgi apparatus and the cis and traps Golgi
networks. In
addition, Applicants have demonstrated that POSH is necessary for proper
secretion,
localization or processing of a variety of proteins, including phospholipase
D, HIV
Gag, HIV Nef, Rapsyn and Src. Many of these proteins are myristoylated,
indicating that POSH plays a general role in the processing and proper
localization
of myristoylated proteins. N-myristoylation is an acylation process, which
results in
covalent attachment of myristate, a 14-carbon saturated fatty acid to the N-
terminal
glycine of proteins (Farazi et al., J. Biol. Chem. 276: 39501-04 (2001)). N-
myristoylation occurs co-translationaly and promotes weak and reversible
protein-
membrane interaction. Myristoylated proteins are found both in the cytoplasm
and
associated with membrane. Membrane association is dependent on protein
configuration, i.e., surface accessibility of the myristoyl group may be
regulated by
protein modifications, such as phosphorylation, ubiquitination etc. Modulation
of
intracellular transport of myristoylated proteins in the application includes
effects on
transport and localization of these modified proteins.
As described herein, POSH and POSH-APs are involved in viral maturation,
including the production, post-translational processing, assembly and/or
release of
proteins in a viral particle. Accordingly, viral infections may be ameliorated
by
inhibiting an activity (e.g., ubiquitin ligase activity or target protein
interaction) of
POSH or a POSH-AP (e.g., inhibition of kinase activity or ubiquitin ligase
activity),
and in preferred embodiments, the virus is a retroid virus, an RNA virus or an
envelope virus, including HIV, Ebola, HBV, HCV, HTLV, West Nile Virus (WNV)
or Moloney Murine Leukemia Virus (MMuLV). Additional viral species are
described in greater detail below. In certain instances, a decrease of a POSH
function is lethal to cells infected with a virus that employs POSH in release
of viral
particles.
In certain aspects, the application describes an hPOSH interaction with Rac,
a small GTPase and the POSH associated kinases MLK, MKK and JNK. Rho, Rac
and Cdc42 operate together to regulate organization of the actin cytoskeleton
and the
MLK-MKK-JNK MAP kinase pathway (referred to herein as the "JNK pathway" or
"Rac-JNK pathway" (Xu et al., 2003, EMBO J. 2: 252-6I). Ectopic expression of
mouse POSH ("mPOSH") activates the JNK pathway and causes nuclear
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localization of NF-oB. Overexpression of mPOSH in fibroblasts stimulates
apoptosis. (Tapon et al. (1998) EMBO 3. 17:1395-404). In Drosophila, POSH may
interact with, or otherwise influence the signaling of, another GTPase, Ras.
(Schnorr et al. (2001) Genetics 159: 609-22). The JNK pathway and NF-~cB
regulate a variety of key genes involved in, for example, immune responses,
inflammation, cell proliferation and apoptosis. For example, NF-~cB regulates
the
production of interleukin 1, interleukin 8, tumor necrosis factor and many
cell
adhesion molecules. NF-KB has both pro-apoptotic and anti-apoptotic roles in
the
cell (e.g., in FAS-induced cell death and TNF-alpha signaling, respectively).
NF-KB
is negatively regulated, in part, by the inhibitor proteins IoBa and hcB(3
(collectively
termed "IrcB"). Phosphorylation of IKB permits activation and nuclear
localization
of NF-icB. Phosphorylation of IKB triggers its degradation by the ubiquitin
system.
In an additional embodiment, a POSH polypeptide promotes nuclear localization
of
NF-KB. In further embodiments, manipulation of POSH levels and/or activities
may
be used to manipulate apoptosis. By upregulating POSH or a POSH-AP, apoptosis
may be stimulated in certain cells, and this will generally be desirable in
conditions
characterized by excessive cell proliferation (e.g., in certain cancers). By
downregulating POSH or a POSH-AP, apoptosis may be diminished in certain
cells,
and this will generally be desirable in conditions characterized by excessive
cell
death, such as myocardial infarction, stroke, degenerative diseases of muscle
and
nerve (particularly Alzheimer's disease), and for organ preservation prior to
transplant. In a further embodiment, a POSH polypeptide associates with a
vesicular
trafficking complex, such as a clathrin- or coatomer- containing complex, and
particularly a trafficking complex that localizes to the nucleus and/or Golgi
apparatus.
As described in W003/078601A2 (application no. W02003US0008194),
POSH is overexpressed in a variety of cancers, and downregulation of POSH is
associated with a decrease in proliferation in at least one cancer cell line.
Accordingly, agents that modulate POSH itself or a POSH-AP may be used to
treat
POSH associated cancers. POSH associated cancers include those cancers in
which
POSH is overexpressed and/or in which downregulation of POSH leads to a
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decrease in the ,proliferation or survival of cancer cells. POSH-associated
cancers
are described in more detail below. In addition, it is notable that many
proteins
shown herein to be affected by POSH downregulation are themselves involved in
cancers. Phospholipase D and SRC are both aberrantly processed in a POSH-
impaired cell, and therefore modulation of POSH and/or a POSH-AP may affect
the
wide range of cancers in which PLD and SRC play a significant role.
As described in W003/095971A2 (application no. W02002US0036366) and
W003/078601A2 (application no. W02003US0008194), POSH polypeptides
function as E3 enzymes in the ubiquitination system. Accordingly,
downregulation
or upregulation of POSH ubiquitin ligase activity can be used to manipulate
biological processes that are affected by protein ubiquitination. Modulation
of
POSH ubiquitin ligase activity may be used to affect POSH-APs and related
biological processes, and likewise, modulation of POSH-APs may be used to
affect
POSH ubiquitin ligase activity and related processes. Downregulation or
1 S upregulation may be achieved at any stage of POSH formation and
regulation,
including transcriptional, translational or post-translational regulation. For
example,
POSH transcript levels may be decreased by RNAi targeted at a POSH gene
sequence. As another example, POSH ubiquitin ligase activity may be inhibited
by
contacting POSH with an antibody that binds to and interferes with a POSH RING
domain or a domain of POSH that mediates interaction with a target protein (a
protein that is ubiquitinated at least in part because of POSH activity). As a
further
example, small molecule inhibitors of POSH ubiquitin ligase activity are
provided
herein. As another example, POSH activity may be increased by causing
increased
expression of POSH or an active portion thereof. POSH, and POSH-APs that
modulate POSH ubiquitin ligase activity may participate in biological
processes
including, far example, one or more of the various stages of a viral
lifecycle, such as
viral entry into a cell, production of viral proteins, assembly of viral
proteins and
release of viral particles from the cell. POSH may participate in diseases
characterized by the accumulation of ubiquitinated proteins, such as demential
(e.g.,
Alzheimer's and Piclc's), inclusion body myositis and myopathies, polyglucosan
body myopathy, and certain forms of amyotrophic lateral sclerosis. POSH may
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participate in diseases characterized by excessive or inappropriate
ubiquitination
and/or protein degradation.
3. POSH Associated Proteins
In certain aspects, the application relates to the discovery of novel
associations between POSH proteins and other proteins (termed POSH-APs), and
related methods and compositions. In certain aspects, the application relates
to
novel associations among certain disease states, POSH nucleic acids and
proteins,
and POSH-AP nucleic acids and proteins. POSH-APs may interact either directly
or
indirectly with POSH. In certain embodiments, a POSH-AP binds directly to a
POSH polypeptide.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with one subunit of Protein Kinase A (PISA; CAMP-
dependent
protein kinase). In one aspect, the application relates to the discovery that
POSH
binds directly with PRKARlA. This interaction was identified by Applicants in
a
yeast 2-hybrid assay. Exemplary PKA subunits may include, but are not limited
to,
a regulatory subunit (e.g., PI~K.AR1A) and a catalytic subunit (e.g., PRI~ACA
or
PRKACB). PKA is an essential enzyme in the signaling pathway of the second
messenger cyclic AMP (CAMP). Through phosphorylation of target proteins, PISA
controls many biochemical events in the cell including regulation of
metabolism, ion
transport, and gene transcription. The PKA holoenzyme is composed of two
regulatory and two catalytic subunits and dissociates from the regulatory
subunits
upon binding of CAMP. The PKA enzyme is inactive in the absence of cAMP.
Activation of PKA occurs when two cAMP molecules bind to each regulatory
subunit, eliciting a reversible conformational change that releases active
catalytic
subunits.
A number of human PKA subunits have been characterized, including a
regulatory subunit (type I alpha: PRKAR1) and two catalytic subunits (C-alpha:
PRKA.CA; and C-beta: PRKACB). Boshart et al. identified the regulatory
subttnit
PRKAR1 of PKA as the product of the TSE1 locus (Boshart, M et al. (1991) Cell
66: 849-859). The evidence consisted of concordant expression of PRKAR1 mRNA
and TSE 1 g enetic activity, high r esolution physical mapping of the two g
enes on
human chromosome 17, and the ability of transfected PRKAR1 cDNA to generate a
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phenocopy of TSE1-mediated extinction. Jones et al. independently established
identity of TSE1 and the RI-alpha submit (Jones, KW et al. (1991) Cell 66: 861-
872).
Other than a role of PKA in metabolism, PKA subunits have recently been
implicated in multiple diseases. For example, a specific role for localized
PRKAR1
has been demonstrated in human T lymphocytes, where type I PKA localizes to
the
activated TCR complex and is required for attenuation of signals propagated
through
this complex (Skalhegg, BS et al. (1992) J Biol Chem 267:15707-15714;
Skalhegg,
BS et al. (1994) Science 263: 84-87). The importance of type I PKA-mediated
effects in attenuation of T cell replication has led to its consideration as a
therapeutic
target in combined variable immunodeficiency (CVI) and acquired immune
deficiency syndrome (AIDS). Furthermore, type I PKA in T cells may also serve
as
a potential therapeutic target in systemic lupus erythematosis (SLE). For
example, a
series of recently published articles has uncovered the first human disease
mapping
to a PKA subunit-Carnet' complex (Casey, M et al. (2000) J Clin Invest 106:
R31-
38; Kirschner, LS et al. (2000) Nat Genet 26: 89-92). Carnet' complex (CNC) is
a
multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac
and
skin myxomas, endocrine tumors, and psammomatous melanotic schwannomas.
CNC maps to two genomic loci, 17q24 and 2p16. Familial cases mapping to the
17q24 locus reveal deletions/mutations in the PRKARl coding exons leading to
frameshifts and premature stop codons-no mRNA and protein from the mutant
alleles has been observed.
Accordingly, in certain aspects of the present disclosure, POSH participates
in the formation of PKA complexes, including human PKA-containing complexes.
Certain P OSH p olypeptides m ay b a i nvolved i n d isorders o f t he i mmune
s ystem,
e.g., autoimmune disorders. Certain POSH polypeptides may be involved in the
regulation of T-cell activation. In certain aspects, POSH participates in the
ubiquitination of PI3K. In certain aspects, PKA subunit polypeptides
participate in
POSH-mediated processes.
Additionally, the disclosure relates in part to the discovery that PKA
phosphorylates POSH, and further, that this phosphorylation inhibits the
interaction
of POSH with small GTPases, such as Rac. Small GTPases are important in
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vesicular trafficking, and therefore the findings disclosed herein demonstrate
that
POSH phosphorylation regulates the formation of complexes between POSH and
proteins involved in the secretory system, such as Rac, TCL, TC10, Cdc42, Wrch-
l,
Rac2, Rac3 or RhoG. Applicants have shown that inhibition of PKA and POSH has
similar effects, indicating that inhibition of PKA will achieve an effect
similar to
that of inhibition of POSH. However, given the effect of PKA on POSH
interaction
with proteins in the secretory pathway, it is expected that PKA regulates the
timing
of cyclical interactions that are needed to effect vesicular trafficking.
Accordingly,
it is expected that significant inhibition or activation of PKA will cause a
disruption
in POSH function.
The term "PKA subunit" is used herein to refer to a full-length human PKA
subunit which includes a regulatory subunit (e.g., PRKAR1A) and a catalytic
subunit (e.g., PRI~ACB or PRI~ACA), as well as an altern tive PKA subunit
composed of separate PKA subunit sequences (e.g., nucleic acid sequences) that
may be a splice variant. The term "PKA subunit" is used herein to refer as
well to
various naturally occurring PISA subunit homologs, as well as functionally
similar
variants and fragments that retain at least 80%, 90%, 95%, or 99% sequence
identity
to a naturally occurring PKA subunit (e.g., SEQ ID NOs: 264-265, 111-122, 395
396). The term specifically includes human PKA subunit nucleic acid and amino
acid sequences and the sequences presented in Figure 36.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with human UNC84B, a human homolog of C. elegans Unc-
84. Accordingly, the application provides complexes comprising POSH and
UNC84B. In one aspect, the application relates to the discovery that POSH
binds
directly with UNC84B. This interaction was identified by Applicants in a yeast
2-
hybrid assay. In C. elegans, Unc-84 is involved in the cellular positioning of
the
nucleus. UNC84/SUN is positioned at the nuclear membrane and recruits
SyneIANC-1, which directly tethers the nuclear envelope to the actin
cytoskeleton.
Accordingly, in certain aspects, POSH participates in formation of a UNC84
complexes, including human UNC84B-containing complexes, and in the
connections between the nucleus and the cytoskeleton. In certain aspects,
UNC84
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polypeptides participate in POSH-mediated processes. See, for example, Starr
and
Han, 2003, J Cell Sci 116(Pt 2):211-6.
The term UNC84 is used herein to refer to various naturally occurnng Unc-
84 homologs, a s well as functionally similar variants and fragments that
retain a t
least 8 0%, 9 0%, 9 5%, o r 9 9% se quence i dentity t o a n aturally o
ccurring UNC84
(e.g., SEQ ID NOs: 314, 211-213 ). The term specifically includes human UNC84B
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with human GOCAPl. Certain GOCAPl polypeptides are
cytoplasmic proteins associated with the Golgi complex. Accordingly, the
application provides complexes comprising POSH and GOCAP1. In one aspect, the
application relates to the discovery that POSH binds directly with GOCAPl.
This
interaction was identified by Applicants in a yeast 2-hybrid assay. In certain
aspects, these complexes associate with the Golgi complex. GOCAPl is
synonymous with GCP60. Certain GCP60 polypeptides interact with the Golgi
complex integral membrane protein, giantin. Certain GCP60 polypeptides are
involved in the maintenance of the Golgi structure through interaction with
giantin
and affect protein transport between the endoplasmic reticulum and the Golgi
complex (Sohda, M, et al. (2001) J Biol Chem 276:45298-306). In certain
aspects,
GOCAP1 polypeptides participate in POSH-mediated processes.
The term GOCAP1 is used herein to refer to various naturally occurring
GOCAP1 homologs, as well as functionally similar variants and fragments that
retain at least 80%, 90%, 95%, or 99% sequence identity to a naturally
occurring
GOCAP1 (e.g., SEQ ID NOs: 240-243, 61-68). The term specifically includes
human GOCAP1 nucleic acid and amino acid sequences and the sequences
presented in Figure 36.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with human PTPN12, a protein tyrosine phosphatase.
Accordingly, t he application p rovides c omplexes c omprising P OSH a nd
PTPN12.
In one aspect, the application relates to the discovery that POSH binds
directly with
PTPN12_ This interaction was identified by Applicants in a yeast 2-hybrid
assay.
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PTPN12 polypeptides are synonymous with the protein tyrosine phosphatase, PTP-
PEST. PTP-PEST polypeptides contain proline-rich sequences and are rich in
proline, glutamate, serine, and threonine residues at their carboxyl terminus,
features
characteristic of PEST motifs. Certain PTP-PEST polypeptides interact with
paxillin, a scaffolding protein to which focal adhesion proteins bind, leading
to the
formation of the focal adhesion contact (Shen, Y et al. (1998) J Biol Chem
273:6474-81). Certain PTP-PEST polypeptides associate with the focal adhesion
protein, p130cas (Garton, AJ et al. (1997) Oncogene 15:8?7-85). Certain PTP-
PEST polypeptides have also been shown to associate with JAK2, PSTPIP and
WASP, gelsolin, cell adhesion kinase beta, Csk, Hef 1 or Sin , Hic-S, or Shc
(See,
for example, Horsch, et aI (2001) Mol Endocrinol 15:2182-96; Cote, et al
(2002) J
Biol Chem 277:2973-86; Chellaiah, et al (2001) J Biol Chem 276:47434-44;
Lyons,
et al (2001) J Biol Chem 276:24422-31; Davidson, et al (1997) J Biol Chem
21:1077-88; Cote, JF et al (1998) Biochemistry 37:13128-37; Nishiya, N (1999)
J
Biol Chem 274:9847-53; Habib, T et al (1994) J Biol Chem 269:25243-6). Certain
PTP-PEST polypeptides are involved in inactivation of the Ras pathway
(Davidson,
D and Veillette, A (2001) EMBO J 20:3414-26). The expression level of certain
PTP-PEST polypeptides can modulate the activity of the GTPase, Racl (Sastry,
et al
(2002) J Cell Sci 115(Pt 22): 4305-16). Certain PTP-PEST polypeptides are
involved in the regulation of cell motility (Garton, AJ and Tonks, NIA. (1999)
J Biol
Chem 274:3811-8; Angers-Loustau, et al (1999) J Cell Biol 144:1019-31; and
Sastry, et al. (2002) J Cell Sci 155(Pt 22): 4305-16). Accordingly, certain
POSH
polypeptides are involved in inactivation of the Ras pathway. Certain POSH
polypeptides are involved in the regulation of cell motility.
Certain PTP-PEST polypeptides are involved in amyloid(3-induced neuronal
dystrophy, a pathological hallmark of Alzheimer's disease (Grace, EA and
Busciglio, J (2003) J Neurosci. 23:493-502). Accordingly, certain POSH
polypeptides may be involved in Alzheimer's disease. Certain PTP-PEST
polypeptides function as negative regulators of lymphocyte activation
(Davidson, D
and Veillette, A (2001) EMBO J 20:3414-26). Accordingly, certain POSH
polypeptides may be involved in the regulation of lymphocyte activation. In
certain
aspects, PTPN12 polypeptides participate in POSH-mediated processes.
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The term PTPN12 is used herein to refer to various naturally occurnng
PTPN12 homologs, as well as functionally similar variants and fragments that
retain
at least 80%, 90%, 9S%, or 99% sequence identity to a naturally occurring
PTPN12
(e.g., SEQ ID NOs: 266-268, 123-129). The term specifically includes human
PTPN12 nucleic acid and amino acid sequences and the sequences presented in
Figure 36.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with HERPUD1, a "homocysteine-inducible, endoplasmic
reticulum stress-inducible, ubiquitin-like domain member 1" protein.
Accordingly,
the application provides complexes comprising POSH and HERPUD 1. In one
aspect, the application relates to the discovery that POSH binds directly with
HERPUDl. This interaction was identified by Applicants in a yeast 2-hybrid
assay.
HERPUD1 is synonymous with Herp. In part, the present application relates to
the
discovery that a POSH-AP, HERPUDl, is involved in the maturation of an
envelope
1S virus, such as HIV.
Certain HERPUDI polypeptides are involved in JNK-mediated apoptosis,
particularly in vascular endothelial cells, including cells that are exposed
to high
levels of homocysteine. Certain HERPUDl polypeptides are involved in the
Unfolded Protein Response, a cellular response to the presence of unfolded
proteins
in the endoplasmic reticulum. Certain HERPUD1 polypeptides are involved in the
regulation of sterol biosynthesis. Accordingly, certain POSH polypeptides are
involved in the Unfolded Protein Response and sterol biosynthesis.
In other aspects, certain HERPUDl polypeptides enhance presenilin-
mediated amyloid (3-protein generation. For example, HERPUD1 polypeptides,
2S when overexpressed in c ells, increase the level of amyloid (3 generation,
and it is
observed that HERPUD 1 polypeptides interact With the presenilin proteins,
presenilin-1 and presenilin-2. (See Sai, X. et al (2002) J. Biol. Chem.
277:12915-
12920). Accordingly, in certain aspects, POSH polypeptides may modulate the
level
of amyloid (3 generation. Additionally, POSH polypeptides may interact with
presenilin 1 and presenilin 2. Therefore, it is believed certain POSH
polypeptides
modulate nresenilin-mediated amyloid (3 generation. The accumulation of
amyloid
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(3 is one hallmark of Alzheimer's disease. Accordingly, these POSH
polypeptides
may be involved in the pathogenesis of Alzheimer's disease. At sites such as
late
.intracellular compartment sites including the traps-Golgi network, certain
mutant
presenilin-2 polypeptides up-regulate production of amyloid (3 peptides ending
at
position 42 (A(342). (See Iwata, H. et al (2001) J. Biol. Chem. 276: 21678-
21685).
Accordingly, POSH polypeptides regulate production of A(342 through mutant
presenilin-2 at late intracellular compartment sites including the traps-Golgi
network. Furthermore, elevated homocysteine levels have been found to be a
risk
factor associated with Alzheimer's disease and cerebral vascular disease. Some
risk
factors, such as elevated plasma homocysteine levels, may accelerate or
increase the
severity of several central nervous system (CNS) disorders. Elevated levels of
plasma homocysteine were fond in young male patients with schizophrenia
suggesting that elevated homocysteine levels could be related to the
pathophysiology of aspects of schizophrenia (Levine, J. et al (2002) Am. J.
Psychiatry 159:1790-2). Accordingly, certain POSH polypeptides may be involved
in neurological disorders. Neurological disorders include disorders associated
with
increased levels of plasma homocysteine, increased levels of amyloid [3
production,
or aberrant presenilin acitivity. Neurological disorders include CNS
disorders, such
as Alzheimer's disease, cerebral vascular disease and schizophrenia. Certain
POSH
polypeptides may be involved in cardiovascular diseases, such as
thromboembolic
vascular disease, and particularly the disease characteristics associated with
hyperhomocysteinemia. See, for example, Kokame et al. 2000 J. Biol. Chem.
275:32846-53; Zhang et al. 2001 Biochem Biophys Res Commun 289:718-24.
The t erm H ERPUD 1 i s used h erein t o r efer t o v arious n aturally o
ccurnng
HERPUD1 homologs, as well as functionally similar variants and fragments that
retain at least 80%, 90%, 95%, or 99% sequence identity to a naturally
occurring
HERPUD1 (e.g., SEQ ID NOs: 249-252, 77-86). The term specifically includes
human HERPUDl nucleic acid and amino acid sequences and the sequences
presented in Figure 36.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with one or more Cbl-b polypeptides. Accordingly, the
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application provides complexes comprising POSH and Cbl-b. In one aspect, the
application relates to the discovery that POSH binds directly with Cbl-b. This
interaction was identified by Applicants in a yeast 2-hybrid assay. Cbl-b
polypeptides contain an amino-terminal variant SH2 domain, a RING finger, and
a
carboxyl-terminal proline-rich domain with potential tyrosine phosphorylation
sites.
Cbl-b is highly homologous to the mammalian Cbl and the nematode Sli-1
proteins.
This application provides four Cbl-b variants and shows that the POSH
polypeptide
interacts with one or more of these variants. In one aspect, the POSH
polypeptide
interacts with a human Cbl-b (IlniGene No.: Hs.3144). In another aspect, the
POSH
,polypeptide i nteracts w ith an a lternative h uman Cbl-b ( UniGene N o.: H
s.381921)
that may be a splice variant of Cbl-b. In yet another aspect, the POSH
polypeptide
interacts with a human Cbl-b polypeptide that is a splice variant represented
by the
amino acid sequence depicted in SEQ ID NO: 361, which is encoded by the
nucleic
acid sequence depicted in SEQ ID NO: 359. In yet another aspect, the POSH
polypeptide interacts with a human Cbl-b polypeptide that is a splice variant
represented by the amino acid sequence depicted in SEQ ~ NO: 398, which is
encoded by the nucleic acid sequence depicted in SEQ ID NO: 360.
Certain Cbl-b polypeptides have been shown to function as adaptor proteins
by interacting with other signaling molecules, e.g., interaction with cell
surface
receptor tyrosine kinases, e.g., EGFR (Ettenberg, SA et al (2001) J Biol Chem
276:77-84) or with proteins such as Syk (Elly, C et al (1999) Oncogene 18:1147-
56), Crk-L (Elly, C et al (1999) Oncogene 18:1147-56), PI3K (Fang, D et al.
(2001)
J Biol Chem 16:4872-8), Grb2 (Ettenberg, SA et al (1999) Oncogene 18:1855-66),
or Vav (Bustelo, XR et al. (1997) Oncogene 15:2511-20). Certain Cbl-b
polypeptides have been demonstrated to interact directly with the nucleotide
exchange f actor, V av ( Bustelo, X R a t a 1. ( 1997) O ncogene 15:2511-20).
C attain
Cbl-b polypeptides have been shown to function as an E3 ubiquitin ligase that
recognizes tyrosine phosphorylated substrates through its SH2 domain and
through
its RING domain, recruits a ubiquitin-conjugating enzyme, E2 (Joazeiro, C et
al.
(1999) S cience 2 86:309-312) A dditionally, certain C bl-b p olypeptides have
b een
shown to associate directly with the p85 subunit of PI3K and to function as an
E3
ligase in the ubiquitination of PI3K (Fang, D et al. (2001) J Biol Chem
16:4872-8).
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Certain Cbl-b polypeptides are negative regulators of T-cell activation. Cbl-
b-deficient mice become very susceptible to experimental autoimmune
encephalomyelitis (Chiang, YJ et al. (2000) Nature 403:216-220). Also, Cbl-b-
deficient mice develop spontaneous autoimmunity (Bachmaier, K, et al (2000)
Nature 403:211-216). Furthermore, Cbl-b is a major susceptibility gene for rat
type
1 diabetes mellitus (Yokoi, N et al (2002) Nature Genet. 31:391-394).
Accordingly, in certain aspects, POSH participates in the formation of Cbl-b
complexes, including human Cbl-b-containing complexes. Certain POSH
polypeptides may be involved in disorders of the immune system, e.g.,
autoimmune
disorders. Certain POSH polypeptides may be involved in the regulation of T-
cell
activation. In certain aspects, POSH participates in the ubiquitination of
PI3K. In
certain aspects, Cbl-b polypeptides participate in POSH-mediated processes.
The term Cbl-b is used herein to refer to full-length, human Cbl-b (UniGene
No.: Hs.3144) as well as an alternative Cbl-b (UniGene No.: Hs.381921)
composed
of two separate Cbl-b sequences (e.g., nucleic acid sequences) that may be a
splice
variant. The term Cbl-b is used herein to refer as well to the human Cbl-b
splice
variant represented by the amino acid sequence of SEQ ID NO: 361, which is
encoded by the nucleic acid sequence of SEQ ID NO: 359 and to the human Cbl-b
splice variant represented by the amino acid sequence of SEQ ID NO: 398, which
is
encoded by the nucleic acid sequence of SEQ ID NO: 360. The term Cbl-b is used
herein to refer as well to various naturally occurring Cbl-b homologs, as well
as
functionally similar variants and fragments that retain at least 80%, 90%,
95%, or
99% sequence identity to a naturally occurring Cbl-b (e.g., SEQ ID NOs: 361,
398,
227-230, 353-360 ). The term specifically includes human Cbl-b nucleic acid
and
amino acid sequences and the sequences presented in Figure 36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with GOSR2. Accordingly, the application provides
complexes comprising POSH and GOSR2. In one aspect, the application relates to
the discovery that POSH binds directly with GOSR2. This interaction was
identified by Applicants in a yeast 2-hybrid assay. Certain GOSR2 polypeptides
are
synonymous with GS27 (for Golgi SNARE of 27K) and are involved in trafficking
membrane proteins between the endoplasmic reticulum and the Golgi and between
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Golgi subcompartments such as between the cis-,medial- and trans-Golgi
network.
(See, for example, Lowe, SL et al (1997) Nature 389:881-4 and Bui, TD et al
(1999)
57:285-8). Accordingly, certain POSH polypeptides are involved in the
trafficking
of membrane proteins between the endoplasmic reticulum and the Golgi and
between Golgi subcompartments.
The term GOSR2 is used herein to refer to various naturally occurring
GOSR2 homologs, as well as functionally similar variants and fragments that
retain
at least 80%, 90%, 95%, or 99% sequence identity to a naturally occurring
GOSR2
(e.g., SEQ ID NOs: 244-248, 69-76). The term specifically includes human GOSR2
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with RALA. Accordingly, the application provides
complexes
comprising POSH and RALA. W one aspect, the application relates to the
discovery
that POSH binds directly with RALA. This interaction was identified by
Applicants
in a yeast 2-hybrid assay. RALA polypeptides are GTP-binding polypeptides.
RALA polypeptides are members of the Ras family of proteins and are GTPases.
Certain RALA polypeptides may be synonymous with RalA polypeptides. RaIA
polypeptides are small GTPases. RaIA polypeptides have been shown to interact
with phospholipase D and to effect phospholipase D activity. Additionally,
RaIA
polypeptides may be involved in tumor formation and cell transformation. (See,
for
example, Kim, JH et al (1998) FEBS Lett 430:231-5; Aguirre-Ghiso, JA et al
(1999)
Oncogene 18:4718-25; Lu, Z et al (2000) Mol Cell Biol 20:462-7; Gildea, JJ et
al
(2002) Cancer Res 62:982-5; Lucas, L et al (2002) Int J Oncol 21:477-85; and
Xu, L
et al (2003) Mol Cell Biol 23:645-54). Accordingly, certain POSH polypeptides
may i nteract w ith P LD and m odulate i is activity, and certain P OSH p
olypeptides
may be involved in tumor formation and cell transformation. In other aspects,
certain RaIA polypeptides interact with calmodulin and may be involved in
calcium/calmodulin-mediated intracellular signaling pathways (Clough, RR et al
(2002) J Biol Chem 277:28972-80). Certain RaIA polypeptides are involved in
controlling actin cytoskeletal remodeling and vesicle transport in mammalian
cells.
Certain RaIA polypeptides interact with the exocyst complex, which is involved
in
exocytosis. (See, for example, Sugihara, K et al (2002) Nat Cell Biol 4:73-8;
Polzin,
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A et al (2002) Mo1 Cell Biol 22:1714-22; and Lipschutz, JH and Mostov, KE
(2002)
Curr Biol 12(6):8212-4). Accordingly, certain POSH polypeptides are involved
in
vesicle transport.
The term RALA is used herein to refer to various naturally occurring RALA
homologs, as well as functionally similar variants and fragments that retain
at Ieast
80%, 90%, 95%, or 99% sequence identity to a naturally occurring RALA (e.g.,
SEQ ID NOs: 269-270, 130-134). The term specifically includes human RALA
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with SMN1. Accordingly, the application provides
complexes
comprising POSH and SMNI. In one aspect, the application relates to the
discovery
that POSH binds directly with SMNl. This interaction was identified by
Applicants
in a yeast 2-hybrid assay. SMN1 polypeptides are encoded by the nucleic acid
of
the survival motor neuron gene 1 (SMN1). Mutations in this gene (such as its
homozygous absence) cause spinal muscular atrophy (SMA), a common autosomal
recessive disorder characterized by degeneration of motor neurons in the
spinal cord,
leading to progressive paralysis with muscular atrophy. Accordingly, POSH may
be
involved in the pathogenesis of SMA. SMNl is part of a multiprotein complex
that
is required for biogenesis of the Sm class of small nuclear ribonucleoproteins
(Sm
snRNPs). SMNl associates with a number of proteins, such as Gemin2 to Gemin6,
to form a large complex found in both the cytoplasm and in the nucleus. SMN1
also
associates with Snurportin I, an adaptor protein that recognizes the nuclear
localization signal of Sm snRNPs. (See, for example, Lefebvre, S et al (1995)
Cell
80:155-65; Narayanan, U et a1 (2002) Hum Mol Genet 11:1785-95; Massenet, S et
al
(2002) 22:6533-41; and Monani, UR et al (1999) Hum Mol Genet 8:1177-83).
Accordingly, certain POSH polypeptides may be involved in the biogenesis of
snRNPs. Certain SMN1 polypeptides interact with the large nonstnictural
protein
NS1 of the autonomous parvovirus minute virus of mice (MVM). NS1 is essential
for viral replication, and it is a potent transcriptional activator (Young, PJ
et al
(2002) J Virol 76:3892-904). Certain SMNl polypeptides interact with the
protein
NS2 of MVM. NS2 is also required for efficient viral replication. Certain SMNl
polypeptides colocalize with NS2 in infected nuclei and at late times
following
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MVM infection. (See Young, PJ et al (2002) J Virol 76:6364-9). Accordingly,
POSH polypeptides are involved in viral replication.
