Note: Descriptions are shown in the official language in which they were submitted.
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Methods and Rea~ents for Mod~ tinF Apoptosis
. Background of the Invention
This invention relates to apoptosis.
Apoptosis, which is also referred to as programmed cell death, is a
form of cell death characterized by membrane blebbing and nuclear DNA
fragmentation. Apoptotic cell death is morphologically distinct from necrotic
cell death and is important in the normal development and maintenance of
multicellular organisms. Dysregulation of apoptosis has been implicated in a
number of human diseases. An inapplo~liate suppression of apoptosis in a cell
may lead to the uncontrolled propagation of that cell. Such an event would
favor, for example, the development of cancer. In contrast, a failure to controlthe extent of apoptotic cell death may lead to degeneration of specific tissues
and cell-types. For example, an inappro~liately high level of apoptosis in
leukocytes may result in acquired immunodeficiency. Likewise, certain
neurodegenerative disorders may result from an inap~ro~liately high level of
apoptosis in neuronal cells and tissues.
Although apoptotic cell death is initially triggered by a specific death
signal received, for example, by ligation of the Fas cell surface molecule,
execution of the apoptotic pathway occurs only upon the activation of members
of the Ced-3/ICE (caspase) family of cysteine proteases. There are at least 10
known members of the caspase family whose activities lead to site-specific
cleavage and consequent activation/inactivation of various target molecules.
FLICE and related caspases may initiate apoptosis by activating a downstream
caspase cascade, including CPP32 (caspase-3).
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The decision to engage the apoptotic execution pathway in response
to specific death signals depends on the status of various cellular regulators of
apoptosis, including pS3 and the Bcl-2/Bax set point. The latter set point arises
through heterodimerization between the Bc1-2/Bcl-XL family of suppressors
and promoters, respectively, in which the ratio of the heterodimerizing partnersdetermines the outcome - cell death or cell survival - in response to various
death signals. Bad, a more distantly related family member, is a direct
regulator of the set point, by a mechanism that is governed by phosphorylation.
The phosphorylation may, in turn, be affected by Bcl-2-dependent recruitment
of Raf-l kinase.
Although it is now known that Bcl-2/Bcl-XL controls the apoptotic
execution pathway at a point that is either at or upstream of pro-enzyme
activation of the caspases, how this is achieved remains to be elucidated. Therethus remains a need to identify Bc1-2 binding proteins that are functionally
linked to apoptosis. There also remains a need to identify factors that interactwith Bc1-2 and modulate the apoptotic sign~llin~ pathway. Further, it would be
useful to identify factors which enable a signalling of the apoptotic pathway
from Bc1-2 and interacting factors to thc caspases involved therein.
Summary of the Invention
We have discovered that p28 Bap3 1 polypeptides, nucleic acids, and
antibodies may be used for the detection and treatment of conditions involving
apoptosis and for the identification of therapeutic molecules.
In a first aspect, the invention features a substantially pure p28
Bap3 1 polypeptide fragrnent that modulates apoptosis.
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In a second aspect, the invention features a substantially purified
nucleic acid molecule encoding a substantially pure p28 Bap31 polypeptide
fragment that modulates apoptosis.
ln various embodiments of the first two aspects of the invention, the
fragment includes a domain that is required for an association of p28 Bap31
with pro-FLICE, or a domain that is re4uired for an association of p28 Bap31
with a Bc1-2 protein (e.g., Bc1-2 or BCI-XL). In other preferred embodiments,
the fragment either increases apoptosis or inhibits apoptosis. In yet another
embodiment of the first and second aspccts of the invention, the fragment is
1() from a m~mm~l (e.g., a human or a mouse).
In an third aspect, the invention features a substantially pure
polypeptide that modulates apoptosis, the polypeptide having 50% or greater
amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. In
preferred embodiments, the polypcptide has 70% or greater ammo acid
sequence identity to the amino acid sequence of SEQ ID NO: 1, or has 80% or
greater amino acid sequence identity to the amino acid sequence of SEQ ID
NO: 1.
In a fourth aspect, the invention features a method for modulating
apoptosis in a ccll that includes administering to the cell a compound that alters
p28 Bap31 biological activity, the compound being a~rnini.stered at a dosage
which is sufficient to modulate the p28 Bap31 biological activity. In various
preferred embodiments of this aspect of the invention, the p28 Bap31 biological
activity may be cleavage of the p28 Bap31 polypeptide to produce a p20
product, formation of a complex of the p28 Bap31 polypeptide with pro-
FLICE, formation of a complex of the p28 Bap31 polypeptide with a Bc1-2
protein (e.g., Bc1-2 or BCI-XL), binding of the p28 Bap31 polypeptide by an
antibody that specifically binds to p28 Bap31, or expression of the p28 Bap31
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polypeptide in the cell. In various other embodiments, the cell is from a
m~n~m~l (e.g, a human or a rodent).
In another embodiment of the fourth aspect of the invention, the
modulating is inhibiting. In another embodiment, where the modul~ting is
inhibiting, the cell is in a m~mm~l with a degenerative disease (e.g., a
neurodegenerative disease, cirrhosis of the liver, a myelodysplastic syndrome,
an ischemic injury, an infection with HIV, or a bone degenerative disease). In
yet another embodiment, where the mocl~ ting is inhibiting, the compound
may be a p28 Bap3 1 antisense nucleic acid molecule, an antibody that
specifically binds to p28 Bap3 1 (e.g., a p28 Bap3 1 neutralizing antibody), a
p20 antisense nucleic acid molecule, an antibody that specifically binds to p20
(e.g., a p20 neutralizing antibody), a p20-inhibiting amount of a Bc1-2 protein,or a p20-inhibiting amount of a nucleic acid molecule encoding a Bc1-2
polypeptide, where the nucleic acid molecules are positioned for expression in
said cell.
In another embodiment of the fourth aspect of the invention, the
modulating is increasing. In another embodiment, where the modulating is
increasing, the cell is in a mammal with a neoplasia. In another embodiment,
where the modulating is incrcasing, the compound may be a p28 Bap3 l
polypeptide, a p20 product that is a cleavage product of p28 Bap3 1, or a nucleic
acid molecule encoding a p28 Bap3 I polypeptide, where the nucleic acid
molecule is positioned for expression in the cell.
In a fifth aspect, the invention features a method for detecting a
compound that modulates apoptosis that includes the steps of: (a) providing a
cell having: (i) a reporter gene operably linked to a DNA-binding-protein
recognition site; (ii) a first fusion gene capable of expressing a first fusion
protein, the first fusion protein including a polypeptide fragrnent ofp28 Bap3 1
. . .
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covalently bonded to a binding moiety, the binding moiety capable of
specifically binding to the DNA-binding-protein recognition site; and (iii) a
second fusion gene capable of expressing a second fusion protein, the second
fusion protein including a polypeptide fragment of a second protein covalently
bonded to a gene activating moiety; (b) exposing the cell to the compound; and
(c) measuring reporter gene expression in the cell, a change in the reporter gene
expression identifying a compound that modulates apoptosis. ~n one
embodiment of this aspect, the cell is a yeast cell.
In a sixth aspect, the invention features a method for detecting a
compound that modulates apoptosis that includcs the steps of: (a) providing a
cell having: (i) a reporter gene operably linked to a DNA-binding-protein
recognition site; (ii) a first fusion gene capable of expressing a first fusion
protein, the first fusion protein including a polypeptide fragment of a second
protein covalently bonded to a binding moiety, the binding moiety capable of
specifically binding to the DNA-binding-protein recognition site; and (iii) a
second fusion gene capable of expressing a second fusion protein, the second
fusion protein including a polypeptide fragment of p28 Bap3 1 covalently
bonded to a gene activating moiety; (b) exposing the cell to the compound; and
(c) measuring reporter gene expression in the cell, a change in the reporter gene
cxpression identifying a compound that modulates apoptosis. In one
embodiment of this aspect, the cell is a yeast cell.
In a seventh aspect, the invention features a method for identifying a
compound that modulates apoptosis that includes the steps of: (a) providing a
first polypeptide including a region of p28 Bap3 1, the region of p28 Bap3 1
including a first domain that interacts with a second protein; (b) allowing an
interaction of the first polypeptide with a second polypeptide that includes a
region of the second protein, the region of the second protein including a
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second domain that interacts with the p28 Bap31; (c) contacting the interaction
of the first polypeptide and the second polypeptide with a candidate compound;
and (d) measuring the interaction of the first polypeptide and the second
polypeptide, a change in the interaction of the first polypeptide and the secondpolypeptide in the presence of the candidate compound relative to an
interaction of the first polypeptide and the second polypeptide not contacted
with the candidate compound identifying the presence of a compound that
modulates apoptosis.
In various embodiments of the fifth, sixth, and seventh aspects of the
invention, the second protein is a Bc1-2 protein (e.g., Bc1-2 or Bcl-XI ).
In an eighth aspect, the invention features a method for identifying a
compound that modulates apoptosis that includes: (a) providing a cell
expressing p28 Bap31 polypeptidc; and (b) contacting the cell with a candidate
compound and monitoring the level of p28 Bap31 biological activity, a change
in the level of p28 Bap31 biological activity in response to the candidate
compound relative to a level of p28 Bap31 biological activity in a cell not
contacted with the candidate compound identifying a compound that modulates
apoptosis.
In various preferred embodiments of the eighth aspect of the
invention, the p28 biological activity may be cleavage of the p28 Bap31
polypeptide to produce a p20 product, formation of a complex of the p28
Bap31 polypeptide with pro-FLICE, formation of a complex of the p28 Bap31
polypeptide with a Bc1-2 protein (e.g., Bc1-2 or BCI XL), binding of the p28
Bap31 polypeptide by an antibody that specifically binds to p28 Bap31, or
expression of the p28 Bap31 polypeptide in the cell. In various other
embodiments, the cell is from a m~mm~l (e.g, a human or a rodent).
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In another embodiment of the fifth, sixth, seventh, and eighth aspects
of the invention, where the change is a decrease, the compound is useful for
inhibiting apoptosis. In another embodiment, where the change is a decrease,
the compound may be used to treat an animal with a degenerative disease (e.g.,
a neurodegenerative disease, cirrhosis of the liver, a myelodysplastic syndrome,an ischemic injury, an infection with HIV, or a bone degenerative disease). In
yet another embodiment, where the change is an increase, the compound is
useful for increasing apoptosis. In yet another embodiment, where the change
is an increase, the compound may be used to treat an animal with neoplasia
(e.g., a cancer, a hyperplastic disorder, or a benign tumor).
ln a ninth aspect, the invention features a method for diagnosing a
m~mm~l for the presence of a disease involving altered apoptosis or an
incrcased likelihood of developing the disease, where the method includes
measuring the level of p28 Bap31 biological activity in a sample from the
m~mm~l, a change in the level of p28 Bap31 biological activity in the sample
relative to a level of p28 Bap31 biological activity in a sample from an
unaffected mammal being an indication that the m~mm~l has the disease or
increased likelihood of developing the disease.
ln various preferred embodiments of the ninth aspect of the
invention, the p28 biological activity may be cleavage of the p28 Bap31
polypeptide to produce a p20 product, formation of a complex of the p28
Bap31 polypeptide with pro-FLlCE, formation of a complex of the p28 Bap31
~ polypeptide with a Bc1-2 protein (e.g., Bc1-2 or BCI-XL), binding of the p28
Bap31 polypeptide by an antibody that specifically binds to p28 Bap31, or
expression of the p28 Bap31 polypeptide in the cell. In another embodiment,
the m~mm~l is a human or a rodent.
. . .
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In another embodiment of the ninth aspect of the invention, where
the change is a reduction indicates that the m~rnm~l has the disease or
increased likelihood of developing the disease, wherein the disease is caused bydecreased apoptosis. In another embodiment, where the change is a reduction,
the disease may be neoplasia (e.g., a cancer, a hyperplastic disorder, or a benign
tumor).
In yet another embodiment of the ninth aspect of the invention,
where the change is an increase indicates that the m~mm~l has the disease or
increased likelihood of developing the disease, wherein the disease is caused byincreased apoptosis. ln another embodiment, where the change is an increase,
the disease may be a degenerative disease (e.g., a neurodegenerative disease,
cirrhosis of the liver, a myelodysplastic syndrome, an ischemic injury, an
infection with HIV, or a bone degenerative disease).
In a tenth aspect, the invention features a method for identifying a
l S nucleic acid molecule encoding a p28 Bap3 1 polypeptide that includes: (a)
providing a cell; (b) introducing by transforrnation into the cell a candidate
nucleic acid molecule, the nucleic acid molecule being positioned for
expression in the cell; and (c) determining whether the transformed cell
exhibits an increased level of apoptosis relative to a cell not transformed withthe candidate nucleic acid molecule, wherein the increased level of apoptosis inthe transformed cell identifies a nucleic acid molecule encoding a p28 Bap3 1
polypeptide.
In an eleventh aspect, the invention features a method for identifying a
nucleic acid molecule encoding a p28 Bap3 1 polypeptide that includes: (a)
providing a cell; (b) introducing by transforrnation into the cell a candidate
nucleic acid molecule, the nucleic acid molecule being positioned for
expression in the cell; and (c) det~,",;ll;"~ whether the transformed cell
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exhibits an increased level of p28 Bap3 1 biological activity relative to a cell not
transformed with the candidate nucleic acid molecule, wherein the increased
level of p28 Bap31 biological activity in the transformed cell identifies a
nucleic acid molecule encoding a p28 Bap3 1 polypeptide.
S In a twelfth aspect, the invention features a method for identifying a
nucleic acid molecule encoding a protease that cleaves p28 Bap3 1 into p20 that
includes: (a) providing a cell expressing a p28 Bap3 1 polypeptide; (b)
introducing by transformation into the cell a candidate nucleic acid molecule,
the nucleic acid molecule being positioned for expression in the cell; and (c)
determining whether the transformed cell exhibits an increased level of p20
expression relative to a cell not transformed with the candidate nucleic acid
molecule, wherein the increased level of p20 expression in the transformed cell
idcntifies a nucleic acid molecule encoding a protcase that cleaves p28 Bap3 1
into p20.
In a thirteenth aspect, the invention features a method for identifying a
nucleic acid molecule encoding a protease that cleaves p28 Bap3 1 into p20 that
includes: (a) providing a cell expressing a p28 Bap3 1 polypeptide; (b)
introducing by transformation into thc cell a candidate nucleic acid molccule,
the nucleic acid molecule being positioned for exprcssion in the cell; and (c)
determining whether the transformed cell exhibits an increascd levcl of
apoptosis relative to a cell not transformed with thc candidate nucleic acid
molecule, wherein the increased level of apoptosis in the transforrned cell
identifies a nucleic acid molecule encoding a protease that cleaves p28 Bap3 1
into p20.
In a preferred embodiment of the tenth, eleventh, twelfth, and thirteenth
aspects of the invention, the cell is from a m~m~l (e.g., a human or a rodent).
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In a fourteenth aspect, the invention features a kit for diagnosing a
m~mm~l for the presence of a disease involving altered apoptosis or an
increased likelihood of developing the disease, where the kit includes a
substantially pure antibody that specifically binds a p28 Bap3 1 polypeptide,
where the antibody modulates apoptosis. In one embodiment of this aspect of
the invention, the kit further includes a means for detecting the binding of theantibody to the p28 Bap3 1 polypeptide.
In a fifteenth aspcct, the invention features a substantially pure antibody
that specifically binds to the p28 Bap3 1 polypeptide, where the antibody
1 () modulates apoptosis. In prcferred embodiments, the antibody is a polyclonal
antibody, a monoclonal antibody, or a neutralizing antibody.
ln a sixteenth aspect, thc invention features a transgenic cell (e.g., an
embryonal cell) that has a knockout mutation of an endogenous p28 Bap3 1
gene. In one embodiment, the mutation includes an insertion of exogenous
DNA.
In a seventeenth aspect, the invention features a transgenic animal
generated from a transgenic cell (e.g, an embryonal cell) that has a knockout
mutation of an endogenous p28 Bap3 1 gene, where the endogenous p28 Bap3 1
gene is not expressed in the transgenic animal. In one embodiment of this
aspect, the gerrn-line cells and somatic cells of the transgenic animal do not
express the endogenous p28 Bap3 1 gene. ln another cmbodiment, the
transgenic animal includes germ-line cells and somatic cells expressing a
nucleic acid molecule encoding a truncated or a mutated p28 Bap3 1
polypeptide.
In an eighteenth aspect, the invention features a vector including a
knockout mutation in a DNA sequence encoding a p28 Bap3 1 gene, where the
gene includes: (a) a first region corresponding to a S' sequence of the p28
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Bap3 1 gene, wherein the initiator methionine codon of the p28 Bap3 1 gene is
absent from the sequence; (b) a second region including DNA including a drug-
resistance cassette, the DNA capable of conferring resistance to the drug in a
cell when the DNA is present in the cell; and (c) a third region corresponding to
a 3' sequence of the p28 Bap3 1 gene.
In a nineteenth aspect, the invention features a vector including a
knockout mutation in a DNA sequence encoding a p28 Bap3 1 gene, where the
gene includes: (a) a first region corresponding to a 5' sequence of the p28
Bap3 l gene, wherein the initiator methionine codon of the p28 Bap3 1 gene is
absent from the sequence; (b) a second region including DNA including a drug-
resistance cassette, the DNA capable of conferring resistance to the drug in a
cell when the DNA is present in the cell; and (c) a third region corresponding to
a 3' sequence of the p28 Bap3 1 gene.
ln a twentieth aspect, the invention features a method of producing a
transgenic m~mm~l lacking expression of an endogenous p28 Bap31
polypeptide that includes: (a) introducing by homologous recombination a
nucleic acid molecule encoding a knockout mutation of a p28 Bap3 I gene into
a locus occupied by an endogenous p28 Bap31 gene, the locus present in the
genome of an embryonal cell of the m~mm~l; and (b) growing the embryonal
cell to produce the transgenic mz~mm~l.
In a twenty-first aspect, the invention features a therapeutic composition
that includes, as an active ingredient, a p20 cleavage product of p28 Bap3 l, the
active ingredient being formulated in a physiologically acceptable carrier,
where the composition modulates apoptosis.
In a twenty-second aspect, the invention features a therapeutic
composition that includes, as an active ingredient, a p28 Bap3 1 polypeptide, the
.
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active ingredient being formulated in a physiologically acceptable carrier,
where the composition modulates apoptosis.
In a twenty-third aspect, the invention features a therapeutic composition
that includes, as an active ingredient, an antibody that specifically binds p28
Bap3 1, the active ingredient being formulated in a physiologically acceptable
carrier, where the composition modulates apoptosis.
In a twenty-fourth aspect, the invention features a therapeutic
composition that includes, as an active ingredient, an antisense nucleic acid
molecule that corresponds to p28 Bap3 1, the active ingredient being formulated
in a physiologically acceptable carrier, where the composition modulates
apoptosis.
In a twenty-fifth aspect, the invention features the use of a p28 Bap3 1
polypeptide for the manufacture of a medicament for the modulation of
apoptosis.
In a twenty-sixth aspect, the invention features the use of an antibody
that specifically binds to p28 Bap3 1 for the manufacture of a medicament for
the modulation of apoptosis.
In a t~venty-seventh aspect, the invention features the use of an antisense
nucleic acid molecule corresponding to p28 Bap3 1 for the manufacture of a
medicament for the modulation of apoptosis.
In a twenty-eighth aspect, the invention features the use of a p20
cleavage product of p28 Bap3 1 for the manufacture of a medicament for the
modulation of apoptosis.
In a twenty-ninth aspect, the invention features the use of an antibody
that specifically binds to a p20 cleavage product of p28 Bap3 1 for the
manufacture of a medicament for the modulation of apoptosis.
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In a thirtieth aspect, the invention features the use of an antisense
nucleic acid molecule corresponding to a p20 cleavage product of p28 Bap31
for the manufacture of a medicament for the modulation of apoptosis.
As summarized above, a p28 Bap31 nucleic acid molecule, polypeptide,
or antibody may be used to modulate apoptosis. Furthermore, a p28 Bap31
nucleic acid molecule, polypeptide, or antibody may be used in the discovery
and/or manufacture of a medicament for the modulation of apoptosis.
By "p28 Bap31," "p28 Bap31 protein," or "p28 Bap31 polypeptide" is
meant a protein, or a polypeptidc fragment thereof, that can interact with a Bcl-
2 protein (including, without limitation, Bc1-2 and BCI XL) or a pro-FLlCE
protein, that can participate in apoptosis, or that can be cleaved to produce a
p20 product (i.e., an approximately 20 kDa product), that can induce apoptosis
when expressed in a cell. Preferably, a p28 Bap31 protein has an amino acid
sequence that is at least 50% identical to thc amino acid sequence of human
Bap31 (GenBank accession number X81817) or to the amino acid sequence of
human CDM (GenBank accession number Z31696), more preferably at least
60% identical, more prcferably at least 70% idcntical, still more preferably at
Icast 80% identical, and most preferably at least 90% identical to at least one of
these sequences. lt will be understood that a fragment thereof has a p28 Bap31
biological activity that is observed with full length p28 Bap31 protein;
preferably, the p28 Bap31 biological activity is at least 50% as observed with
full length p28 Bap31.
Polypeptide fragments of p28 Bap31 that are a part of the invention
include those fragments that bind Bc1-2 polypeptides, those fragments that are
capable of selecting an antibody which specifically binds p28 Bap31, and those
fragments that can modulate apoptosis in a cell.
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By "p2B Bap31 gene" is meant a gene encoding p28 Bap31. Preferably,
sequences include sequences which encode polypeptide fragments of the p28
Bap31, as defined above. In preferred embodiments, included as a part of the
gene are the nucleic acid sequences fl~nking the 5' and 3' regions of the codingregion of the p28 Bap31 gene sequence. M~m~lian p28 Bap31 genes include
nucleotide sequences isolated from any mamm~ n source. Preferably, the
m~mm~l is a human.
By "purified antibody" is meant antibody which is at ieast 60%, by
weight, free from proteins and naturally occurring organic molecules with
which it is naturally associated. Preferably, the preparation is at least 75%,
more preferably 90%, and most preferably at least 99~/O, by weight, antibody,
e.g., a p28 Bap31 specific antibody. A purified antibody may be obtained, for
example, by affinity chromatography using recombinantly-produced
polypeptide or conserved motif peptides and standard techniques.
By "specifically binds" is meant an antibody that recognizes and binds a
polypeptide but that does not substantially recognize and bind other molecules
in a sample, e.g., a biological sample, that naturally includes protein. One
preferred antibody specifically binds to the p28 Bap31 polypeptide.
By "p28 Bap31 biological activity," as used herein in reference to p28
Bap31, is meant any one of the biological activitics of a p28 Bap31 protein (or
a polypeptide fragment thereof). P28 Bap31 biological activities include,
without limitation, an ability of p28 Bap31 to be cleaved to produce a p20
product, an ability to form a complex with a Bc1-2 protein (e.g., Bc1-2 or Bcl-
XL), an ability to form a complex with pro-FLICE, an ability to be bound by an
antibody that specifically binds p28 Bap31, an ability to be expressed, and an
ability to participate in apoptosis.