The term SMN1 is used herein to refer to various naturally occurring SMN1
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurring SMNl (e.g.,
SEQ ID NOs: 273-275, 142-146). The term specifically includes human SMN1
nucleic acid and amino acid sequences and the sequences~presented in Figure
36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with SMN2. Accordingly, the application provides
complexes
comprising POSH and SMN2. In one aspect, the application relates to the
discovery
that POSH binds directly with SMN2. This interaction was identified by
Applicants
in a yeast 2-hybrid assay. The SMN2 gene is an almost identical copy of the
SMNl
gene that causes SMA. A critical difference between the two genes is a 1
nucleotide
base change inside exon 7 that affects the splicing pattern of the genes. The
majority of the SMN2 transcript lacks exon 7. Certain SMN2 polypeptides
influence the severity of SMA. (See, for example, Monani, UI~ et al (1999) Hum
Mol Genet 8: 1177-83; Cartegni, L and Krainer, AR (2002) Nat Genet 30:377-84;
and Feldkotter, M et al (2002) Am J Hum Genet 70: 358-68). Accordingly,
certain
POSH polypeptides rnay influence the severity of SMA.
The term SMN2 is used herein to refer to various naturally occurnng SMN2
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurring SMN2 (e.g.,
SEQ ID NOs: 276-280, 147-151). The term specifically includes human SMN2
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with SIAH1. Accordingly, the application provides
complexes
comprising POSH and SIAH1. In one aspect, the application relates to the
discovery
that POSH binds directly with SIAHl. This interaction was identified by
Applicants
in a yeast 2-hybrid assay. Certain SIAH1 polypeptides bind ubiquitin-
conjugating
enzymes and target proteins for proteasome-mediated degradation. Certain SIAH1
polypeptides are involved in targeting beta-catenin for degradation
(Matsuzawa, S
JC (2001) Molec Cell 7: 915-926 and Liu, J et al (2001) Molec Cell 7:
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927-936). Accordingly, certain POSH polypeptides are involved in the targeting
of
beta-catenin for degradation. Certain SIAH1 polypeptides are E3 ubiquitin
ligases
and regulate the ubiquitination and degradation of synaptophysin (Wheeler, TC
et al.
(2002) J Biol Chem 277: 10273-92). Accordingly, certain POSH polypeptides are
involved in the ubiquitination and degardation of synaptophysin. Certain SIAH1
polypeptides regulate the protein, DCC (deleted in colorectal cancer), via the
ubiquitin-proteosome pathway (Hu, G et al. (1997) Genes Dev 11: 2701-14).
Accordingly, certain POSH polypeptides are involved in the ubiquitination and
degardation of DCC. Certain SIAH1 polypeptides are a target of activation of
p53
and are upregulated by p53, and certain SIAHl polypeptides are involved in
apoptosis, tumor suppression, as well as vertebrate development (Maeda, A et
al
(2002) FEBS Lett 512: 223-226; Hu, G et a1 (1997) Genomics 46:103-111; and
Nemani, M a t al ( 1996) P roc N atl A cad S ci U SA 9 3: 9 039-9042).
Accordingly,
certain POSH polypeptides may be a target of p53 activation, and certain POSH
polypeptides may be involved in apoptosis and tumor suppression.
The term SIAH1 is used herein to refer to various naturally occurring SIAH1
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurring SIAHl (e.g.,
SEQ >D NOs: 271-272, 135-141). The term specifically includes human SIAH1
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with SYNE1. Accordingly, the application provides
complexes comprising POSH and SYNE1. In one aspect, the application relates to
the discovery that POSH binds directly with SYNE1. This interaction was
identified
by Applicants in a yeast 2-hybrid assay. SYNE1 ,polypeptides are synonymous
with
Syne-1, myne-1, and nesprin-1 polypeptides. Syne-1 polypeptides are associated
with nuclear envelopes in skeletal, cardiac, and smooth muscle cells. Syne-1
polypeptides contain multiple spectrin repeats. In muscle, myne-1 expression
is
observed in the inner nuclear envelope, and myne-1 has been shown to interact
with
the inner nuclear membrane protein lamin A/C. Syne-1 also associates with the
nuclear envelope protein, emerin. Syne-I polypeptides may be involved in
maintaining nuclear organization and structural integrity, and certain Syne-1
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polypeptides may be involved in the migration of myonuclei in myotubes and/or
their anchoring at the postsynaptic apparatus. (See, for example, Apel et al
(2000) J
Biol Chem 275:31986-95; Zhang, Q et al (2001) J Cell Sci 114:4485-98; Zhang, Q
et al (2002) Genomics 80:473-81; and Mislow, JM et al (2002) J Cell Sci 115
(Pt
1):61-70). Accordingly, certain POSH polypeptides may interact with the lamin
A/C polypeptides and/or emerin polypeptides. Also, certain POSH polypeptides
may 'be involved in maintaining nuclear organization and structural integrity,
and
certain POSH polypeptides may be involved in the migration of myonuclei in
myotubes and/or their anchoring at the postsynaptic apparatus.
The term SYNE1 is used herein to refer to various naturally occurring
SYNE1 homologs, as well as functionally similar variants and fragments that
retain
at least 80%, 90%, 95%, or 99% sequence identity to a naturally occiu-ring
SYNEl
(e.g., SEQ ID NOs: 295-307, 183-201). The term specifically includes human
SYNE1 nucleic acid and amino acid sequences and the sequences presented in
Figure 36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with TTC3. Accordingly, the application provides
complexes
comprising POSH and TTC3. In one aspect, the application relates to the
discovery
that POSH binds directly with TTC3 This interaction was identified by
Applicants
in a yeast 2-hybrid assay. Certain TTC3 polypeptides are synonymous with the
proteins, TPRDI, TPRDII, TRPDIII, TPRD and DCRRl and may be involved in the
pathogenesis o f certain characteristics o f D own syndrome, s uch a s m
orphological
features, hypotonia, and mental retardation (Tsukahar, F et al (1996) J
Biochem
(Tokyo) 120: 820-827; Ohira, M et al (1996) DNA Res 3: 9-16; Dahmane, N et al
(1998) Genomics 48: 12-23; and Eki, T et al (1997) DNA Seq 7:153-164).
The term TTC3 is used herein to refer to various naturally occurring TTC3
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurring TTC3 (e.g.,
SEQ
ID NOs: 308-312, 202-207). The term specifically includes human TTC3 nucleic
acid and amino acid sequences and the sequences presented in Figure 36.
In certain embodiments, the application relates to the discovery that a POSH
nc,lvnentide interacts with VCY2IP1. Accordingly, the application provides
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complexes comprising POSH and VCY2IP1. In one aspect, the application relates
to the discovery that POSH binds directly with VCY2IP1. This interaction was
identified by Applicants in a yeast 2-hybrid assay. VCY2IP1 is synonymous with
VCYZIP-1, which has been shown to interact with the testis-specific protein,
VCY2.
VCY21P1 is also synonymous with Cl9orf5, which has been shown to interact with
the tumor suppressor, RASSF1, suggesting a role for Cl9orf5 in apoptosis and
tumor suppression (In Vitro Cell Dev Biol Anim (2002) 38:582-94). Cl9orf5 also
demonstrates a strong homology to microtubule-associated proteins (Genomics
(2002} 79:124-6). Accordingly, POSH may play a role in apoptosis and tumor
suppression.
The term VCY2IP1 is used herein to refer to various naturally occurring
VCY2IP1 homologs, as well as functionally similar variants and fragments that
retain at least 80%, 90%, 95%, or 99% sequence identity to a naturally
occurring
VCY2IP1 (e.g., SEQ ID NOs: 315-323, 214-222). The term specifically includes
human VCY2IP1 nucleic acid and amino acid sequences and the sequences
presented in Figure 36.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with MSTP028. In one aspect, the application relates to
the
discovery that POSH binds directly with MSTP028. This interaction was
identified
by Applicants in a yeast 2-hybrid assay. In part, the present application
relates to the
discovery that a POSH-AP, MSTP028, is involved in the maturation of an
envelope
virus, such as HIV. Certain MSTP028 polypeptides contain one or more BTB/POZ
domains that are g enerally involved in dimerization. Accordingly t he
application
provides complexes comprising POSH and MSTP028, optionally in a dimeric form.
The term MSTP028 is used herein to refer to various naturally occurring
MSTP028
homologs, as well as fimctionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurring MSTP028
(e.g.,
SEQ ID NOs: 255-256, 9 0-94). The term specifically includes human M STP028
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with SNX1. Accordingly, the application provides
complexes
comprising POSH and SNX1. In one aspect, the application relates to the
discovery
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WO 2004/078130 PCT/US2004/006308
that POSH binds directly with SNXl. This interaction was identified by
Applicants
in a yeast 2-hybrid assay. SNXl is a member of the sorting nexin (SNX) protein
family, which is implicated in regulating membrane traffic. SNXI is a membrane
associated p rotein t hat h as b een s hown t o b a i evolved w ith t argeting
r eceptors t o
lysosomal degradation. SNX1 has been shown to bind to the C-terminal tail of
the
DS dopamine receptor (Mol Cell Biol (1998) 18: 7278-87). Accordingly, in
certain
aspects POSH may associate with the DS dopamine receptor. SNX1 is involved in
regulating the targeting of internalized epidermal growth factor receptors for
lysosomal d egradation ( Science ( 1996) 2 72:1008-1010). In c ertain a
spects, P OSH
may be involved in targeting proteins for degradation to the lysosome. SNX1
has
also been found to be involved in sorting PAR1, a G-protein coupled receptor
for
thrombin (Mol Cell Biol (2002) 13:1965-76). It has further been demonstrated
that
SNXl functions in regulating trafficking in the endosome compartment via
recognition of phosphorylated phosphatidylinositol through the phox homology
domain (PX domain} of SNXl (Proc Natl Acad Sci (2002) 99:6767-72).
The term SNXI is used herein to refer to various naturally occm-ring SNXl
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurnng SNXl (e.g.,
SEQ
ID NOs: 281-286, 152-161). The term specifically includes human SNX1 nucleic
acid and amino acid sequences and the sequences presented in Figure 36.
In additional embodiments, the application relates to the discovery that a
POSH polypeptide interacts with SNX3. Accordingly, the application provides
complexes comprising POSH and SNX3. In one aspect, the application relates to
the discovery that POSH binds directly with SNX3. This interaction was
identified
by Applicants in a yeast 2-hybrid assay. SNX3 is also a member of the SNX
protein
family. SNX3 has been shown to associate with the early endosome through its
PX
domain, a domain capable of interaction with phosphatidylinositol-3-phosphate
(Nat
Cell Biol (2002) 3:658-66). Accordingly, POSH may be involved in membrane
traffic at the early endosome.
The term SNX3 is used herein to refer to various naturally occurring SNX3
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurnng SNX3 (e.g.,
SEQ
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ID NOS: 287-290, 162-174). The term specifically includes human SNX3 nucleic
acid and amino acid sequences and the sequences presented in Figure 36.
In further embodiments, the application relates to the discovery that a POSH
polypeptide interacts with ATP6VOC. Accordingly, the application provides
complexes comprising POSH and ASTP6VOC. In one aspect, the application relates
to the discovery that POSH binds directly with ATP6VOC. This interaction was
identified by Applicants in a yeast 2-hybrid assay. ATP6VOC, vacuolar-H(+)-
ATPase, is a 'large multimeric protein composed of at least twelve distinct
subunits
and it is involved in the H(+) transport across cellular membranes. ATP6VOC is
synonymous with ATP6L. Treatment with anticancer agents has been shown to
enhance ATP6L expression (Cytogenet Genome Res (2002) 97:111-5; J Biol Chem
(2002) 277:36534-43).
The term ATP6VOC is used herein to refer to various naturally occurring
ATP6VOC homologs, as well as functionally similar variants and fragments that
retain at least 80%, 90%, 95%, or 99% sequence identity to a naturally
occurring
ATP6VOC (e.g., SEQ III NOs: 225-226, 345-351). The term specifically includes
human ATP6V0C nucleic acid and amino acid sequences and the sequences
presented in Figure 36.
Iri certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with PPP1CA. Accordingly, the application provides
complexes comprising POSH and PPP1CA. In one aspect, the application relates
to
the discovery that POSH binds directly with PPP 1 CA. This interaction was
identified by Applicants in a yeast 2-hybrid assay. PPP 1 CA is the protein
phosphatase type 1 alpha catalytic subunit. The genetic and expression status
of the
PPP1CA gene was examined in 55 human cancer cell lines and found to be
ubiquitously expressed and lacking in genetic variation, suggesting an
essential role
for PPP1CA in the growth of cancer cells (Int J Oncol (2001) 18:817-24).
The term PPP1CA is used herein to refer to various naturally occurring
PPP1CA homologs, as well as functionally similar variants and fragments that
retain
at least 80%, 90%, 95%, or 99% sequence identity to a naturally occurring PPP1
CA
(e.g., SEQ ID NOs: 261-263, 101-110). The term specifically includes human
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PPP 1 CA nucleic acid and amino acid sequences and the sequences presented in
Figure 36.
The application further relates to the discovery that a POSH polypeptide
interacts with DDEFl. Accordingly, the application provides complexes
comprising
POSH and DDEFl. In one aspect, the application relates to the discovery that
POSH
binds directly with DDEF1. This interaction was identified by Applicants in a
yeast
2-hybrid assay. DDEF1 is a putative candidate gene associated with Meckel-
Gruber
syndrome (MKS), the most common monogenic cause of neural tube defects (Hum
Genet (2002) 111:654-61).
The term DDEF1 is used herein to refer to various naturally occurring
DDEF1 homologs, as well as fimctionally similar variants and fragments that
retain
at least 80%, 90%, 95%, or 99% sequence identity to a naturally occurring
DDEF1
(e.g., SEQ ID NOs: 233-237, 48-54). The term specifically includes human DDEFl
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
1 S In certain embodiments, the application relates to the discovery that a
POSH
polypeptide interacts with PALS-1. Accordingly, the application provides
complexes comprising POSH and PACS-1. In one aspect, the application relates
to
the discovery that POSH binds directly with PALS-1. This interaction was
identified by Applicants in a yeast 2-hybrid assay. PACS-1 is a cytosolic
sorting
protein that directs localization of membrane proteins in the TGN/endosomal
system. PACE-1 is a cytosolic protein involved in controlling the correct
subcellular
localization of integral membrane proteins that contain acidic cluster sorting
motifs,
such as furin and HIV-1 Nef, and PACS-1 has been shown to interact with the
adaptor complexes AP-1 and AP-3 (EMBO J (2003) 22:6234-44; EMBO J (2001)
20:2191-201). Furthermore, PACS-1 polypeptides have been shown to interact
with
Nef and through this interaction, by a PI3K-dependent proces, MHC class I
molecules are downregulated by Nef (Cell (2002) 11:853-66). Accordingly, POSH
may be involved in Nef mediated downregulation of MHC class I molecules in a
cell infected with HIV-1. Additionally, PALS-1 interacts with the HIV-1
protein,
Vpu. Vpu expresses an acidic amino acid sorting motif that is required for TGN
localization through a retroviral process mediated by PALS-1 (Wan, L et al
(1998)
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Cell 94:205-216). Accordingly, in certain aspects, POSH may associate with Vpu
through its interaction with PACS-1.
The term PACS-1 is used herein to refer to various naturally occurring
PACS-1 homologs, as well as functionally similar variants and fragments that
retain
at least 80%, 90%, 95%, or 99% sequence identity to a naturally occurnng PACS-
1
(e.g., SEQ 1D NOs: 362-366, 95-100). The term specifically includes human PACS-
1 nucleic acid and amino acid sequences and the sequences presented in Figure
36.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with EPS8L2. Accordingly, the application provides
complexes comprising POSH and EPS8L2. In one aspect, the application relates
to
the discovery that POSH binds directly with EPS8L2. This interaction was
identified by Applicants in a yeast 2-hybrid assay. EPSSLZ is an eps8-related
protein. Eps8 forms a multimeric complex with Sos-l, Abil and PI3K that is
required for Rac activation leading to actin remodelling. EPS8L2 has been
shown to
interact with Abil and Sos-1. EPS8L2 also has been shown to localize to PDGF-
induced F-actin-rich raffles and to restore r eceptor tyrosine kinase mediated
actin
remodeling when expressed in eps8-/- fibroblasts (Mol Biol Cell (2004) 15:91-
8).
The term EPS8L2 is used herein to refer to various naturally occurring
EPS8L2 homologs, as well as functionally similar variants and fragments that
retain
at least 80%, 90%, 95%, or 99% sequence identity to a naturally occurring
EPS8L2
(e.g., SEQ ~ NOs: 239, 58-60). The term specifically includes human EPS8L2
nucleic acid and amino acid sequences and the sequences presented in Figl~re
36.
The application additionally relates to the discovery that a POSH polypeptide
interacts with HIP55. Accordingly, the application provides complexes
comprising
POSH and HIP55. In one aspect, the application relates to the discovery that
POSH
binds directly with HIP55. This .interaction was identified by Applicants in a
yeast
2-hybrid assay. HIP55 is a cytoplasmic adaptor protein that has been shown to
bind
to t he c ytoplasmic tail o f t he C D2v p rotein o f A frican s wine f ever v
irus ( J G en
Virol (2004) 85:119-30). HIP55 (synonymous with mAbpl and SH3P7) comprises
an SH3 domain and through its SH3 domain, associates with dynamin (J Cell Biol
(2001) 153:351-66; Biochem Biophys Res Commun (2003) 301:704-10).
Accordingly, in certain aspects, POSH may associate with dynamin through its
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interaction with HIP55. HIP55 has also been shown to be important for receptor
mediated a ndocytosis o f t he t ransferrin r eceptor ( Biochem Biophys R es C
ommun
(2003) 301:704-10).
The term HIP55 is used herein to refer to various naturally occurring HIP55
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurnng HIP55 (e.g.,
SEQ
ID NOs: 390-394, 377-385). The term specifically includes human HIl'S5 nucleic
acid and amino acid sequences and the sequences presented in Figure 36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with CENTBl. Accordingly, the application provides
complexes comprising POSH and CENTB1. In one aspect, the application relates
to
the discovery that POSH binds directly with CENTB1. This interaction was
identified by Applicants in a yeast 2-hybrid assay. CENTBl is synonymous with
ACAP1. ACAP1 is an ARF GTPase activating protein (ARF GAP). ACAPl can
function as a GAP for ARF 1 and ARF6 (J Biol Chem (2002) 277:7962-9).
The term CENTB 1 is used herein to refer to various naturally occurnng
CENTB1 homologs, as well as functionally similar variants and fragments that
retain at least 80%, 90%, 95%, or 99% sequence identity to a naturally
occurring
CENTBl (e.g., SEQ ID NOs: 231-232, 37-47). The term specifically includes
human CENTB 1 nucleic acid and amino acid sequences and the sequences
presented
in Figure 36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with EIF3S3. Accordingly, the application provides
complexes comprising POSH and EIF3S3. In one aspect, the application relates
to
the discovery that POSH binds directly with EIF3S3. This interaction was
identified
by Applicants in a yeast 2-hybrid assay. EIF3S3 is elevated in certain
hepatocellular
carcinomas and in prostate cancer (Hepatology (2003) 38:1242-9; Am J Pathol
(2001) 159:2081-84). It has also been demonstrated that EIF3S3 is often
amplified
and o verexpressed i n b reast c ancer ( Genes C hromosomes C ancer. (2000) 2
8:203-
210).
The term EIF3S3 is used herein to refer to various naturally occurring
EIF3S3 homologs, as well as functionally similar variants and fragments that
retain
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at least 80%, 90%, 95%, or 99% sequence identity to a naturally occurring
EIF3S3
(e.g., SEQ ID NOs: 238, 55-57). The term specifically includes human EIF3S3
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
In certain embodiments, the application relates to the discovery that a POSH
polypeptide interacts with SRA1. Accordingly, the application provides
complexes
comprising POSH and SRA1. In one aspect, the application relates to the
discovery
that POSH binds directly with SRA1. This interaction was identified by
Applicants
in a yeast 2-hybrid assay. SRAl is a transcriptional coactivator, steroid
receptor
RNA activator 1. SRA is selective for steroid hormone receptors and mediates
transactivation via their amino-terminal activation function (Cell (1999)
9?:17-27).
The term SRA1 is used herein to refer to various naturally occurring SRA1
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurring SRAl (e.g.,
SEQ
ID NOs: 291-294, 175-182). The term specifically includes human SR.A1 nucleic
acid and amino acid sequences and the sequences presented in Figure 36.
The application additionally relates to the discovery that a POSH polypeptide
interacts with WASFl. Accordingly, the application provides complexes
comprising POSH and WASF1. In one aspect, the application relates to the
discovery that POSH binds directly with WASFl. This interaction was identified
by
Applicants in a yeast 2-hybrid assay. WASF1 is a member of the Wiskott-Aldrich
syndrome protein (WASP) family of proteins. WASF-1 bas been shown to regulate
cortical actin filament reorganization in response to extracellular stimuli.
WASF1 is
synonymous with WAVE1 and is an actin regulatory protein. It has been shown
that
Ras and the adaptor protein Nck activate actin nucleation through WAVE1
(Nature
(2002) 418:790-3).
The term WASFl is used herein to refer to various naturally occurring
WASFl homologs, as well as functionally similar variants and fragments that
retain
at least 80%, 90%, 95%, or 99% sequence identity to a naturally occurring
WASF1
(e.g., SEQ ID NOs: 389, 375-376). The term specifically includes human WASF1
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
The application additionally relates to the discovery that a POSH polypeptide
interacts with SPG20. Accordingly, the application provides complexes
comprising
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POSH and SPG20. In one aspect, the application relates to the discovery that
POSH
binds directly with SPG20. This interaction was identified by Applicants in a
yeast
2-hybrid assay. SPG20 is synonymous with spartin, and mutation in the gene has
been implicated in Troyer syndrome, an autosomal recessive complicated
hereditary
spastic paraplegia. Comparative sequence analysis has shown that spartin
shares
similarity with molecules involved in endosomal trafficking (Nat Genet (2002)
31:347-8).
The term SPG20 is used herein to refer to various naturally occurring SPG20
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurring SPG20 (e.g.,
SEQ ID NOs: 386-388, 367-374). The term specifically includes human SPG20
nucleic acid and amino acid sequences and the sequences presented in the
Figure 36.
In further embodiments, the application relates to the discovery that a POSH
polypeptide interacts with HLA-A. Accordingly, the application provides
complexes comprising POSH and HLA-A. In one aspect, the application relates to
the discovery that POSH binds directly with HLA-A. This interaction was
identified
by Applicants in a yeast 2-hybrid assay. In additional aspects, the
application relates
to the discovery that a POSH polypeptide interacts with HLA-B. Accordingly,
the
application provides complexes comprising POSH and HLA-B. In one aspect, the
application relates to the discovery that POSH binds directly with HLA-B. This
interaction was identified by Applicants in a yeast 2-hybrid assay. HLA-A and
HLA-B are MHC class I molecules. HLA-A and HLA-B molecules are
downregulated in the progression of A)DS, and this downregulation is a
ssociated
with the activity of HIV-1 Nef.
The term HLA-A is used herein to refer to various naturally occurring HLA-
A homologs, as well as functionally similar variants and fragments that retain
at
least 8 0%, 9 0%, 9 5%, o r 9 9% se quence i dentity t o a n aturally o
ccurnng H LA-A
(e.g., SEQ ID NOs: 253, 87-88). The term specifically includes human HLA-A
nucleic acid and amino acid sequences and the sequences presented in Figure
36.
The term HLA-B is used herein to refer to various naturally occurring HLA-
B homologs, as well as functionally similar variants and fragments that retain
at
least 80%, 90%, 95%, or 99% sequence identity to a naturally occurring HLA-B
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(e.g., SEQ ID NOs: 254, 89). The term specifically includes human HLA-B
nucleic
acid and amino acid sequences and the sequences presented in Figure 36.
In certain aspects, the application relates to the discovery that a POSH
polypeptide interacts with a ubiquitin-conjugating enzyme (E2). An exemplary
E2
may include, but are not limited to, UBCSa, UBCSc, UBC6, and UBC13. UBC13 is
often found in a heterodimer complex with a Ub conjugating enzymer variant
(UEV)
protein, such as, for example, UEV 1 a. (See Hofinann and Pickart,
Noncanonical
MMS2-Encoded Ubiquitira-Conjugating Enzyme Functions in Assembly of Novel
Ubiquitira Chains for DNA Repair, Cell 96: 645-653 (1999), McKenna et al.,
2002,
Energetics afad Specificity of Interactions within Ub-Uev-Ubcl3 Human
Ubiquitirz
Conjugating Complexs, Biochemistry. Vol. 42. pp.7922-7930, and Ulrich, 2003,
Protein-Protein Interactions within an E2-RING Finger Complex, The Jurnal of
Biological Chemistry, Vol. 278. No 9. pp. 7051-7058). UVE proteins share
significant sequence and structural similarities with E2s, yet lack the
requisite active
site cystine of the classical E2 protein family.
Generally, UBCS conjugates ubiquitin to Lysine 4.8 in a target protein, a
signal that marks the protein for degredation by the 26 S proteosome. In
constrast,
UBC 13/LJEV 1 a conjugates ubiquitin to Lysine 63 residue in a target protein,
which
is not a degradation signal. Instead, ubiquitin conjugated at Lysine 63 has
been
implicated in diverse biological processes, including, for example, DNA damage
repair, endocytosis, ribosome biogenesis, mitochondrial inheritance, and NF~cB
signaling (See Ulrich, 2003). The UBC13/UEVIa has been shown to work with two
,
other RING-ubiquitin ligases, TRAF6 and RADS. (See Ulrich, 2003). TRAF6-
UBC 13-UEV 1 a complex ubiquitinates TRAF6 (self ubiquitination), thus
enabling it
to activate a kinase cascade.
Without being bound to theory, it appears that UBCSa, UBCSc and UBC6
may work with POSH in one pathway, while UBC13/IJEVla work with POSH in
another distinct pathway. This is supported by the fact that UBCSl6 marks POSH
for degradation by conjugating ubiquitin at Lysine 48, whereas UBC13/UEVla
marks POSH for purposes other than degradation by conjugating ubiquitin at
Lysine
63. T his t henry i s further s upported b y t he fact t hat U BCSa, U BCSc a
nd U BC6
share high sequence similarities.
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Accordingly, in certain aspects, the present application relates to an
isolated,
purified or recombinant complex comprising a POSH polypeptide and a UBC13. In
certain aspects, the present application relates to an isolated, purified or
recombinant
complex comprising: a polypeptide comprising a domain that is at least 90%
identical to a POSH RING domain, and a POSH-AP comprising an E2. An
exemplary POSH associated protein E2 include, for example, is UBC13. UBC13
may b a i n a h eterodimer c omplex w ith a Ub c onjugating a nzyrner v ariant
( UEV)
protein, such as, for example, UEV 1 a.
The t erm "UBC 13" a nd i s a sed h erein t o r efer to full-length U BC 13,
any
splice variants thereof, various naturally occurnng UBC13 homologs, as well as
functionally similar variants and fragments that retain at least 80%, 90%,
95%, or
99% sequence identity to a naturally occurring UBC13 (e.g., SEQ ID NOs: 313,
208-210). The term specifically includes UBC13 nucleic acid and amino acid
sequences and the sequences presented in Figure 36.
I S In certain embodiments, the application relates to the interaction between
an
ARFS polypeptide and a POSH polypeptide. ARFS is a member of the ARF gene
family. The ARF proteins stimulate the in vitro ADP-ribosyltransferase
activity of
cholera toxin. ARF proteins play a role in vesicular trafficking in vivo. ARFs
are
members of the Ras GTPase superfamily. ARFs activate specific PLDs.
Mammalian ARFs are divided into three classes based on size, amino acid
sequence,
gene structure, aald phylogenetic analysis. ARF1 is in class I, and ARFS is in
class
II.
In certain embodiments, the application relates to the interaction between an
ARF1 polypeptide and a POSH polypeptide. ARF1 is a small G protein involved in
vesicular trafficking. The assembly/disassembly cycle of the coat protein I
(COPI)
on Golgi membranes is coupled to the GTP/GDP cycle of ARF1 (Nature (2003)
426:563-6). ARF1 has been implicated in mitotic Golgi disassembly, chromosome
segregation, and cytokinesis (Proc Natl Acad Sci (2003) 100:13314-9). ARF1 has
been shown to bind to the 5-HT2A receptor, a G protein coupled receptor (GPCR)
(Mol Pharmacol (2003) 64:1239-50).
The term ARF-1 is used herein to refer to various naturally occurring ARF-1
homologs, as well as functionally similar variants and fragments that retain
at least
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80%, 90%, 95%, or 99% sequence identity to a naturally occurring ARF-1 (e.g.
SEQ
ID NOs: 223, 325-339). The term specifically includes human ARF-1 nucleic acid
and amino acid sequences and the sequences presented in Figure 36.
The term ARF-5 is used herein to refer to various naturally occurring ARF-5
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurring ARF-5 (e.g.,
SEQ ID NOs: 224, 340-344). The term specifically includes human ARF-5 nucleic
acid and amino acid sequences and the sequences presented in Figure 36.
In certain embodiments, the application relates to the inhibition of viral
maturation by modulation of an activity associated with a dynamin II
polypeptide.
Dynamin I I is a large GTP-binding protein that is involved in endocytosis and
in
vesicle formation at the trans-Golgi network. Dynamin II contains a pleckstrin
homology domain (PHD) and a proline-rich domain (PRD). Dynamin II plays an
important role in vesicle formation at the plasma membrane, trans-Golgi
network,
and various other intracellular organelles. Accordingly, disrupting the
activity of a
dynamin II polypeptide or the interaction between a POSH polypeptide and a
dynamin II polypeptide (e.g., by reducing POSH protein levels or
alternatively,
reducing dynamin II protein levels, through RNAi) may disrupt the activity of
dynamin II in the secretory pathway and prevent the secretion of viral
proteins, such
as, for example, HBV proteins. Dynamin II participates in the transport and
secretion of HBV proteins (Abdulkarim, AS et al (2003) J. Hepat. 38:76-83).
Accordingly, in certain embodiments, inhibition of POSH adversely effects the
transport and release of HBV proteins.
In certain embodiments, the application relates to the inhibition of dynamin
activity, in particular the inhibition of the activity of dynamin II, a member
of the
dynamin family of proteins. In certain embodiments, the application relates to
inhibition of dynamin II activity, which inhibition disrupts the transport and
secretion of HBV proteins. The term dynamin II is used herein to refer to full
length, human dynamin II as well as various naturally occurring dynamin II
homologs, as well as functionally similar variants and fragments that retain
at least
80%, 90%, 95%, or 99% sequence identity to a naturally occurnng dynamin II
(e.g.,
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public gi number: 1196422, public gi number: 1706539, public gi number:
1196423,
and public gi number: 1363934).
In certain embodiments, the application relates to the inhibition of viral
maturation by modulation of an activity associated with a Vpu polypeptide. Vpu
is
an HIV-1 encoded ion channel, which, among other tasks in the HIV-1 life
cycle, is
necessary f or a fficient v ims b udding ( Schubert, U a t a 1 ( 1995) J. V
irol. 69:7699-
7711). Vpu may function at the trans Golgi network (TGN). Vpu expresses an
acidic amino acid sorting motif that is required for TGN localization through
a
retroviral p rocess m ediated b y t he P OSH-AP, P ACS-1 ( Wan, L et a 1 (
1998) C ell
94:205-216). Moreover, the phenotype conferred by human POSH knockdown is
similar to that observed in cells expressing HIV-1 lacking Vpu where viruses
also
accumulate in intracellular membranes (Klimkait, T et al (1990) J. Virol.
64:621-
629).
Vpu regulates virus release from a post-endoplasmic reticulum compartment,
such as possibly the TGN, by an ion channel activity mediated by its
transmembrane
anchor. Vpu also induces the selective down regulation of host cell receptor
proteins such as CD4 and major histocompatibility complex class I molecules,
in a
process involving its cytoplasmic tail. Furthermore, Vpu-mediated degradation
of
CD4 is dependent on an intact ubiquitin-conjugating system. (See Schubert, U
et al
(1998) J. Virol. 72:2280-8). In certain embodiments of the present invention,
Vpu-
mediated degradation of a protein such as CD4 may involve a ubiquitin-
conjugating
system that includes a POSH polypeptide or a POSH-AP, such as, for example,
Cbl-
b.