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By a "compound that alters p28 Bap31 biological activity," is meant a
compound that increases or reduces any biological activity of p28 Bap31 (or a
fragment thereof), as defined above. Such a compound may be, without
limitation, a compound that increases the transcription of p28 Bap31, a
compound that increases p28 Bap31 protein expression levels, a p28 Bap31
antisense nucleic acid molecule, an antibody that specifically binds to p28
Bap31, a p20 antisense nucleic acid molecule, an antibody that specifically
binds to p20, a p20-inhibiting amount of a Bc1-2 protein, a p20-inhibiting
amount of a Bcl-2-expressing nucleic acid molecule, a p28 Bap31 protein (or
fragment thereof), a p20 product (or fragment thereof), and a nucleic acid
molecule encoding a p28 Bap31 polypeptide (or fragment thereof).
By a "neutralizing antibody," as used herein in reference to an antibody
that specifically binds p28 Bap31, is meant an antibody that interferes with anyof the biological activities of the p28 Bap31 protein. For example, a p28 Bap31
neutralizing antibody may interfere with the ability of p28 Bap31 to participatein apoptosis, for example, by inhibiting cleavage of p28 Bap31 to produce the
p20 product. A p28 Bap31 neutralizing antibody may also interfered with the
ability of p28 Bap31 to form a complex with a Bc1-2 protein (e.g., Bc1-2 or Bcl-X,) or to form a complex with pro-FLICE. Such p28 Bap31 neutralizing
antibodies may reduce the ability of p28 Bap31 and polypeptide fragments
thereof to participate in apoptosis and to associate with pro-FLICE and/or a
Bc1-2 protein by, preferably 50~/u, more preferably by 70%, and most preferably
by 90% or more. Standard assays of apoptosis and protein:protein interactions,
including those described herein, may be used to assess potentially neutralizingp28 Bap31 antibodies.
By "modulating apoptosis" or "altering apoptosis" is meant increasing
or decreasing the number of cells that would otherwise undergo apoptosis in a
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given cell population. Preferably, the cell population is selected from a group
including T cells, neuronal cells, fibroblasts, myocardial cells, or any other cell
line known to undergo apoptosis in a laboratory setting, for example, human
epithelial KB cells infected with adenovirus type 5 lacking expression of ElB
1 9K (pm 1716/2072). It will be appreciated that the degree of modulation
provided by p28 Bap3 1 or p28 Bap3 1 modulating compounds in a given assay
will vary, but that one skilled in the art can determine the statistically
significant change in the level of apoptosis which identifies a p28 Bap3 1 or a
compound which modulates this proteins.
By "increasing apoptosis" is meant any increase in the number of cells
that undergo apoptosis relative to an untreated control. Preferably, the increase
is at least 25%, more preferably the increase is at least 50%, and most
preferably the increase is at least one-fold.
By "inhibiting apoptosis" is meant any decrease in the number of cells
that undergo apoptosis relative to an untreated control. Preferably, the decrease
is at least 25%, more preferably the decrease is at least 50%, and most
preferably the decrease is at least one-fold.
By "protein" or "polypeptide" is meant any chain of more than two
amino acids, regardless of post-translational modification such as glycosylationor phosphorylation.
By "substantially identical" is meant a polypeptide or nucleic acid
exhibiting at least 50%, preferably at least 85%, more preferably at least 90%,
and most preferably at least 95% identity to a reference amino acid or nucleic
acid sequence. For polypeptides, the length of comparison sequences will
generally be at least 16 amino acids, preferably at least 20 amino acids, more
preferably at least 25 amino acids, and most preferably at least 35 amino acids.For nucleic acids, the length of comparison sequences will generally be at least
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50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75
nucleotides, and most preferably at least 1 10 nucleotides.
Sequence identity is typically measured using sequence analysis
software with the default parameters specified therein (e.g., Se~uence Analysis
Software Package of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This
software program matches similar sequences by assigning degrees of homology
to various substitutions, deletions, and other modifications. Conservative
substitutions typically include substitutions within the following groups:
glycine, alaninc, valine, isoleucine, leucine; aspartic acid, glutamic acid,
asparagine, glutamine; serine threonine; Iysine, arginine; and phenylalanine,
tyrosine.
By "substantially pure polypeptide" is meant a polypeptide that has been
separated from the components that naturally accompany it. Typically, the
polypeptide is substantially pure when it is at least 60%, by weight, free from
the proteins and naturally-occurring organic molecules with which it is
naturally associated. Preferably, the polypeptide is a p2~ Bap3 1 polypeptide
that is at least 75%, more preferably at least 90%, and most preferably at least99~/O, by weight, pure. A substantially pure p28 Bap3 1 polypeptide may be
obtained, for example, by extraction from a natural source (e.g., a fibroblast,
neuronal cell, or Iymphocyte) by expression of a recombinant nucleic acid
encoding a p28 Bap3 l polypeptide, or by chemically synthcsizing the protein.
Purity can be measured by any ap~lopliate method, e.g., by column
chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A polypeptide is substantially free of naturally associated components
when it is separated from those cont~min~nt~ which accompany it in its natural
state. Thus, a polypeptide which is chemically synthesized or produced in a
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-18-
cellular system different from the cell from which it naturally originates will be
substantially free from its naturally associated components. Accordingly,
substantially pure polypeptides include those which naturally occur in
eukaryotic organisms but are synthesized in E. coli or other prokaryotes, or aresynthesized in viruses.
By "substantially pure nucleic acid" is meant nucleic acid that is free of
the genes which, in the naturally-occurring genome of the organism from which
the nucleic acid of the invention is derived, flank the nucleic acid. The term
therefore includes, for example, a recombinant nucleic acid which is
incorporated into a vector; into an autonomously replicating plasmid or virus;
or into the genomic nucleic acid of a prokar,vote or a eukaryote cell; or which
exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment
produccd by PCR or restriction endonuclease digestion) independent of other
sequences. It also includes a recombinant nucleic acid which is part of a hybridgene encoding additional polypeptide sequence.
By "embryonal cell" is meant a cell that is capable of being a progenitor
to all the somatic and gerrn-line cells of an organism. Embryonal cells are alsoreferred to as embryonic stem cells, or ES cells. Preferably, the embryonal
cells of the invention are mammalian embryonal cells.
By "endogenous," as used herein in reference to a gene or a polypeptide,
is meant a gene or polypeptide that is normally present in an organism.
By "germ-line cell" is meant a cell, progenitor, or progeny thereof,
which is a product of a meiotic cell division.
By "transformed cell" is meant a cell into which (or into an ancestor of
which) has been introduced, by means of recombinant nucleic acid techniques,
a nucleic acid molecule encoding (as used herein) a p28 Bap3 1 polypeptide.
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By "transformation" is meant any method for introducing foreign
molecules into a cell. Lipofection, calcium phosphate precipitation, retroviral
delivery, electroporation, and biolistic transformation are just a few of the
teachings which may be used. For example, biolistic transformation is a
method for introducing foreign molecules into a cell using velocity driven
micro projectiles such as tungsten or gold particles. Such velocity-driven
methods originate from pressure bursts which include, but are not limited to,
helium-driven, air-driven, and gunpowder-driven techniques. Biolistic
transformation may be applied to the transformation or transfection of a widc
variety of cell typcs and intact tissues including, without limitation,
intraccllular organelles (e.g., mitochondria ), bacteria, yeast, animal tissue, and
cultured cells.
By "transgene" is meant any piece of nucleic acid which is inscrted by
artifice into a cell, and becomes part of the genome of the organism that
develops from that cell. Such a transgene may include a gene which is partly or
entirely heterologous (i.e., foreign) to the transgenic organism, or may
represent a gene homologous to an endogenous gene of the organism.
By "transgenic" is mcant any cell which includes a nucleic acid
sequence which is inserted by artifice into a cell and becomes part of thc
genome of thc transgenic organism which devclops from that cell. Such a
transgene may be partly or entirely heterologous to the transgenic animal.
Although transgenic mice represent a preferred embodiment of the invention,
other transgenic m~mm~l~ including, without limitation, transgenic rodents (for
example, hamsters, guinea pigs, rabbits, and rats), and transgenic pigs, cattle,sheep, and goats may be constructed by standard techniques and are included in
the invention. Preferably, the transgene is inserted by artifice into the nuclear
genome.
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By "knockout mutation" is meant an alteration in the nucleic acid
sequence that reduces the biological activity of the polypeptide normally
encoded therefrom by at least 80% relative to the unmutated gene. The
mutation may, without limitation, be an insertion, deletion, frameshift mutation,
or a mis-sense mutation. Preferably, the mutation is an insertion or deletion
(e.g., the p28 Bap3 1-NEO knockout gene described herein), or is a frameshift
mutation that creates a stop codon.
By "positioned for expression" is meant that the nucleic acid molecule is
operably linked to a nucleic acid sequence which directs transcription and
translation of the sequence (i.e., facilitates the production of, e.g., a p28 Bap3 1
polypeptide, recombinant polypeptide, or an RNA molecule), such that the
nucleic acid molecule that is positioned for expression in a cell is expressed in
the cell.
By "promoter" is meant a minim~l sequence sufficient to direct
transcription of a desired nucleic acid molecule. Also included in the inventionare those promoter elements which are sufficient to render promoter-dependent
nucleic acid molecule expression controllable for cell type-specific, tissue-
specific or inducible by external signals or agents; such elements may be
located in the 5' or 3' regions of the native gene, and may be placed, by standard
recombinant DNA manipulations, adjacent to or within the desired nucleic acid
molecule.
By "operably linked" is meant that a nucleic acid molecule and one or
more regulatory sequences are connected in such a way as to permit expression
of the nucleic acid molecule when the ~ O~UIiate molecules (e.g.,
transcriptional activator proteins) are bound to the regulatory sequences.
By "reporter gene" is meant any gene that encodes a product whose
expression is detectable. A reporter gene product may have one of the
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following attributes, without restriction: fluorescence (e.g., green fluorescentprotein), enzymatic activity (e.g., luciferase or chloramphenicol acetyl
transferase), toxicity (e.g., ricin), or an ability to be specifically bound by a
second molecule (e.g., biotin or a detectably labelled antibody).
By "conserved region" is meant any stretch of six or more contiguous
amino acids exhibiting at least 30%, preferably 50%, and most preferably 70%
amino acid sequence identity between two or more of the p28 Bap3 1 family
members, (e.g., between human p28 Bap31 and murine p28 Bap31).
By "detectably-labelled" is meant any means for marking and
identifying the presence of a molecule, e.g., an oligonucleotide probe or primer,
a gene or fragment thereof, or a cDNA or RNA moleculc. Methods for
detectably-labeling a moleculc arc well known in the art and include, without
limitation, radioactive labeling (e.g., with an isotope such as 32p or 35S) and
nonradioactive labeling (e.g., chemiluminescent labeling, e.g., fluorescein
labeling).
By "antisense," as used herein in reference to nucleic acids, is meant a
nucleic acid sequence that is complementary to the coding strand of a gene,
preferably, a p28 Bap3 I gene. Preferably the antisense nucleic acid molecule
decreases the amount of transcription from the gene; more preferably, the
decrease is at least 10%, and most prcferably, the decrease is at least 50% when~lmini~tered at the maximally effective dose.
By "pharmaceutically acceptable carrier" is meant a carrier that is
physiologically acceptable to the treated mammal while retaining the
therapeutic properties of the compound with which it is ~flministered. One
exemplary pharmaceutically acceptable carrier is physiological saline solution.
Other physiologically acceptable carriers and their formulations are known to
one skilled in the art and described, for example, in Remin~ton's
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Pharmaceutical Sciences, (1 8th edition), ed. A. Gennaro, 1990, Mack Publishing
Company, Easton, PA.