Vpu nucleic acid and the corresponding amino acid sequence encoded
thereby are exemplified by the Vpu discussed in Strebel, K et al (1988)
241:1221-
1223. The term Vpu is used herein to refer as well to Vpu of other HIV-1
isolates,
such as the Vpu disclosed in GenBank, accession number U51190, and the Vpu
disclosed in GenBank, accession number U52953. The term Vpu is used herein to
refer as well to various naW rally occurring Vpu homologs, as well as
functionally
similar variants and fragments that retain at least 80%, 90%, 95%, or 99%
sequence
identity to a naturally occurring Vpu.
thods and Compositions for Treating POSH-associated Diseases
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In certain aspects, the application provides methods and compositions for
treatment of POSH-associated diseases (disorders), including cancer and viral
disorders, as well as disorders associated with unwanted apoptosis,-including,
for
example a variety of neurodegenerative disorders, such as Alzheimer's disease.
In certain embodiments, the application relates to viral disorders (e.g.,
viral
infections), and particularly disorders caused by retroid viruses, RNA viruses
and/or
envelope viruses. In view of the teachings herein, one of skill in the art
will
understand that the methods and compositions of the application are applicable
to a
wide range of viruses such as, for example, retroid viruses, RNA viruses, and
envelope viruses. In a preferred embodiment, the present application is
applicable to
retroid viruses. In a more preferred embodiment, the present application is
further
applicable to retroviruses (retroviridae). In another more preferred
embodiment, the
present application is applicable to lentivirus, including primate lentivirus
group. In
a most preferred embodiment, the present application is applicable to Human
Immunodeficiency virus (HIV), Human Immunodeficiency virus type-1 (HIV-1),
Hepatitis B Virus {HBV) and Human T-cell Leukemia Virus (HTLV).
While not intended to be limiting, relevant retroviruses include: C-type
retrovirus which causes lymphosarcoma in Northern Pike, the C-type retrovirus
which infects mink, the caprine lentivirus which infects sheep, the Equine
Infectious
24 Anemia Virus (EIA.V), the C-type retrovirus which infects pigs, the Avian
Leukosis
Sarcoma Virus (ALSV), the Feline Leukemia Virus (FeLV), the Feline Aids Virus,
the Bovine Leukemia Virus (BLV), Moloney Murine Leukemia Virus (MMuLV),
the Simian Leukemia Virus (SLV), the Simian Immuno-deficiency Virus (SIV), the
Human T-cell Leukemia Virus type-I (HTLV-I), the Human T-cell Leukemia Vines
type-II (HTLV-II), Human Immunodeficiency virus'type-2 (HIV-2) and Human
Immunodeficiency virus type-1 (HIV-1).
The method and compositions of the present application are further
applicable to RNA viruses, including ssRNA negative-strand viruses and ssRNA
positive-strand viruses. The ssRNA positive-strand viruses include Hepatitis C
Vines (HCV). In a preferred embodiment, the present application is applicable
to
mononegavirales, including filoviruses. Filoviruses further include Ebola
viruses
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and Marburg viruses. In another preferred embodiment, the present invention is
applicable to flaviviruses, including West Nile Virus (WNV).
Other RNA viruses include picornaviruses such as enterovinis, poliovirus,
coxsackievirus and hepatitis A virus, the caliciviruses, including Norwalk-
like
viruses, the rhabdoviruses, including rabies virus, the togaviruses including
alphaviruses, Semliki Forest virus, denguevirus, yellow fever virus and
rubella virus,
the orthomyxoviruses, including Type A, B, and C influenza viruses, the
bunyaviruses, including the Rift Valley fever virus and the hantavirus, the
filovimses such as Ebola virus and Marburg virus, and the paramyxoviruses,
including mumps virus and measles vims. Additional viruses that may be treated
include herpes viruses.
The methods and compositions of the present application are further
applicable to hepatotrophic viruses, including HAV, HBV, HCV, HDV, and HEV.
In certain aspects, the application relates to a method of inhibiting a
hepatotrophic
virus, comprising administering a POSH inhibitor to a subject in need thereof
In
further aspects, the application relates to a method of treating a viral
hepatitis
infection, comprising administering a POSH inhibitor to a subject in need
thereof
A viral hepatitis infection may be caused by a hepatotxophic virus, such as
HAV,
HBV, HCV, HDV, or HEV. In certain embodiments, the application relates to a
method of treating an HBV infection by administering a POSH inhibitor to a
subject
in need thereof.
In other embodiments, the application relates to methods of treating or
preventing cancer diseases. The terms "cancer," "tumor," and "neoplasia" are
used
interchangeably herein. As used herein, a cancer (tumor or neoplasia) is
characterized by one or more of the following properties: cell growth is not
regulated by the normal biochemical and physical influences in the
environment;
anaplasia (e.g., lack of normal coordinated cell differentiation); and in some
instances, metastasis. Cancer diseases include, for example, anal carcinoma,
bladder
carcinoma, breast carcinoma, cervix carcinoma, chronic lymphocytic leukemia,
chronic myelogenous leukemia, endometrial carcinoma, hairy cell leukemia, head
and neck carcinoma, lung (small cell) carcinoma, multiple myeloma, non-
Hodgkin's
lymphoma, follicular lymphoma, ovarian carcinoma, brain tumors, colorectal
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carcinoma, hepatocellttlar carcinoma, Kaposi's sarcoma, lung (non-small cell
carcinoma), melanoma, pancreatic carcinoma, prostate carcinoma, renal cell
carcinoma, and soft tissue sarcoma. Additional cancer disorders can be found
in, for
example, Isselbacher et al. (1994) Harnson's Principles ofInternal Medicine
1514-
177, herein incorporated by reference.
In a specific embodiment, anticancer therapeutics of the application are used
in treating a POSH-associated cancer. As described herein, POSH-associated
cancers include, but are not limited to, the thyroid carcinoma, liver cancer
(hepatocellular cancer), lung cancer, cervical cancer, ovarian cancer, renal
cell
carcinoma, lymphoma, osteosacoma, liposarcoma, leukemia, breast carcinoma, and
breast adeno-carcinoma.
Preferred antiviral and anticancer therapeutics of the application can
function
by disrupting the biological activity of a POSH polypeptide or POSH complex in
viral maturation. Certain therapeutics of the application function by
disrupting the
activity of a POSH-AP (e.g., HERPUDl) in viral maturation. Certain
therapeutics
of the application function by disrupting the activity of POSH by inhibiting
the
ubiquitin ligase activity of a POSH polypeptide. In certain embodiments of the
application, a therapeutic of the application inhibits the ubiquitination of a
POSH-
AP, such as fox example the ubiquitination of HERPUD1.
In other embodiments, the application relates to methods of treating or
preventing n eurological disorders. In o ne a spect, t he i nvention p rovides
m ethods
and compositions for the identification of compositions that interfere with
the
function of a POSH or a POSH-AP, which function may relate to aberrant protein
processing associated with a neurodegenerative disorder, such as for example,
the
processing of amyloid beta precursor protein associated with Alzheimer's
disease.
Neurological disorders include disorders associated with increased levels of
amyloid
~i production, such as for example, Alzheimer's disease. Neurological
disorders also
include Parkinson's disease, Huntington's disease, schizophrenia, Niemann-
Pick's
disease, and prion-associated diseases
Exemplary therapeutics of the application include nucleic acid therapies such
as, for example, RNAi constnicts (small inhibitory RNAs), antisense
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oligonucleotides, ribozyme, and DNA enzymes. Other therapeutics include
polypeptides, peptidomimetics, antibodies and small molecules.
Antisense therapies of the application include methods of introducing
antisense nucleic acids to disrupt the expression of POSH polypeptides or
proteins
that are necessary for POSH function.
RNAi therapies include methods of introducing RNAi constructs to
downregulate the expression of POSH polypeptides or POSH-APs (e.g.,
HERPUDl). In certain embodiments, RNAi therapeutics are delivered to the liver
(e.g., to hepatocytes). Exemplary RNAi therapeutics include any one of SEQ ID
NOs: 15, 16, 18, 19, 21, 22, 24 and 25.
Therapeutic polypeptides may be generated by designing polypeptides to
mimic certain protein domains important in the formation of POSH: POSH-AP
complexes, such as, for example, SH3 or RING domains. For example, a
polypeptide comprising a POSH SH3 domain such as, for example, the SH3 domain
as set forth in SEQ ID NO: 30 will compete for binding to a POSH SH3 domain
and
will therefore act to disrupt binding of a partner protein. In one embodiment,
a
binding partner may be a Gag polypeptide. In another embodiment, a binding
partner may be Rac. In a further embodiment, a polypeptide that resembles an L
domain may disrupt recruitment of Gag to the POSH complex.
In view of the specification, methods for generating antibodies directed to
epitopes of POSH and POSH-APs are known in the art. Antibodies may be
introduced into cells by a variety of methods. One exemplary method comprises
generating a nucleic acid encoding a single chain antibody that is capable of
disrupting a POSH:POSH-AP complex. Such a nucleic acid may be conjugated to
antibody that binds to receptors on the surface of target cells. It is
contemplated that
in certain embodiments, the antibody may target viral proteins that are
present on the
surface of infected cells, and in this way deliver the nucleic acid only to
infected
cells. Once bound to the target cell surface, the antibody is taken up by
endocytosis,
and the conjugated nucleic acid is transcribed and translated to produce a
single
chain antibody that interacts with and disnipts the targeted POSH:POSH-AP
complex. Nucleic acids expressing the desired single chain antibody may also
be
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introduced into cells using a variety of more conventional techniques, such as
viral
transfection (e.g., using an adenoviral system) or liposome-mediated
transfection.
Small molecules of the application may be identified for their ability to
modulate the formation of POSH:POSH-AP complexes.
Certain embodiments of the disclosure relate to use of a small molecule as an
inhibitor of POSH. Examples of such small molecules include the following
compounds:
Compound CAS 27430-18-8:
S~N O
N ~ i
0
0
Compound CAS 1631-29-4:
Compound CAS 503065-65-4:
O
N
S N
Compound CAS 414908-08:
0
N w \ \ N+-.~
10_
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Compound CAS 415703-60-5:
Br
B O
N
-N
Br
Compound CAS 77367-94-3:
O ~ .O
+:
N N ~ / N -
CI ~ O
CI
Compound CAS 154184-27-7:
CI
O
N ~"~ O \
In certain embodiments, compounds useful in the instant compositions and
methods include heteroarylmethylene-dihydro-2,4,6-pyrimidinetriones and their
thione analogs. Preferred heteroaryl moieties include 5-membered rings such as
thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, and imidazolyl moieties.
In certain embodiments, compounds useful in the instant compositions and
methods include N-arylmaleimides, especially N-phenylmaleimides, in which the
phenyl group may be substituted or unsubstituted.
In certain embodiments, compounds useful in the instant compositions and
methods include arylallylidene-2,4-imidazolidinediones and their thione
analogs.
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Preferred aryl groups are phenyl groups, and both the aryl and allylidene
portions of
the molecule may be substituted or unsubstituted.
In certain embodiments, compounds useful in the instant compositions and
methods include substituted distyryl compounds and aza analogs thereof such as
substituted 1,4-diphenylazabutadiene compounds.
In certain other embodiments, compounds useful in the instant compositions
and methods include substituted styrenes and aza analogs thereof, such as 1,2-
diphenylazaethylenes and 1-phenyl-2-pyridyl-azaethelenes.
In yet other embodiments, compounds useful in the instant compositions and
methods include N-aryl-N'-acylpiperazines. I n such compounds, the aryl ring,
the
acyl substituent, and/or the piperazine ring may be substituted or
unsubstituted.
In additional embodiments, compounds useful in the instant compositions
and methods include aryl esters of (2-oxo-benzooxazol-3-yl)-acetic acid, and
analogs thereof in which one or more oxygen atoms are replaced by sulfur
atoms.
In certain embodiments, the present application contemplates use of known
PKA modulators (e.g., inhibitors or activators) in the methods of ihibiting
viral
infection and in the methods of treating or preventing cancer. Such PKA
modulators
include any compound, peptide, nucleotide derivative, nucleoside derivative,
polysaccharide, sugar or other substance that can inhibit the activity of
protein
kinase A. Many PKA inhibitors are available and may be used. For example, many
examples of PKA inhibitors including chemical structures, methods for
administration and pharmacological effects are listed at the Calbiochem
website at
calbiochem.com. In general, inhibitors that also significantly inhibit protein
kinase C
activity are avoided.
In some embodiments, the PKA inhibitor is a nucleotide or nucleoside
derivative. Specific examples of nucleoside or nucleotide derivatives that act
as
PKA inhibitors and that can be utilized in the disclosure include adenosine
3',5'
cyclic monophosphorothioate. The H-89 inhibitor is a potent PKA inhibitor that
can
be used in the disclosure. The chemical name for the H-89 inhibitor is N-[2-
((Pbromocinnamyl) amino)ethyl] isoquinolinesulfonamide. The KT5720 inhibitor
from Calbiochem can also be used in the disclosure. Other PKA inhibitors which
are
available at from Calbiochem and can be used in the disclosure include ellagic
acid
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(also named 4,4',5,5',6,6'-hexahydroxydiphenic acid 2,6,2',6'-ditactone),
piceatannol,
1-(5-Isoquinolinesulfonyl) methylpiperazine (H-7), N-[2-(methylamino)ethyl]
isoquinolinesulfonamide (H-8), N-(2-aminoethyl) isoquinolinesulfonamide (H-9),
and (5-isoquinolinesulfonyl)piperazine, 2HCI (H-100).
The PKA inhibitor can also be a peptide inhibitor (PKI). Such a peptide
inhibitor c an b a a ny p eptide t hat i s r ecognized and b ound b y P KA b
ut that P KA
cannot phosphorylate. An example of a peptide inhibitor is a peptide with a
"consensus sequence" for PKA recognition but with alanine in place of serine,
for
example, a peptide with the following sequence: Xaa-Arg-Arg-Xaa-Ala-Xaa,
wherein Xaa is any amino acid, which specifically binds to the pseudoregion of
the
regulatory domain of PKA. Myristoylated PKA inhibitor amide (14-22, Cell-
Permeable) having the sequence Myr-N-Gly-Arg-Thr-G1y-Arg-Arg-Asn-Ala-Ile-
NHZ is another example of a peptide inhibitor that can be utilized in the
disclosure.
A variety of other PKI peptides can be used as an inhibitor of protein kinase
A in the
practice of the disclosure. For example, several PKI peptides can be found in
the
NCBI protein database. See website at ncbi.nlm.nih.gov/
GenbanklGenbankOverview. One example of a human PKI peptide can be found at
Genbank Accession No. P04541 (gi: 417194). Another example of a human PKI
peptide is at Genbank Accession No. NP 008997 (gi: 5902020). Another PKI that
can be used as an inhibitor has the following sequence:
Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-11 e-Leu-Val-SerSer-
Ala. See published PCT application WO 03/080649.
Further examples of protein kinase A inhibitors are provided in the following
.references: Muniz et al., Proceedings of the National Academy of Sciences USA
1997 Dec 23; 94(26) 14461-66; Baude et al., Journal of Biological Chemistry
Vol.
269 issue 27 18128-18133 (Jul. 1994); Scott et al.
Applicants found that POSH is phosphorylated by PKA and phosphorylation
of POSH by PKA can inhibit POSH function, for example dissociating POSH from
POSH interacting proteins (e.g, Rac). Therefore, in certain embodiments, the
present
disclosure also cotemplates use of PKA activators in treating or preventing a
POSH-
associated disease (e.g., viral infection or cancer). Exemplary PKA activators
include, but are not limited to, forslcolin, 8-Br-cAMP, and rolipram.
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In additional embodiments of the application, compounds useful in the
present application include phosphatase inhibitors. Phosphatase inhibitors
useful in
the subject application include sodium phosphate, sodium vanadate, and okadaic
acid. In certain embodiments, the present application contemplates use of
known
phosphatase inhibitors in the methods of inhibiting viral infection, in the
methods of
treating or preventing cancer, and in the methods of inhibiting the
progression of a
neurodenerative disorder. Phosphatase inhibitors may be useful in inhibiting
the
activity of a POSH-AP, such as for example, PTPN 12.
For POSH-APs that are GTPases, inhibitors such as GTPgamma35S would
be effective at inhibiting the GTPase activity of the POSH-AP. For example,
inhibition of ARFl or ARFS could be accomplished with the use of a GTPase
inhibitor such as GTPgamma35S, a non-hydrolyzable form of GTP.
The generation o f n ucleic a cid b ased t herapeutic a gents d irected t o P
OSH
and POSH-APs is described below.
Methods for identifying and evaluating further modulators of POSH and
POSH-APs are also provided below.
5. RNA Interference, Ribozymes, Antisense and Related Constructs
In certain aspects, the application relates to RNAi, ribozyme, antisense and
other nucleic acid-related methods and compositions for manipulating
(typically
decreasing) a POSH activity. Exemplary RNAi and ribozyme molecules may
comprise a sequence as shown in any of SEQ ID Nos: 15, 16, 18, 19, 21, 22, 24
and
25.
in certain aspects, the application relates to RNAi, ribozyme, antisense and
other nucleic acid-related methods and compositions for manipulating
(typically
decreasing) a POSH-AP activity. Specific instances of nucleic acids that may
be
used to design nucleic acids for RNAi, ribozyme, antisense are provided in
Figure
36. Additionally, nucleic acids of POSH-APs listed in Table 8 may be used to
design nucleic acids for RNAi, ribozyme, antisense.
Certain embodiments of the application make use of materials and methods
for a ffecting k nockdown o f o ne o r m ore P OSH or P OSH-AP genes b y means
o f
RNA interference (RNAi). RNAi is a process of sequence-specific post-
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transcriptional gene repression which can occur in eukaryotic cells. In
general, this
process involves degradation of an mRNA of a particular sequence induced by
double-stranded RNA (dsRNA) that is homologous to that sequence. For example,
the expression of a long dsRNA corresponding to the sequence of a particular
single-
s stranded mRNA (ss mRNA) will labilize that message, thereby "interfering"
with
expression of the corresponding gene. Accordingly, any selected gene may be
repressed by introducing a dsRNA which corresponds to all or a substantial
part of
the m RNA f or t hat gene. It a pp ears t hat w hen a 1 ong d sRNA i s
expressed, i t i s
initially processed by a ribonuclease III into shorter dsRNA oligonucleotides
of as
few as 21 to 22 base pairs in length. Furthermore, Accordingly, RNAi may be
effected by introduction or expression of relatively short homologous dsRNAs.
Indeed the use of relatively short homologous dsRNAs may have certain
advantages
as discussed below.
Mammalian cells have at least two pathways that are affected by double-
stranded RNA ( dsRNA). I n the RNAi ( sequence-specific) pathway, the
initiating
dsRNA i s first b roken i nto s hort i nterfering ( si) R NAs, a s d ascribed
above. T he
siRNAs have sense and antisense strands of about 21 nucleotides that form
approximately 19 nucleotide si RNAs with overhangs of two nucleotides at each
3'
end. Short interfering RNAs are thought to provide the sequence information
that
allows a specific messenger RNA to be targeted for degradation. In contrast,
the
nonspecific pathway is triggered by dsRNA of any sequence, as long as it is at
least
about 30 base pairs in length. The nonspecific effects occur because dsRNA
activates two enzymes: PKR, which in its active form phosphorylates the
translation
initiation factor eIF2 to shut down all protein synthesis, and 2', 5'
oligoadenylate
synthetase (2', S'-AS), which synthesizes a molecule that activates Rnase L, a
nonspecific enzyme that targets all mRNAs. The nonspecific pathway may
represent a host response to stress or viral infection, and, in general, the
effects of
the nonspecific pathway are preferably minimized under preferred methods of
the
present application. Significantly, longer dsRNAs appear to be required to
induce
the nonspecific pathway and, accordingly, dsRNAs shorter than about 30 bases
pairs
axe preferred to effect gene repression by RNAi (see Hunter et al. (1975) J
Biol
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Chem 250: 409-17; Manche et al. (1992) Mol Cell Biol 12: 5239-48; Minks et aI.
(I979) J Biol Chem 254: 10180-3; and Elbashir et al. (2001) Nature 411: 494-
8).
RNAi has been shown to be effective in reducing or eliminating the
expression of genes in a number of different organisms including
Caenorhabditiis
elegans (see e.g., Fire et al. (1998) Nature 391: 806-lI), mouse eggs and
embryos
(Wianny et al. (2000) Nature Cell Biol 2: 70-5; Svoboda et al. (2000)
Development
127: 4147-56), and cultured RAT-1 fibroblasts (Bahramina et al. (1999) Mol
Cell
Biol 19: 274-83), and appears to be an anciently evolved pathway available in
eukaryotic plants and animals (Sharp (2001) Genes Dev. 15: 48S-90). RNAi has
IO proven to be an effective means of decreasing gene expression in a variety
of cell
types including HeLa cells, NTH/3T3 cells, COS cells, 293 cells and BHK-21
cells,
and typically decreases expression of a gene to lower levels than that
achieved using
antisense techniques and, indeed, frequently eliminates expression entirely
(see Bass
(2001) Nature 4I1: 428-9). In mammalian cells, siRNAs are effective at
concentrations that are several orders of magnitude below the concentrations
typically used in antisense experiments (Elbashir et al. (200I) Nature 411:
494-8).
The double stranded oligonucleotides used to effect RNAi are preferably less
than 30 base pairs in length and, more preferably, comprise about 25, 24, 23,
22, 21,
20, 19, 18 or 17 base pairs of ribonucleic acid. Optionally the dsRNA
oligonucleotides o f t he application m ay i nclude 3 ' o verhang ends. E
xemplary 2
nucleotide 3' overhangs may b a composed of ribonucleotide residues of any
type
and may even be composed of 2'-deoxythymidine resides, which lowers the cost
of
RNA synthesis and may enhance nuclease resistance of siRNAs in the cell
culture
medium and within transfected cells (see Elbashir et al. (2001) Nature 4I1:
494-8).
Longer dsRNAs of 50, 75, 100 or even 500 base pairs or more may also be
utilized
in certain embodiments of the application. Exemplary concentrations of dsRNAs
for
effecting RNAi are about 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 1.5 nM, 25 nM or 100
nM, although other concentrations may be utilized depending upon the nature of
the
cells treated, the gene target and other factors readily discernable the
skilled artisan.
Exemplary dsRNAs may be synthesized chemically or produced in vitro or in vivo
using appropriate expression vectors. Exemplary synthetic RNAs include 21
nucleotide RNAs chemically synthesized using methods known in the art (e.g.,
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Expedite RNA phophoramidites and thymidine phosphoramidite (Proligo,
Germany). S ynthetic o ligonucleotides are p referably d eprotected and gel-
purified
using methods known in the art (see e.g., Elbashir et al. (2001) Genes Dev.
15: 188-
200). Longer RNAs may be transcribed from promoters, such as T7 RNA
polymerase promoters, known in the art. A single RNA target, placed in both
possible orientations downstream of an in vitro promoter, will transcribe both
strands of the target to create a dsRNA oligonucleotide of the desired target
sequence. Any of the above RNA species will be designed to include a portion
of
nucleic acid sequence represented in a POSH or POSH-AP nucleic acid, such as,
for
example, a nucleic acid that hybridizes, under stringent and/or physiological
conditions, to any of SEQ ID Nos: 1, 3, 4, 6, 8 and 10 and complements thereof
or
any of the POSH-AP sequences presented in Figure 36.
The specific sequence utilized in design of the oligonucleotides may be any
contiguous sequence of nucleotides contained within the expressed gene message
of
the target. Programs and algorithms, known in the art, may be used to select
appropriate target sequences. In addition, optimal sequences may be selected
utilizing programs designed to predict the secondary structure of a specified
single
stranded nucleic acid sequence and allowing selection of those sequences
likely to
occur in exposed single stranded regions of a folded mRNA. Methods and
compositions for designing appropriate oligonucleotides may be found, for
example,
in U.S. Patent Nos. 6,251,588, the contents of which are incorporated herein
by
reference. Messenger RNA (mRNA) is generally thought of as a linear molecule
which contains the information for directing protein synthesis within the
sequence of
ribonucleotides, however studies have revealed a number of secondary and
tertiary
structures that exist in most mRNAs. Secondary structure elements in RNA are
formed largely by Watson-Crick type interactions between different regions of
the
same RNA molecule. Important secondary stnictural elements include
intramolecular d ouble s tranded r egions, h airpin I oops, b ulges i n d
uplex RNA a nd
internal loops. Tertiary stnictural elements are formed when secondary
structural
elements come in contact with each other or with single stranded regions to
produce
a more complex three dimensional structure. A number of researchers have
measured the binding energies of a large number of RNA duplex strictures and
have
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derived a set of rules which can be used to predict the secondary structure of
RNA
(see e.g., Jaeger et al. (1989) Proc. Natl. Acad. Sci. USA 86:7706 (1989); and
Turner et al. (1988) Annu. Rev. Biophys. Biophys. Chem. 17:167) . The rules
are
useful in identification of RNA structural elements and, in particular, for
identifying
single stranded RNA regions which may represent preferred segments of the mRNA
to target for silencing RNAi, ribozyme or antisense technologies. Accordingly,
preferred segments of the mRNA target can be identified for design of the RNAi
mediating dsRNA oligonucleotides as well as for design of appropriate ribozyme
and hammerheadribozyme compositions of the application.
The dsRNA oligonucleotides may be introduced into the cell by transfection
with an heterologous target gene using carrier compositions such as liposomes,
which are known in the art- e.g., Lipofectamine 2000 (Life Technologies) as
described by the manufacturer for adherent cell lines. Transfection of dsRNA
oligonucleotides for targeting endogenous genes may be carried out using
Oligofectamine (Life Technologies). Transfection efficiency may be checked
using
fluorescence microscopy for mammalian cell lines after co-transfection of hGFP-
encoding pAD3 (Kehlenback et al. (I998) J Cell Biol 141: 863-74). The
effectiveness of the RNAi may be assessed by any of a number of assays
following
introduction of the dsRNAs. These include Western blot analysis using
antibodies
which recognize the POSH or POSH-AP gene product following sufficient time for
turnover o f t he a ndogenous p ool a $er n ew p rotein s ynthesis i s r
epressed, r averse
transcriptase polymerase chain reaction and Northern blot analysis to
determine the
level of existing POSH or POSH-AP target mRNA.
Further compositions, methods and applications of RNAi technology are
provided in U.S. Patent Application Nos. 6,278,039, 5,723,750 and 5,244,805,
which are incorporated herein by reference.
Ribozyme molecules designed to catalytically cleave POSH or POSH-AP
mRNA transcripts can also be used to prevent translation of suject POSH or
POSH-
AP mRNAs and/or expression of POSH or POSH-APs (see, e.g., PCT International
Publication W090/11364, published October 4, 1990; Sarver et al. (1990)
Science
247:1222-1225 and U.S. Patent No. 5,093,246). Ribozymes are enzymatic RNA
molecules capable of catalyzing the specific cleavage of RNA. (For a review,
see
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Rossi (1994) Current Biology 4: 469-471). The mechanism of ribozyme action
involves sequence specific hybridization of the ribozyme molecule to
complementary target RNA, followed by an endonucleolytic cleavage event. The
composition of ribozyme molecules preferably includes one or more sequences
complementary to a POSH or POSH-AP mRNA, and the well known catalytic
sequence responsible for mRNA cleavage or a functionally equivalent sequence
(see, e.g., U.S. Pat. No. 5,093,246, which is incorporated herein by reference
in its
entirety).
While r ibozymes t hat cleave m RNA a t s ite s pecific r ecognition s
equences
can be used to destroy target mRNAs, the use of hammerhead ribozymes is
preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking
regions that form complementary base pairs with the target mRNA. Preferably,
the
target mRNA has the following sequence of two bases: 5'-UG-3'. The
construction
and production of hammerhead ribozymes is well known in the art and is
described
more fully in Haseloff and Gerlach ((1988) Nature 334:585-591; and see PCT
Appln. No. WO89/05852, the contents of which are incorporated herein by
reference). H ammerhead r ibozyme s equences c an b a a mbedded i n a s table
R NA
such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo
(Perriman et
al. (1995) Proc. Natl. Acad. Sci. USA, 92: 6175-79; de Feyter, and Gaudron,
Methods in Molecular Biology, Vol. 74, Chapter 43, "Expressing Ribozyrnes in
Plants", E dited b y T urner, P . C , H umana P ress Inc., T otowa, N .J). In
p articL~lar,
RNA polymerase III-mediated expression of tRNA fusion ribozymes are well
known in the art ( see Kawasaki et al. (1998) Nature 393: 284-9; Kuwabara et
al.
(1998) Nature Biotechnol. 16: 961-5; and Kuwabara et al. (1998) Mol. Cell 2:
617-
27; Koseki et al. (1999) J~Virol 73: 1868-77; Kuwabara et al. (1999) Proc Natl
Acad
Sci USA 96: 1886-91; Tanabe et al. (2000) Nature 406: 473-4). There are
typically
a number of potential hammerhead ribozyme cleavage sites within a given target
cDNA sequence. Preferably the ribozyme is engineered so that the cleavage
recognition site is located near the 5' end of the target mRNA- to increase
efficiency
and minimize the intracellular accumulation of non-functional mRNA
transcripts.
Furthermore, the use of any cleavage recognition site located in the target
sequence
encoding different portions of the C-terminal amino acid domains of, for
example,
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long and short forms of target would allow the selective targeting of one or
the other
form of the target, and thus, have a selective effect on one form of the
target gene
product.
Gene targeting ribozymes necessarily contain a hybridizing region
complementary to two regions, each of at least 5 and preferably each 6, 7, 8,
9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides in length of a
POSH
or POSH-AP mRNA, such as an mRNA of a sequence represented in any of SEQ ID
Nos: l, 3, 4, 6, 8 or 10 or a POSH-AP presented in Figure 3.6. In addition,
ribozymes possess highly specific endoribonuclease activity, which
autocatalytically
cleaves the target sense mRNA. The present application extends to ribozymes
which hybridize to a sense mRNA encoding a POSH gene such as a therapeutic
drug
target candidate gene, thereby hybridising to the sense mRNA and cleaving it,
such
that it is no longer capable of being translated to synthesize a functional
polypeptide
product.
The ribozymes of the present application also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which
occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS
RNA)
and which has been extensively described by Thomas Cech and collaborators
(Zaug,
et al. (1984) Science 224:574-578; Zaug, et al. (1986) Science 231:470-475;
Zaug,
et al. (1986) Nature 324:429-433; published International patent application
No.
W088/04300 by University Patents Inc.; Been, et al. (1986) Cell 47:207-216).
The
Cech-type ribo~ymes have an eight base pair active site which hybridizes to a
target
RNA sequence whereafter cleavage of the target RNA takes place. The
application
encompasses those Cech-type ribozymes which target eight base-pair active site
sequences that are present in a target gene or nucleic acid sequence.
Ribozymes can be composed of modified oligonucleotides (e.g., for
improved stability, targeting, etc.) and should be delivered to cells which
express the
target gene in vivo. A preferred method of delivery involves using a DNA
construct
"encoding" the ribozyme under the control of a strong constitutive pol III or
pol II
promoter, so that transfected cells will produce sufficient quantities of the
ribozyme
to destroy endogenous target messages and inhibit translation. Because
ribozymes,
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unlike antisense molecules, are catalytic, a lower intracellular concentration
is
required for efficiency.
In certain embodiments, a ribozyme may be designed by first identifying a
sequence portion sufficient to cause effective knockdown by RNAi. The same
sequence portion may then be incorporated into a ribozyme. In this aspect of
the
application, the gene-targeting portions of the ribozyme or RNAi axe
substantially
the same sequence of at least 5 and preferably 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16,
17, 18, 19 or 20 or more contiguous nucleotides of a POSH nucleic acid, such
as a
nucleic acid of any of SEQ ID Nos: 1, 3, 4, 6, 8, or 10 or POSH-AP nucleic
acid, as
presented in Figure 36. In a long target RNA chain, significant numbers of
target
sites are not accessible to the ribozyme because they are hidden within
secondary or
tertiary structures (Birikh et al. (1997) Eur J Biochem 245: 1-16). To
overcome the
problem of target RNA accessibility, computer generated predictions of
secondary
structure are typically used to identify targets that are most likely to be
single-
stranded or have an "open" configuration (see Jaeger et al. (1989) Methods
Enzymol
183: 281-306). Other approaches utilize a systematic approach to predicting
secondary structure which involves assessing a huge number of candidate
hybridizing oligonucleotides molecules (seeMilner et al. ( I 997) Nat
Biotechnol 15:
537-41; and Patzel and Sczakiel (1998) Nat Biotechnol 16: 64-8). Additionally,
U.S.
Patent No. 6,251,588, the contents of which are hereby incorporated herein,
describes methods for evaluating oligonucleotide probe sequences so as to
predict
the potential for hybridization to a target nucleic acid sequence. The method
of the
application provides for the use of such methods to select preferred segments
of a
target mRNA sequence that are predicted to be single-stranded and, further,
for the
opportunistic utilization of the same or substantially identical target mRNA
sequence, preferably comprising about 10-20 consecutive nucleotides of the
target
mRNA, in the design of both the RNAi oligonucleotides and ribozymes of the
application.
A further aspect of the application relates to the use of the isolated
"antisense" nucleic acids to inhibit expression, e.g., by inhibiting
transcription
and/or translation of a POSH or POSH-AP nucleic acid. The antisense nucleic
acids
may hind to the potential drug target by conventional base pair
complementarity, or,
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for example, in the case of binding to DNA duplexes, through specific
interactions
in the major groove of the double helix. In general, these methods refer to
the range
of techniques generally employed in the art, and include any methods that rely
on
specific binding to oligonucleotide sequences.
S An antisense construct of the present application can be delivered, for
example, as an expression plasmid which, when transcribed in the cell,
produces
RNA w hich i s complementary t o at 1 east a a pique p onion o f t he cellular
m RNA
which encodes a POSH or POSH-AP polypeptide. Alternatively, the antisense
construct is an oligonucleotide probe, which is generated ex vivo and which,
when
introduced into the cell causes inhibition of expression by hybridizing with
the
mRNA and/or genomic sequences ,of a POSH or POSH-AP nucleic acid. Such
oligonucleotide probes are preferably modified oligonucleotides, which are
resistant
to endogenous nucleases, e.g., exonucleases andlor endonucleases, and are
therefore
stable in vivo. Exemplary nucleic acid molecules for use as antisense
oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate
analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775).
Additionally, general approaches to constructing oligomers useful in antisense
therapy have been reviewed, for example, by Van der Krol et al. (I988)
BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659- 2668.
With respect to antisense DNA, oligodeoxyribonucleotides derived from the
translation initiation site, e.g., between the -10 and +10 regions of the
target gene,
are preferred. Antisense approaches involve the design of oligonucleotides
(either
DNA or RNA) that are complementary to mRNA encoding a POSH or POSH-AP
polypeptide. The antisense oligonucleotides will bind to the xnRNA transcripts
and
prevent translation. Absolute complementarity, although preferred, is not
required.
In the case of double-stranded antisense nucleic acids, a single strand of the
duplex
DNA may thus be tested, or triplex formation may be assayed. The ability to
hybridize will depend on both the degree of complementarity and the length of
the
antisense nucleic acid. Generally, the longer the hybridizing nucleic acid,
the more
base mismatches with an RNA it may contain and still form a stable duplex (or
triplex, as the case may be). One skilled in the art can ascertain a tolerable
degree of
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mismatch by use of standard procedures to determine the melting point of the
hybridized complex.
Oligonucleotides that are complementary to the 5' end of the mRNA, e.g., the
5' untranslated sequence up to and including the AUG initiation codon, should
work
most efficiently at inhibiting translation. However, sequences complementary
to the
3' a ntranslated s equences o f m RNAs h ave r ecently b een s hown t o b a a
ffective a t
' inhibiting translation of mRNAs as well. (Wagner, R. 1994. Nature 372:333).
Therefore, oligonucleotides complementary to either the 5' or 3' untranslated,
non-
coding regions of a gene could be used in an antisense approach to inhibit
translation
of that mRNA. Oligonucleotides complementary to the 5' untranslated region of
the
mRNA should include the complement of the AUG start codon. Antisense
oligonucleotides complementary to mRNA coding regions are less efficient
I' inhibitors of translation but could also be used in accordance with the
application.
Whether designed to hybridize to the 5', 3' or coding region of mRNA,
antisense
nucleic acids should be at least six nucleotides in length, and are preferably
less that
about 100 and more preferably less than about 50, 25, 17 or 10 nucleotides in
length.
It is preferred that in vitro studies are first performed to quantitate the
ability
of the antisense oligonucleotide to inhibit gene expression. It is preferred
that these
studies utilize controls that distinguish between antisense gene inhibition
and
nonspecific biological effects of oligonucleotides. It is also preferred that
these
studies compare levels of the target RNA or protein with that of an internal
control
RNA or protein. Results obtained using the antisense oligonucleotide may be
compared with those obtained using a control oligonucleotide. It is preferred
that
the control oligonucleotide is of approximately the same length as the test
oligonucleotide and that the nucleotide sequence of the oligonucleotide
differs from
the antisense sequence no more than is necessary to prevent specific
hybridization to
the target sequence.
The antisense oligonucleotides can be DNA or RNA or chimeric mixtures or
derivatives o r m odified versions t hereof, s ingle-stranded o r d ouble-
stranded. T he
oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate
backbone, for example, to improve stability of the molecule, hybridization,
etc. The
oligonucleotide may include other appended groups such as peptides (e.g., fox
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targeting host cell receptors), or agents facilitating transport across the
cell
membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.
86:6553-
6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication No.
W088109810, published December 15, 1988) or the blood- brain barrier (see,
e.g.,
PCT Publication No. W089!10134, published April 25, 1988), hybridization
triggered cleavage agents. (See, e.g., Krol et al., 1988, BioTechniques 6:958-
976)
or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To
this end,
the oligonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent, hybridization-
triggered
cleavage agent, etc.
The antisense oligonucleotide may comprise at least one modified base
moiety which is selected from the group including but not limited to 5-
fluorouracil,
5- bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-
acetylcytosine, 5- (carboxyhydroxytiethyl) uracil, 5-carboxymethylaminomethyl-
2-
thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-
galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-
methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-
methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-
methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-
mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-
N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil,
queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid
(v), 5-
methyl-2-thiouracil, 3-(3-amino-3- N-2-carboxypropyl) uracil, (acp3)w, and 2,6-
diaminopurine.
The antisense oligonucleotide may also comprise at least one modified sugar
moiety selected from the group including but not limited to arabinose, 2-
fluoroarabinose, xylulose, and hexose.
The antisense oligonucleotide can also contain a neutral peptide-like
backbone. Such molecules are termed peptide nucleic acid (PNA)-oligomers and
are described, e.g., in Perry-O'Keefe et al. (1996) Prac. Natl. Acad. Sci.
U.S.A.
93:14670 and in Eglom et al. (1993) Nature 365:566. One advantage of PNA
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oligomers is their capability to bind to complementary DNA essentially
independently from the ionic strength of the medium due to the neutral
backbone of
the DNA. I n y et another embodiment, the antisense oligonucleotide comprises
at
least one modified phosphate backbone selected from the group consisting of a
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof.
In yet a further embodiment, the antisense oligonucleotide is an alpha
anomeric oligonucleotide. An alpha-anomeric oligonucleotide forms specific
double-stranded hybrids with complementary RNA in which, contrary to the usual
antiparallel orientation, the strands run parallel to each other (Gautier et
al., 1987,
Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2'-0-
methylribonucleotide
(moue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA
analogue (moue et al., 1987, FEBS Lett. 215:327-330).
While antisense nucleotides complementary to the coding region of a POSH
or POSIT-AP mRNA sequence can be used, those complementary to the transcribed
untranslated region may also be used.
In certain instances, it may be difficult to achieve intracellular
concentrations
of the antisense sufficient to suppress translation on endogenous mRNAs.
Therefore
a preferred approach utilizes a recombinant DNA construct in which the
antisense
oligonucleotide is placed under the control of a strong pol III or pol II
promoter.
The use of such a construct to transfect target cells will result in the
transcription of
sufficient amounts of single stranded RNAs that will form complementary base
pairs
with the endogenous potential drug target transcripts and thereby prevent
translation.
For example, a vector can be introduced such that it is taken up by a cell and
directs
the transcription of an antisense RNA. Such a vector can remain episomal or
become chromosomally integrated, as long as it can be transcribed to produce
the
desired antisense RNA. Such vectors can be constructed by recombinant DNA
technology methods standard in the art. Vectors can be plasmid, viral, or
others
known in the art, used for replication and expression in mammalian cells.
Expression o f t he s equence a ncoding t he a ntisense R NA c an b a b y a ny
p romoter
known in the art to act in mammalian, preferably human cells. Such promoters
can
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be inducible or constitutive. Such promoters include but are not limited to:
the
SV40 a arly p romoter r egion ( Bernoist a nd C hambon, 1981, N ature 2 90:304-
310),
the promoter contained in the 3' long terminal repeat of Rous sarcoma virus
(Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter
(Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the
regulatory
sequences of the metallothionein gene (Brinster et al, 1982, Nature 296:39-
42), etc.
Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the
recombinant DNA construct, which can be introduced directly into the tissue
site.
Alternatively, POSH or POSH-AP gene expression can be reduced by
targeting deoxyribonucleotide sequences complementary to the regulatory region
of
the gene (i.e., the promoter and/or enhancers) to form triple helical
structures that
prevent transcription of the gene in target cells in the body. (See generally,
Helene,
C. 1991, Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann.
N.Y.
Acad. Sci., 660:27-36; and Maher, L.J., 1992, Bioassays 14(12):807-15).
Nucleic acid molecules to be used in triple helix formation for the inhibition
of transcription are preferably single stranded and composed of
deoxyribonucleotides. The base composition of these oligonucleotides should
promote triple helix formation via Hoogsteen base pairing rules, which
generally
require sizable stretches of either purines or pyrimidines to be present on
one strand
of a duplex. Nucleotide sequences may be pyrimidine-based, which will result
in
TAT and CGC triplets across the three associated strands of the resulting
triple
helix. The pyrimidine-rich molecules provide base complementarity to a purine-
rich
region of a single strand of the duplex in a parallel orientation to that
strand. I n
addition, nucleic acid molecules may be chosen that are purine- rich, for
example,
containing a stretch of G residues. These molecules will form a triple helix
with a
DNA duplex that is rich in GC pairs, in which the majority of the purine
residues are
located on a single strand of the targeted duplex, resulting in CGC triplets
across the
three strands in the triplex.
Alternatively, POSH or POSH-AP sequences that can be targeted for triple
helix formation may be increased by creating a so called "switchback" nucleic
acid
molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5'
manner, such that they base pair with first one strand of a duplex and then
the other,
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eliminating the necessity for a sizable stretch of either purines or
pyrimidines to be
present on one strand of a duplex.
A further aspect of the application relates to the use of DNA enzymes to
inhibit expression of a POSH or POSH-AP gene. DNA enzymes incorporate some
of the mechanistic features of both antisense and ribozyme technologies. DNA
enzymes are designed so that they recognize a particular target nucleic acid
sequence, much like an antisense oligonucleotide, however much like a ribozyme
they are catalytic and specifically cleave the target nucleic acid.
There are currently two basic types of DNA enzymes, and both of these were
identified by Santoro and Joyce (see, for example, US Patent No. 6110462). The
10-23 DNA enzyme comprises a loop structure which connect two arms. The two
arms provide specificity by recognizing the particular target nucleic acid
sequence
while the loop structure provides catalytic function under physiological
conditions.
Briefly, to design an ideal DNA enzyme that specifically recognizes and
cleaves a target nucleic a cid, one of skill in the art must first identify
the unique
target sequence. This can be done using the same approach as outlined for
antisense
oligonucleotides. Preferably, the unique or substantially sequence is a G/C
rich of
approximately 18 to 22 nucleotides. High G/C content helps insure a stronger
interaction between the DNA enzyme and the target sequence.
When synthesizing the DNA enzyme, the specific antisense recognition
sequence that will target the enzyme to the message is divided so that it
comprises
the two arms of the DNA enzyme, and the DNA enzyme loop is placed between the
two specific arms.
Methods of making and administering DNA enzymes can be found, for
example, in US 6110462. Similarly, methods of delivery DNA ribozymes in vitro
or
in vivo include methods of delivery RNA ribozyme, as outlined in detail above.
Additionally, one of skill in the art will recognize that, like antisense
oligonucleotide, DNA enzymes can be optionally modified to improve stability
and
improve resistance to degradation.
Antisense RNA and DNA, ribozyme, RNAi and triple helix molecules of the
application may be prepared by any method known in the art for the synthesis
of
r»ta any RNA molecules. These include techniques for chemically synthesizing
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oligodeoxyribonucleotides and oligoribonucleotides well known in the art such
as
for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of DNA
sequences
encoding the antisense RNA molecule. Such DNA sequences may be incorporated
into a wide variety of vectors which incorporate suitable RNA polymerase
promoters s uch as t he T 7 o r S P6 p olymerase p romoters. A lternatively,
antisense
cDNA constructs that synthesize antisense RNA constitutively or inducibly,
depending on the promoter used, can be introduced stably into cell lines.
Moreover,
various well-known modifications to nucleic acid molecules may be introduced
as a
means of increasing intracellular stability and half life. Possible
modifications
include but are not limited to the addition of flanking sequences of
ribonucleotides
or deoxyribonucleotides to the 5' andlor 3' ends of the molecule or the use of
phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within
the
oligodeoxyribonucleotide backbone.
6. Drub Screening Assays
In certain aspects, the present application provides assays for identifying
therapeutic agents which either interfere with or promote POSH or POSH-AP
function. In certain aspects, the present application also provides assays for
identifying therapeutic agents w hich either interfere w ith or promote the
complex
formation between a POSH polypeptide and a POSH-AP polypeptide.
In certain embodiments, agents of the application are antiviral agents,
optionally interfering with viral maturation, and preferably where the virus
is an
envelope virus, and optionally a retroid virus or an RNA virus. In other
embodiments, agents of the application are anticancer agents. In further
embodiments, agents of the application inhibit the progression of a
neurodegenerative disorder. In certain embodiments, an antiviral or anticancer
agent
or an agent that inhibits the progression of a neurodegenerative disorder
interferes
with the ubiquitin ligase catalytic activity of POSH (e.g., POSH auto-
ubiquitination
or transfer to a target protein). In other embodiments, agents disclosed
herein inhibit
or promote POSH and POSH-AP mediated cellular processes such as apoptosis and
protein processing in the secretory pathway.
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In certain preferred embodiments, an antiviral agent interferes with the
interaction b etween P OSH and a P OSH-AP p olypeptide, for example an
antiviral
agent may disrupt or render irreversible interaction 'between a POSH
polypeptide
and POSH-AP polypeptide (as in the case of a POSH dimer, a heterodimer of two
different POSH polypeptides, homomultimers and heteromultimers). In further
embodiments, agents of the application are anti-apoptotic agents, optionally
interfering with TNK and/or NF-KB signaling. In yet additional embodiments,
agents of the application interfere with the signaling of a GTPase, such as
Rac or
Ras, optionally disrupting the interaction between a' POSH polypeptide and a
Rac
protein. In certain embodiments, agents of the application modulate the
ubiquitin
ligase activity of POSH and may be used to treat certain diseases related to
ubiquitin
ligase activity. In certain embodiments, agents of the application interfere
with the
trafficking of a protein through the secretory pathway.
In certain embodiments, the application provides assays to identify, optimize
or otherwise assess agents that increase or decrease a ubiquitin-related
activity of a
POSH polypeptide. Ubiquitin-related activities of POSH polypeptides may
include
the self ubiquitination activity of a POSH polypeptide, generally involving
the
transfer of ubiquitin from an E2 enzyme to the POSH polypeptide, and the
ubiquitination of a target protein, generally involving the transfer of a
ubiquitin from
a POSH polypeptide to the target protein. In certain embodiments, a POSH
activity
is mediated, at least in part, by a FOSH RII\TG domain.
In certain embodiments, an assay comprises forming a mixture comprising a
POSH polypeptide, an E2 polypeptide and a source of ubiquitin (which may be
the
E2 polypeptide pre-complexed with ubiquitin). Optionally the mixture comprises
an
E1 polypeptide and optionally the mixture comprises a target polypeptide.
Additional components of the mixture may be selected to provide conditions
consistent with the ubiquitination of the POSH polypeptide. One or more of a
variety of parameters may be detected, such as POSH-ubiquitin conjugates, E2-
ubiquitin thioesters, free ubiquitin and target polypeptide-ubiquitin
complexes. The
term "detect" is used herein to include a determination of the presence or
absence of
the subject of detection (e.g., POSH-ubiqutin, E2-ubiquitin, etc.), a
quantitative
measure of the amount of the subject of detection, or a mathematical
calculation of
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the presence, absence or amount of the subject of detection, based on the
detection
of other parameters. The term "detect" includes the situation wherein the
subject of
detection is determined to be absent or below the level of sensitivity.
Detection may
comprise detection of a label (e.g., fluorescent label, radioisotope label,
and other
S described below), resolution and identification by size (e.g., SDS-PAGE,
mass
spectroscopy), purification and detection, and other methods that, in' view of
this
specification, will be available to one of skill in the art. For instance,
radioisotope
labeling may be measured by scintillation counting, or by densitometry after
exposure to a photographic emulsion, or by using a device such as a
Phosphorimager. Likewise, densitometry may be used to measure bound ubiquitin
following a reaction with an enzyme label substrate that produces an opaque
product
when an enzyme label is used. In a preferred embodiment, an assay comprises
detecting the POSH-ubiquitin conjugate.
In certain embodiments, an assay comprises forming a mixture comprising a
POSH polypeptide, a target polypeptide and a source of ubiquitin (which may be
the
POSH polypeptide pre-complexed with ubiquitin). Optionally the mixture
comprises an E1 and/or E2 polypeptide and optionally the mixture comprises an
E2
ubiquitin thioester. Additional components of the mixture may be selected to
provide conditions consistent with the ubiquitination of the target
polypeptide. One
or more of a variety of parameters may be detected, such as POSH-ubiquitin
conjugates and target polypeptide-ubiquitin conjugates. In a preferred
embodiment,
an assay comprises detecting the target polypeptide-ubiquitin conjugate. In
another
preferred embodiment, an assay comprises detecting the POSH-ubiquitin
conjugate.
An assay described above may be used in a screening assay to identify agents
that modulate a ubiquitin-related activity of a POSH polypeptide. A screening
assay
will generally involve adding a test agent to one of the above assays, or any
other
assay designed to assess a ubiquitin-related activity of a POSH polypeptide.
The
parameters) detected in a screening assay may be compared to a suitable
reference.
A suitable reference may be an assay run previously, in parallel or later that
omits
the test agent. A suitable reference may also be an average of previous
measurements in the absence of the test agent. In general the components of a
screening assay mixture may be added in any order consistent with the overall
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activity to be assessed, but certain variations may be preferred. For example,
in
certain embodiments, it may be desirable to pre-incubate the test agent and
the E3
(e.g., the POSH polypeptide), followed by removing the test agent and addition
of
other components to complete the assay. In this manner, the effects of the
agent
S solely on the POSH polypeptide may be assessed. In certain preferred
embodiments, a screening assay for an antiviral agent employs a target
polypeptide
comprising an L domain, and preferably an HIV L domain.
In certain embodiments, an assay is performed in a high-throughput format.
For example, one of the components of a mixture may be affixed to a solid
substrate
and one or more of the other components is labeled. For example, the POSH
polypeptide may be affixed to a surface, such as a 96-well plate, and the
ubiquitin is
in solution and labeled. An E2 and El are also in solution, and the POSH-
ubiquitin
conjugate formation may be measured by washing the solid surface to remove
uncomplexed labeled ubiquitin and detecting the ubiquitin that remains bound.
1S Other variations may be used. For example, the amount of ubiquitin in
solution may
be detected. In certain embodiments, the formation of ubiquitin complexes may
be
measured by an interactive technique, such as FRET, wherein a ubiquitin is
labeled
with a first label and the desired complex partner (e.g., POSH polypeptide or
target
polypeptide) is labeled with a second label, wherein the first and second
label
interact when they come into close proximity to produce an altered signal. In
FRET, the first and second labels are fluorophores. FRET is described in
greater
detail below. The formation of polyubiquitin complexes may be performed by
mixing two or more pools of differentially labeled ubiquitin that interact
upon
formation of a polyubiqutin (see, e.g., US Patent Publication 20020042083).
High-
2S throughput may be achieved by performing an interactive assay, such as
FRET, in
solution a s w ell. In a ddition, i f a p olypeptide in t he m fixture, s uch
a s the P OSH
polypeptide or target polypeptide, is readily purifiable (e.g., with a
specific antibody
or via a tag such as biotin, FLAG, polyhistidine, etc.), the reaction may be
performed in solution and the tagged polypeptide rapidly isolated, along with
any
polypeptides, such as ubiquitin, that are associated with the tagged
polypeptide.
Proteins may also be resolved by SDS-PAGE for detection.
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In certain embodiments, the ubiquitin is labeled, either directly or
indirectly.
This typically allows for easy and rapid detection and measurement of ligated
ubiquitin, m along t he a ssay useful f or h igh-throughput sc reening a
pplications. A s
descrived above, certain embodiments may employ one or more tagged or labeled
proteins. A "tag" is meant to include moieties that facilitate rapid isolation
of the
tagged polypeptide. A tag may be used to facilitate attachment of a
polypeptide to a
surface. A "label" is meant to include moieties that facilitate rapid
detection of the
labeled polypeptide. Certain moieties may be used both as a label and a tag
(e.g.,
epitope tags that are readily purified and detected with a well-characterized
antibody). Biotinylation of polypeptides is well known, for example, a large
number
of biotinylation agents are known, including amine-reactive and thiol-reactive
agents, for the biotinylation of proteins, nucleic acids, carbohydrates,
carboxylic
acids; see chapter 4, Molecular Probes Catalog, Haugland, 6th Ed. 1996, hereby
incorporated by reference. A biotinylated substrate can be attached to a
biotinylated
component via avidin or streptavidin. Similarly, a large number of
haptenylation
reagents are also known.
An "E1" is a ubiquitin activating enzyme. In a preferred embodiment, E1 is
capable of transfernng ubiquitin to an E2. In a preferred embodiment, El forms
a
high energy thiolester bond with ubiquitin, thereby "activating" the
ubiquitin. An
"E2" is a ubiquitin carrier enzyme (also known as a ubiquitin conjugating
enzyme).
In a preferred embodiment, ubiquitin is transferred from E1 to E2. In a
preferred
embodiment, the transfer results in a thiolester bond formed between E2 and
ubiquitin. In a p referred a mbodiment, E 2 i s c apable o f t ransferring a
biquitin t o a
POSH polypeptide.
In an alternative embodiment, a POSH polypeptide, E2 or target polypeptide
is bound to a bead, optionally with the assistance of a tag. Following
ligation, the
beads may be separated from the unbound ubiquitin and the bound ubiquitin
measured. In a preferred embodiment, POSH polypeptide is bound to beads and
the
composition used includes labeled ubiquitin. In this embodiment, the beads
with
bound ubiquitin may be separated using a fluorescence-activated cell sorting
(FAGS) machine. Methods for such use are described in IJ.S. patent application
Ser.
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No. 09/047,119, which is hereby incorporated in its entirety. The amount of
bound
ubiquitin can then be measured.
In a screening assay, the effect of a test agent may be assessed by, for
example, assessing the effect of the test agent on kinetics, steady-state
and/or
endpoint of the~reaction.
The components of the various assay mixtures provided herein may be
combined in varying amounts. In a preferred embodiment, ubiquitin (or E2
complexed ubiquitin) is combined at a final concentration of from 5 to 200 ng
per
100 microliter reaction solution. Optionally El is used at a final
concentration of
from 1 to 50 ng per 100 microliter reaction solution. Optionally E2 is
combined at a
final concentration of 10 to 100 ng per 100 microliter reaction solution, more
preferably 10-50 ng per 100 microliter reaction solution. In a preferred
embodiment,
POSH polypeptide is combined at a final concentration of from 1 to 500 ng. per
100
microliter reaction solution.
Generally, an assay mixture is prepared so as to favor ubiquitin ligase
activity and/or ubiquitination activity. Generally, this will be physiological
conditions, such as 50 - 200 mM salt (e.g., NaCI, KCl), pH of between 5 and 9,
and
preferably between 6 and 8. Such conditions may be optimized through trial and
error. Incubations m ay be p erformed a t any t emperature which facilitates o
ptimal
activity, typically between 4 and 40 °C. Incubation periods are
selected for optimum
activity, but may also be optimized to facilitate rapid high through put s
Greening.
Typically between 0.5 and 1.5 hours will be sufficient. A variety of other
reagents
may be included in the compositions. These include reagents like salts,
solvents,
buffers, neutral proteins, e.g., albumin, detergents, etc. which may be used
to
facilitate optimal ubiquitination enzyme activity and/or reduce non-specific
or
background interactions. Also reagents that otherwise improve the efficiency
of the
assay, such as protease inhibitors, nuclease inhibitors, anti-microbial
agents, etc.,
may be used. The compositions will also preferably include adenosine tri-
phosphate
(ATP). The mixture of components may be added in any order that promotes
ubiquitin ligase activity or optimizes identification of candidate modulator
effects. In
a preferred embodiment, ubiquitin is provided in a reaction buffer solution,
followed
by addition of the ubiquitination enzymes. In an alternate preferred
embodiment,
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ubiquitin is provided in a reaction buffer solution, a candidate modulator is
then
added, followed by addition of the ubiquitination enzymes.
In general, a test agent that decreases a POSH ubiquitin-related activity may
be used to inhibit POSH function in vivo, while a test agent that increases a
POSH
ubiquitin-related activity may be used to stimulate POSH function in vivo.
Test
agent may be modified for use in vivo, e.g., by addition of a hydrophobic
moiety,
such as an ester.
In certain embodiments, a ubiquitination assay as described above for POSH
can similarly be conducted for a Cbl-b, a SIAHl, or a TTC3 polypeptide. In
certain
embodiments, the application provides assays to identify, optimize or
otherwise
assess agents that increase or decrease a ubiquitin-related activity of a Cbl-
b, a
SIAHl, or a TTC3 polypeptide. Ubiquitin-related activities of Cbl-b, SIAHl, or
TTC3 polypeptides may include the self ubiquitination activity of a Cbl-b,
SIAHl,
or TTC3 polypeptide, generally involving the transfer of ubiquitin from an E2
I5 enzyme to the respective Cbl-b, SIAHI, or TTC3 polypeptide, and the
ubiquitination
of a target protein, e.g., the p85 subunit of PI3K, e.g, synaptophysin,
generally
involving the transfer of a ubiquitin from a Cbl-b, SIAH1, or TTC3 polypeptide
to
the target protein, e.g, the p85 subunit of PI3I~, e.g., synaptophysin, e.g.,
HERPUD1. In certain embodiments, a Cbl-b, a SIAH1, or a TTC3 activity is
mediated, at least in part, by a RING domain of a Cbl-b, a SIAH1, or a TTC3,
respectively.
An additional POSH-AP may be added to a POSH ubiquitination assay t o
assess the effect of the POSH-AP (e.g., PRKARlA, PRKACA, or PRKACB) on
POSH-mediated ubiquitination and/or to assess whether the POSH-AP is a target
for
POSH-mediated ubiquitination (e.g., HERPUD1, e.g., PKA).
Certain embodiments of the application relate to assays for identifying agents
that b ind t o a P OSH o r P OSH-AP p olypeptide, o ptionally a p articular
domain o f
POSH such as an SH3 or RING domain or a particular domain of a POSH-AP,
particularly a kinase catalytic domain or ATP binding domain. In preferred
embodiments, a POSH polypeptide is a polypeptide comprising the fourth SH3
domain of hPOSH (SEQ ID NO: 30). A wide variety of assays may be used for this
purpose, including labeled in vitro protein-protein binding assays,
electrophoretic
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mobility shift assays, immunoassays for protein binding, and the like. The
purified
protein may a lso be used for determination of three-dimensional c rystal
stntcture,
which can be used for modeling intermolecular interactions and design of test
agents. In one embodiment, an assay detects agents which inhibit interaction
of one
or more subject POSH polypeptides with a POSH-AP. In another embodiment, the
assay detects agents which modulate the intrinsic biological activity of a
POSH
polypeptide or POSH complex, such as an enzymatic activity, binding to other
cellular components, cellular compartmentalization, and the like.
Certain embodiments of the application relate to assays for identifying agents
that modulate a POSH-AP polypeptide such as a 'PKA subunit polypeptide.
Preferred PKA subunit polypeptides include PRKARlA, PRKA.CA, and PRKACB.
Exemplary assays used for this purpose may include detecting phosphorylation
of
PKA subunit, kinase activity of the PKA subunit, ability of the PKA subunit to
elicit
downstream signaling of the PKA pathway, and the like. For example, activity
of
protein kinase A can be assayed either in vitro or .in vivo. PKA activity can
be
determined b y d etecting p osphorylation o f a P IAA s pacific s ubstrate. T
he s pacific
PKA substrate can be any convenient peptide with a serine that is recognized
as a
phosphorylation site by PISA. For example, the peptide substrate can have the
sequence: Leu-Arg-Arg-Ala- Ser-Leu-Gly.
In one aspect, the application provides methods and compositions for the
identification of compositions that interfere with the function of POSH or
POSH-AP
polypeptides. Given the xole of POSH polypeptides in viral production,
compositions that perturb the formation or stability of the protein-protein
interactions between POSH polypeptides and the proteins that they interact
with,
such as POSH-APs, and particularly POSH complexes comprising a viral protein,
are candidate pharmaceuticals for the treatment of viral infections.
While not wishing to be bound to mechanism, it is postulated that POSH
polypeptides promote the assembly of protein complexes that are important in
release of virions and other biological processes. Complexes of the
application may
include a combination of a POSH polypeptide and a POSH-AP. Exemplary
complexes may comprise one or more of the following: a POSH polypeptide (as in
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the case of a POSH dimer, a heterodimer of two different POSH, homomultimers
and heteromultimers); a HERPUDl polypeptide; or an MSTP028 polypeptide.
In an assay for an antiviral or antiapoptotic agent, the test agent is
assessed
for its ability to disrupt or inhibit the .formation of a complex of a POSH
polypeptide
and a small GTPase, such as a Rac polypeptide, particularly a human Rac
polypeptide, such as Racl.
A variety of assay formats will suffice and, in light of the present
disclosure,
those not expressly described herein will nevertheless be comprehended by one
of
ordinary skill in the art. Assay formats which approximate such conditions as
formation of protein complexes, enzymatic activity, and even a POSH
polypeptide-
mediated membrane reorganization or vesicle formation activity, may be
generated
in many different forms, and include assays based on cell-free systems, e.g.,
purified
proteins or cell lysates, as well as cell-based assays which utilize intact
cells.
Simple binding assays can also be used to detect agents which bind to POSH.
Such
binding assays may also identify agents that act by disrupting the interaction
between a POSH polypeptide and a POSH interacting protein, or the binding of a
POSH polypeptide or complex to a substrate. Agents to be tested can be
produced,
for example, by bacteria, yeast or other organisms (e.g., natural products),
produced
chemically (e.g., small molecules, including peptidomimetics), or produced
recombinantly. In a preferred embodiment, the test agent is a small organic
molecule, e.g., other than a peptide or oligonucleotide, having a molecular
weight of
less than about 2,000 daltons.
In many drug screening programs which test libraries of compounds and
natural extracts, high throughput assays are desirable in order to maximize
the
number of compounds surveyed in a given period of time. Assays of the present
application which are performed in cell-free systems, such as may be developed
with
purified or semi-purified proteins or with lysates, are often preferred as
"primary"
screens in that they can be generated to permit rapid development and
relatively easy
detection of an alteration in a molecular target which is mediated by a test
compound. Moreover, the effects of cellular toxicity andlor bioavailability of
the
test compound can be generally ignored in the in vitro system, the assay
instead
being focused primarily on the effect of the drug on the molecular target as
may be
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manifest in an alteration of binding affinity with other proteins or changes
in
enzymatic properties of the molecular target.
In preferred in vitro embodiments of the present assay, a reconstituted POSH
complex c omprises a r econstituted m fixture o f a t 1 east s emi-purified p
roteins. B y
semi-purified, it is meant that the proteins utilized in the reconstituted
mixture have
been previously separated from other cellular or viral proteins. For instance,
in
contrast to cell lysates, the proteins involved in POSH complex formation are
present in the mixture to at least 50% purity relative to all other proteins
in the
mixture, and more preferably are present at 90-95% purity. In certain
embodiments
of the subject method, the reconstituted protein mixture is derived by mixing
highly
purified proteins such that the reconstituted mixture substantially lacks
other
proteins (such as of cellular or viral origin) which might interfere with or
otherwise
alter the ability to measure POSH complex assembly and/or disassembly.
Assaying POSH complexes, in the presence and absence of a candidate
inhibitor, can be a ccomplished in any v essel suitable for containing the
reactants.
Examples include microtitre plates, test tubes, and micro-centrifuge tubes.