Other features and advantages of the invention will be apparent from the
following description of the preferred embodiments.
Brief Description of the Drawin~s
Fig. lA is a graph showing the viability (as judged by exclusion of
trypan blue at indicated times post-infection) of KB cells expressing neomycin-
resistance, either alone (Neo~ or together with Bc1-2, infected with adenovirus
dlS20ElB- (expressing 12S ElA and no ElB products).
Fig. 1 B is a graph showing the viability (as }udged by exclusion of
trypan blue at indicated timcs post-infection) of KB cells expressing neomycin-
resistance, either alone (Neo) or together with Bc1-2, infected with adenovirus
pml760/2072 (expressing 12S and 13S ElA but not ElB l9K).
Fig. 1 C is a Far Western blotting analysis showing the appearance of a
Bc1-2 interacting polypeptide (probed with 32P-Bc1-2~c21 /his6/HMK) in total
cellular protein prepared from KB cells expressing neomycin-resistance, either
alone (Neo; lower panel) or together with Bc1-2 (upper panel)~ following
infection at indicated times with either adenovirus dl520ElB- (expressing 12S
El A and no ElB products; lanes I -4) or adenovirus pm 1760/2072 (expressing
12S and 13S ElA but not ElB 19K; lanes 5-8). The probe-reactive bands on
the blots were visualized by phosphorimaging. The radioactive band associated
with a polypeptide of Mr 20 kDa is labelled p20, whereas that which co-
migrates with Bax is labelled p21 Bax.
Fig. 2A shows a preparative SDS PAGE analysis (and a quantifying
graph thereof) of differentially solubilized protein from KB cells 60 hours post-
infection with adenovirus pm 1760/2072. Aliquots of fractions eluted from the
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gel were assayed by 32P-Bc1-2~c21/his6/HMK Far Western blotting analysis,
and the radioactive bands corresponding to p20 and p2 1 Bax were detected and
quantified by Phosphorimager. The levels relative to the maximal signal
detected (set at 100) were plotted as a bar graph (upper panel). Equal aliquots
from the same fractions were also subjected to analytical 12% SDS PAGE, and
the gels stained with Coomassie brilliant blue (lower panel). The positions of
molecular mass marker proteins are indicated. The black bars on the graph
indicate p20. The white stippled bars on the graph indicate p2 I Bax.
Fig. 2B is a reverse phase HPLC analysis (A280 profile; upper panel) and
corresponding graph (lower panel) showing proteins eluted between 190 and
220 ml from thc preparativc SDS PAGE of Fig. 2A. Equal aliquots from all
fractions were assayed for 32P-Bc1-2~c21/his6/HMK interacting protein by Far
Western blotting analysis, for which only p20 was detected. Amounts relative
to the maximal signal detected (set at 100) were plotted as a bar graph (lower
panel). Fractions 52, 53, 54 (peak activity), and 55 were individually subjectedto NH~-terminal peptide sequence analysis.
Fig. 2C shows the polypeptide sequence of p28 Bap31/CDM (SEQ ID
NO: l ). Peptide sequencing of p20 revealed a perfect match with amino acids
2-10 of human Bap3 1 (underlined) (GenBank accession number X8 1817). This
was the only detectable sequence in fraction 54, and was detectable together
with other sequences in fraction 53, but was not detected in fractions 52 and 55.
Predicted transmembrane (TM) segments are boxed and contain charged amino
acids in TM1 and TM3 (asterisks). The predicted caspase recognition sites,
AAVDG (SEQ ID NO: 2), are highlighted and cleavage denoted by arrows
following asp at positions 164 and 238. A potential leucine zipper located
between the caspase recognition sites is denoted by bold letters, as is the
KKXX (SEQ ID NO: 3) ER retention signal at the COOH-terminus.
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Fig. 2D shows an amino acid alignment of the region between the two
AAVDG sites in of p28 Bap3 1 with the death effector domains in indicated
proteins. The sequences, given in the single letter amino acid code, were
obtained from GenBank, and their relative positions in the molecule shown in
parenthesis. Sequences were aligned using the PILEUP program of the GCG
software package, and optimized by spacing (shown as dashes).
Fig. 3A is an SDS-PAGE analysis showing the insertion of p28 Bap3 1
into endoplasmic reticulum (ER) microsomes. The positions of p28 Bap3 1,
pre-~-lactamase (pre-~-L), and processed ~-lactamase (~-L) are indicated, as is
the gel front. "c" indicates the marker translation product.
Fig. 3B is a schematic representation showing thc deduced topology and
domain structure of Flag-tagged p28 Bap3 1 in the ER mem~rane. The
orientation of the three transmembrane segments of Bap3 1 predicts a
N(lumen)-C(cytosol) topology in the ER membrane. The region of the 13 kDa
cytosolic domain conLaini~g putative death effector homology (D) and leucine
zipper (L) domains, flanked on either side by caspase-8 recognition sites
(asterisks), is boxed. Amino acid positions used for generating deletion
mutants are indicated. The Flag epitope was inserted immediately upstream of
the C-terminal KKEE ER retrieval signal.
Fig. 4A shows duplicate SDS-PAGE blots (upper panel) of resolved p28
Bap3 l-GST filsion proteins, one stained with Ponceau S (left blot), and the
other subjected to Far Western blotting analysis using
32P-Bc1-2Ac22/his6/HMK as a probe. Lanes are: GST (lane 1 ); GST fused to
p28 Bap3 1 amino acids 165-246 (lane 2); GST fused to p28 Bap3 1 amino acids
122-164 (lane 3); GST fused to p28 Bap3 1 amino acids 1-164 (lane 4); and
GST fused to p28 Bap3 1 amino acids 1-246 (lane 5) expressed in bacteria,
purified, and transferred to nitrocellulose in duplicate following SDS PAGE.
.. .. . . .. . . . ..
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Constructs and results are summarized in the lower panel of the figure as, from
top to bottom: GST-p28 (165-246); GST-p28 (122-164); GST-p28 (1-164);
and GST-p28 (1-246).
Fig. 4B shows a series of Western blotting analyses from cell Iysates and
Flag-imml-noprecipitates from 293T cells transfected with Myc-tagged Bcl-X,,
HA-tagged pro-FLICE, HA-tagged Bc1-2, and/or Flag-tagged p28 Bap31, as
indicated (pluses and minuses) probed with anti-Myc or anti-HA antibodies.
lmmunoreactive bands are visualized by electro-chemiluminescence. "Ig HC"
indicates immunoglobulin heavy chain.
Fig. 4C shows a series of Western blotting analyses from cell Iysates and
Flag-immunoprecipitates from transfected 293T cells. The analyses were
similar to those described in the above brief description of Fig. 4B, except that
Bax was included in the 293T cell co-transfections, as indicated.
Fig. SA are SDS-PAGE blots showing resolved products produced by
incubating the 35S-labelled transcription-translation product of p28 Bap31
cDNA with increasing concentrations of CPP32 or ICE. Units of enzyme
added per 25 ~I reaction mixture were: none (lane 1), 0.0056 (lane 2), 0.98
(lane 3), 1.95 (lane 4), 3.9 (lane 5), 7.8 (lane 6), 15.6 (lane 7), 31.2 (lane 8),
62.5 (lane 9), and 125 (lane 10). The positions of polypeptide molecular mass
markers are shown. Arrows designated "a" and "b" denote cleavagc products
whose sizes are consistent with cleavage of p28 Bap31 at the sites indicated by
"a" and "b" in the schematic at the bottom of the figure.
Fig. SB is a graph showing the analysis as described in the brief
description of Fig. 5A, where p28 Bap31 was incubated with purified ICE
(caspase- 1) or FLICE (caspase-8), and the resulting p20 cleavage product was
quantitated using a Phosphorimager. One unit of caspase enzyme activity is
equivalent to 1 pmol AMC liberated from fluorogenic tetrapeptide-AMC per
.. , .. . . ~
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-26-
min at 25~C at saturating substrate concentrations (according to the methods
described in Nicholson et a/., Nature 376: 37-43, 1995).
Fig. 6A shows two Western blotting analyses of cell extracts that were
obtained from KB cells that had either been infected for 60 hours with
adenovirus pm 1716/2072 lacking expression of El B 19K or had been mock-
infected (+ or - apoptosis, respectively), probed with affinity-purified chickenantibody against p28 Bap31 amino acids 165-246 (o~ p28-C; left blot) or p28
Bap31 amino acids 122-164 (o~ p28-M; right blot). Bands corresponding to p28
Bap31 are indicated. Arrows labelled "a" and "b" denote products whose sizes
are consistent with cleavage of p28 Bap31 at the sites designated "a" and "b" inthe schematic shown in the lower panel below the blots.
Fig. 6B shows a series of Western blotting analyses of cellular extracts
prepared from KB cells expressing neomycin-resistance either alonc (minus
Bc1-2, lanes 6- 10) or together with Bc1-2 (plus Bc1-2, lanes 1 -5) infected with
adenovirus pm 1716/2072 lacking expression of E1 B 19K, and probed with
antibody against p28-M (upper two blots) or against the 17 kDa subunit of
CPP32 (lower t~,vo blots; Boulakia et al., Oncogene 12: 529-535, 1996). The
positions of p28 Bap31 and the cleavage products "a" and "b" are indicated in
thc upper two blots. The arrow in the upper left blot denotes a cross-reacting
product whose appearance is variable (e.g., it did not appear in Fig. 6A). The
positions of full-length pro-CPP32 and the processed 17 kDa subunit (pl7) and
putativc 29 kDa processing inte~nediate (asterisk) are indicated in the lower
two blots.
Fig. 7A is a graph showing the relative luciferase expression in CHO
LR73 cells expressing neomycin-resistance, either alone (- Bc1-2) or together
with Bc1-2 (+ Bc1-2), that were co-transfected with a luciferase reporter plasmid
and Rc/RSV expressing either full-length p28 Bap31 or p28 Bap31 amino acids
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-27-
1-164 (i.e., p20). After 2 days, cells were recovered, analyzed for luciferase
activity, and the enzyme activity expressed relative to the values obtained in the
presence of p28 Bap31 (arbitrarily set at 100). The results shown are the
average of two separate experiments.
Fig. 7B are a set of photographs showing CHO cells transfected with the
p28 Bap3 l and p20 expression plasmids together with pHook (Invitrogen). 24
hours later, the transfected cells were recovered with Capture-Tec beads,
cultured on coverslips, stained with 4',6'-diamidino-2-phenyl indole (DAPI),
and visualized under a microscope.
Fig. 7C is a schematic interpretation of the results of Figs. 7A and 7B
showing p28 Bap31 as part of a hypothetical complex with an interacting
target, T.
Figs. 8A, 8B, and 8C arc schematic diagrams showing the construction
of the BP3 l TV plasmid, which may be used to generate a p28 Bap31 knock
out mouse.
Fig. 9 is a schematic representation of a model for p28 Bap31 in the Bcl-
2/caspase-apoptotic pathway.
Detailed Description
The large pro-rcgions of initiator caspases contain a death effector
domain that physically links these pro-enzymes to an apoptotic signalling
complex. In the case of Fas (also known as CD95 or Apo-1) and the tumor
necrosis receptor 1 (TNFR1) complexes, recruitment of pro-FLICE (FLICE has
also been referred to as MACH or caspase-8) involves the adaptor molecule,
FADD. This recruitment occurs via interactions bet~,veen the death effector
domains within the two molecules (Boldin et al., Cell 85: 803-815, 1996;
Muzio et al., Cell 85: 817-827, 1996). Bc1-2 family memberproteins are
~ ,. . . ....... . .. .... .......