In one embodiment of the present application, drug screening assays can be
generated which detect inhibitory agents on the basis of their ability to
interfere with
assembly or s tability o f the P OSH c omplex. In a n exemplary binding assay,
t he
compound of interest is contacted with a mixture comprising a POSH polypeptide
and at least one interacting polypeptide. Detection and quantification of POSH
complexes provides a means for determining the compound's efficacy at
inhibiting
(or potentiating) interaction between the two polypeptides. The efficacy of
the
compound can be assessed by generating dose response curves from data obtained
using various concentrations of the test compound. Moreover, a control assay
can
also be performed to provide a baseline for comparison. In the control assay,
the
formation of complexes is quantitated in the absence of the test compound.
Complex formation between the POSH polypeptides and a substrate
polypeptide may be detected by a variety of techniques, many of which are
effectively described above. For instance, modulation in the formation of
complexes
can be quantitated using, for example, detectably labeled proteins (e.g.,
radiolabeled,
fluorescently labeled, or enzymatically labeled), by immunoassay, or by
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chromatographic detection. Surface plasmon resonance systems, such as those
available from B iacore International A B ( Uppsala, S weden), m ay a lso b a
a sed t o
detect protein-protein interaction
Often, it will be desirable to immobilize one of the polypeptides to
facilitate
separation of complexes from uncomplexed forms of one of the proteins, as well
as
to accommodate automation of the assay. In an illustrative embodiment, a
fusion
protein can be provided which adds a domain that permits the protein to be
bound to
an insoluble matrix. For example, GST-POSH fusion proteins can be adsorbed
onto
glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione
derivatized microtitre plates, which are then combined with a potential
interacting
protein, e.g., an ASS-labeled polypeptide, and the test compound and incubated
under
conditions c onducive t o c omplex formation. F ollowir~g i ncubation, t he b
eads are
washed to remove any unbound interacting protein, and the matrix bead-bound
radiolabel determined directly (e.g., beads placed in scintillant), or in the
supernatant
after the complexes are dissociated, e.g., when microtitre plate is used.
Alternatively, after washing away unbound protein, the complexes can be
dissociated from the matrix, separated by SDS-PAGE gel, and the level of
interacting polypeptide found in the matrix-bound fraction quantitated from
the gel
using standard electrophoretic techniques.
In a further embodiment, agents that bind to a POSH or POSH-AP may be
identified by using an immobilized POSH or POSH-AP. In an illustrative
embodiment, a fusion protein can be provided which adds a domain that permits
the
protein to be bound to an insoluble matrix. For example, GST-POSH fusion
proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
Louis, MO) or g lutathione derivatized microtitre plates, which a re then c
ombined
with a potential labeled binding agent and incubated under conditions
conducive to
binding. Following incubation, the beads are washed to remove any unbound
agent,
and the matrix bead-bound label determined directly, or in the supernatant
after the
bound agent is dissociated.
In yet another embodiment, the POSH polypeptide and potential interacting
polypeptide can be used to generate an interaction trap assay (see also, U.S.
Patent
NW 5_283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J
Biol
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Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and
Twabuchi a t a 1. ( 1993) O ncogene 8 :1693-1696), for s ubsequently d
etecting a gents
which disrupt binding of the proteins to one and other.
In particular, the method makes use of chimeric genes which express hybrid
proteins. To illustrate, a first hybrid gene comprises the coding sequence for
a
DNA-binding domain of a transcriptional activator can be fused in frame to the
coding sequence for a "bait" protein, e.g., a POSH polypeptide of sufficient
length to
bind to a potential interacting protein. The second hybrid protein encodes a
transcriptional activation domain fused in frame to a gene encoding a "fish"
protein,
e.g., a p otential i nteracting p rotein o f s ufficient length t o i nteract
with t he P OSH
polypeptide portion of the bait fusion protein. If the bait and fish proteins
are able to
interact, e.g., form a POSH complex, they bring into close proximity the two
domains o f t he t ranscriptional a ctivator. T his p roximity c auses t
ranscription o f a
reporter gene which is operably linked to a transcriptional regulatory site
responsive
to the transcriptional activator, and expression of the reporter gene can be
detected
and used to score for the interaction of the bait and fish proteins.
One aspect of the present application provides reconstituted protein
preparations including a POSH polypeptide and one or more interacting
polypeptides.
In still further embodiments of the present assay, the POSH complex is
generated in whole cells, taking advantage of cell culture techniques to
support the
subject assay. For example, as described below, the POSH complex can be
constituted in a eukaryotic cell culture system, including mammalian and yeast
cells.
Often it will be desirable to express one or more viral proteins (e.g., Gag or
Env) in
such a cell along with a subject POSH polypeptide. It may also be desirable to
infect the cell with a virus of interest. Advantages to generating the subj
ect assay in
an intact cell include the ability to detect inhibitors which are functional
in an
environment more closely approximating that which therapeutic use of the
inhibitor
would require, including the ability of the agent to gain entry into the cell.
Furthermore, certain of the in vivo embodiments of the assay, such as examples
given below, are amenable to high through-put analysis of candidate agents.
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The components of the POSH complex can be endogenous to the cell
selected to support the assay. Alternatively, some or all of the components
can be
derived from exogenous sources. F or instance, fusion proteins can be
introduced
into the cell by recombinant techniques (such as through the use of an
expression
vector), as well as by microinjecting the fusion protein itself or mRNA
encoding the
fusion protein.
In many embodiments, a cell is manipulated after incubation with a
candidate agent and assayed for a POSH or POSH-AP activity. hi certain
embodiments, a POSH-AP, such as PTPN12, is a tyrosine phosphatase. Tyrosine
phosphatase activity may be assessed by incubating a cell lysate, which has
optionally been treated with pervanadate to stimulate tyrosine
phosphorylation, with
a POSH-AP that has tyrosine phosphatase activity, immunoprecipitating the
substrate protein and immunoblotting for the presence of phosphorylated
tyrosine.
Alternatively, tyrosine phosphatase activity may be assessed by the substrate
trapping method. The substrate trapping method employs catalytically inactive
mutants of a tyrosine phosphatase (e.g., a POSH-AP such as PTPN12). The
catalytically inactive phosphatase mutant is immobilized on a solid matrix
(e.g.,
AG25-protein A-Sepharose beads) and incubated with a substrate protein. The
solid
matrix to which the catalytically inactive phosphatase is bound is isolated
and
subjected to SDS-PAGE and immunoblotting for the presence of the substrate
protein. The proteins employed in a phosphatase assay may optionally be
purified
proteins. (Lyons, PD et al (2001) J Biol Chem 246:24422-31; Garton, AJ et al
(1996) Mol Cell Biol 16:6408-18).
In many embodiments, a cell is manipulated after incubation with a candidate
agent
and assayed for a POSH or POSH-AP activity. In certain embodiments a POSH or
POSH-AP activity is represented by production of virus like particles. As
demonstrated herein, an agent that disrupts POSH or POSH-AP activity can cause
a
decrease in the production of virus like particles. Other bioassays for POSH
or
POSH-AP activities may include apoptosis assays (e.g., cell survival assays,
apoptosis reporter gene assays, etc.) and NF-kB nuclear localization assays
(see e.g.,
Tapon et al. (1998) EMBO J. 17: 1395-1404). One apoptosis assay that may be
used
to assess TGN-associated protein activity is the TLJNEL assay, which is used
to
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detect the presence of apoptotic cell death. In the TUNEL assay, the enzyme
terminal deoxynucleotidyl transferase labels 3'-OH DNA ends (which are
generated
during apoptosis) with biotinylated nucleotides. The biotinylated nucleotides
are
then detected by immunoperoxidase staining. Another apoptosis assay that may
be
used to assess TGN-associated protein activity is the caspase assay, in which
caspase
activity is measured using a blue fluorescent substrate. Cleavage of the
substrate by
caspase 3 r eleases t he fluorochrome, which t hen fluoresces green. A n assay
t hat
may be employed to monitor cell proliferation associated with a TGN-associated
protein is the MTT cell proliferation assay. The MTT cell proliferation assay
is a
colorimetric assay which measures the reduction of a tetrazolium component
(MTT)
into an insoluble formazan product by the mitochondria of viable cells. After
incubation of the cells with the MTT reagent, a detergent solution is added to
lyse
the cells and solubilize the colored crystals. The samples may be read using
an
ELISA plate reader. The amount of color produced is directly proportional to
the
number of viable cells.
In certain embodiments, POSH or POSH-AP activities may include, without
limitation, complex formation, ubiquitination and membrane fusion events (eg.
release of viral buds or fusion of vesicles). POSH-AP activity may be assessed
by
the presence of phosphorylated substrate, such as, in the case of PKA,
phosphorylated POSH. The interaction of POSH with a small GTPase such as Rac
may also be indicative of the absence of phosphorylation of POSH by PKA. POSH
complex formation may be assessed by immunoprecipitation and analysis of co
immunoprecipiated proteins or affinity plu-ification and analysis of co-
purified
proteins. Fluorescence R esonance Energy Transfer (FRET)-based assays or other
energy transfer assays may also be used to determine complex formation.
The effect of an agent that modulates the activity of POSH or a POSH-AP
may b a a valuated f or a ffects o n t he t rafficking o f a p rotein t hrough
t he s ecretory
system. For example, the effects of the agent on the trafficking of the
protein may
be assessed by detecting the glycosylation of the protein in the presence and
absence
of the agent, for instance, through the use of antibodies specific for sugar
moieties.
For example, cell lysates from cells treated in the absence and presence of an
agent
that modulates the activity of POSH or a POSH-AP may be subjected to
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immunoprecipitation and immunoblotting with antibodies directed to the
glycoprotein of interest and the glycosylation state of the protein then
compared.
Additional bioassays for assessing POSH and POSH-AP activities may
include assays to detect the improper processing of a protein that is
associated with a
neurological disorder. One assay that may be used is an assay to detect the
presence,
including a n i ncrease o r a d ecrease i n t he amount, o f a p rotein
associated w ith a
neurological disorder. For example, the use of RNAi may be employed to
knockdown the expression of a POSH or POSH-AP in cells (e.g., CHO cells or COS
cells). The production of a secreted protein such as for example, amyloid
beta, in
the cell culture media, can then be assessed and compared to production of the
secreted protein from control cells, which may be cells in which the POSH or
POSH-AP activity has not been inhibited. The production of secreted proteins
may
be assessed, such as amyloid beta protein, which is associated with
Alzheimer's
disease. In some instances, a label may be incorporated into a secreted
protein and
the presence of the labeled secreted protein detected in the cell culture
media.
Proteins secreted from any cell type may be assessed, including for example,
neural
cells.
The effect of an agent that modulates the activity of POSH or a POSH-AP
may be evaluated for effects on mouse models of various neurological
disorders.
For example, mouse models of Alzheimer's disease have been described. See, for
example, United States Patent No. 5,612,486 for "Transgenic Animals Harboring
APP Allele Having Swedish Mutation,"Patent No. 5,850,003 (the '003 patent) for
"Transgenic Rodents Harboring APP Allele Having Swedish Mutation,"and United
States Patent No. 5,455,169 entitled "Nucleic Acids for Diagnosing and
Modeling.
Alzheimer's Disease". Mouse models of Alzheimer's disease tend to produce
elevated levels of beta-amyloid protein in the brain, and the increase or
decrease of
such protein in response to treatment with a test agent may be detected. In
some
instances, it may also be desirable to assess the effects of a test agent on
cognitive or
behavioral characteristics of a mouse model for Alzheimer's disease, as well
as
mouse models for other neurological disorders.
In a further embodiment, transcript levels may be measured in cells having
higher or lower levels of POSH or POSH-AP activity in order to identify genes
that
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are regulated by POSH or POSH-APs. Promoter regions for such genes (or larger
portions of such genes) may be operatively linked to a reporter gene and used
in a
reporter gene-based assay to detect agents that enhance or diminish POSH- or
POSH-AP-regulated gene expression. Transcript levels may be determined in any
way known in the art, such as, for example, Northern blotting, RT-PCR,
microarray,
etc. Increased POSH activity may be achieved, for example, by introducing a
strong
POSH expression vector. Decreased POSH activity may be achieved, for example,
by RNAi, antisense, ribozyme, gene knockout, etc.
In general, where the screening assay is a binding assay (whether protein-
protein binding, agent-protein binding, etc.), one or more of the molecules
may be
joined to a label, where the label can directly or indirectly provide a
detectable
signal. Various labels include radioisotopes, fluorescers, chemiluminescers,
enzymes, specific binding molecules, particles, e.g., magnetic particles, and
the like.
Specific b finding m olecules i nclude p airs, s uch as b iotin a nd s
treptavidin, d igoxin
and antidigoxin etc. For the specific binding members, the complementary
member
would normally be labeled with a molecule that provides for detection, in
accordance with known procedures.
In further embodiments, the application provides methods for identifying
targets for therapeutic intervention. A polypeptide that interacts with POSH
or
participates in a POSH-mediated process (such as viral maturation) may be used
to
identify candidate therapeutics. Such targets may be identified by identifying
proteins that associated with POSH (POSH-APs) by, for example,
immunoprecipitation with an anti-POSH antibody, in silico analysis of high-
throughput binding data, two-hybrid screens, and other protein-protein
interaction
assays described herein or otherwise known in the art in view of this
disclosure.
Agents t hat b find t o s uch t argets o r d isrupt p rotein-protein i
nteractions t hereof, o r
inhibit a biochemical activity thereof may be used in such an assay. Targets
that
have been identified by such approaches include POSH-APs provided in Tables '~
and 8 and in Figure 36.
A variety of other reagents may be included in the screening assay. These
include reagents like salts, neutral proteins, e.g., albumin, detergents, etc
that are
used to facilitate optimal protein-protein binding and/or reduce nonspecific
or
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background interactions. Reagents that improve the efficiency of the assay,
such as
protease inhibitors, nuclease inhibitors, anti- microbial agents, etc. may be
used. The
mixture of components are added in any order that provides for the requisite
binding. Incubations are performed at any suitable temperature, typically
between 4
°C and 40 °C. Incubation periods are selected for optimum
activity, but may also be
optimized to facilitate rapid high-throughput screening.
In certain embodiments, a test agent may be assessed for antiviral or
anticancer activity by assessing effects on an activity (function) of a 'POSH-
AP.
Activity (function) may be affected by an agent that acts at one or more of
the
transcriptional, translational or post-translational stages. For example, an
siRNA
directed to a POSH-AP encoding gene will decrease activity, as will a small
molecule that interferes with a catalytic activity of a POSH-AP. In certain
embodiments, the agent inhibits the activity of one or more polypeptides
selected
from among HERPUD1 and MSTP028.
7. Exemplars% Nucleic Acids and Expression Vectors
In certain aspects, the application relates to nucleic acids encoding POSH
polypeptides, such as, for example, SEQ ID Nos: 2, 5, 7, 9, 11, 26, 27, 28, 29
and
30. Nucleic acids of the application are further understood to include nucleic
acids
that comprise variants of SEQ ID Nos:l, 3, 4, 6, 8, 10, 31, 32, 33, 34, and
35.
Variant nucleotide sequences include sequences that differ by one or more
nucleotide substitutions, additions or deletions, such as allelic variants;
and will,
therefore, include coding sequences that differ from the nucleotide sequence
of the
coding sequence designated in SEQ ID Nos:l, 3, 4, 6, 8 10, 31, 32, 33, 34, and
35,
e.g., due to the degeneracy of the genetic code. In other embodiments,
variants will
also include sequences that will hybridize undex highly stringent conditions
to a
nucleotide sequence of a coding sequence designated in any of SEQ ID Nos:l, 3,
4,
6, 8 10, 31, 32, 33, 34, and 35. Preferred nucleic acids of the application
are human
POSH sequences, including, for example, any of SEQ ID Nos: l, 3, 4, 6, 31, 32,
33,
34, 35 and variants thereof and nucleic acids encoding an amino acid sequence
selected from among SEQ ID Nos: 2, 5, 7, 26, 27, 28, 29 and 30.
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In certain aspects, the application relates to nucleic acids encoding POSH-AP
polypeptides. For example, POSH-APs of the disclosure are listed in Table 7.
Nucleic acid sequences encoding these POSH-APs are provided in Figure 36.
Additional examples of POSH-APs of the disclosure are provided in Table 8. In
certain embodiments, variants will also include nucleic acid sequences that
will
hybridize under highly stringent c onditions to a nucleotide sequence of a
coding
sequence of a POSH-AP. Preferred nucleic acids of the application are human
POSH-AP sequences and variants thereof.
One of ordinary skill in the art will understand readily that appropriate
stringency conditions which promote DNA hybridization can be varied. For
example, one could perform the hybridization at 6.0 x sodium chloridelsodium
citrate (SSC) at about 45 °C, followed by a wash of 2.0 x SSC at 50
°C. For
example, the salt concentration in the wash step can be selected from a low
stringency of about 2.0 x SSC at 5.0 °C to a high stringency of about
0.2 x SSC at 50
°C. In addition, the temperature in the wash step can be increased from
low
stringency conditions at room temperature, about 22 °C, to high
stringency
conditions at about 65 °C. Both temperature and salt may be varied, or
temperature
or salt concentration may be held constant while the other variable is
changed. In
one embodiment, the application provides nucleic acids which hybridize under
low
stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x
SSC at room temperature.
Isolated nucleic acids which differ from the POSH nucleic acid sequences or
from the POSH-AP nucleic acid sequences due to degeneracy in the genetic code
are
also within the scope of the application. For example, a number of amino acids
are
designated by more than one triplet. Codons that specify the same amino acid,
or
synonyms (for example, CAU and CAC are synonyms for histidine) may result in
"silent" mutations which do not affect the amino acid sequence of the protein.
However, it is expected that DNA sequence polymorphisms that do lead to
changes
in t he a mino a cid s equences o f t he s ubj ect p roteins w ill a xist a
mong m ammalian
cells. One skilled in the art will appreciate that these variations in one or
more
nucleotides (up to about 3-5% of the nucleotides) of the nucleic acids
encoding a
particular protein may exist among individuals of a given species due to
natural
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allelic variation. Any and all such nucleotide variations and resulting amino
acid
polymorphisms are within the scope of this application.
Optionally, a POSH or a POSH-AP nucleic acid of the application will
genetically complement a partial or complete loss of function phenotype in a
cell:
For example, a POSH nucleic acid of the application may be expressed in a cell
in
which endogenous POSH has been reduced by RNAi, and the introduced POSH
nucleic acid will mitigate a phenotype resulting from the RNAi. An exemplary
POSH loss of function phenotype is a decrease in virus-like particle
production in a
cell transfected with a viral vector, optionally an HIV vector.
Another aspect of the application relates to POSH and POSH-AP nucleic
acids that are used for antisense, RNAi or ribozymes. As used herein, nucleic
acid
therapy refers to administration or ifa situ generation of a nucleic acid or a
derivative
thereof which specifically hybridizes (e.g., binds) under cellular conditions
with the
cellular mRNA andlor genomic DNA encoding one of the POSH or POSH-AP
polypeptides so as to inhibit production of that protein, e.g., by inhibiting
transcription and/or translation. The binding may be by conventional base pair
complementarity, or, for example, in the case of binding to DNA duplexes,
through
specific interactions in the major groove of the double helix.
A nucleic acid therapy construct of the present application can be delivered,
for example, as an expression plasmid which, when transcribed in the cell,
produces
RNA w hich i s c omplementary t o a t 1 east a a pique p onion o f t he c
ellular m RNA
which encodes a POSH or POSH-AP polypeptide. Alternatively, the the construct
is
an oligonucleotide which is generated ex vivo and which, when introduced into
the
cell causes inhibition of expression by hybridizing with the mRNA and/or
genomic
sequences encoding a POSH or POSH-AP polypeptide. Such oligonucleotide
probes a re o ptionally m odified o ligonucleotide which a re resistant t o
endogenous
nucleases, e.g., exonucleases and/or endonucleases, and is therefore stable
ifa vivo.
Exemplary nucleic acid molecules for use as antisense oligonucleotides are
phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also
U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general
approaches to constructing oligomers useful in nucleic acid therapy have been
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reviewed, for example, by van der Krol et al., (1988) Biotechniques 6:958-976;
and
Stein et al., (1988) Cancer Res 48:2659-2668.
Accordingly, the modified oligomers of the application are useful in
therapeutic, diagnostic, and research contexts. In therapeutic applications,
the
oligomers are utilized in a manner appropriate for nucleic acid therapy in
general.
In another aspect of the application, the subject nucleic acid is provided in
an
expression vector comprising a nucleotide sequence encoding a POSH or POSH-AP
polypeptide and operably linked to at least one regulatory sequence.
Regulatory
sequences are art-recognized and are selected to direct expression of the POSH
or
POSH-AP polypeptide. Accordingly, the term regulatory sequence includes
promoters, enhancers and other expression control elements. Exemplary
regulatory
sequences are described in Goeddel; Gene Expression Technology: Methods irZ
Enzyrnolooy, Academic Press, San Diego, CA (1990). For instance, any of a wide
variety of expression control sequences that control the expression of a DNA
sequence when operatively linked to it may be used in these vectors to express
DNA
sequences encoding a POSH or POSH-AP polypeptide. Such useful expression
control sequences, include, for example, the early and late promoters of SV40,
tet
promoter, adenovirus or cytomegalovirus immediate early promoter, the lac
system,
the trp system, the TAC or TRC system, T7 promoter whose expression is
directed
by T7 RNA polymerase, the major operator and promoter regions of phage lambda
,
the control regions for fd coat protein, the promoter for 3-phosphoglycerate
kinase
or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS,
the
promoters of the yeast a-mating factors, the polyhedron promoter of the
baculovims
system and other sequences known to control the expression of genes of
prokaryotic
or eukaryotic cells or their viruses, and various combinations thereof. It
should be
understood that the design of the expression vector may depend on such factors
as
the choice of the host cell to be transformed and/or the type of protein
desired to be
expressed. Moreover, the vector's copy number, the ability to control that
copy
number and the expression of any other protein encoded by the vector, such as
antibiotic markers, should also be considered.
As will be apparent, the subject gene constructs can be used to cause
expression of the POSH or POSH-AP polypeptides in cells propagated in culture,
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e.g., to produce proteins or polypeptides, including fusion proteins or
polypeptides,
for purification.
This application also pertains to a host cell transfected with a recombinant
gene including a coding sequence for one or more of the POSH or POSH-AP
polypeptides. The host cell may be any prokaryotic or eukaryotic cell. For
example, a polypeptide of the present application may be expressed in
bacterial cells
such as E. coli, insect cells (e.g., using a baculovims expression system),
yeast, or
mammalian cells. Other suitable host cells are known to those skilled in the
art.
Accordingly, t he p resent application further p ertains t o m ethods o f p
roducing t he
POSH or POSH-AP polypeptides. For example, a host cell transfected with an
expression vector encoding a POSH polypeptide can be cultured under
appropriate
conditions to allow expression of the polypeptide to occur. The polypeptide
may be
secreted and isolated from a mixture of cells and medium containing the
polypeptide. Alternatively, the polypeptide may be retained cytoplasmically
and the
cells harvested, lysed a nd the protein isolated. A cell culture includes h
ost cells,
media and other byproducts. Suitable media for cell culture are well known in
the
art. The polypeptide can be isolated from cell culture medium, host cells, or
both
using techniques known in the art for purifying proteins, including ion-
exchange
chromatography, gel filtration chromatography, ultrafiltration,
electrophoresis, and
immunoaffinity p urification w ith a ntibodies s pecific f or p articular
epitopes o f t he
polypeptide. In a preferred embodiment, the POSH or POSH-AP polypeptide is a
fusion protein containing a domain which facilitates its purification, such as
a
POSH-GST fusion protein, POSH-intein fusion protein, POSH-cellulose binding
domain fusion protein, POSH-polyhistidine fusion protein etc.
A recombinant POSH or POSH-AP nucleic acid can be produced by ligating
the cloned gene, or a portion thereof, into a vector suitable fox expression
in either
prokaryotic cells, eukaryotic cells, or both. Expression vehicles for
production of a
recombinant POSH or POSH-AP polypeptides include plasmids and other vectors.
For instance, suitable vectors for the expression of a POSH polypeptide
include
plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-
derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for
expression in prokaryotic cells, such as E. coli.
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The preferred mammalian expression vectors contain both prokaryotic
sequences to facilitate the propagation of the vector in bacteria, and one or
more
eukaryotic transcription units that are expressed in eukaryotic cells. The
pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,
pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of
mammalian expression vectors suitable for transfection of eukaryotic cells.
Some of
these vectors are modified with sequences from bacterial plasmids, such as
pBR322,
to facilitate replication and drug resistance selection in both prokaryotic
and
eukaryotic cells. Alternatively, derivatives of viruses such as the bovine
papilloma
virus (BPV-1), or Epstein-Barr vints (pHEBo, pREP-derived and p205) can be
used
for transient expression of proteins in eukaryotic cells. Examples of other
viral
(including retroviral) expression systems can be fotmd below in the
description of
gene therapy delivery systems. The various methods employed in the preparation
of
the plasmids and transformation of host organisms are well known in the art.
For
other suitable expression systems for both prokaryotic and eukaryotic cells,
as well
as general recombinant procedures, see Nfolecular Cloning A Labof-at~ry
lllaraual,
2nd E d., a d. b y S ambrook, F ritsch a nd M aniatis ( Cold S pring H arbor
Laboratory
Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to
express
the recombinant POSH or POSH-AP polypeptide by the use of a baculovirus
expression system. Examples of such baculovirus expression systems include pVL-
derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors
(such as pAcUWl), and pBlueBac-derived vectors (such as the 13-gal containing
pBlueBac III).
Alternatively, the coding sequences for the polypeptide can be incorporated
as a part of a fusion gene including a nucleotide sequence encoding a
different
polypeptide. This type of expression system can be useful under conditions
where it
is d esirable, a .g., t o p roduce an i mmunogenic fragment o f a P OSH o r P
OSH-AP
polypeptide. For example, the VP6 capsid protein of rotavirus can be used as
an
immunologic carrier protein for portions of polypeptide, either in the
monomeric
form or in the form of a viral particle. The nucleic acid sequences
corresponding to
the p ortion o f t he P OSH o r P OSH-AP p olypeptide t o w hich antibodies
are t o b a
raised can be incorporated into a fusion gene construct which includes coding
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sequences for a 'late vaccinia virus stnictural protein to produce a set of
recombinant
viruses expressing fusion proteins comprising a portion of the protein as part
of the
virion. The Hepatitis B surface antigen can also be utilized in this role as
well.
Similarly, chimeric constructs coding for fusion proteins containing a portion
of a
POSH polypeptide and the poliovirus capsid protein can be created to. enhance
immunogenicity (see, for example, EP Publication NO: 0259149; and Evans et
al."
(1989) Nature 339:385; Huang et al., (1988) J. Virol. 62:3855; and Schlienger
et al.,
( 1992) J. Virol. 66:2).
The Multiple Antigen Peptide system for peptide-based immunization can be
utilized, wherein a desired portion of a POSH or POSH-AP polypeptide is
obtained
directly from organo-chemical synthesis of the peptide onto an oligomeric
branching
lysine core (see, for example, Posnett et al., (1988) JBC 263:1719 and
Nardelli et
al., (1992) J. Immu~rol. 148:914). Antigenic determinants of a POSH or POSH-AP
poIypeptide can also be expressed and presented by bacterial cells.
In another embodiment, a fusion gene coding for a purification leader
sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-
terminus of the desired portion of the recombinant protein, can allow
purification of
the expressed fusion protein by affinity chromatography using a Ni2+ metal
resin.
The purification leader sequence can then be subsequently removed by treatment
with enterokinase to provide the purified POSH or POSH-AP polypeptide (e.g.,
see
Hochuli et al., (1987) J. Chromatob aphy 411:177; and Janknecht et al., PNAS
LISA
88:8972).
Techniques for making fusion genes are well known. Essentially, the joining
of various DNA fragments coding for different polypeptide sequences is
performed
in accordance with conventional techniques, employing blunt-ended or stagger
ended termini for ligation, restriction enzyme digestion to provide for
appropriate
termini, filling-in of cohesive ends as appropriate, alkaline phosphatase
treatment to
avoid undesirable joining, and enzymatic ligation. In another embodiment, the
fusion gene can be synthesized by conventional techniques including automated
DNA synthesizers. Alternatively, PCR amplification of gene fragments can be
earned out using anchor primers which give rise to complementary overhangs
between two consecutive gene fragments which can subsequently be annealed to
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generate a chimeric gene sequence (see, for example, Curr-erzt Pf~otocols in
Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
Table 2: Exemplary POSH nucleic acids
Sequence Name Organism Accession Number
cDNA FLJ11367 fis, Homo sapiens AK021429
clone
HEMBA1000303
Plenty of SH3 domainsMus musculus NM_021506
(POSH) mRNA
Plenty of SH3s (POSH)Mus musculus AF030131
mRNA
Plenty of SH3s (POSH)Drosophila melanogasterNM 079052
mRNA
Plenty of SH3s (POSH)Drosophila melanogasterAF220364
mRNA
Table 3: Exemplary POSH polypeptides
Sequence Name Or ag nism Accession Number
SH3 domains- Mus musculus T09071
containing protein
POSH
plenty of SH3 domainsMus musculus NP-067481
Plenty of SH3s; POSHMus musculus AAC40070
Plenty of SH3s Drosophila melanogasterAAF37265
LD45365p Drosophila melanogasterAAK93408
POSH gene product Drosophila melanogasterAAF57833
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Plenty of SH3s I Drosophila melanogaster ~ NP-523776
In addition the following Tables provide the nucleic acid sequence and
related SEQ ID NOs for domains of human POSH protein and a summary of POSH
sequence identification numbers used in this application.
Table 4. Nucleic Acid Sequences and related SEQ ID NOs for domains in human
POSH
Name of Sequence SEQ ID
the
sequence NO.
~
RING domainTGTCCGGTGTGTCTAGAGCGCCTTGATGCTTCTGCGAAGGTCT31
TGCCTTGCCAGCATACGTTTTGCAAGCGATGTTTGCT
GGGGATCGTAGGTTCTCGAAATGAACTCAGATGTCCCGAGT
1St SH3 CCATGTGCCAAAGCGTTATACAACTATGAAGGAAAAGAGCCTG32
domain GAGACCTTAAATTCAGCAAAGGCGACATCATCATTTT
GCGAAGACAAGTGGATGAAAATTGGTACCATGGGGAAGTCAAT
GGAATCCATGGCTTTTTCCCCACCAACTTTGTGCAGA
TTATT
2nd SH3 CCTCAGTGCAAAGCACTTTATGACTTTGAAGTGAAAGACAAGG33
domain AAGCAGACAAAGATTGCCTTCCATTTGCAAAGGATGA
TGTTCTGACTGTGATCCGAAGAGTGGATGAAAACTGGGCTGAA
GGAATGCTGGCAGACAAAATAGGAATATTTCCAATTT
CATATGTTGAGTTTAAC
3rd SH3 AGTGTGTATGTTGCTATATATCCATACACTCCTCGGAAAGAGG34
domain ATGAACTAGAGCTGAGAAAAGGGGAGATGTTTTTAGT
GTTTGAGCGCTGCCAGGATGGCTGGTTCAAAGGGACATCCATG
CATACCAGCAAGATAGGGGTTTTCCCTGGCAATTATG
TGGCACCAGTC
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SH3 ~GAAAGGCACAGGGTGGTGGTTTCCTATCCTCCTCAGAGTGAGG ~35
domain ~CAGAACTTGAACTTAAAGAAGGAGATATTGTGTTTGT
TCATAAAAAACGAGAGGATGGCTGGTTCAAAGGCACATTACAA
CGTAATGGGAAAACTGGCCTTTTCCCAGGAAGCTTTG
TGGAAAACA
Table 5. Summary of POSH sequence Identification Numbers
Sequence Tnformation Sequence
Identification
Number
(SEQ
ID
NO)
Human POSH Coding Sequence SEQID No:1
Human POSH Amino Acid SequenceSEQID No:2
Human POSH cDNA Sequence SEQID No:3
5' cDNA Fragment of Human SEQID No:4
POSH
N-terminus Protein Fragment SEQID No:5
of
Human POSH
3' mRNA Fragment of Human SEQID No:6
POSH
C-terminus Protein Fragment SEQID No:7
of
Human POSH
Mouse POSH mRNA Sequence SEQID No:8
Mouse POSH Protein Sequence SEQID No:9
Drosophila melanogaster POSH SEQID No:10
mRNA Sequence
Drosophila melanogaster POSH SEQID No:11
Protein Sequence
Human POSH RING Domain Amino SEQID No:26
Acid Sequence
Human POSH 1st SH3 Domain SEQID No:27
Amino
Acid Sequence
Human POSH 2d SH3 Domain AminoSEQID No:28
Acid Sequence
Human POSH 3rd SH3 Domain SEQID No:29
Amino
Acid Sequence
Human POSH 4th SH3 Domain SEQID No:30
Amino
Acid Sequence
Human POSH RING Domain NucleicSEQID No:31..
Acid Sequence
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Human POSH SH3DomainNucleic SEQ ID No: 32
1St
Acid Sequence
Human POSH SH3DomainNucleic SEQ ID No: 33
2d
Acid Sequence
Human POSH SH3DomainNucleic SEQ ID No: 34
3rd
Acid Sequence
Human POSH SH3DomainNucleic SEQ ID No: 35
4th
Acid Sequence
8. Exemplary Polypeptides
In certain aspects, the present application relates to POSH polypeptides,
which are isolated from, or otherwise substantially free of, other
intracellular
proteins which might normally be associated with the protein or a particular
complex including the protein. In certain embodiments, POSH polypeptides have
an
amino acid sequence that is at least 60% identical to an amino acid sequence
as set
forth in any of SEQ ID Nos: 2, 5, 7, 9, 11, 26, 27, 28, 29 and 30. In other
embodiments, the polypeptide has an amino acid sequence at least 65%, 70%,
75%,
80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence
as set forth in any of SEQ II? Nos: 2, 5, 7, 9, 1 l, 26, 27, 28, 29 and 30.
In certain aspects, the application also relates to POSH-AP polypeptides
(e.g., a POSH-AP provided in Table 7). Amino acid sequences of the POSH-APs
listed in Table 7 are provided in Figure 36. Additional POSH-AP polypeptides
are
provided in Table 8. In certain embodiments, POSH-AP polypeptides have an
amino acid sequence that is at least 60% identical to an amino acid sequence
as set
forth in Figime 36. In other embodiments, the POSH-AP polypeptide has an amino
acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or
100% identical to an amino acid sequence as set forth in Figure 36.
Optionally, a POSH or POSH-AP polypeptide of the application will
function in place of an endogenous POSH or POSH-AP polypeptide, for example by
mitigating a partial or complete loss of function phenotype in a cell. For
example, a
POSH polypeptide of the application may be produced in a cell in which
endogenous POSH has been reduced by RNAi, and the introduced POSH
polypeptide will mitigate a phenotype resulting from the RNAi. An exemplary
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POSH loss of function phenotype is a decrease in virus-like particle
production in a
cell transfected with a viral vector, optionally an HIV vector. In certain
embodiments, a POSH polypeptide, when produced at an effective level in a
cell,
induces apoptosis.
In another aspect, the application provides polypeptides that are agonists or
antagonists of a POSH or POSH-AP polypeptide. Variants and fragments of a
POSH or POSH-AP polypeptide may have a hyperactive or constitutive activity,
or,
alternatively, act to prevent POSH or POSH-AP polypeptides from performing one
or more functions. For example, a truncated form lacking one or more domain
may
have a dominant negative effect.
Another aspect of the application relates to polypeptides derived from a full-
length P OSH o r P OSH-AP p olypeptide. Isolated p eptidyl p ortions o f t he
s ubject
proteins c an b a o btained b y screening p olypeptides r ecombinantly
produced from
the corresponding fragment of the nucleic acid encoding such polypeptides. In
addition, fragments can be chemically synthesized using techniques known in
the art
such as conventional Mernfield solid phase f Moc or t-Hoc chemistry. For
example,
any one of the subject proteins can be arbitrarily divided into fragments of
desired
length with no overlap of the fragments, or preferably divided into
overlapping
fragments of a desired length. The fragments can be produced (recombinantly or
by
chemical synthesis) and tested to identify those peptidyl fragments which can
function as either agonists or antagonists of the formation of a specific
protein
complex, or more generally of a POSH:POSH-AP complex, such as by
microinjection assays.
It is also possible to modify the structure of the POSH or POSH-AP
polypeptides for such puzposes as enhancing therapeutic or prophylactic
efficacy, or
stability (e.g., ex vivo shelf life and resistance to proteolytic degradation
in vivo).
Such modified polypeptides, when designed to retain at least one activity of
the
naturally-occurring form of the protein, are considered functional equivalents
of the
POSH or POSH-AP polypeptides described in more detail herein. Such modified
polypeptides can be produced, for instance, by amino acid substitution,
deletion, or
addition.
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For instance, it is reasonable to expect, for example, that an isolated
replacement of a leucine with an isoleucine or valine, an aspartate with a
glutamate,
a threonine with a serine, or a similar replacement of an amino acid with a
structurally related amino acid (i.e,. conservative mutations) will not have a
major
effect on the biological activity of the resulting molecule. Conservative
replacements
are those that take place within a family of amino acids that are related in
their side
chains. Genetically encoded amino acids are can be divided into four families
(see,
for example, Biochemistry, 2nd ed., Ed. by L. Stryer, W.H. Freeman and Co.,
1981).
Whether a change in the amino acid sequence of a polypeptide results in a
functional
homolog can be readily determined by assessing the ability of the variant
polypeptide to produce a response in cells in a fashion similar to the wild-
type
protein. For instance, such variant forms of a POSH polypeptide can be
assessed,
e.g., for their ability to bind to another polypeptide, e.g., another POSH
polypeptide
or another protein involved in viral maturation. Polypeptides in which more
than
one replacement has taken place can readily be tested in the same manner.
This application further contemplates a method of generating sets of
combinatorial mutants of the POSH or POSH-AP polypeptides, as well as
truncation
mutants, and is especially useful for identifying potential variant sequences
(e.g.,
homologs) that are functional in binding to a POSH or 'POSH-AP polypeptide.
The
purpose of screening such combinatorial libraries is to generate, for example,
POSH
homologs which can act as either agonists or antagonist, or alternatively,
which
possess novel activities all together. Combinatorially-derived homologs can be
generated which have a selective potency relative to a naturally occurring
POSH or
POSH-AP polypeptide. Such proteins, when expressed from recombinant DNA
constructs, can be used in gene therapy protocols.
Likewise, mutagenesis can give rise to homologs which have intracellular
half lives dramatically different than the corresponding wild-type protein.
For
example, the altered protein can be rendered either more stable or less stable
to
proteolytic degradation or other cellular process which result in destruction
of, or
otherwise inactivation of the POSH or POSH-AP polypeptide of interest. Such
homologs, and the genes which encode them, can be utilized to alter POSH or
POSH-AP levels by modulating the half life ofthe protein. For instance, a
short
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half life can give rise to more transient biological effects and, when part of
an
inducible expression system, can allow tighter control of recombinant POSH or
POSH-AP levels within the cell. As above, such proteins, and particularly
their
recombinant nucleic acid constricts, can be used in gene therapy protocols.
In similar fashion, POSH or POSH-AP homologs can be g enerated by t he
present combinatorial approach to act as antagonists, in that they are able to
interfere
with the ability of the corresponding wild-type protein to function.
In a representative embodiment of this method, the amino acid sequences for
a population of POSH or POSH-AP homologs are aligned, preferably to promote
the
highest homology possible. Such a population of variants can include, for
example,
homologs from one or more species, or homologs from the same species. but
which
differ due to mutation. Amino acids which appear at each position of the
aligned
sequences are selected to create a degenerate set of combinatorial sequences.
In a
preferred embodiment, the combinatorial library is produced by way of a
degenerate
library of genes encoding a library of polypeptides which each include at
least a
portion of potential POSH or POSH-AP sequences. For instance, a mixture of
synthetic oligonucleotides can be enzymatically ligated into gene sequences
such
that the degenerate set of potential POSH or POSH-AP nucleotide sequences are
expressible as individual polypeptides, or alternatively, as a set of larger
fusion
proteins (e.g., for phage display).
There are many ways by which the library of potential homologs can be
generated from a degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be carned out in an automatic DNA synthesizer,
and
the synthetic genes then be ligated into an appropriate gene for expression.
The
purpose of a degenerate set of genes is to provide, in one mixW re, all of the
sequences encoding the desired set of potential POSH or POSH-AP sequences. The
synthesis of degenerate oligonucleotides is well known in the art (see for
example,
Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA,
Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam:
Elsevier pp273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura
et
al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477).
Such
techniaues have been employed in the directed evolution of other proteins
(see, for
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example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS
USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al.,
(1990) PNAS USA 87: 6378-6382; as well as U.S. Patent Nos: 5,223,409,
5,198,346, and 5,096,815).
Alternatively, other forms of mutagenesis can be utilized to generate a
combinatorial 1 ibrary. F or a xample, P OSH o r P OSH-AP h omologs ( both a
gonist
and antagonist forms) can be generated and isolated from a library by
screening
using, f or example, a lanine s canning m utagenesis a nd t he 1 ike ( Ruf et
a l., ( 1994)
Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099;
Balint a t al., ( 1993) G ene 137:109-118; G rodberg a t a l., ( 1993) Eur. J.
B iochem.
218:597-601; Nagashima et al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et
al., (1991) Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science
244:1081-1085), by linker scanning mutagenesis (Gustin et al., (1993) Virology
193:653-660; Brown et al., (1992) Mol. Cell Biol. 12:2644-2652; McKnight et
al.,
(1982) Science 232:316); by saturation mutagenesis (Meyers et al., (1986)
Science
232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol 1:11-
19); or by random mutagenesis, including chemical mutagenesis, etc. (Miller et
al.,
(1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor,
NY;
and Greener et al., (1994) Strategies in Mol Biol 7:32-34). Linker scanning
mutagenesis, particularly in a combinatorial setting, is an attractive method
for
identifying truncated (bioactive) forms of POSH or POSH-AP polypeptides.
A wide range of techniques are known in the art for screening gene products
of combinatorial libraries made by point mutations and truncations, and, for
that
matter, for screening cDNA libraries for g ene products having a c ertain p
roperty.
Such techniques will be generally adaptable for rapid screening of the gene
libraries
generated by the combinatorial mutagenesis of POSH or POSH-AP homologs. The
most widely used techniques for screening large gene libraries typically
comprises
cloning the gene library into replicable expression vectors, transforming
appropriate
cells with the resulting library of vectors, and expressing the combinatorial
genes
under conditions in which detection of a desired activity facilitates
relatively easy
isolation of the vector encoding the gene whose product was detected. Each of
the
illustrative assays described below are amenable to high through-put analysis
as
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necessary to screen large numbers of degenerate sequences created by
combinatorial
mutagenesis techniques.
In an illustrative embodiment of a screening assay, candidate combinatorial
gene products of one of the subject proteins are displayed on the surface of a
cell or
virus, and the ability of particular cells or viral particles to bind a POSH
or POSH-
AP polypeptide is detected in a "panning assay". For instance, a library of
POSH
variants can be cloned into the gene for a surface membrane protein of a
bacterial
cell (Ladner et al." WO 88/06630; Fuchs et al., (199I) Bio/Technology 9:1370-
1371; and Goward et al., (1992) TIES 18:136-140), and the resulting fusion
protein
detected by panning, e.g., using a fluorescently labeled molecule which b rods
the
POSH polypeptide, to score for potentially functional homologs. Cells can be
visually inspected and separated tinder a fluorescence microscope, or, where
the
morphology of the cell permits, separated by a fluorescence-activated cell
sorter.
In similar fashion, the gene library can be expressed as a fusion protein on
the surface of a viral particle. For instance, in the filamentous phage
system, foreign
peptide sequences can be expressed on the surface of infectious phage, thereby
conferring two significant benefits. First, since these phage can be applied
to
affinity matrices at very high concentrations, a large number of phage can be
screened at one time. Second, since each infectious phage displays the
combinatorial gene product on its surface, if a particular phage is recovered
from an
affnity matrix in low yield, the phage can be amplified by another round of
infection. The group of almost identical E. coli filamentous phages M13, fd,
and fl
are most often used in phage display libraries, as either of the phage gIII or
gVIII
coat proteins can be used to generate fusion proteins without disrupting the
ultimate
packaging of the viral particle (Ladner et al., PCT publication WO 90/02909;
Garrard et al., PCT publication WO 92109690; Marks et al., (1992) J. Biol.
Chem.
267:16007-16010; Griffiths et al., (1993) EMBO J. 12:725-734; Clackson et al.,
(1991) Nature 352:624-628; and Barbas et al., (1992) PNAS USA 89:4457-4461).
The application also provides for reduction of the POSH or POSH-AP
polypeptides to generate mimetics, e.g., peptide or non-peptide agents, which
are
able to mimic binding of the authentic protein to another cellular partner.
Such
muta~enic techniques as described above, as well as the thioredoxin system,
are also
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particularly useful fox mapping the determinants of a POSH or POSH-AP
polypeptide which participate in protein-protein interactions involved in, for
example, binding of proteins involved in viral maturation to each other. To
illustrate, the critical residues of a POSH or POSH-AP polypeptide which are
involved in molecular recognition of a substrate protein can be determined and
used
to generate its derivative peptidomimetics which bind to the substrate
protein, and
by inhibiting POSH or POSH-AP binding, act to inhibit its biological activity.
By
employing, for example, scanning mutagenesis to map the amino acid residues of
a
POSH polypeptide which are involved in binding to another polypeptide,
peptidomimetic compounds can be generated which mimic those residues involved
in binding. For instance, non-hydrolyzable peptide analogs of such residues
can be
generated using benzodiazepine (e.g., see Freidinger et al., in Peptides:
Chemistry
and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
azepine (e.g., see Huffinan et aL, in Peptides: Chemistry and Biology, G.R.
Marshall
ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam
rings
(Garvey et al., in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM
Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson
et
al., (1986) J. Med. Chem. 29:295; and Ewenson et al., in Peptides: Structure
and
Function (Proceedings of the 9th American Peptide Symposium) Pierce C hemical
Co. Rockland,IL, 1985), b-turn dipeptide cores (Nagai et al., (1985)
Tetrahedron
Lett 26:647; and Sato et al., (1986) J Chem Soc Perkin Trans 1:1231), and b-
aminoalcohols (cordon et al., (I985) Biochem Biophys Res Commun 126:419; and
Dann et al., (1986) Biochem Biophys Res Commun 134:71).
The following table provides the sequences of the RING domain and the
various SH3 domains of POSH.
Table 6. Amino Acid Sequences and related SEQ ID NOs for domains in human
POSH
Name of Sequence SEQ
ID
the N0.
sequence
RING CPVCLERLDASAKVLPCQHTFCKRCLLGIVGSRNELRCPEC26
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domain
1St SH3 PCAKALYNYEGKEPGDLKFSKGDIIILRRQVDENWYHGEVNGIHGF27
domain FPTNFVQIIK
2nd SH3 PQCKALYDFEVKDKEADKDCLPFAKDDVLTVIRRVDENWAEGMLAD28
domain KIGIFPISYVEFNS
3rd SH3 SVYVAIYPYTPRKEDELELRKGEMFLVFERCQDGWFKGTSMHTSKI29
domain GVFPGNYVAPVT
4th 5H3 ERHRVWSYPPQSEAELELKEGDIVFVHKKREDGWFKGTLQRNGKT30
domain GLFPGSFVENI
The following table provides a list of selected POSH-APs and their related
SEQ m NOs.
Table 7 - Selected POSH APs
Protein Protein Sequence mRNA Sequence
(SEA II) I~~:) (SEA II~ lid~:)
ARFl 223 325-339
ARFS 224 340-344
ATP6VOC 225-226 345-351
CBL-B 361; 398; 227-230 353-360
CENTB 1 231-232 37-47
DDEFl 233-237 48-54
EIF3S3 238 55-57
EPS8L2 239 58-60
GOCAP 1 240-243 61-68
GOSRZ 244-248 69-76
HERPUD 1 249-252 77-86
HLA-A 253 87-88
HLA-B 254 89
MSTP028 255-256 90-94
PALS-1 362-366 95-100
PPP1CA 261-263; 395 101-110
PRKAR1A 264-265 111-122; 396-397
PTPN12 266-268 123-129
RALA 269-270 130-134
SIAH1 271-272 135-141
SMN1 273-275 142-146
SMN2 276-280 147-151
SNX1 281-286 152-161
SNX3 287-290 162-174
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'Protein Protein Sequence mRNA Sequence
(SEQ ID NO:) (SEQ ID NO:)
SRA.1 291-294 175-182
SYNE1 295-307 183-201
TTC3 308-312 202-207
UBE2N 313 208-210
UNC84B 314 211-213
VCY2IP 1 315-323 214-222
SPG20 386-388 367-374
WASFl 389 375-376
HIP55 390-394 377-385
Table 8 below provides a list of POSH-APs that bound POSH in a 2-hybrid
assay. Nucleic acid and amino acid sequences of the POSH-APs listed in Table 8
were filed in a U.S. provisional application filed in the name of Daniel N.
Taglicht,
Iris Alroy, Yuval Reiss, Liora Yaar, Danny Ben-Avraham, Shmuel Tuvia, and
Tsvika Greener entitled "Posh Interacting Proteins and Related Methods", filed
on
March 2, 2004 (Attorney Docket No. PROL-P79-024), which Provisional
Application is incorporated herein by reference in its entirety.
Table 8 - POSH-APs
Protein and Variant Protein Sequence mRNA Sequence
( ublic i No.) ( ublic i No.)
BCL9 - var 1 4757846 4757845
BRD4 - var 1 19718731 19718730
BRD4 - var 2 7657218 . 7657217
DRP2 - var 1 4503393 4503392
MAPlA - var 1 21536458 21536457
SH2D2A - var 1 4503633 31543620
BAT3 - var 1 18375630 18375633
BAT3 - var 2 18375634 18375631
BAT3 - var 3 * 18375629
BCARl - var 1 7656924 7656923
DAP - var 1 4758120 4758119
EVPL - var 1 4503613 4503612
FLJ13231 - var 1 38604073 38604072
FL53657 - var 1 13376230 13376229
HSPC142-var 1 7661802 7661801
LOCI 18987 - var 29789403 31341089
1
NAP4 - var 1 2443367 2443366
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Protein and Variant Protein Sequence mRNA Sequence
( ublic i No.) ( ublic i No.)
RBAF600 - var 1 24416002 24416001
XTP3TPB - var 1 20070264 20070263
Hs.31535 - var 1 37546355 37546354
ASF1B - var 1 8922549 8922548
ATP5A1- var 1 4757810 23346425
C6 or fl 1- var 1 9954875 39725662
C6 or f60 - var 1 24431997 24431996
CDT1- var 1 16418337 19923847
CIC - var 1 16507208 16507207
CLKZ - var 1 4557477 4557476
CLI~2 - var 2 4502883 4502882
DNM2 - var 1 4826700 4826699
EEF1A1- var 1 4503471 25453469
EIF4EBP1-var 1 4758258 20070179
FLJ13479 - var 1 24432013 39725704
GC20 - var 1 5031711 5031710
GLUL - var 1 19923206 21361767
HEBP2 = var 1 7657603 7657602
ITGB- var 1 4504779 4504778
LAMAS - var 1 21264602 21264601
LOC90987 - var 1 29734345 29734344
MRPL36 - var 1 23111040 20806105
Hs.380933 - var 1 30149441 37550602
NQ02 - var 1 4505417 4505416
PCBPl -var 1 5453854 14141164
PCNT2 - var 1 22035674 35493922
PGD - var 1 984325 984324
RAP80 - var 1 21361593 21361592
RIVH - var 1 21361547 21361546
RPL - var 1 4506597 15431291
RPS20 - var 1 4506697 14591915
RPS27A - var 1 4506713 27436941
SETDB1-var 1 6912652 6912651
SF3A2 - var 1 21361376 32189413
UBB - var 1 11024714 22538474
ARHV - var 1 20070360 20070359
KIAAl 111- var 1 32698700 32698699
ZNF147 - var 1 4827065 15208652
PAWR-var 1 4505613 4505612
TPX2 - var 1 20127519 31542258
HSPA1B - var 1 4885431 26787974
DLGS - var 1 3043690 3650451
DLGS - var 2 28466997 28466996
DLGS - var 3 3650452 16549841
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Protein and Variant Protein Sequence mRNA Sequence
( ublic i No.) ( ublic i No.)
DLGS - var 4 * 16807129
DLGS - var 5 * 22539637
DLGS - var 6 * 15929207
DLGS - var 7 * 3043689
KIAA1598 - var 1 7023592 7023591
KIAA1598 - var 2 10047271 701 8519
KIAAI 598 - var 3 * 21314680
KIAA1598 - var 4 * 10047270
KIAA1598 - var 5 * 21755030
KIAA1598 - var 6 * 21755023
KIAA1598 - var 7 * 21754670
KIAA1598 - var 8 * 21750902
KIAA1598 - var 9 * 21749984
KIAA1598 - var 10 * 21749775
KIA.A1598 - var 11 * 21749737
CGI-27 - var 1 7705720 23270696
CGI-27 - var 2 * 22902234
CGI-27 - var 3 * 17046302
CGI-27 - var 4 * 16553689
CGI-27 - var 5 * 10433504
CGI-27 - var 6 * 4680692
CGI-27 - var 7 * 20127543
BIA2 - var 1 5262640 5262639
BIA2 - var 2 21591225 21591224
BIA2 - var 3 * 21755615
COLTA1-var 1 180392 407589
COLIA1- var 2 180857 30015
COLIAI -var 3 1418928 30092
COLTAl -var 4 22328092 7209641
COLIAI -var 5 762938 22328091
COLIAI - var 6 30016 1418927
COLIAI -var 7 407590 180856
COLIA1- var 8 ~= 180391
COLIAl - var 9 * 14719826
DKFZp761A052 - var 10434104 10434103
1
DKFZ 761A052 - var 10439058 10439057
2
DKFZ 761A052 - var 14602829 14602828
3
DKFZ 761A052 - var 20380411 15079884
4
DKFZp761A052 - var 6808165 20380410
DKFZp761A052 - var * 6808164
6
TLE1-var 1 14603281 16041735
TLEl - var 2 307510 14603280
TLE1 -var 3 * 307509
EGLN2 - var 1 8922130 23273571
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Protein and Variant Protein Sequence mRNA Sequence
( ublic i No.) ( ublic i No.)
EGLN2 - var 2 12804603 10437903
EGLN2 - var 3 14547148 21733075
EGLN2 - var 4 18031805 21758140
EGLN2 - var 5 * 18677002
EGLN2 - var 6 * 18031804
EGLN2 - var 7 * 18141576
EGLN2 - var 8 * 14547147
EGLN2 - var 9 * 12804602
EGLN2 - var 10 * 10439822
EGLN2 - var 11 * 8922129
STC2 - var 1 3335144 3335143
STC2 - var 2 * 3702223
-
STC2 - var 3 * 4050037
STC2 - var 4 * 4104014
STC2 - var 5 * 13623494
STC2 - var 6 * 14042507
STC2 - var 7 * 14042032
STC2 - var 8 * 21755241
STC2 - var 9 * 21755207
STC2 - var 10 * 22761473
STC2 - var 11 * 12653744
OPTN - var I 20149572 16550123
OPTN - var 2 21619683 3387890
OPTN - var 3 3329431 3127082
OPTN - var 4 3127083 3329430
OPTN - var 5 * 21619682
OPTN - var 6 * I 8644681
OPTN - var 7 * 18644683
OPTN - var 8 * 18644685
OPTN - var 9 * 20149571
FLJ37147 - var 1 21753535 21753534
FLJ37147 - var 2 30153743 30153742
KH-DRBS 1- var 1 21749696 189499
KHDRBS1-var 2 1841747 12653852
KHDRBS 1- var 3 189500 17512262
KHDRBS 1- var 4 * 14714433
KHDRBS 1- var 5 * 1841746
KHDRB S 1- var 6 * 21749695
SLC2Al -var 1 3387905 3387904
SLC2A1- var 2 5730051 5730050
SLC2A1- var 3 14268550 14268549
DKFZp434B1231-var 6808117 6808116
1
NUMA1-var 1 27694103 5453819
NUMA1- var 2 35119 13278785
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Protein and Variant Protein Sequence mRNA Sequence
( ublic i No.) ( ublic i No.)
NUMAl - var 3 14249928 14249927
NUMA1 - var 4 13278786 15991876
NUMA1-var 5 5453820 296118
NUMAl - var 6 * 296119
NUMAl - var 7 * 296120
NUMAl - var 8 * 35118
NUMAl - var 9 * 20073234
NL1MA1- var 10 * 22477305
NUMAl - var 11 * 22749583
NUMA1- var 12 * 27694102
HSPC016 - var 1 68.41310 12654536
HSPC016 - var 2 12654537 6841309
HSPC016 - var 3 * 4679017
HSPC016 - var 4 * 10834763
UBC - var 1 5912028 3360475
UBC - var 2 340058 2647407
UBC - var 3 340068 24657521
UBC - var 4 14286308 21751700
UBC - var 5 15928840 21757163
UBC - var 6 16552475 21758959
UBC - var 7 * 16552474
UBC - var 8 * 15928839
UBC - var 9 * 14286307
UBC - var 10 * 12653358
UBC - var 11 * 10439801
UBC - var 12 * 340067
UBC - var 13 * 340057
LTBC - var 14 ~~ 5912027
ZFM1- var 1 785999 785998
PIASY - var 1 14603164 - 3643110
PIASY - var 2 5533373 5533372
PIASY - var 3 24850133 10433892
PIASY - var 4 3643111 14603163
PIASY - var 5 * 20987516
PIASY - var 6 * 14709019
XM 208944 - var 1 30153743 30153742
J03930 - var 1 178442 178441
MT2A - var 1 187528 37120
MT2A - var 2 37121 263506
MT2A - var 3 * 13937856
MT2A - var 4 * 1495465
MT2A - var 5 * 187527
EWSRl - var 1 7669490 21734132
EWSR1 - var 2 12653511 547565
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Protein and Variant Protein Sequence mRNA Sequence
( ublic i No.) ( ublic i No.)
EWSR1- var 3 15029675 21756356
EWSRl - var 4 16552153 16551673
EWSRl - var 5 16551674 16552152
EWSRl - var 6 31280 15029674
EWSRl - var 7 * 13435962
EWSRl - var 8 * 12653510
EWSRl - var 9 * 10439073
EWSR1- var 10 * 7669489
MADH6 - var 1 2828712 1654326
MADH6 - var 2 2736316 20379504
MADH6 - var 3 1654327 2736315
MADH6 - var 4 * 2828711
MADH6 - var 5 * 15278059
THOC2 - var 1 20799318 10435649
THOC2 - var 2 10435650 20799317
THOC2 - var 3 * 7023224
ZNF151- var 1 676873 2230870
ZNF151- var 2 2230871 676872
DDX31- var 1 10435700 14042193
DDX31- var 2 10440004 15215272
DDX31- var 3 20336298 16566549
DDX31- var 4 16566550 20336297
DDX31- var 5 15215273 20336296
DDX31- var 6 14042194 10440003
DDX31- var 7 * 10435699
POLR2J2 - var 1 11595478 21704271
POLR2J2 - var 2 21704.274 21704270
POLR2J2 - var 3 19401711 19401710
POLR2J2 - var 4 14702175 21704273
POLR2J2 - var 5 21704272 16878085
POLR2J2 - var 6 * 11595475
POLR2J2 - var 7 * 11595477
POLR2J2 - var 8 * 11595473
BANF1- var 1 3002951 11038645
BANF1- var 2 4502389 13543576
BANF1-var 3 * 14713907
BANF1- var 4 * 3002950
BANFl - var 5 * 4321975
BANFl - var 6 * 3220254
CBX4 - var 1 1945453 1945452
CBX4 - var 2 15929016 2317722
CBX4 - var 3 2317723 15929015
ARIH2 - var 1 3925604 3925603
ARIH2 - var 2 9963793 3930777
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.Protein and VariantProtein Sequence mIRNA Sequence
( ublic i No.) ( ublic i No.)
ARIH2 - var 3 12653307 3986675
ARIH2 - var 4 * 3986676
ARIH2 - var 5 * 3986677
ARIH2 - var 6 * 7328049
ARIH2 - var 7 * 6855602
ARIH2-var 8 * 21749565
ARIHZ - var 9 * 33875424
ARIH2 - var 10 * 9963792
ARIH2 - var 11 * 5453556
ARIH2 - var 12 * ' S 817100
ARIH2 - var 13 * 3930775
SRPK2 - var 1 1857944 21752284
SRl'K2 - var 2 23270876 21749007
SRPK2 - var 3 * 23270875
SRPK2 - var 4 * 1857943
SIAH2 - var 1 2673968 16549991
SIAH2 - var 2 2664283 34189635
SIA.H2 - var3 * 2664282
SIAH2 - var 4 * 2673967
KIAA0191-var 1 27480017 29387261
KIAA0191 - var 2 1228035 10438300
KIAA0191- var 3 29387262 1228034
KIAA0191- var 4 * 21755057
KIAA0191 - var 5 * 27480016
KIAA0191- var 6 * 19387907
KIAA0191- var 7 * 15636651
KIAA0191- var 8 * 23273514
PAl-R~P1-var 1 5262551 22760761
PAl-RBPl - var 2 4929579 20072477
PAl-RBP1-var 3 12804377 17939456
PAl-RBP1- var 4 12803339 18088243
PAl-RBP1-var 5 14029171 16924316
PA1-RBP1-var 6 18088244 33872286
PAl-RBPl -var 7 22760762 14029170
PAl-RBP1 -var 8 * 33876749
PAl-RBPl -var 9 * 12804376
PAl-RBPl -var 10 * 4929578
PAl-RBPl -var 11 * 4406639
PAl-RBPl - var 12 * 5262550
FAT - var 1 2281025 1107686
FAT - var 2 1107687 15214611
FAT - var 3 * 2281024
FAT - var 4 * 598748
VCL - var 1 24657579 ~ 7669551 '
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Protein and Variant Protein Sequence mRNA Sequence
( ublic i No.) ( ublic i No.)
VCL - var 2 340237 7669549
VCL - var 3 7669550 340236
VCL - var 4 * 21732673
VCL - var 5 * 15426616
VCL - var 6 * 246657578
SSR4 - var 1 15929882 30583222
SSR4 - var 2 13097213 1071680
SSR4 - var 3 * 22749791
SSR4 - var 4 * 21753447
SSR4 - var 5 * 16552704
SSR4 - var 6 * 15929881
SSR4 - var 7 * 13097212
SSR4 - var 8 * 2398656
PRDXS - var 1 6166493 27484966
PRDXS - var 2 6746355 9802047
PRDXS - var 3 9802048 8745393
PRDXS - var 4 2748496? 6746354
PRDXS - var 5 * 6563211
PRDXS - var 6 * 6103723
PRDXS - var 7 * 6166492
PRDXS - var 8 * 6523288
PRDXS - var 9 * 32455258
FLJ10120 - var 1 8922239 27469671
FLJ10120 - var 2 * 8922238
PROL4 - var 1 22208536 22208535
PROL4 - var 2 6005802 1050982
CL25084 - var 1 15341891 4406555
CL25084 - var 2 7023472 4406692
CL25084 - var 3 4406693 7023471
CL25084 - var 4 4406556 15341890
C 11 orfl 7 - var 22761313 21361869
1
C 11 orfl 7 - var 21105773 20149226
2
C 11 orfl 7 - var 20149225 20149224
3
C 11 orfl 7 - var 20149227 21105772
4
C 11 orfl 7 - var 21361870 21410957
C 11 orfl 7 - var * 22761312
6
POLQ - var 1 3510695 13892060
POLQ - var 2 4163931 13892060
POLQ - var 3 13892061 4163930
POLQ - var 4 * 3510694
MBD2 - var 1 3170202 3800812
MBD2 - var 2 3800801 5817231
MBD2 - var 3 7710145 21595775
MBD2 - var 4 21595776 21464120
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Protein and Variant Protein Sequence mRNA Sequence
( ublic i No.) ( ublic i No.)
MBD2 - var 5 * 21464121
MBD2 - var 6 * 3800800
MBD2 - var 7 * 3800792
MBD2 - var 8 * 3170201
FSTL1- var 1 12658309 536897
FSTLl - var 2 12652619 16924272
FSTLl - var 3 * 33990756
FSTL1- var 4 * 12658308
FSTL1- var 5 * 10438502
FSTL1- var 6 * 4884472
* denotes a polypeptide sequence that can be deduced from the corresponding
mRNA sequence.
9. Effective Dose
Toxicity and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental animals,
e.g.,
for determining The LD50 (the dose lethal to 50% of the population) and the
ED50
(the dose therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and it can be
expressed
as the ratio LD50/ED50. Compounds which exhibit large therapeutic induces are
preferred. While compounds that exhibit toxic side effects may be used, care
should
be taken to design a delivery system that targets such compounds to the site
of
affected tissue in order to minimize potential damage to uninfected cells and,
thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used
in formulating a range of dosage for use in humans. The dosage of such
compounds
lies p referably w ithin a range o f c irculating c oncentrations t hat i
nclude t he E D50
with little or no toxicity. The dosage may vary within this range depending
upon the
dosage form employed and the route of administration utilized. For any
compound
used in the method of the application, the therapeutically effective dose can
be
estimated initially from cell culture assays. A dose may be formulated in
animal
models to achieve a circulating plasma concentration range that includes the
IC50
(i.e., the concentration of the test compound which achieves a half maximal
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inhibition of symptoms) as determined in cell culture. Such information can be
used
to more accurately determine useful doses in humans. Levels in plasma may be
measured, for example, by high performance liquid chromatography.