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-28-
located in the endoplasmic reticulum (ER) / nuclear envelope and
mitochondrial outer membrane (Krajewski et a/., Cancer Res. 53: 4701-4714,
1993; Nguyen et al., J. Biol. Chem. 268: 25265-25268, 1993; Gonzalez-Garcia
et a/., Development 120: 3033-3042, 1994) and, in the latter location, appear toprevent activation of downstream effector caspases such as procaspase-3 in
response to diverse death signals.
We have now identified a Bcl-2/Bcl-XL-interacting, polytopic integral
membrane protein of the endoplasmic reticulum, p28 Bap31, that bears a
canonical COOH-terminal ER retention motif in its cytosolic domain and that
is part of a complex that contains Bc1-2 proteins and procaspase-8 (pro-FLICE).
In the absence of Bc]-2, p28 Bap31 itsclf becomes a target of a ICE/FLICE-
related caspase upon induction of apoptosis.
Appearance of a Bc1-2 Interacting Polypeptide Following lnduction of
Apoptosis
To detect potential Bcl-2-interacting polypeptides by Far Western
blotting analysis, a 32P-labelled probe was constructed by expressing a modifiedversion of the cytosolic domain of Bc1-2 in E. coli. To generate the modified
Bc1-2 cytosolic domain, the last 21 amino acids of Bc1-2 werc deleted and
substituted with hexahistidine (his6) plus a heart muscle kinase (HMK)
recognition peptide to facilitate purification and 32P-labeling, respcctively.
Isolation conditions were developed in which the recombinant protein was
purified without the use of harsh denaturants, yielding a soluble product that
included a mixture of monomers and dimers at pH 7.4, as judged by FPLC
molecular sieve chromatography. The 32P-labelled probe (32P-Bcl-
2~c21/his6/HMK) readily detected either recombinant Bc1-2 or Bax as the only
radioactive products on a Far Western blot of total bacterial lysate (not shown).
., .
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-29-
When used as a probe to analyse potential Bcl-2-interacting polypeptides in
cells induced to undergo apoptosis in response to various stimuli (including
- adenovirus ElA expression or treatment with puromycin), a product of
approximately 20 kDa in size (p20) as judged by SDS PAGE was consistently
observed.
Figs. 1 A, 1 B, and I C show the appearance of a Bcl-2-interacting
polypeptide during E1 A-induced apoptosis. Samples of neo- and Bc1-2-
expressing human KB cells were assessed for viability by exclusion of trypan
blue (Figs. I A and IB) or prepared for Far Western blotting analysis using 32p_Bc1-2Ac21/his6/HMK as a probe (Fig. lC) at various times following infection.
The neo- and Bcl-2-expressing human KB cells were infected with cither
adenovirus dl520ElB-, which cxpresses 12S ElA, but lacks expression of all
E 1 B products (encodes the 243R E 1 A protein; Fig. 1 A; lanes I -4 of Fig. 1 C),
or adenoviruspml760/2072, which expresses 12S and 13S E~IA, but lacks
expression of the dominant suppressor of apoptotic cell death, ElB 19 kDa
protein (19K) (encodes both the 243R and 289R EIA proteins; Fig. lB; lanes
5-8 of Fig. lC). Induction of apoptotic cell death by either virus (Nguyen et
a/., J. Biol. Chem. 269: 16521-16524, 1994; Teodoro et al., Oncogene 11:
467-474, 1995) was accompanied by thc appearance of p20 Bcl-2-binding
activity. The radioactive band associated with a polypeptide of Mr 20 kDa is
labelled p20 in Fig.1 C, whereas the band that co-migrates with Bax is
designated p21 Bax. Bax co-migration was determined using a blot cut along
the vertical mid-line of a protein lane, and developing one half by Western
blotting analysis with anti-human Bax (Chen et al., J. Biol. Chem. 271:
24221-24225, 1996) and the other by Far Western blotting analysis with 32p_
Bc1-2~c21/his6/HMK (results not shown). Apparent binding of ~2P-Bcl-
2~c21/his6/HMK to Bax did not alter significantly over the time course of
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infection (Fig. 1 C). Of note, however, stable expression of Bc1-2 in KB cells
countered cell death and prevented the appearance of p20 Bcl-2-binding
activity following viral infection (upper blot, Fig. 1 C).
Identification of Bc1-2 Interacting p28 Bap31 and its Cleavage Product,p20
Identification of the p20 Bcl-2-binding polypeptide was obtained by
NH2-terminal peptide sequence analysis of p20 following its isolation by a
combination of differential solubilization in detergent, preparative SDS PAGE,
and reverse phase HPLC (Figs. 2A and 2B). Several individual HPLC fractions
were subjected to peptide sequence analysis in order to detect a polypeptide
sequence whose appearance correlated with the appearance of p20 Bc1-2
binding activity (Figs. 2A and 2B). One candidate sequence emerged, and was
the only sequence that was detected in the peak fraction of Bc1-2 binding
activity (fraction 54, Fig. 2B). It showed a perfect match with amino acids 2-
10 of human Bap31 (GenBank accession number X81817) / CDM (GenBank
accession number Z31696), suggesting that p20 derives from the NH2-terminus
of Bap31/CDM, a 27,991 kDa (p28) protein (Fig. 2C). CDM was discovered
because of its proximity to the adrenoleukodystrophy locus (Mosser et al.,
Gcnomics 22: 469-471 ~ 1994) and Bap31 because it was one of several
polypeptidcs that were found in immunoprecipitates of the B-cell receptor
complex obtained from detergent solubilized cells (Kim et al., EMBO J. 13:
3793-3800, 1994;Adachi etal., EMBOJ. 15: 1534-1541, 1996). RTPCR
analysis of the p28 Bap31 coding region, using total RNA obtained from KB
cells following induction of apoptosis, showed no evidence that p20 arose by
differential splicing of p28 mRNA (data not shown). As demonstrated below,
Bc1-2 also associates with full length p28 Bap31 in vitro and in vivo; failure to
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observe this interaction in the original ligand blot analyses (Fig. lC) was likely
the result of relatively inefficient transfer of p28 Bap31 to nitrocellulose blots.
Characterization of p28 Bap31
Fig. 2C highlights several predicted motifs in the human p28 Bap31
sequence. There are 3 potential transmembrane (TM) segments located in the
NH2-terminal half of the molecule (as detected using the method described in
Kyte and Doolittle, J. Mol. Biol. 157: 105-132, 1982). TMI and TM3 each
contain charged residues. Additionally, two potential caspase cleavage sites,
comprised of identical P 1 -P4 tctrapeptide recognition scquences (ala-ala-val-
asp) plus a preferred small amino acid (gly) in the Pl' position, are located atpositions 164 and 238 in the polypeptide, on either side of a predicted leucine
zipper domain (Fig.2C) and overlapping homology to death effector domains
found in such proteins as procaspase-8, procaspase-10, and FADD (Fig. 2D).
Cleavage at the proximal caspase recognition site would generate a product
(calculated Mt (molecular mass) ofl8.8 kDa) similar in size to p20.
Interestingly, the distal caspase recognition site is lacking in the mouse p28
Bap31 sequence, however the proximal caspase recognition site, whose
cleavage would generate an p20 product, is present in murine p28 Bap31 (Fig.
2D). Finally, the p28 Bap31 molecule terrninates in Iys-lys-glu-glu which
conforrns to a canonical KKXX COOH-terminal signal that retains integral ER
proteins containing COOH-termini exposed to the cytosol within the ER, thus
preventing their exit into the distal secretory pathway (Jackson et al., J. CellBiol. 121: 317-333,1993).
As shown in Fig.3A, p28 Bap31 was efficiently inserted co-
translationally into dog pancreas microsomes. Pre-~-lactamase (Fig.3A, lanes
1 -4) and p28 Bap31 (Fig.3A, lanes 5-8) mRNA was translated in a rabbit
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-32-
reticulocyte lysate system in the presence of 35S-methionine, and in the
presence (Fig.3A, lanes 2-4 and 6-8) or absence (Fig. 3A, lanes I and 5) of
ribosome-stripped canine pancreas microsomes (prepared according to the
methods of Walter and Blobel, Meth. Enzymol. 96: 84-93, 1983). At the end of
the reaction, microsomes were recovered and analyzed by SDS PAGE and
autoradiography either directly (Fig. 3A, lanes 2 and 6) or following isolation
of alkali-insoluble (NaCO3, pH 11.5) product (Fig. 3A, lanes 3 and 7)
(according to methods described in Nguyen et al., J. Biol. Chem. 268:
25265-25268, 1993), or following treatment with proteinase K (Fig. 3A, lanes 4
and 8) (according to methods described in McBride et al., J. Cell Biol. 119:
1451-1457, 1992). In contrast to ~-lactamase, which was translocated across
the ER membrane and deposited in thc lumen as a soluble protein, p28 Bap31
was recovered as an integral protein following release from the ribosome.
Whereas the processed form of ~-lactamase was protected from external
protease (Fig. 3A, lane 4) and liberated from microsomes by alkaline extraction
(Fig. 3A, lane 3), p28 Bap31 was resistant to alkaline extraction (Fig. 3A, lane7) and exhibited sensitivity to external protease (Fig. 3A, lane 8), resulting in
the generation of proteolytic fragments which would be expected for a multi-
spanning integral protein with an exposed cytosolic domain. Unlike ~-
lactamasc, whose NH,-terminal signal sequence was removed during
translocation (Fig. 3At compare lanes 1 and 4), proccssing of p28 Bap31 was
not observed (Fig. 3A, compare lanes S and 6) suggesting that insertion into themicrosomal membrane is initiated by an uncleaved signal anchor. Though not
studied in detail, the observed properties of p28 Bap31 (Fig. 3A) together with
predictions for the orientation of transmembrane segments in the ER based on
charge-difference rules (as defined in von Heijne, G., J., Mol. Biol. 192:
287-290, 1986; Hartmann et al., Proc. Natl. Acad. Sci. U~A 86: 5786-5790,
CA 022~2422 1998-10-20
W O 98/39434 PCT~B98/00706
1989), suggests a topology for p28 Bap31 in the ER membrane in which the
NH2-terminus of this triple sp~nning polypeptide faces the lumen, leaving an
approximately 13 kDa COOH-terminal fragment containing the predicted
leucine zipper / death effector homology domain (amino acids 265-238)
flanked on either side by sites that are cleaved by caspase-8 or related caspaseduring adenovirus ElA-induced apoptosis, and ER retention motif exposed to
the cytosol. Fig.3B shows a schematic diagram of p28 Bap31 that has been
modified by the insertion of a Flag tag between pro 240 and met 241 of p28
Bap31 (i.e., the Flag tag was inserted immediately upstream of the C-terminal
KKEE ER retrieval signal). Both biochemical fractionation and cryo-
immunocytochemical electron microscopy confirmed that p28 Bap31 is
predominantly located in the ER in rat hepatocytes (not shown).
Recombinant p28 Bap31 and p20 Interact with Bc1-2
Various p28 Bap31 fusion proteins were constructed in which
glutathione S-transferase (GST) was linked to p28 Bap31 amino acids 1-246
(full length p28 Bap31), 1-164 (p20),122-164, and 165-246. These constructs,
together with GST itself, were purified and equal amounts examined for their
ability to bind to the cytosolic domain of Bc1-2 in a Far Western blotting
analysis assay. As shown in Fig. 4A, reactivity was observed for both GST-
p28 Bap31 (Fig.4A, lane 5) and GST-p20 (Fig.4A, lane 4), with weak activity
possibly registering with the COOH-terminal 165-246 amino acid domain (Fig.
4A, lane 2), and none detected for the middle 122-164 amino acid domain (Fig.
4A, lane 3) or for GST alone (Fig. 4A, lane 1).
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Bc1-2 Proteins and Procasr~ 8 (pro-FLICE) Associate with p28 Bap31 In
Vivo
P2~ Bap31 and its cleavage product, p20, associate with Bc1-2 in vitro.
Thus, we attempted to detect the presence of a such a complex in vivo.
Standard recombinant DNA manipulations were used to create cDNAs
encoding Bcl-XI tagged at the C-terminus with the myc epitope,
EQKLISEED~ (SEQ ID NO: 16; Chinnayan et al., Science 275: 1122-1126,
1997); pro-FLICE tagged at the C-terrninus with the Hemagglutinen (HA)
epitope, YPYDVPDYA (SEQ ID NO: 17; Chinnayan et al., Science 275:
1122-1126, 1997); Bc1-2 tagged at the N-terminus with the HA epitope
(Nguyen et al., J. Biol. Chem. 269: 16521-16524, 1994); and p28 Bap31 tagged
with the Flag epitope, in which the Flag se4uence, MDYKDDDDKA (SEQ ID
NO: 18), was inserted between pro 240 and met 241 of p28 Bap31 to avoid
interference with the function of the ER retention signal in p28 Bap31 (i.e.~ the
Flag tag was inserted immediately upstream of the C-terminal KKEE ER
retrieval signal in p28 Bap31). The recombinant cDNAs encoding myc-tagged
Bcl-XL HA-tagged pro-FLICE, or Flag-tagged p28 Bap31, or Flag DNA alone
(Control-Flag), were inserted into thc expression vector, pcDNA3
(commercially available from Invitrogen, Carlsbad, CA). The cDNA encoding
HA-tagged Bc1-2 was inserted into the expression vector, RcRSV
(commercially available from Pharmacia, Uppsala, Sweden). The resulting
cxpression constructs were transfected into 293T cells, as is indicated in Fig.
4B (pluses and minuses), using the following procedure: 293T cells at 50-60%
confluency in 10 cm culture plates were transfected by calcium phosphate
precipitation with 15 ~lg total plasmid DNA and shocked with 15% glycerol 24
hours following transfection. Approximately 30 hours following transfection,
the cells were washed in phosphate buffered saline (PBS) and homogenized in
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1 ml Iysis buffer (SOmM Hepes, pH 7.4, 150 mM NaC1, lmM ethylenediamine
tetraacetate (EDTA), 0.5% v/v NP40, 10 rLlg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 10 ,ug/ml leupeptin) per 10 cm
culture plate. After centrifugation at 11,000 g, the supernatant was incubated
with 50 ~1 of a 1:1 slurry of protein G sepharose for 1 hour at 4"C. The
sepharose was removed and the supernatant was incubated with mouse M2
anti-Flag antibody (commercially available from IBI Kodak) for 6-8 hours at
4~C, whereupon 20 ~11 of a 1:1 slurry of protein G sepharose was added. After
another hour at 4~C, the beads were recovered, washed, and boiled in SDS
sample buffer. Immunoprecipitates (ip) and boiled Iysates (cell Iysates prior toimunoprecipitation with the anti-Flag antibody) were resolved by SDS-PAGE,
transfered to nitrocellulose, and probed with the indicated antibody directed
toward either myc or HA, and are commercially available from Babco
(Berkeley, CA). Immunoreactive bands were visualized by
electrochemiluminescence of the immunoglobulin heavy chain (Ig HC).
Shown in Fig. 4B are the results of the Western blotting analysis of the
Iysates and immunoprecipitations probed with anti-Myc or anti-HA antibodies.
As judged by the co-immunoprecipitation, Bcl-XI, pro-FLICE, and Bc1-2 each
demonstrated a specific association with p28 Bap3 1, as shown in Fig. 4B, lanes
3, 8, and 14, respectively. Pro-FLICE was observed as a doublet band that
migrated immediately below the Ig heavy chain; transcription translation of the
cDNA in vitro was found to likewise generate a doublet of similar size (not
shown). Interestingly, BC1-XL and pro-FLICE, when expressed in combination,
did not mutually antagonize each other's ability to associate with p28 Bap3 1
(see Fig. 4B, lanes 5 and 10; note the lower input levels of BCI XL in the cell
lysate in lane 5). Although some activation of wild-type procaspase-8 might be
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expected, the full length pro-enzyme was readily detectable at similar levels inboth the presence and absence of BC1 XL (Fig. 4B, lanes 8 and 10).
Finally, the pro-apoptotic member of the Bc1-2 family, Bax (Oltvai et
al., Cell 74: 609-619, 1993), did not co-immunoprecipitate with p28 Bap3 1
following their co-expression in 293T cells (Fig. 4C, lane 5), despite the fact
that significant expression levels of transfected Bax were recorded (Fig. 4C,
lanes 2 and 3). However, Bax prevented Bc1-2 from associating with p28
Bap31 (Fig. 4C, compare lanes 11 and 12). Although the level of Bc1-2 in cell
Iysates was somewhat lower in transfectants containing Bax (Fig. 4C, compare
lanes 8 and 9), such a level of Bc1-2 would otherwise have been sufficient to
readily detect co-immunoprecipitation of Bc1-2 and p28 Bap3 1.
p28 Bap31 is Cleaved to p20 following induction of apoptosis
Although we demonstrated that p28 Bap3 1 was cleaved into a product,
p20, in a cell that had been induced to undergo apoptosis, the exact mechanism
by which this cleavage occurred was unknown. To determine whether or IlOt
the caspase recognition sequences (AAVDG) in p28 Bap3 1 were in fact
recognized by one or more of the caspases, a 35S-labelled p28 transcription-
translation product of p28 Bap3 1 cDNA was generated by 35S-methionine
labclling in vitro translated proteins according to the methods known in the art(see, for example, Ausubel et al., Current Protocols in Molecular Bio]o~y, John
Wiley & Sons, New York, NY, 1994) using 35S-labelled methionine
cornmercially available from Dupont/NEN. This 35S-labelled p28 was
incubated with increasing concentrations of caspase-3 (CPP32) or caspase- 1
(ICE) in vitro (according to the methods of Nicholson et al., Nature 376: 37-43,1995), and the products examined following resolution on SDS-PAGE. As can
be seen in Fig. SA (upper blot), little reactivity was observed for caspase-3 over
.. . . . . . .. , _ .
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a wide range of enzyme concentration. ICE (caspase-1), on the other hand,
generated two products (denoted "a" and "b" in the schematic diagram at the
bottom of Fig. 5A) whose apparent sizes in SDS gels (approximately 27 kDA
and 20 kDa, respectively) were more consistent with cleavage occurring at both
AAVDG caspase recognition sequences (Fig. 5A, lower blot). Similar analysis
was made by incubating ~sS-methionine labelled p28 with FLICE and ICE,
although in this experiment, the amount of the resulting p20 cleavage product
was quantitated on a Phosphorimager. As shown in Fig. SB, it is noteworthy
that p28 Bap31 was more sensitive to cleavage by FLICE (caspase-8) than ICE
(caspase- 1).
Two cleavage products of p28 Bap31, similar in size to those seen in
vitro (see Fig. SA), were observed in KB cells that had been induced to undergo
apoptotic cell death in response to infection by 19K-defective adenovirus (Fig.
6A). In Fig. 6A, p28 Bap31 cleavage during apoptosis in vivo was analyzed
using antibodies raised in chicken to either of two regions of the protein: p28
Bap31 amino acids 122-164 (Ol p28-M) and 165-246 (oc p28-C) Following
resolution of cellular extract proteins by 12% SDS PAGE and transfer to
nitrocellulose, the blots were incubatcd with the primary chicken antibody, and
then developed with secondary antibody conjugated either to horseradish
peroxidase and visualized by electrochemiluminescence (Amersham Intl.,
Arlington Heights, IL) (for o~ p28-M, right blot) or to alkaline phosphatase andvisualized with NBT/BCIP (Boehringer Mannheim Biochemicals, Indianapolis,
~N) (cc p28-C; left blot), according to the manufacturer's instructions. Of note,
cleavage products were detected with ~ p28-M but not with a p28-C, a finding
consistent with the suggestion from peptide sequence analysis that the p20
cleavage product derives from the NH2-terminus of p28 Bap31 (see Fig. 2C). oc
p28-C also failed to detect the larger of the two cleavage products (designated
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W098/39434 PCT~B98/00706
"a" in Figure 6A) despite the predicted overlap of this product with the proteinsequence injected into chickens. Presumably, this means that the extreme 8
amino acids of p28 Bap31 are critically important for epitope recognition by
this antibody. Finally, protein electrophoretic blots were developed from
apoptotic cell extracts, cut in half along the vertical midline of a protein lane,
and one half probed with o~ p28-M and the other with 32P-Bcl-
2~c21/his6/HMK. p20 detected by Far Western blotting analysis migrated
exactly with p20 detected by o~ p28-M immunoblotting (data not shown).
In Fig. 6B, the effect of Bc1-2 on the appearance of p28 Bap31 cleavage
products following cell infection with 19K-deficient adenovirus was examined
using the a p28-M antibody. Cellular extracts were prepared at 0 hours (Fig.
6B, lanes I and 6),24 hours (Fig.6B, lanes 2 and 7),36 hours (Fig. 6B, lanes 3
and 8), 48 hours (Fig.6B, lanes 4 and 9), and 60 hours (Fig. 6B, lanes 5 and 10)post-infection (p.i.) from KB cells expressing neo plus Bc1-2 (Fig. 6B, lanes 1-S) or neo only (Fig.6B, lanes 6- 10). Aliquots (15 ~Lg protein) were subjected to
12% SDS-PAGE, transferred to nitrocellulose, and probed, and visualized as
described above for the results pictured in Fig.6A. In the absence of Bc1-2
expression, the time course of appearance of these products closely followed
the time course for activation of pro-CPP32, as judged by processing of the
pro-enzyme to the p 17 subunit of CPP32 (Fig.6B, lanes 6- 10). However, both
p28 Bap31 cleavage and pro-CPP32 processing were blocked in virus-infected
cells that express Bc1-2 (Fig. 6B, lanes 1-5).
Ectopic Expression of p20 Induces Apoptosis in Transfected KB Cells
CHO (neo) cells were transiently co-transfected with a luciferase
reporter gene together with RcRSV expressing either p2~ Bap31 or p20.
Subsequent measurements revealed that co-expression of p20 with the reporter
,
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severely depressed the amount of luciferase activity obtained relative to co-
expression with p28 Bap3 1 (Fig. 7A). p28 Bap3 1, on the other hand, had no
deleterious effect on the recovery of luciferase activity compared to a control
RcRSV plasmid that did not encode protein (not shown). If these same
transfections were conducted in KB cells stably expressing Bc1-2, however,
Bc1-2 largely overcame the dominant negative influence of p20 on luciferase
activity (Fig. 7A), presumably because p20 could no longer interfere with
normal p28 Bap3 I function. Because of this protective effect by Bc1-2, we
concluded that the negative influence of p20 on luciferase activity was the
result of induction of apoptotic cell death. This was confirmed by microscopic
analysis, which revealed dying cells with condenscd apoptotic nuclei in p20-,
but not p28 Bap3 1-transfected cells (Fig. 7B). The findings described in Fig.
7A, and the above-mentioned microscopic analysis (Fig. 7B) havc been
consistently observed many times and in different cell types. Hence the p20
Bap3 1 product is a potent inducer of apoptosis when expressed ectopically in
otherwise normal cells, presumably because it has a dominant-negative effect
on endogenous p28 Bap3 1.
A p28 Bap31 Knockout ES Cells and Mouse Models: Construction of a
Transgenic Animal
P28 Bap3 1 gene characterization provides information that is necessary
for p28 Bap3 1 knockout animal models to be developed by homologous
recombination. Preferably, the models are m:~lmm~ n ~nim~ls, most
preferably mice. Similarly, an animal model of p28 Bap31 ovel~plession may
be generated by integrating one or more p28 Bap3 1 gene sequences into the
genome, according to standard transgenic techniques.