10. Formulation and Use
Pharmaceutical compositions for use in accordance with the present
application may be formulated in conventional manner using one or more
physiologically acceptable carriers or excipients. Thus, the compounds and
their
physiologically acceptable salts and solvates may be formulated foi
administration
by, for example, injection, inhalation or insufflation (either through the
mouth or the
nose) or oral, buccal, parenteral or rectal administration.
An exemplary composition of the application comprises an RNAi mixed
with a delivery system, such as a liposome system, and optionally including an
acceptable excipient. In a preferred embodiment, the composition is formulated
for
topical administration for, e.g., herpes virus infections.
For such therapy, the compounds of the application can be formulated for a
variety of loads of administration, including systemic and topical or
localized
administration. Techniques and formulations generally may be found in
Remmington's P harmaceutical S ciences, M eade P ublishing C o., E aston, P A.
F or
systemic administration, injection is preferred, including intramuscular,
intravenous,
intraperitoneal, and subcutaneous. For injection, the compounds of the
application
can be formulated in liquid solutions, preferably in physiologically
compatible
buffers such as Hank's solution or Ringer's solution. In addition, the
compounds
may be formulated in solid form and redissolved or suspended immediately prior
to
use. Lyophilized forms are also included.
For oral administration, the pharmaceutical compositions may take the form
of, for example, tablets or capsules prepared by conventional means with
pharmaceutically acceptable excipients such as binding agents (e.g.,
pregelatinised
maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g.,
lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants
(e.g.,
magnesium stearate, talc or silica); disintegrants (e.g., potato starch or
sodium starch
glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may
be
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coated by methods well known in the art. Liquid preparations for oral
administration may take the form of, for example, solutions, syrups or
suspensions,
or they may be presented as a dry product for constitution with water or other
suitable vehicle before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such as
suspending
agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats);
emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g.,
ationd oil,
oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives
(e.g.,
methyl o r p ropyl-p-hydroxybenzoates o r s orbic acid). T he p reparations m
ay also
contain buffer salts, flavoring, coloring and sweetening agents as
appropriate.
Preparations for oral administration may be suitably formulated to give
controlled release of the active compound. For buccal administration the
compositions may take the form of tablets or lozenges formulated in
conventional
manner. For administration by inhalation, the compounds for use according to
the
present application are conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebuliser, with the use of a suitable
propellant, e.g., dichlorodifluoromethane; trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a
pressurized aerosol the dosage unit may be determined by providing a valve to
deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in
an
inhaler or insufflator may be formulated containing a powder mix of the
compound
and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion. Formulations for
injection
may be presented in unit dosage form, e.g., in ampoules or in multi-dose
containers,
with an added preservative. The compositions may take such forms as
suspensions,
solutions or emulsions in oily or aqueous vehicles, and may contain
formulatory
agents such as suspending, stabilizing andlor dispersing agents.
Alternatively, the
active i ngredient m ay b a i n p owder form f or c onstitution w ith a s
uitable v ehicle,
e.g., sterile pyrogen-free water, before use.
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The compounds may also be formulated in rectal compositions such as
suppositories o r r etention a nemas, a .g., c ontaining c onventional s
uppository b ases
such as cocoa butter or other glycerides.
In addition to the formulations described previously, the compounds may
also be formulated as a depot preparation. Such long acting formulations may
be
administered by implantation (for example subcutaneously or intramuscularly)
or by
intramuscular injection. Thus, for example, the compounds may be formulated
with
suitable polymeric or hydrophobic materials (for example as an emulszon in an
acceptable oil) or ion exchange resins, or as sparingly soluble derivatives,
for
example, as a sparingly soluble salt.
Systemic administration can also be by transmucosal or transdermal means.
For transmucosal or transdermal administration, penetrants appropriate to the
barrier
to be permeated are used in the formulation. Such penetrants are generally
known in
the art, and include, for example, for transmucosal administration bile salts
and
fusidic acid derivatives. in addition, detergents may be used to facilitate
permeation.
Transmucosal administration may be through nasal sprays or using
suppositories.
For topical administration, the oligomers of the application are formulated
into
ointments, salves, gels, or creams as generally known in the art. A wash
solution
can be used locally to treat an injury or inflammation to accelerate healing.
The compositions may, if desired, be presented in a pack or dispenser device
which may contain one or more unit dosage forms containing the active
ingredient.
The pack may for example comprise metal or plastic foil, such as a blister
pack. The
pack or dispenser device may be accompanied by instructions for
administration.
For therapies involving the administration of nucleic acids, the oligomers of
the application can be formulated for a variety of modes of administration,
including
systemic and topical or localized administration. Techniques and formulations
generally may be found in Remmington's Pharmaceutical Sciences, Meade
Publishing Co., Easton, PA. For systemic administration, injection is
preferred,
including intramuscular, intravenous, intraperitoneal, intranodal, and
subcutaneous
for injection, the oligomers of the application can be formulated in liquid
solutions,
preferably in physiologically compatible buffers such as Hanlc's solution or
Ringer's
solution. In addition, the oligomers may be formulated in solid form and
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redissolved or suspended immediately prior to use. Lyophilized forms are also
included.
Systemic administration can also be by transmucosal or transdermal means,
or the compounds can be administered orally. For transmucosal or transdermal
administration, penetrants appropriate to the barrier to be permeated are used
in the
formulation. Such penetrants are generally known in the art, and include, for
example, for transmucosal administration bile salts and fusidic acid
derivatives. In
addition, detergents may be used to facilitate permeation. Transmucosal
administration may be through nasal sprays or using suppositories. For oral
administration, the oligomers are formulated into conventional oral
administration
forms such as capsules, tablets, and tonics. For topical administration, the
oligomers
of the application are formulated into ointments, salves, gels, or creams as
generally
known in the art.
The application now being generally described, it will be more readily
understood by reference to the following examples, which are included merely
for
purposes of illustration of certain aspects and embodiments of the present
application, and are not intended to limit the application.
EXAMPLES
Example 1. Role of POSH in vines-like particle (VLP buddies
1. Obj ective:
Use RNAi to inhibit POSH gene expression and compare the efficiency of
viral budding and GAG expression and processing in treated and untreated
cells.
2. Study Plan:
HeLa SS-6 cells are transfected with mRNA-specific RNAi in order to
knockdown the target proteins. Since maximal reduction of target protein by
RNAi
is achieved after 48 hours, cells are transfected twice - first to reduce
target mRNAs,
and subsequently to express the viral Gag protein. The second transfection is
performed with pNLenv (plasmid that encodes HIV) and with low amounts of RNAi
to maintain the knockdown of target protein during the time of gag expression
and
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budding of VLPs. Reduction in mRNA levels due to RNAi effect is verified by RT-
PCR amplification of target mRNA.
3. Methods, Materials, Solutions
a. Methods
i. Transfections according to manufacturer's protocol and as described in
procedure.
ii. Protein determined by Bradford assay.
iii. SDS-PAGE in Hoeffer miniVE electrophoresis system. Transfer in Bio-
Rad mini-proteawII wet transfer system. Blots visualized using Typhoon system,
and ImageQuant software (ABbiotech)
b. Materials
Material Manufacturer Catalog Batch #
#
Lipofectamine 2000Life Technologies11668-019 1112496
(LF2000)
OptiMEM Life Technologies31985-047 3063119
RNAi Lamin AIC Self 13
RNAi TSG101 688 Self 65
RNAi Posh 524 Self 81
plenvll PTAP Self 148
plenvll ATAP Self 149
Anti-p24 polyclonalSeramun A-0236/5-
antibody 10-O1
Anti-Rabbit Cy5 Jackson 144-175-11548715
conjugated antibody
10% acrylamide Life TechnologiesNP0321 1081371
Tris-
Glycine SDS-PAGE
gel
Nitrocellulose Schleicher 401353 BA-83
membrane &
Schuell
NuPAGE 20X transferLife TechnologiesNP0006-1 224365
buffer
0.45 ~.m filter Schleicher 10462100 CS 1 Ol
& 8-1
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c. Solutions
Lysis BufferCompound Concentration
Tris-HCl pH 7.6 SOmM
MgCl2 1 SmM
NaCI 150mM
Glycerol 10%
EDTA 1 mM
EGTA 1 mM
ASB-14 (add immediately1
before use)
6X Sample Tris-HCl, pH=6.8 1M
Buffer Glycerol 30%
SDS 10%
DTT ~ 9.3%
Bromophenol Blue 0.012%
TBS-T Tris pH=7.6 20mM
NaCI 137mM
Tween-20 0.1
4. Procedure
a. Schedule
D
-
~
1 2 3 ~ 4 5
PlateTransfectionPassageTransfection Extract
II RNA
cellsI cells (RNAi and ~ for RT-PCR
(RNAi only)(1:3) pNlenv) (post
(12:00, PM) txansfection)
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Extract RNA Harvest VLPs
for
RT-PCR and cells
(pre-transfection)
b. Day 1
Plate HeLa SS-6 cells in 6-well plates (35mm wells) at concentration of 5 X10
cells/well.
c. Day 2
2 hours before transfection replace growth medium with 2 ml growth medium
without antibiotics.
Transfection I:
RNAi
RNAi [20~M]OPtiMEMLF2000
[nM] mix
ReactionRNAi nameTAGDA# Reactions u1 (u1) (~I)
1 Lamin 13 2 50 12.5 500 500
A/C
2 Lamin 13 1 50 6.25 250 250
A/C
3 TSG10168865 2 20 5 500 500
5 Posh 524 81 2 50 12.5 500 500
Transfections:
Prepare LF2000 mix: 250 ~,l OptiMEM + 5 ~l LF2000 for each reaction. Mix by
inversion, 5 times. Incubate 5 minutes at room temperature.
Prepare RNA dilution in OptiMEM (Table 1, column A). Add LF2000 mix
dropwise to diluted RNA (Table l, column B). Mix by gentle vortex. Incubate at
room temperature 25 minutes, covered with aluminum foil.
Add 500 ~.l transfection mixture to cells dropwise and mix by rocking side to
side.
Incubate overnight.
d. Day 3
Split 1:3 after 24 hours. (Plate 4 wells for each reaction, except reaction 2
which
is plated into 3 wells.)
e. Day 4
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2 hours pre-transfection replace medium with DMEM growth medium without
antibiotics.
Transfection II
A B C D
RNAi
Plasmid [20~M]
for
RNAi TAG Reaction for 10nM OPtiMEM LF2000
2.4 ~g mix
name DA# Plasmid# (u1) (N1) (N1) (~I)
Lamin 3.4
A/C 13 PTAP 3 3.75 750 750
Lamin 2.5
A/C 13 ATAP 3 3.75 750 750
TSG101 3.4
688 65 PTAP 3 3.75 750 750
Posh 81 PTAP 3 3.4 3.75 750 750
524
Prepare LF2000 mix: 250 ~.1 OptiMEM + 5 ~.l LF2000 for each reaction. Mix by
inversion, 5 times. Incubate 5 minutes at room temperature.
Prepare RNA+DNA diluted in OptiMEM (Transfection II, A+B+C)
Add LF2000 mix (Transfection II, D) to diluted RNA+DNA dropwise, mix by
gentle vortex, and incubate 1h while protected from light with aluminum foil.
Add LF2000 and DNA+RNA to cells, 500~llwell, mix by gentle rocking and
incubate overnight.
f. Day 5
Collect samples for VLP assay (approximately 24 hours post-transfection) by
the
following procedure (cells from one well from each sample is taken for RNA
assay, by RT-PCR).
g. Cell Extracts
i. Pellet floating cells by centrifugation (Smin, 3000 rpm at 4 °C),
save
supernatant (continue with supernatant immediately to step h), scrape
remaining cells in the medium which remains in the well, add to the
corresponding floating cell pellet and centrifuge for 5 minutes, 1800rpm at
4°C.
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ii. Wash cell pellet twice with ice-cold PBS.
iii. Resuspend cell pellet in 100 ~d lysis buffer and incubate 20 minutes on
ice.
iv. Centrifuge at 14,000 rpm for 15 min. Transfer supernatant to a clean
tube. This is the cell extract.
v. Prepare 10 ~,1 of cell extract samples for SDS-PAGE by adding SDS-
PAGE sample buffer to 1X, and boiling for 10 minutes. Remove an aliquot
of the remaining sample for protein determination to verify total initial
starting material. Save remaining cell extract at -80 °C..
h. Purification of VLPs from cell media
i. Filter the supernatant from step g through a 0.45m f lter.
ii. Centrifuge supernatant at 14,000 rpm at 4 °C for at least 2 h.
iii. Aspirate supernatant carefully.
iv. Re-suspend VLP pellet in hot (100 °C warmed for 10 min at least) 1X
sample buffer.
v. Boil samples for 10 minutes, 100 °C.
i. Western Blot analysis
i. Run all samples from stages A and B on Tris-Glycine SDS-PAGE 10%
( 120V for 1.5 h).
ii. Transfer samples to nitrocellulose membrane (65V for 1.5 h).
iii. Stain membrane with ponceau S solution.
iv. Block with 10% low fat milk in TBS-T fox 1 h.
v. Incubate with anti p24 rabbit 1:500 in TBS-T o/n.
vi. Wash 3 times with TBS-T for 7 min each wash.
vii. Incubate with secondary antibody anti rabbit cy5 1:500 for 30 min.
viii. Wash five times for 10 min in TBS-T.
ix. View in Typhoon gel imaging system (Molecular Dynamics/APBiotech)
for fluorescence signal.
Results are shown in Figures 11-13.
Example 2. Exemplary POSH RT-PCR primers and siRNA duplexes
RT-PCR primers
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Name PositionSequence
Sense primerPOSH=271 271 5' CTTGCCTTGCCAGCATAC 3' (SEQ
ID N0:12)
Anti-sense POSH=926c926C 5' CTGCCAGCATTCCTTCAG 3' (SEQ
primer ID NO:13)
siRNA duplexes:
siRNA No: 153
siRNA Name: POSH-230
Position in mRNA 426-446
Target sequence: 5' AACAGAGGCCTTGGAAACCTG 3' SEQ ID N0:
siRNA sense strand: 5' dTdTCAGAGGCCUUGGAAACCUG 3' SEQ I D NO
siRNA anti-sense strand: 5'dTdTCAGGUUUCCAAGGCCUCUG 3' SEQ ID N0:
siRNA No: 155
siRNA Name: POSH-442
Position in mRNA 638-658
Target sequence: 5' AA.AGAGCCTGGAGACCTTAAA 3' SEQ ID N0:
siRNA sense strand: 5' ddTdTAGAGCCUGGAGACCUUAAA 3' SEQ ID NO:
siRNA anti-sense strand: 5' ddTdTUUUAAGGUCUCCAGGCUCU 3' SEQ ID N0:
siRNA No: 157
siRNA Name: POSH-Ul 11
Position in mRNA 2973-2993
Target sequence: 5' AAGGATTGGTATGTGACTCTG 3' SEQ ID NO:
siRNA sense strand: 5' dTdTGGAUUGGUAUGUGACUCUG 3' SEQ ID N0:
siRNA anti-sense strand: 5' dTdTCAGAGUCACAUACCAAUCC 3' SEQ ID N0:
siRNA No: 159
siRNA Name: POSH-U410
Position in mRNA 3272-3292
Target sequence: 5' AAGCTGGATTATCTCCTGTTG 3' SEQ ID N0:
siRNA sense strand: 5' ddTdTGCUGGAUUAUCUCCUGWG 3' SEQ ID N0:
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siRNA anti-sense strand: 5' ddTdTCAACAGGAGAUAAUCCAGC 3' SEQ ID N0:
siRNA No.: 187
siRNA Name: POSH-control
Position in mRNA: None. Reverse to #153
Target sequence: 5' AAGTCCAAAGGTTCCGGAGAC 3' SEQ ID
NO: 36
3. Knock-down of hPOSH entraps HIV virus particles in intracellular vesicles
HIV virus release was analyzed by electron microscopy following siRNA
and full-length HIV plasmid (missing the envelope coding region) transfection.
Mature viruses were secreted by cells transfected with HIV plasmid and non-
relevant siRNA (control, lower panel). Knockdown of Tsg101 protein resulted in
a
budding defect, the viruses that were r eleased had an immature phenotype
(upper
panel). Knockdown of hPOSH levels resulted in accumulation of viruses inside
the
cell in intracellular vesicles (middle panel). Results, shown in Figure 28,
indicate
that inhibiting hPOSH entraps HIV virus particles in intracellular vesicles.
As
accumulation of HIV virus particles in the cells accelerate cell death,
inhibition of
hPOSH therefore destroys HIV reservoir by killing cells infected with HIV.
Example 4. In-vitro assay of Human POSH self ubiquitination
Recombinant hPOSH was incubated with ATP in the presence of E1, E2 and
ubiquitin as indicated in each lane. Following incubation at 37 °C for
30 minutes,
reactions w ere t erminated b y a ddition o f S DS-PAGE s ample b uffer. T he
s amples
were subsequently r esolved on a 10% polyacrylamide g e1. The separated
samples
were then transferred to nitrocellulose and subjected to immunoblot analysis
with an
anti ubiquitin polyclonal antibody. The position of migration of molecular
weight
marlcers is indicated on the right.
Poly-Ub: Ub-hPOSHconjugates, detected as high molecular weight adducts only in
reactions containing E1, E2 and ubiquitin. hPOSH-176 and hPOSH-178 are a short
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and a longer derivatives (respectively) of bacterially expressed hPOSH; C,
control
E3.
Preliminary steps in a high-throughput screen
Materials
1. E1 recombinant from bacculovirus
2. E2 UbchSc from bacteria
3. Ubiquitin
4. POSH #178 (1-361) GST fusion-purified but degraded
5. POSH # 176 (1-269) GST fusion-purified but degraded
6. hsHRD 1 soluble ring containing region
5. Bufferxl2 (Tris 7.6 40 mM, DTT lmM, MgCl2 SmM, ATP 2uM)
6. Dilution buffer (Tris 7.6 40mM, DTT lmM, ovalbumin lug/ul)
protocol
O.lug/ulO.Sug/ulSug/ul0.4ug/ul2.Sug/u/0.8ug/uI
El E2 Ub 176 178 Hrdl Bxl2
-.
-E1 (E2+176) _______0.5 0.5 1 ______ ______ 10
-E2 (El+176) 1 ______ 0.5 1 ______ ______ 9.5
-ub (El+E2+176) 1 0.5 -------1 ------ ------ 9.5
E1+E2+176+Ub 1 0.5 0.5 1 ------
-El (E2+178) ______ 0.5 0.5 ______ 1 _______10
-E2 (El+178) 1 ----- 0.5 ------ 1 -------9.5
-ub (E1+E2+178) 1 0.5 ----- ------ 1 -------9.5
E1+E2+178+Ub 1 0.5 0.5 ------ 1 ------19
Hrdl, E1+E2+Ub 1 0.5 0.5 ------ ----- 1 8.5
1. Incubate for 30 minutes at 37 °C.
2. Run 12% SDS PAGE gel and transfer to nitrocellulose membrane
3. Incubate with anti-Ubiquitin antibody.
Results, shown in Figure 19, demonstrate that human POSH has
ubiquitin ligase activity.
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Example 5. Co-immunoprecipitation of hPOSH with myc-ta~~ed activated (V 12)
and dominant-negative (N17) Racl
HeLa cells were transfected with combinations of myc-Racl Vl2 or N17 and
hPOSHdeIRING-V5. 24 horns after transfection (efficiency 80% as measured by
GFP) cells were collected, washed with PBS, and swollen in hypotonic lysis
buffer
(10 mM HEPES pH=7.9, 15 mM KCI, 0.1 mM EDTA, 2 mM MgCl2, 1 mM DTT,
and protease inhibitors). Cells were lysed by 10 strokes with Bounce
homogenizer
and centrifuged 3000xg for 10 minutes to give supernatant (Fraction 1) and
nucleii.
Nucleii were washed with Fraction 2 buffer (0.2% NP-40, 10 mM HEPES pH=7.9,
40 mM KCI, 5% glycerol) to remove peripheral proteins. Nucleii were spun-down
and supernatant collected (Fraction 2). Nuclear proteins were eluted in
Fraction 3
buffer (20 mM HEPES pH=7~.9, 0.42 M KCI, 25% glycerol, 0.1 mM EDTA, 2 mM
MgCl2, 1 mM DTT) by rotating 30 minutes in cold. Tnsoluble proteins were spun-
down 14000xg and solubilized in Fraction 4 buffer (1% Fos-Choline 14, 50 mM
HEPES pH=7.9, 150 mM NaCI, 10% glycerol, 1mM EDTA, 1.5 mM MgCla, 2 mM
DTT). Half of the total extract was pre-cleared against Protein A sepharose
for 1.5
hours and used for IP with 1 ~,g anti-myc (9E10, Roche 1-667-149) and Protein
A
sepharose for 2 hours. Immune complexes were washed extensively, and eluted in
SDS-PAGE sample buffer. Gels were run, and proteins electro-transferred to
nitrocellulose for immunoblot as in Figure 20. Endogenous POSH and transfected
hPOSHdeIRING-VS are precipitated as a complex with Myc-Racl V12/N17.
Results, s hown i n F figure 2 0, d emonstrate t hat POSH c o-
immunoprecipitates w ith
Rac 1.
Example 6. POSH reduction results in decreased secretion of phospholipase D
PLD
Hela SS6 cells (two wells of 6-well plate) were transfected with POSH
siRNA or control siRNA (100 nM). 24 hours later each well was split into 5
wells of
a 24-well plate. The next day cells were transfected again with 100 nM of
either
POSH siRNA or control siRNA. The next day cells were washed three times with
lxPBS and than 0.5 ml of PLD incubation buffer (118 mM NaCl, 6 mM KCI, 1 mM
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CaCl2, 1.2 mM MgS04, 12.4 mM HEPES, pH7.5 and 1 % fatty acid free bovine
serum albumin) were added.
48 hours later medium was collected and centrifuged at 800xg for 15
minutes. The medium was diluted with SxPLD reaction buffer (Amplex red PLD
kit)
and assayed for PLD by using the Amplex Red PLD kit (Molecular probes, A-
12219). The assay results were quantified and presented below in as a bar
graph.
The cells were collected and lysed in 1 % Triton X-100 lysis buffer (20 mM
HEPES-
NaOH, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1% Triton X-100 and
lx protease inhibitors) for 15 minutes on ice. Lysates were cleared by
centrifugation
and protein concentration was determined. There were equal protein
concentrations
between the two transfectants. Equal amount of extracts were
immunoprecipitated
with anti-POSH antibodies, separated by SDS-PAGE and immunoblotted with anti-
POSH antibodies to assess the reduction of POSH levels. There was
approximately
40% reduction in POSH levels (Figure 21).
Example 7. Effect of hPOSH on Gad-EGFP intracellular distribution
HeLa SS6 were transfected with Gag-EGFP, 24 hours after an initial
transfection with either hPOSH-specific or scrambled siRNA (control) (100nM)
or
with plasmids encoding either wild type hPOSH or hPOSH C(12,55)A. Fixation
and staining was preformed 5 hours after Gag-EGFP transfection. Cells were
fixed,
stained with Alexa fluor 647-conjugated Concanavalin A (ConA) (Molecular
Probes), permeabilized and then stained with sheep anti-human TGN46. After the
primary antibody incubation cells were incubated with Rhodamin-conjugated goat
anti-sheep. Laser scanning confocal microscopy was performed on LSM510
confocal microscope (Zeiss) equipped with Axiovert 100M inverted microscope
using x40 magnification and 1.3-numerical-aperture oil-immersion lens for
imaging.
For co-localization experiments, 10 optical horizontal sections with i
ntervals of 1
~,m were taken through each preparation (Z-stack). A single median section of
each
preparation is shown. See Figure 22.
Example 8. POSH-Regulated Intracellular Transport of Myristoylated Proteins
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The localization of myristoylated proteins, Gag (see Figure 22), HIV-1 Nef,
Src and Rapsyn, in cells depleted of hPOSH were analyzed by
immunofluorescence.
In control cells, HIV-1 Nef was found in a perinuclear region co-localized
with
hPOSH, indicative of a TGN localization (Figure 23). When hPOSH expression was
reduced by siRNA treatment, Nef expression was weaker relative to control and
nef
lost its TGN, perinuclear localization. Instead it accumulated in punctated
intracellular loci segregated from the TGN.
Src is expressed at the plasma membrane and in intracellular vesicles, which
are found close to the plasma membrane (Figure 24, H187 cells). However, when
hPOSH levels were reduced, Src was dispersed in the cytoplasm and loses its
plasma
membrane proximal localization detected in control (H187) cells (Figure 24,
compare H153-1 and H187-2 panels).
Rapsyn, a peripheral membrane protein expressed in skeletal muscle, plays a
critical role in organizing the structure of the nicotinic postsynaptic
membrane
(Saves and Lichtman, Annu. Rev. Neurosci. 22: 389-442 (1999)). Newly
synthesized Rapsyn associates with the TGN and than transported to the plasma
membrane (Marchand et al., J. Neurosci. 22: 8891-O1 (2002)). In hPOSH-depleted
cells (H153-1) Rapsyn was dispersed in the cytoplasm, while in control cells
it had a
punctuated pattern and plasma membrane localization, indicating that hPOSH
influences its intracellular transport (Figure 25).
Materials and Methods Used:
Antibodies:
Src antibody was purchased from Oncogene research products( Darmstadt,
Germany). Nef antibodies were pusrchased from ABI (Columbia, MA) and
Fitzgerald Industries Interantional (Concord, MA). Alexa Fluor conjugated
antibodies were pusrchased from Molecular Probes Inc. (Eugene, OR).
hPOSH antibody: Glutathione S-transferase (GST) fusion plasmids were
constructed by PCR amplification of hPOSH codons 285-430. The amplified PCR
products was cloned into pGEX-6P-2 (Amersham Pharmacia Biotech,
Buclcingharnshire, UK). The truncated hPOSH protein was generated in E. coli
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BL21. Bacterial cultures were grown in LB media with carbenicillin (100
p,g/ml)
and recombinant protein production was induced with 1 mM IPTG for 4 hours at
30
°C. Cells were lysed by sonication and the recombinant protein was then
isolated
from the cleared bacterial lysate by affinity chromatography on a glutathione-
sepharose resin (Amersham Pharmacia Biotech, Buckinghamshire, UK). The
hPOSH portion of the fusion protein was then released by incubation with
PreScission protease (Amersham Pharmacia Biotech, Buckinghamshire, UK)
according to the manufacturer's instructions and the GST portion was then
removed
by a second glutathione-sepharose affinity chromatography. The purified
partial
hPOSH polypeptide was used to immunize New Zealand white rabbits to generate
antibody 15B (Washington Biotechnology, Baltimore, Maryland).
Construction of siRNA retroviral vectors:
hPOSH scrambled oligonucleotide (5'- CACACACTGCCG TCAACT
GTTCAAGAGAC AGTTGACGGCAGTGTGTGTTTTTT -3'; and 5'-
AATTAAAAAACACA CACTGCCGTCAACTGTC TCTTGAACAGTTGA
CGGCAGTGTGTGGGCC -3') were annealed and cloned into the Apal-EcoRI
digested .pSilencer 1.0-US (Ambion) to generate pSIL-scrambled. Subsequently,
the
U6-promoter and RNAi sequences were digested with BamHI, the ends filled in
and
the insert cloned into the Olil site in the retroviral vector, pMSVhyg
(Clontech),
generating pMSCVhyg-U6-scrambled. hPOSH oligonucleotide encoding RNAi
against hPOSH (5'-AACAGAGGCCTTGGAAA CCTGGAAGC TTGCAGGTTT
CCAAGGCCTCTGTT -3'; and 5'- GATCAACAGAG GCCTTGGAAACCTGC
AAGCTTCCAGGTTTCCAA GGCCTCTGTT -3') were annealed and cloned into
the BamHI-EcoRI site of pLIT-U6, generating pLIT-U6 hPOSH-230. pLIT-U6 is an
shRNA vector containing the human U6 promoter (amplified by PCR from human
genomic DNA with the primers, 5'-GGCCCACTAGTCA AGGTCG GGCA
GGAAGA- 3' and S'- GCCGAATT CAAA.A.A.GGATC CGGCGATATCCGG
TGTTTCGTCCTTTCCA -3') cloned into pLITMUS38 (New England Biolabs)
digested with SpeI-EcoRI. Subsequently, the U6 promoter-hPOSH shRNA (pLIT-
U6 hPOSH-230 digested with SnaBI and PvuI) was cloned into the Olil site of
pMSVhyg (Clontech), generating pMSCVhyg U6-hPOSH-230.
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~ Generation of stable clones:
HEK 293T cells were transfected with retroviral RNAi plasmids
(pMSCVhyg-U6-POSH-230 and pMSCVhyg-U6-scrambled and with plasmids
encoding VSV-G and moloney gag-pol. Two days post transfection, medium
containing retrovinises was collected and filtered and polybrene was added to
a final
concentration of 8~,g/ml. This was used to infect HeLa SS6 cells grown in 60
mm
dishes. Forty-eight hours post-infection cells were selected for RNAi
expression by
the addition of hygromycin to a final concentration of 300 ~g/ml. Clones
expressing
RNAi against hPOSH were named H153, clones expressing scrambled RNAi were
named H187.
~ Transfection and immunofluorescent analysis:
Gag-EGFP experiments are described in Figure 22.
H153 or H187 cells were transfected with Src or Rapsyn-GFP (Image clone
image: 3530551 or pNLenv-1). Eighteen hours post transfection cells were
washed
with P BS and i ncubated o n i ce with A Iexa F luor 6 47 c onjugated C on A t
o 1 abet
plasma membrane glycoproteins. Subsequently cells were fixed in 3%
paraformaldehyde, blocked with PBS containing 4% bovine serum albumin and 1%
gelatin. Staining with rabbit anti-Src, rabbit anti-hPOSH (15B) or mouse anti-
nef
was followed with secondary antibodies as indicated.
Laser scanning confoeal microscopy was performed on LSM510 confocal
microscope (Zeiss) equipped with Axiovert 100M inverted microscope using x40
magnification and 1.3-numerical-aperture oil-immersion lens for imaging. For
co
localization experiments, 10 optical horizontal sections with intervals of 1
~.m were
taken through each preparation (Z-stack). A single median section of each
preparation is shown.
Example 9. POSH Reduction by siRNA Abrogates West Nile Virus ("WNV")
Infectivity.
HeLa SS6 cells were transfected with either control or POSH-specif c
siRNA. Cells were subsequently infected with WNV (4x104 PFU/well). Viruses
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were harvested 24 hours and 48 hours post-infection, serially diluted, and
used to
infect Vero cells. As a control WNV (4x104 PFU/well), that was not passed
through
HeLa SS6 cells, was used to infect Vero cells. Virus titer was determined by
plaque
assay in Vero cells.
Virus titer was reduced by 2.5-log in cells treated with POSH-specific
siRNA relative to cells transfected with control siRNA, thereby indicating
that
WNV requires POSH for virus secretion. See Figure 26.
Experimental Procedure:
~ Cell culture, transfections and infection:
Hela SS6 cells were grown in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% heat-inactivated fetal calf serum and 100
units/ml
penicillin and 100 ~g/ml streptomycin. For transfections, HeLa SS6 cells were
grown to 50% confluency in DMEM containing 10% FCS without antibiotics. Cells
were then transfected with the relevant double-stranded siRNA (100 nM) using
lipofectamin 2000 (Invitrogen, Paisley, UK). On the day following the initial
transfection, cells were split 1:3 in complete medium and transfected with a
second
portion of double-stranded siRNA (50 nM). Six hours post-transfection medium
was
replaced and cells infected with WNV (4x104 PFU/well). Medium was collected
from infected HeLa SS6 cells twenty-four and forty-eight post-infection (200
~1),
serially diluted, and used to infect Vero cells. Virus titer was determined by
plaque
assay (Ben-Nathan D, Lachmi B, Lustig S, Feuerstien G (1991) Protection of
dehydroepiandrosterone (DHEA) in mice ifected with viral encephalitis. Arch
Viro;
120, 263-271).