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A replacement-type l~ ;ling vector, which may be used to create a
knockout model, may be constructed using an isogenic genomic clone, for
example, from a mouse strain such as 129/Sv (Stratagene Inc., La Jolla, CA).
The talgelillg vector may be introduced into a suitably-derived line of
embryonic stem (ES) cells by electroporation to generate ES cell lines that
carry a profoundly truncated form of a p28 Bap3 1 gene. The targeted cell lines
may then be injected into a mouse blastula stage embryo to generate chimeric
founder mice. ~Ieterozygous offspring may be interbred to homozygosity.
Knockout mice provide the means, in vivo, to screen for therapeutic compounds
that modulate apoptosis via a pathway that involves p28 Bap3 1. Making such
mice may require use of loxP sites if there are multiple copies of p28 Bap3 1 onthe chromosome (see Sauer and Henderson, Nucleic Aids Res. 17: 147-61,
1 989).
Hence, to further assess the role of p28 Bap3 1 at an org~ni~m~l level,
the generation of a mouse lacking the p28 Bap3 1 -encoding gene is desired. To
achieve this, a plasmid bearing a "knock-out" version of the murine p28 Bap3 1
gene, such as the plasmid shown as schematic diagrams in Figs. 8A, 8B, and
8C, may be used. The resulting plasmid, BP31TV, is shown in a schematic
diagram in Fig. 8C, and bears a gene, "p2B Bap3 1 -NEO" that encodes a protein
capable of resisting the presence of neomycin (i.e., G418).
The BP3 I TV was then transfected into embryonic stem cells (ES cells)
from a mouse. Once homologous recombination of the BP3 I TV DNA with
genomic DNA is detected, the clone of ES cells bearing this homologous
recombination is used to produce mice that are heterozygous and homozygous
for a Bap3 1 knock-out mutation. It will be appreciated that the ES cells are,
themselves, useful, e.g., for identifying compounds which mimic p28 Bap3 1 in
culture.
.. . . . . .
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It will be understood that once such an ES cell genetically engineered to
lack an endogenous p28 Bap31 gene is generated, the cell may be further
manipulated to express a mutated or truncated version of p28 Bap31. Nucleic
acid encoding such a truncation or deletion may be generated by standard
techniques, and inkoduced into a plasmid. The plasmid may then be
introduced by artifice into the ES cells bearing the p28 Bap31 -NEO gene at the
locus normally bearing the endogenous p28 Bap31 gene. ES cells both lacking
an endogenous p28 Bap31 gene and expressing a truncated or mutated p28
Bap31 are able to grow in culture media containing both G418 and hygromycin
(commercially available from Sigma). The cells may be analyzed, or used to
generate a transgenic mouse lacking expression of endogenous p28 Bap31 and
expressing a truncated or mutated form of the protein.
The coopcrative associations between BCI XL, procaspase-8, and the p28
Bap31 cytosolic domain described herein suggest that Bap31 may provide a
mechanism by which corresponding molecular complexes in m~mm~lian cells
are linked to the multitude of signals that can trigger activation of procaspases,
and how they interface with the numerous proapoptotic and antiapoptotic
regulators that modulate thesc signals. Without wanting to be limited to a
particular hypothesis, we propose a working model (Fig. 9) in which apoptotic
signalling induced by EIA expression and/or other Bcl-2-inhibiting events
leads to activation of the caspase cascade by a pathway that involves p28
Bap31. One of the activated caspases (e.g., FLICE) then cleaves p28 Bap31 to
produce p20, an event that may act to consolidate and amplify the commitrnent
to apoptosis. By interacting with full length p28 Bap31 (and perhaps other
targets), Bc1-2 abrogates activation of the caspase proteases and blocks
apoptosis.
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Experimental Procedures
Cells and Viruses
Human epithelial KB cells expressing the neomycin resistance gene
(neo) either alone or together with Bc1-2 (Nguyen et al., J. Biol. Chem. 269:
16521-16524, 1994) were cultured in o~-MEM supplemented with 10% fetal
bovine serum, and 100 units/ml streptomycin and penicillin. After reaching
80% confluency, the medium was replaced with fresh medium containing
either no virus or 25-35 plaque forming units (pfu)/cell of adenovirus type 5
lacking expression of E 1 B 19K (pm 1716/2072, McLorie et al., J. Gen. Virol.
72: 1467-1471, 1991) or of adenovirus type 5 expressing only the 243R-forrn
(12S) of ElA and no ElB products (dl520ElB-) (Shepherd et al., J. Virol. 67:
2944-2949, 1993). Following incubation for 1 hour at 37~C, fresh medium was
added and cells were collected at various times for analysis. Both forms of the
virus elicit a cytotoxic response in infected cells which exhibit all of the
hallmark features of apoptosis (Nguyen et al., J. Biol. Chem. 269:
16521 - 16524, 1994; Teodoro et al., Oncogene 11: 467-474, 1995).
Bacterial Expression and Purification of 32P-labelled Bc1-2 Cytosolic Domain
for Ligand Blot (Far Western) Analyses
cDNA encoding the cytosolic domain of human Bc1-2 (i.e., lacking the
COOH-terrninal 21 amino acids) was inserted into the pTrchis vector
(Invitrogen), and standard PCR methodology employed to extend hexahistidine
at the COOH-terminus to include a heart muscle kinase recognition sequence
using the oligonucleotides
5'-CTAGCGCCCGCCGCGCCTCTGTGGAATTCTGAA-3' (SEQ ID NO: 19)
and
.. .. .. .
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WO 98/39434 PCT/IB98/00706
-43 -
5'-AGCTTTCAGAATTCCACAGAGGCGCGGCGGGCG-3' (SEQ ID NO:
20). The final construet encoded Bc1-2 (amino acid residues 1 -218), hexahis,
arg, arg, ala, ser -COOH, and the protein was designated
Bc1-2~c21/his6/HMK. The Bc1-2 portion contained three additional mutations
which were introdueed for reasons not related to this project (met 16 to leu; lys
17 to arg; lys 22 to arg). Stable epithelial cell lines that express full-lengthBc1-2 harboring these mutations were found to be as effeetive as cells
expressing wild-type Bc1-2 in eountering apoptotic death stimuli.
E. coll MC1061 was transformed with pBc1-2/~c21/his6/HMK. When
500 ml cultures reaehed 0.6 ~600 of 0.6, they were treated with 1.0 mM IPTG,
and eells reeovered by eentrifugation 4 hours later. Packed cells (2.5-3.0 ml)
were suspended in 15 ml extraction medium (20 mM Na phosphate, pH 7.4, 0.5
M NaCl, 0.05% v/v Triton X-100, 10 mM ,~-mercaptoethanol, 1.0 mM
phenylmethylsulfonylfluoride, and 1.0 mM benzamidine) and sonieated 8 X
with a Vibra Cell probe sonieator (Sonies and Materials, Inc., Danbury, CT)
operating at setting 7.5 for 15 seconds at 4~C. The sonicate was adjusted to
10% (v/v) glycerol and centrifuged at 25,000 rpm for 30 minutes at 4~C in a
Beckman Ti 50.2 rotor (Beckman lnstruments, lnc., Fullerton, CA). The
supernatant was added to 1.2 ml Ni2~-NTA agarose (commereially available
from QlAGEN Inc., Chatsworth, CA) (a 1: 1 (v/v) mixture with extraetion
medium) and ineubated for 1.5 hours at 4~C. The beads were washed
extensively in extraetion medium eontaining 20% (v/v) glyeerol and 22 mM
imidazole, and Bc1-2~e21/his6/HMK eluted in extraetion medium containing
20% glycerol and 0.3M imidazole. One liter of indueed culture yielded 0.8 -
1.0 mg protein whieh was greater than 95% pure. The purified protein was
labelled with 32p following ineubation with heart musele kinase and 32P-~-ATP,
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W O 98/39434 PCT~B98100706
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yielding 2.0-2.5 x 106 cprn/~lg protein, and was employed for ligand blotting, as
described in Blanar and Rutter (Science 256: 1014-1018, 1992).
Purification and Identification of p20 Fragment
KB cells were cultured in 20 15-cm plates until 80% confluent, and
infected with 25 plaque forrning units (pfu)/ cell of adenovirus type 5 lacking
expression of ElB l9K (pml716/2072). After 60 hours, total cells (65-70%
non-viable, as judged by exclusion of trypan blue) were collected, rinsed, and
the packed cells (approximately 1.5 ml) suspended in ice-cold 6 ml Iysis
medium containing 10 mM Tris HCI, pH 7.5, 140 mM NaCI, 1.5 mM MgCI2,
0.5% Triton X-100 and lmM phenylmethylsulfonylfluoridc) and separated into
3 equal portions. Each was subjected to sonication for 4 x lO sec using an
Artek probe sonicator operating at setting 6.0 (Artek Systems Corp.,
Farmingdale, NY). The combined sonicates were centrifi~ged at 11,000 x g for
20 minutes and the supernatant mixed with 0.25 vol of 5 x SDS sample buffer
(250 mM Tris HCI, pH 6.8, 50% glycerol, 0.5% bromophenol blue, 10% SDS
and lM dithiothreitol). The total volume was subjected to preparative 14%
SDS-PAGE using a Bio Rad Prep Cell 491 system (Bio Rad Laboratories,
Hercules, CA) fitted with a 37 mm diameter resolving gel chamber. Fractions
were collected at a flow rate of 1 ml/min, assayed for the presence of p20 by
ligand blotting using 32P-Bc1-2/\c21/his6/HMK as probe, and the reactive peak
fractions combined and concentrated approximately five-fold in a
Centriprep-10 concentrator (commercially available from Amicon, Inc.,
Beverly, MA). The concentrated sample was mixed with an equal volume of
0.12% trifluoroacetic acid and subjected to reverse phase HPLC in a Hewlett
Packard 1090 System (Hewlett Packard CO., Palo Alto, CA) outfitted with a
Vydac C4 column (0.21 x 20 cm) (The Nest Group, Inc., Southboro, MA)
.
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WO 98/39434 PCT/IB98/00706
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prefixed with t~,vo SDS removal cartridges (2.1 x 20 mm). The column was
developed with a linear gradient of 0 to 80% n-propyl alcohol containing
0.12% trifluoroacetic acid at a flow rate of 0.1 ml/min, and monitored at A280.
Fractions (0.1 ml) were collected and those containing Bcl-2-reactive p20, as
judged by ligand blotting, were individually subjected to NH2-terminal peptide
sequence analysis at Harvard Microchem (Harvard University, Cambridge,
MA).
Clonin~ of p28 Bap31/CDM cDNA
The coding region of p28 Bap31 was cloned by reverse transcriptase-
polymerase chain reaction (RT-PCR) using human fibroblast RNA together
with primers derived from the sequence of human BAP31 (EMBL accession
number X81817). Conditions were exactly as described in Goping et al. (FEBS
373: 45-50, 1995) and used as the anti-sense primer,
5'-TCTCTAGAACAAACAGAAGTACTGGA-3' (SEQ ID NO: 21) and as the
sense primer, 5'-GATCTAGACATCTTCCTGTGGGAA-3' (SEQ ID NO: 22).
Authenticity was confirmed by DNA se~uence analysis.
Glutathione S-Transferase (GST) Fusion Protein~
PCR was employed to generate cDNA fragments colTesponding to p28
Bap31 amino acids 1-246 (full-length), 1-164, 122-164, and 165-246, using
primers that contained either 5'-BamH1 or 3'-EcoR1 overhangs, respectivcly.