Examt~le 10. Analvsis of the effects of POSH knockdown on M-MuLV expression
and budding
Experimental Protocol:
Transfections:-
A day before transfection, Hela SS6 cells were plated in two 6 wells plates at
5 x105 cells per well. 24 hours later the following transfections were
performed:
4 wells were transfected with control siRNA and a plasmid encoding MMuLV.
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4 wells were transfected with POSH siRNA and a plasmid encoding MMuLV.
1 well was a control without any siRNA or DNA transfected.
1 well was transfected with a plasmid encoding MMuLV.
For each well to be transfected 100 nM (12.5 ~.1) POSH siRNA or 100 nM
(12.5 ~1) control siRNA were diluted in 250 ~.l Opti-MEM (Invitrogen).
Lipofectamin 2000 (5 ~,l) (Invitrogen, Cat. 11668-019) was mixed with 250 ~,1
of
OptiMEM per transfected well. The diluted siRNA was mixed with the
lipofectamin
2000 mix and the solution incubated at room temperature for 30 min. The
mixture
was added directly to each well containing 2 ml DMEM +10% FBS (w/o
antibiotics).
24 hours later, four wells of the same siRNA treatment were split to eight
wells, and two wells without siRNA were split to four wells.
24 hours later all wells were transfected with 100 nM control siRNA or 100
nM POSH siRNA with or without a plasmid encoding MMuLV (see table below).
48 hours later virions and cells were harvested.
NO Of RNAi Amount Amount of The volumeApplication
DNA of
Wells of RNAi (wg) per DNA (~.1)
well per
(p1) well
per
well
5 POSH 12.5 MMuLV (2 10 4 wells
~tg) for
100 nM VLPs assay
(15'
and 2"d and 1 well
for
transfection) RT
5 Control 12.5 MMuLV (2 10 4 wells
fig) for
100 nM VLPs assay
(1s'
and 2"d and 1 well
for
transfection) RT
1 _ - - 10 ~l H20 VLPs assay
1 - - MMuLV (2 10 VLPs assay
~.g)
Steady state VLP assay
Cell extracts:-
1. Pellet floating cells by centrifugation (10 min, SOOxg at 4 °C),
save
supernatant (continued at step 7), wash cells once, scrape cells in ice-cold
lxPBS, add to the corresponding cell pellet and centrifuge for 5 min 1800
rpm at 4 °C.
2. Wash cell pellet once with ice-cold lxPBS.
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3. Resuspend cell pellet in 150 p,1 1% Triton X-100 lysis buffer (20 mM
HEPES-NaOH, pH 7.4, I 50 mM NaCI, 1.5 mM MgCl2, I ~mM EDTA, 1
Triton X-100 and lx protease inhibitors) and incubate 20 minutes on ice.
4. Centrifuge at 14,OOOrpm for 15 min. Transfer supernatant to a clean tube.
5. Determine protein concentration by BCA.
6. Prepare samples for SDS-PAGE by adding 2 p.1 of 6xSB to 20 p.g extract
(add lysis buffer to a final volume of 12 p.1), heat to 80 °C for 10
min.
Purification of virions from cell media
7. Filtrate the supernatant through a 0.45 p,m filter.
8. Transfer 1500 p1 of virions fraction to an ultracentrifuge tube (swinging
rotor).
9. Add 300 p.1 of fresh sucrose cushion (20% sucrose in TNE) to the bottom of
the tube.
10. Centrifuge supernatant at 35000 rpm at 4 °C for 2 hr.
1l. Resuspend virion pellet in 50 ~,l hot lx sample buffer each (samples I53-
l,
2, 3, 187-1, 2, 3). Resuspend VLPs pellet (153-4, 5 and 187 4, 5) in 25 p,1
hot
lx sample buffer. Vortex shortly, transfer to an eppendorf tube, unite VLPs
from wells 153-4+5 and 187- 4+5. Heat to 80 °C for 10 min.
12. Load equal amounts of VLPs relatively to cells extracts amounts.
Western Blot analysis
1. Separate all samples on 12% SDS-PAGE.
2. Transfer samples to nitrocellulose membrane (100V for 1.15 hr).
3. Dye membrane with ponceau solution.
4. Block with 10% low fat milk in TBS-T for 1 hour.
5. Incubate membranes with Goat anti p30 (81S-263) (1:5000) in 10% low fat
milk in TBS-T over night at 4 °C. Incubate with secondary antibody
rabbit
anti goat-HRP 1:8000 for 60 min at room temperature.
6. Detect signal by ECL reaction.
7. Following the ECL detection incubate memebranes with Donkey anti rabbit
Cy3 (Jackson Laboratories, Cat 711-165-152) 1:500 and detect signal by
Typhoon scanning and quantitate.
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Results:
As shown in Figure 27, POSH knockdown decreases the release of
extracellular MMuLV particles.
Examble 11. POSH Protein~rotein interactions by yeast two hybrid assa
POSH-associated proteins were identified by using a yeast two-hybrid assay.
Procedure:
Bait plasmid (GAL4-BD) was transformed into yeast strain AH109
(Clontech) and transformants were selected on defined media lacking
tryptophan.
Yeast strain Y187 containing pre-transformed Hela cDNA prey (GAL4-AD) library
(Clontech) was mated according to the Clontech protocol with bait containing
yeast
and plated on defined media lacking tryptophan, leucine, histidine and
containing 2
mM 3 amino triazol. Colonies that grew on the selective media were tested for
beta-
1 S galactosidase activity and positive clones were further characterized.
Prey clones
were identified by amplifying cDNA insert and sequencing using vector derived
primers.
Bait:
Plasmid vector: pGBK-T7 (Clontech)
Plasmid name: pPL269- pGBK-T7 GAL4 POSHdR
Protein sequence: Corresponds to as S3-888 of POSH (R1NG domain deleted)
RTLVGSGVEELPSNILLVRLLDGTKQRPWKPGPGGGSGTNCTNALRSQSSTVANCSSKDL
QSSQGGQQPRVQSWSPPVRGIPQLPCAKALYNYEGKEPGDLKFSKGDIIILRRQVDENWY
HGEVNGIHGFFPTNFVQIIKPLPQPPPQCKALYDFEVKDKEADKDCLPFAKDDVLTVIRR
2S VDENWAEGMLADKIGIFPISYVEFNSAAKQLIEWDKPPVPGVDAGECSSAAAQSSTAPKH
SDTKKNTKKRHSFTSLTMANKSSQASQNRHSMEISPPVLTSSSNPTAAARISELSGLSCS
APSQVHISTTGLIVTPPPSSPVTTGPSFTFPSDVPYQAALGTLNPPLPPPPLLAATVLAS
TPPGATAAAAAAGMGPRPMAGSTDQIAHLRPQTRPSVYVAIYPYTPRKEDELELRKGEMF
LVFERCQDGWFKGTSMHTSKIGVFPGNYVAPVTRAVTNASQAKVPMSTAGQTSRGVTMVS
3O PSTAGGPAQKLQGNGVAGSPSVVPAAVVSAAHTQTSPQAKVLLHMTGQMTVNQARNAVRT
VAAHNQERPTAAVTPIQVQNAAGLSPASVGLSHHSLASPQPAPLMPGSATHTAAISISRA
SAPLACAAAAPLTSPSTTSASLEAEPSGRIVTVLPGLPTSPDSASSACGNSSATKPDKDS
KKEKKGLLKLLSGASTKRKPRVSPPASPTLEVELGSAELPLQGAVGPELPPGGGHGRAGS
CPVDGDGPVTTAVAGAALAQDAFHRKASSLDSAVPIAPPPRQACSSLGPVLNESRPWCE
3S RHRVWSYPPQSEAELELKEGDIVFVHKKREDGWFKGTLQRNGKTGLFPGSFVENI
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Library screened: Hela pretransformed library (Clontech).
POSH-APs identified by yeast two-hybrid assay are provided in Tables 7 and
8. Also, the nucleic acid and amino acid sequences of POSH-APs identified by
yeast two-hybrid assay are provided in Figure 36. In addition, the nucleic
acid and
amino acid sequences of ARF1 and ARFS are provided in Figure 36.
Example 12. Inhibition of PKA Kinase Activity Attenuates HIV-1 Virus
Maturation
HeLa SS6 cells were transfected with pNLenv-lP-~-AP or pNLenv-lATAA (L-
domain mutant). Eighteen hours post-transfection, cells were transferred to 20
°C for
I O two hours in order to inhibit transport of viral particles from the traps-
Golgi (TGN)
to the plasma membrane (PM). Subsequently, the PKA inhibitor, H89 (50 ~M)
(Biosource, Cat. No. PHZ1114) or DMSO were added to the cells and dishes were
transferred to 37 °C to initiate transport from the TGN to the PM.
Reverse
transcriptase activity was assayed from virus-like-particles collected from
cell
supernatant twenty minutes later. H89 treatment resulted in complete
inhibition of
RT activity. Thus, demonstrating that PKA activity is required for HIV-1 viral
maturation.
Materials and methods:
Cell culture and transfections
Hela SS6 cells were grown in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% heat-inactivated fetal calf serum and 100
units/rnl
penicillin and 100 ~.g/ml streptomycin. For transfections, HeLa SS6 cells were
grown to 100% confluency in DMEM containing 10% FCS without antibiotics.
Cells were then transfected with HIV-lNLenm (2 ~.g per 6-well) (Schubert et
al.,
1995).
Assays for virus release by RT activity
Vines and virus-like particle (VLP) release by reverse transcriptase activity
was determined one day after transfection with the pro-viral DNA as previously
described ( Adachi a t a l., 1986; F ulcumori a t a l., 2000; Lenardo a t a
1., 2 002). T he
culture medium of vines-expressing cells was collected and centrifuged at 500
x g
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for 10 minutes. The resulting supernatant was passed through a 0.45 pm-pore
filter
and the filtrate was centrifuged at 14,000 x g for 2 hours at 4 °C. The
resulting
supernatant was removed and the viral-pellet was re-suspended in cell
solubilization
buffer (50 mM Tris-HCI, pH7:8, 80 mM potassium chloride, 0.75 mM EDTA and
0.5% Triton X-100, 2.S mM DTT and protease inhibitors). The corresponding
cells
were washed three times with phosphate-buffered saline (PBS) and then
solubilized
by incubation on ice for 15 minutes in cell solubilization buffer. The cell
detergent
extract was then centrifuged for 15 minutes at 14,000 x g at 4 °C. The
sample of the
cleared extract (normally 1:10 of the initial sample) were resolved on a 12.5%
SDS-
polyacrylamide gel, then transferred onto nitrocellulose paper and subjected
to
immunoblot analysis with rabbit anti-CA antibodies. The CA was detected after
incubation with a secondary anti-rabbit antibody conjugated to Cy5 (Jackson
Laboratories, West Grove, Pennsylvania) and detected by fluorescence imaging
(Typhoon instrument, Molecular D ynamics, Sunnyvale, California). The Pr55 and
CA were then quantified by densitometry. A colorimetric reverse transcriptase
assay
(Ruche Diagnostics GmbH, Mannenheim, Germany) w as used to measure reverse
transcriptase activity in VLP extracts. RT activity was normalized to amount
of Pr55
and CA produced in the cells.
Example 13. hPOSH is~hosphorylated by Protein kinase A (PKA~
PKA is a cAMP-dependent kinase. The holocnzyme is a tetramer of two
catalytic subunits (cPKA.) bound to two regulatory subunits PRKRl or PRKR2.
Activation proceeds by the cooperative binding of two cAMP molecules. to each
R
subunit, which causes the dissociation of each active C subunit from the R
subimit
dimer. The consensus sequence for phosphorylation by the C subunit is,
stringently,
K/R-R-X-S/TY and less stringently, R-X-X-S/TY, where Y tends to be a
hydrophobic r esidue. T he i ntracellular 1 ocalization o f P KA i s c
ontrolled t horough
association w ith A-kinase-anchoring p roteins ( AKAPs). T he r egulatory s
ubunit o f
protein kinase A (PRKR1A) was identified as a POSH interactor by yeast-two
hybrid screen, thereby implicating POSH as an AKAP.
Protein kinase A was demonstrated to be required for the budding of
transport vesicles from the TGN (Muniz et al., 1997, Proc Natl Acad Sci U S A,
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94:14461-6). Furthermore, it was demonstrated that an inhibitor of PKA, H89,
is
able to block HIV-1 release from cells (Cartier et al., 2003, J Biol Chem.,
278:35211-9). Since POSH is localized at the TGN and is implicated as an AKAP,
POSH may regulate PKA-mediated budding at the TGN of vesicles and HIV-1.
Applicants demonstrated that POSH is phosphorylated by PKA. Several
putative PKA phosphorylation sites are found within hPOSH coding sequence
(Figure 30). Phosphorylation of gravin, an AKAP, by PKA modulates its binding
to
the b2-adrenergic r eceptor. This serves to regulate the mobilization of g
ravin and
PKA to the cell membrane and regulation of b2-AR activity by PKA. Two putative
PKA sites are located in the putative-rac-binding region in POSH. Toward this
end,
POSH was subjected to in-vitro phosphorylation and binding to the small GTPase
Racl (Figure 31). Indeed, only unphosphorylated POSH was able to bind
activated,
GTP-loaded, Racl, demonstrating that phosphorylation regulates the binding of
POSH to small GTPases, such as Racl. GTPases of this sort family include TCL,
TC10, Cdc42, Wrch-l, Rac2, Rac3 or RhoG (Aspenstrom et al., 2003, Biochem J.,
377(Pt 2):327-37). Small GTPases of this sort are involved in protein
trafficking in
the secretory system, including the trafficking of viral proteins, such as
those of
HIV.
Materials and methods
PKA-dependent phosphorylation of hPOSH.
Bacterially expressed recombinant maltose-binding-protein (MBP)-hPOSH
(3 ~,g) or GST-c-Cbl were incubated at 30oC for 30 minutes with (*) or without
10
ng PKA catalytic subunit (PKAc) in a buffer containing 40 mM Tris-HCl pH 7.4,
10
mM MgCl2, 4 mM ATP, 0.1 mg/ml BSA, 1 p,M cAMP, 23 mM K3P04, 7 nM DTT,
and PKA peptide protection solution (Promega, Cat.No. V5340). The reaction was
stopped by the addition of SDS-sample buffer, and boiling for 3 minutes.
Samples
were separated by SDS-PAGE on a 10% gel, and transferred to nitrocellulose and
immunoblotted as detailed in the figure.
Binding of Racl to hPOSH
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Bacterially expressed hPOSH (1 pg) or GST (1 ~.g) were phosphorylated as
above. The reaction was terminated by the addition 0.5 ml of ice-cold 200 mM
Tris-
HCl pH 7.4, 5 mM EDTA. hPOSH and GST were then immobilized on NiNTA or
reduced glutathione beads, respectively, by gentle mixing for 30 minutes. The
immobilized proteins were washed three times with wash buffer (50 mM Tris-HCl
pH 7.4, 100 mM NaCI, 5 mM MgCl2, 0.1 mM DTT). Recombinant Rac-1 (0.2 p.g)
(Sigma catalog # 83012) was incubated with or without 0.3 mM GTP~yS (Sigma
Cat.
No. 68638) on ice for 15 minutes. The GTP/mock-loaded Rac-1 was then added to
wash buffer (25 ~1, final) and incubated for 30 minutes at 30 °C. The
beads were
then washed three times with wash buffer containing 0.1% Tween 20. Sample
buffer
was added to the bead pellet and boiled for 3 minutes. Immobilized and
associating
proteins were then separated by SDS-PAGE on a 12% gel and immunobloted with
anti-Rac-1 (Santa Cruz Biotechnology, Cat. No. sc-217). Input is 0.25 p,g of
Rac-1.
Example 14. HERPUD1 Depletion by siRNA Reduces HIV Maturation.
Hela SS6 cells were transfeted with siRNA directed against HERPUDl and
with a plsmid encoding HIV proviral genome (pNLenv-1). Twenty four hours post-
HIV transfection, virus-like particles (VLP) secreted into the medium were
isolated
and reverse transcriptase activity was determined. HIV release of active RT is
an
indication for a release of processed and mature virus. When the levels of
HERPUD1 were reduced RT activity was inhibited by 80%, demonstrating the
importance of HERPUD 1 in HIV-maturation. See Figure 33.
Experimental Outline
~ Cell culture and transfection:
HeLa SS6 were kindly provided by Dr. Thomas Tuschl (the laboratory of
RNA Molecular Biology, Rockefeller University, New York, New York). Cells
were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with
10% heat-inactivated fetal calf serum and 100 U/ml penicillin and 100 p,g/ml
streptomycin. For transfections, HeLa SS6 cells were grown to 50% confluency
in
DMEM containing 10% FCS without antibiotics. Cells were then transfected with
the relevant double-stranded siRNA (50-100nM) (HERPUD1: 5'-
GGGAAGUUCUUCGGAACCUdTdT-3' and 5'-
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dTdTCCCUUCAAGAAGCCUUGGA-5') using lipofectamin 2000 (Invitrogen,
Paisley, UK). A day following the initial transfection cells were split 1:3 in
complete
medium and co-transfected 24 hours later with HIV-lNLenv1 (2 ~g per 6-well)
(Schubert et al., J. Virol. 72:2280-88 (1998)) and a second portion of double-
s stranded siRNA.
~ Assay for virus release
Virus and virus-like particle (VLP) release was determined one day after
transfection with the proviral DNA as previously described (Adachi et al., J.
Virol.
59: 284-91 (1986); Fukumori et al., Vpr. Microbes Infect. 2: 1011-17 (2000);
Lenardo et al., J. Virol. 76: 5082-93 (2002)). The culture medium of virus-
expressing cells was collected and centrifuged at 500 x g for 10 minutes. The
resulting supernatant was passed through a 0.45p,m-pore filter and the
filtrate was
centrifuged at 14,000 x g for 2 hours at 4°C. The resulting supernatant
was removed
and the viral-pellet was re-suspended in SDS-PAGE sample buffer. The
corresponding cells were washed three times with phosphate-buffered saline
(PES)
and then solubilized by incubation on ice for 15 minutes in lysis buffer
containing
the following components: 50 mM HEPES-NaOH, (pH 7.5), 150 mM NaCI, 1.5 mM
MgCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA and
1:200 dilution of protease inhibitor cocktail (Calbiochem, La Jolla,
California). The
cell detergent extract was then centrifuged for 15 minutes at 14,000 x g at
4°C. The
VLP sample and a sample of the cleared extract (normally 1:10 of the initial
sample) were resolved on a 12.5% SDS-polyacrylamide gel, then transferred onto
nitrocellulose paper and subjected to immunoblot analysis with rabbit anti-CA
antibodies. The CA was detected either after incubation with a secondary anti-
rabbit
horseradish peroxidase-conjugated antibody and detected by Enhanced Chemi-
Luminescence (ECL) (Amersham Pharmacia) or after incubation with a secondary
anti-rabbit antibody conjugated to Cy5 (Jackson Laboratories, West Grove,
Pennsylvania) and detected by fluorescence imaging (Typhoon instrument,
Molecular Dynamics, Sunnyvale, CA). The Pr55 and CA were then quantified by
densitometry and the amount of released VLP was then determined by calculating
the ratio between VLP-associated CA and intracellular CA and Pr55 as
previously
rtPCrrihPrl lschubert et al., J. Virol. 72:2280-88 (1998)).
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~ Analysis of reverse transcriptase activity in supernatants
RT activity was determined in pelleted VLP (see above) by using an RT
assay kit (Roche, Germany; Cat.No. 1468120). Briefly, VLP ,pellets were
resuspended in 40 p,1 RT assay lysis buffer and incubated at room temperature
for 30
minutes. At the end of incubation 20 p,1 RT assay reaction mix was added to
each
sample and incubation continued at 37°C overnight. Samples. (60 p,1)
were than
transferred to MTP strip wells and incubated at 37°C for 1 hour. Wells
were washed
five times with wash buffer and DIG-POD added for a one-hour incubation at
37°C.
At the end of incubation wells were washed five times with wash buffer and
ABST
substrate solution was added and incubated until color developed. The
absorbance
was read in an ELISA reader at 405 nm (reference wavelength 492 run). The
resulting signal intensity is directly proportional to RT activity; RT
concentration
was determined by plotting against a known amount of RT enzyme included in
separate wells of the reaction.
Examale 15. MSTP028 Reduction by siRNA Decreases HIV VLP Production.
This example demonstrates the effects of an siRNA-mediated decrease in
MSTP028 expression on the production of HIV virus-like particles in HeLa
cells.
The effects were measured at steady state.
Experiments were performed according to two different protocols.
Experiment 1 proceeded with a second transfection on day 3, while Experiment 2
involved an additional exchange of medium on day 3, and proceeded to the
second
transfection on day 4. The results from Experiment 1 are shown Figure 29A, and
those for Experiment 2 are shown in Figure 29B.
Day 1: Preparing Cells
4.5X105 HeLa SS6 cellslwell, were seeded in 1 x 6 well plates. Cells were
seeded
in transfection medium (growing medium free of antibiotics).
Materials:
Cat. No. Manufacture Reagent Name
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D5796 Sigma DMEM
04-121-lA Beit Haemek FCS
D8537 Sigma PBS
P4333 Sigma PenlStrep
T4049 Sigma 0.25% Trypsin-EDTA
Day 2: Transfection
Materials:
Cat. No. Manufacture Reagent Name
11668-027 Invitrogen LF2000 reagent
31985-047 GibcoBRL OptiMEM
MSTP028 .RNAi constructs:
siRNA target sequence Accession Pos.
MST028 AAGTGCTCACCGACAGTGAAG NM 031954 197
MST028 AAGATACTTATGAGCCTTTCT NM 031954 392
Experimental and Control Conditions:
1- Control siRNA+ pNLEnv-1
2- POSH siRNA + pNLenv-1
3- MSTP028 siRNA + pNLenv-1
1. Two hours before transfection, replace cell media to 2ml/well complete
DMEM without antibiotics.
2. siRNA dilution: for each transfection dilute 100 nm siRNA in 0.25 ml
OptiMEM per well.
3. LF 2000 dilution: for each well dilute 5 ~.l lipofectamine reagent in
0.25m1
OptiMEM.
4. Incubate diluted siRNAs and LF 2000 for 5 minutes at RT.
5. Mix the diluted siRNAs with diluted LF2000 and incubated for 25 minutes at
RT.
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6. Add the mixture to the cells, 0.5 ml/well (drop wise) and incubate for 24
hours at 37°C in CO~ incubator.
Transfections: for each well
(12.5 ~.1 (siRNA)l 0.25 ml OptiMEM) x 3
LF 2000 35 ~.1 / 1.75 ml
Day 3:
Exp. 1: second transfection (as Day 4 below).
Exp. 2: Exchange medium.
Day 4:
Exp. 1: VLP assay (see below).
Exp. 2: Second transfection
1. Two hours before transfection, replace cell media to 2ml/well complete
DMEM without antibiotics.
2. siRNA and DNA dilution: Prepare dilution of plasmid pNLenv-1 0.75 ~.g /
well in 0.25 ml OptiMEM (total of 3 wells). Divide plasmid dilution to
eppendorf tubes (0.25 ml each). To each tube add siRNA 40nM (2.5 ~.l).
3. LF 2000 dilution: for each well dilute 5~.1 lipofectamine reagent in 0.25m1
OptiMEM.
4. Incubate diluted siRNAs and LF 2000 for 5 minutes at RT.
f. Mix the diluted siRNAs with diluted LF2000 and incubated for 1 hour at RT.
6. Add the mixture to the cells, 0.5 ml/well (drop wise) and incubate for 24
hours at 37°C in COZ incubator.
34 Day 5:
Exp. 2: VLP assay.
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Solutions:
Lysis buffer
Tris-HCl pH 7.6 SOmM
MgCl2 l.SmM
NaCI 150mM
Glycerol 10%
NP-40 0.5%
DOC 0.5%
EDTA 1mM
EGTA 1mM
Add PI3C 1:200.
Steady state VLP assay
A. Cell extracts
1. Pellet floating cells by centrifugation (lmin, 14000rpm at 40C), save
supernatant (continue with supernatant immediately to step B), scrape cells
in ice-cold PBS, add to the corresponding floated cell pellet and centrifuge
for 5min 1800rpm at 40C.
2. wash cell pellet once with ice-cold PBS.
3. Resuspend cell pellet (from 6 well) in 100 ~l NP40-DOC lysis buffer and
incubate 10 minutes on ice.
4. Centrifuge at 14,OOOrpm for l5min. Transfer supernatant to a clean
eppendorf.
5. Prepare samples for SDS-PAGE by adding them sample buffer and boil for
l0min - take the same volume for each reaction (15 ~.l).
B. Purification of VLP from cell media
1. Filtrate the supernatant through a 0.45~.m filter.
2. Centrifuge supernatant at 14,OOOrpm at 40C for at least 2h.
3. Resuspend VLP pellet in 50 ~.l 1X sample buffer and boil for 10 min. Load
25 ~,l of each sample.
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C. Western Blot analysis
1. Run all samples from stages A and B on Tris-Gly SDS-PAGE 12.5%.
2. Transfer samples to nitrocellulose membrane (100V for 1.15h.).
3. Dye membrane with ponceau solution.
4. Block with 10% low fat milk in TBS-t for 1h.
5. Incubate with anti p24 rabbit 1:500 in TBS-t 2 hour (room temperature) -
overnight (40C).
6. Wash 3 times with TBS-t for 7min each wash.
7. Incubate with secondary antibody anti rabbit cy5 1:500 for 30min.
8. Wash five times for lOmin in TBS-t
9. View in Typhoon for fluorescence signal (650).
Example 16. POSH-depleted cells have lower levels of Herp and it is not
monoubiquitinated
POSH-depleted cells and their control counterparts were lysed and
immunoblotted with anti-here antibodies. Cells depleted of POSH (H153 RNAi
stables cell lines) cells have lower levels of Herp compared with control
cells (Hl 87
RNAi) (Figure 34A panel A). When cells were trasnfected with a plasmid
encoding
flagged-tagged ubiquitin, and immunoprecipitated with anti-flag antibodies to
immunoprecipitate ubiquitinated proteins, Herp was ubiquitinated only in H187
cells
and not in H153 cells (Figure 34A panel B). When the aforementioned cells were
transfected with Herp-encoding plasmid, exogenous here levels were also
reduced in
H153 cells compared to H187 cells (Figure 34B panel A) and the ubiquitination
of
exogenous here was reduced in the former cells, similar to endogenous Herp.
The
molecular weight of ubiquitinated Herp is as predicated to full-length Herp
and does
not seem as a high molecular weight smear, a characteristic of
polyubiquitinated
proteins. Thus POSH is responsible for the mono-ubiquitination of Herp, and in
the
absence of this modification here is subjected to degradation, which may be
mediated by the proteosome.
Materials and methods
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Plasmid generation
Full-length Herp was cloned from image clone MGC:45131 IMAGE:5575914
(GeneBank Accesion BC032673) into pCMV-SPORT6.
Antibody production
Herpl (amino a cids 1 t o 2 51) w as a mplified from a p lasmid ( 3Gd4) o
btained b y
yeast two hybrid screen for interactors of POSH. The amplified open reading
frame
was cloned into pGEX-6P, expressed in E. coli BL21 by induction with 1 mM IPTG
and purified on glutathione-agarose. The purified protein was cleaved with
PrecisionTM protease (Amersham Biosciences) and the GST moiety removed by
glutathione chromatography. The protein was injected into rabbits (Washington
Biotechnology) to produce anti-Herpl sera.
Transfections and antibody detection
Twenty-four hours prior to transfection POSH-RNAi clones (H153) or control-
RNAi clones (H187) cells were plated in 10 cm dishes in growth medium (DMEM
containing 10% fetal calf serum without antibiotics). Cells were transfected
with
lipofectamin 2000 (Invitrogen Corporation) and either Herp-expression plasmid
(2.5
p.g) or empty vector (2.5 pg) and a vector encoding Flag-tagged ubiquitin (1
p.g).
Twenty-four hours post-trasnfection cells were lysed in lysis buffer (50 mM
Tris-
HCI, pH7.6, 1.5 mM MgCl2, 150 mM NaCL, 10% glycerol, 1 mM EDTA, 1 mM
EGTA, 0.5% NP-40 and 0.5% sodium deoxycholate, containing protease inhibitors)
and subjected to immunoprecipitation with anti-Flag antibodies (Sigma, F7425)
to
precipitate ubiquitinated proteins. Immunoprecipitated material and total cell
lysates
were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes
which were immunoblotted with anti-Herp antibodies.
Generation of H187 and H153 cell lines
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To relieve the necessity for multiple transfections and to improve the
reproducibility
of hPOSH reduction, we have generated two cell lines, H187 and H1S3
constitutively expressing an integrated control and hPOSH siRNA
(respectively).
Construction of shRNA retroviral vectors- hPOSH scrambled oligonucleotide (5'-
S CACACACTGCCGTCAACTGTTCAAGAGACAGTTGACGGCAGTGTGTGTTT
TTT-3'; and S'-AATT~~,AAAAACACACACTGCCGTCAACTGTCTCTTGAACA
GTTGACGGCAGTGTGTGGGCC- 3') were annealed and cloned into the ApaI-
EcoRI digested pSilencer 1.0-U6 (Ambion, Inc.) to generate pSIL-scrambled.
Subsequently, the U6-promoter and RNAi sequences were digested with BamHI,
and blunted by end filling. The insert was cloned into the OIiI site in the
retroviral
vector, pMSCVhyg (BD Biosciences Clontech), generating pMSCVhyg-U6-
scrambled. The hPOSH oligonucleotide encoding RNAi against hPOSH
(S'-AACAGAGGCCTTGGAAACCTGGAAGCTTGCAGGTTTCCAAGGCCTCT
GTT-3'; and
1S S'-GATCAACAGAGGCCTTGGAAACCTGCAAGCTTCCAGGTTTCCAAGGC
CTCTGTT-3') were annealed and cloned into the BamHI-EcoRV site of ALIT-U6,
generating ALIT-U6 hPOSH-230. The ALIT-U6 is an shRNA vector containing the
human U6 promoter (amplified by PCR from human genomic DNA with the
primers, S'-GGCCCACTAGTCAAGGTCGGGCAGGAAGA-3' and
5'-GCCGAATTCAAAAAGGATCCGGCGATATCCGGTGTTTCGTCCTTTCCA-
3') cloned into pLITMUS38 (New England Biolabs, Inc.) digested with Spel-
EcoRI.
Subsequently, the U6 promoter-hPOSH shRNA (ALIT-U6 hPOSH-230 digested
with SnaBI and PvuI) was cloned into the Olil site of pMSCVhyg (BD Biosciences
Clontech) generating pMSCVhyg U6-hPOSH-230.
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Recombinant retrovirus production- HEK 293T cells were transfected with
retroviral RNAi plasmids (pMSCVhyg-U6-POSH-230 and pMSCVhyg-U6-
scrambled and with plasmids encoding VSV-G and Moloney Gag-pol. Two days
post-transfection, the retrovirus-containing medium was collected and
filtered.
Infection and selection- Polybrene (Hexadimethrine bromide) (Sigma) (8ygfml)
was added to the filtered and the treated medium was subsequently used to
infect
HeLa SS6 cells. Forty-eight hours post-infection clones were selected for RNAi
expression by the addition of hygromycin (300 ~,g/ml). Clones expressing the
scrambled and the hPOSH RNAi were termed HI87 and H153 (respectively).
I O Example 17. Inhibition of HBV production
HepG2.2.15 cells were plated on 9cm dishes and allowed to grow in 8% FCS
for 5 days up to 70% confluence. After 5 days, cells were washed twice with
PBS
and re-supplied with fresh DMEM without FCS. In this medium, cells were
treated
every 24 hours with the depicted solutions (3p,1 solution/lml medium) for
another 4
days (4 treatments total). After 4 days, medium was collected from each plate,
viruses were sedimented and analyzed.
As shown in Figure 35, lanes 7 and 8, compounds CAS number 14567-55-4
and CAS number 414908-38-0 inhibit HBV production at a concentration of 3p,M.
Detection of HBV proteins was performed essentially as described in Paran, N
et al
(2001) EMBO J 20(16):4443-4453.
INCORPORATION BY REFERENCE
All publications and patents mentioned herein are hereby incorporated by
reference in their entirety as if each individual publication or patent was
specifically
and individually indicated to be incorporated by reference. In case of
conflict, the
present application, including any definitions herein, will control.
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EQUIVALENTS
While specific embodiments of the subject applications have been discussed,
the above specification is illustrative and not restrictive. Many variations
of the
S applications will become apparent to those skilled in the art upon review of
this
specification and t he c laims b elow. T he full sc ope o f t he applications
should b a
determined by reference to the claims, along with their full scope of
equivalents, and
the specification, along with such variations.
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