The primers were 5'-GCGGATCCATGAGTCTG CAGTGGACT-3' (SEQ ID
NO: 23) and 5'-GCGAATTCTTACTCTTCCTTCTTGTC-3' (SEQ ID NO: 24)
for p28 Bap31 amino acids 1-246;
5'-GCGGATCCATGAGTCTGCAGTGGACT-3' (SEQ ID NO: 25) and
5'-GCGAATTCAGTCA ACAGCAGCTCCCTT-3' (SEQ ID NO: 26) for p28
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Bap31 arnino acids 1 - 164; 5'-GCGCGGATCCCTCATTT CGCAGCAGGCC-
3' (SEQ ID NO: 27) and 5'-GCGAATTCAGTCAACAGCAG CTCCCTT-3'
(SEQ ID NO: 28) forp28 Bap31 amino acids 122-164; and
5'-GCGGATCCGGAGGCAAGTTGGATGTC-3' (SEQ ID NO: 29) and
S'-GCGAATTCTTACTCTTCCTTCTTGTC-3' (SEQ ID NO: 30) for p28
Bap31 amino acids 165-246.
Fragments generated by PCR were digested with BamH1 and EcoRl,
inserted between thc BamH I and EcoR1 sites of pGEX-2T (Pharmacia), and
the recombinant plasmids introduced into E. coli MC 1061. Packed cells from
500 ml of induced culture were recovered, suspended in 25 ml phosphate
buffered saline (PBS) and 0.1% Triton X-100, and sonicated using a Vibra Cell
probe sonicator operating at setting 7.5 for 4 x 15 sec at 4~C. Following
centrifugation at 25,000 rpm in a Beckman Ti 50.2 rotor for 25 min, the
supernatant was recovered, mixed with 750 ~ul of a 1: 1 suspension of
glutathione Sepharose 4B beads, and the mixture rotated at 4~C for 45 min.
Following extensive washing of the beads in PBS and 0.1 % Triton X- 100, GST
fusion protein was eluted with 3 ml of 50 mM Tris HCI, pH 8.0, and 12 mM
reduced glutathione.
Antibodies
GST fusion proteins were injected into chickens and the rcsulting IgY
antibodies recovered from eggs, exactly as described in Goping et a~., FE13S.
373: 45-50, 1995. After adsorption of lgY that reacted with immobilized GST,
antibodies specific for p28 Bap31 sequences were purified by affinity binding
to immobilized GST-p28(165-246) or GST-p28(122-164) fusion protein,
employing the methods described in the Amino Link Plus kit (Pierce Chemical
Co., Rockford, IL).
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Transient Transfections
CHO LR73 cells were seeded at a density of 5 x 105 cells per well in
6-well plates. 24 hours later, cells in each well were transfected by calcium
phosphate precipitation with 0.5 llg luciferase reporter plasmid, 10 ~g
RcRSV-p28 or RcRSV-p20, and 10 ~lg sheared salmon sperm DNA (Goping et
al., Nucl. Acids Res. 23: 1717- 1721, 1995). After 24 hours, cells were shocked
with 15% glycerol, and collected 24 hours later. Cells from each well were
lysed in 0.4 ml 0.5% NP40 and 50 mM Tris HCl, pH 7.8, and aliquots assayed
for luciferase activity as previously described (Goping et al., Nucl. Acids Res.23: 1717-1721, 1995). 293T cells in 10 cm culture plates werc similarly
transfected with 15 ~ug total plasmid DNA when cells reached 50- 60%
confluency.
Co-immunoprecipitations
Approximately 30 hours post-transfection,293T cells were washed in
phosphate buffered saline, and homogenized in 1.0 ml Iysis medium per 10 cm
culture plate (50 mM Hepes, pH 7.4, 150 mM NaCI, 1 mM ethylenediamine
tetraacetate, 0.5% v/v NP40, 10 llg/ml aprotinin, lO llg/ml leupeptin, and 1 mM
phenylmethylsulfonylfluoride). After centrifugation at l l,000 g, the
supernatant was incubated with 50 111 of a 1: 1 slurry of protein G Sepharose for
1 hour at 4~C. The Sepharose was removed and the supernatant incubated with
mouse M2 anti-Flag antibody (IBI-A Kodak Co., New Haven, CT) at 4~C for 6
- 8 hours, at which time 20 ~11 of a 1: 1 slurry of proteinG Sepharose was added.
After 1 hour at 4~C, the beads were recovered, washed, and boiled in SDS
sample buffer. Following SDS PAGE and transfer to nitrocellulose, blots were
developed with either mouse anti-Myc 9ElD antibody or mouse anti-HA
12CA5 antibody (both from Babco, Berkeley, CA) or rabbit anti-Bax sc-526
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-48-
antibody (commercially available from Santa Cruz Biotech., Inc., Santa Cruz,
CA).
Expression Plasmids
cDNAs encoding proteins tagged with a specific epitopes were
constructed in expression vectors. Flag epitope was inserted toward the
C-terminus of Bap3 1, immediately upstream of the KKEE ER retrieval signal;
Myc and HA epitopes were placcd at the C-termini of Bcl-X, and proFLICE,
respectively.
Synthesis of p28 Bap31
The characteristics of the cloned p28 Bap3 1 nucleic acid sequences may
be analyzed by introducing the sequence into various cell types or using in vztro
extracellular systems. The function of p28 Bap3 1 may then be examined under
different physiological conditions. The p28 Bap3 1 nucleic acid sequence may
be manipulated in studies to understand the expression of the gene and gene
product. Alternatively, cell lines may be produced which over-express the gene
product allowing purification of p28 Bap3 l for biochemical characterization,
large-scale production, antibody production, and patient therapy.
For protein expression, eukaryotic and prokaryotic cxpression systems
may be employed in which either the p28 Bap3 I nucleic acid sequence is
introduced into a plasmid or other vector which is then introduced into living
cells. An expression plasmid into which the entire open reading frame of either
p28 Bap3 1 cDNA sequence has been inserted in the correct orientation may be
used for protein ~plession. Alternatively, portions of the sequences, including
wild-type or mutant p28 Bap3 1 sequences, may be inserted. Prokaryotic and
eukaryotic expression systems allow various important functional domains of
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-49-
the protein to be recovered as fusion proteins and then used for binding,
structural and functional studies and also for the generation of ~ro~liate
antibodies. Since p28 Bap3 1 is involved in modulating apoptosis, it may be
desirable to control expression levels by use of an inducible promoter (e.g., tet
or lac).
Typical expression vectors contain promoters that direct the synthesis of
large amounts of mRNA corresponding to the gene. They may also include
sequences allowing for their autonomous replication within the host organism,
sequences that encode genetic traits that allow cells containing the vectors to be
selected, and sequences that increase the efficiency with which the mRNA is
translated. Some vectors contain selectable markers such as neomycin
resistance that permit isolation of cells by growing them under selective
conditions. Stable long-term vectors may be maintained as freely replicating
episomal entities by using regulatory elements of viruses. Cell lines may also
be produced in which the expression vector has been integrated into the cell's
genomic DNA therefore allowing the production of the gene product on a
continuous basis.
Expression of foreign sequences in bacteria such as Escherichia coli
require the insertion of nucleic acid sequences of p28 Bap3 1 polypeptides into
an expression vector, usually a bacterial plasmid. This plasmid vector contains
several elements such as an origin of replication, sequences encoding a
selectable marker that assures maintenance of the vector in the cell, a
controllable transcriptional promoter (i.e., lac) which can produce large
amounts of mRNA from the cloned gene upon induction, translational control
sequences and a polylinker to simplify insertion of the gene in the correct
orientation within the vector. In a simple E. coli expression vector utilizing the
lac promoter, the expression vector plasmid contains a fragment of the E. coli
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-50-
chromosome cont~ining the lac promoter and the neighbouring lacZ gene. In
the presence of the lactose analog IPTG, RNA polymerase normally transcribes
the lacZ gene producing lacZ mRNA which is translated into the encoded
protein"B-galactosidase. The lacZ gene can be cut out of the expression vector
with restriction endonucleases and replaced by p28 Bap31 nucleic acid
se~uences. When this resulting plasmid is transfected into E. coli, addition of
IPTG and subse~uent transcription from the lac promoter produces p28 Bap3 1
mRNA, which is translated into p2~ Bap3 l .
Once the a~lol~l iate expression vectors containing the p28 Bap3 1
nucleic acid sequences are constructed, they may be introduced into an
appropriate host cell by transformation techniques including calcium phosphate
transfection, DEAE-dextran transfection, electroporation, micro-injection,
protoplast fusion, and liposome-mediated transfection. The host cells which
are transfected with the vectors of this invention may be selected from the
group consisting of E. coli, pseudomonas, Bacillus subtillus, or other bacilli,
other bacteria, yeast, fungi, insect (using baculoviral vectors for expression),mouse or other animal or human tissue cells. ~1~mm~ n cells can also be
used to express p28 Bap3 l using a vaccinia virus expression system described
in the art (see, for example, Ausubel et al., supra).
In vitro expression of proteins encoded by cloned DNA is also possible
using the T7 late-promoter expression system. This system depends on the
regulated expression of T7 RNA polymerase which is an enzyme encoded in
the DNA of bacteriophage T7. The T7 RNA polymerase transcribes DNA
beginning within a specific 23-bp promoter se4uence called the T7 late
promoter. Copies of the T7 late promoter are located at several sites on the T7
genome, but none is present in E. coli chromosomal DNA. As a result, in T7
infected cells, T7 RNA polymerase catalyzes transcription of viral genes but
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not of E. coli genes. In this expression system recombinant E. coli cells are
first engineered to carry the gene encoding T7 RNA polymerase next to the lac
promoter. These cells are then transforrned with plasmid vectors that carry a
copy of the T7 late promoter directing the expression of the p28 Bap3 1
proteins. When IPTG is added to the culture medium containing these
transformed E. coli cells, large amounts of T7 RNA polymerase are produced.
The polymerase then binds to the T7 late promoter on the plasmid expression
vectors? catalyzing transcription of the inserted p28 Bap3 I cDNAs at a high
rate. Since each E. coli cell contains many copies of the expression vector,
large amounts of mRNA corresponding to the cloned cDNA can bc produced in
this system and the resu}ting p28 Bap3 I polypeptides can be radioactively
labelled. Plasmid vectors containing late promoters and the corresponding
RNA polymerases from related bacteriophages such as T3, T5, and SP6 may
also be used for in vitro production of p28 Bap3 I polypeptides from cloned
DNA. E. coli can also be used for expression by infection with M13 Phage
mGPI-2. E. coli vectors can also be used with phage lambda regulatory
sequences, by fusion protein vectors, by maltose-binding protein fusions, and
by glutathione-S-transferase fusion proteins.
Eukaryotic expression systems perrnit a~ o}~l-ate post-translational
modifications to expressed proteins. This allows for studies of the p28 Bap3 1
gene and gene product, including determination of proper expression and post-
translational modifications for biological activity, identifying regulatory
elements located in the 5' region of the genes and analyzing their roles in tissue
regulation of gene product expression. It also permits the production of large
amounts of normal and mutant proteins for isolation and purification. Cells
expressing p28 Bap3 1 may be used in a functional assay system for antibodies
generated against the protein and are important vehicles in which to test the
. ~, ,
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effectiveness of pharmacological agents modulating p28 Bap31, to assess the
role of p28 Bap31 as a component of a signal transduction system, and to study
the in vivo function of the normal complete protein, specific portions of the
protein, or naturally occurring polymorphisms and artificially produced
mutated polypeptides. The nucleic acid sequences encoding p28 Bap31 can be
altered using procedures such as restriction endonuclease digestion, DNA
polymerase fill-in, exonuclease deletion, terminal deoxynucleotide transferase
extension, ligation of synthetic or cloned DNA sequences and site-directed
sequence alteration using specific oligonucleotides together with PCR.
A p28 Bap31 may be produced by a stably-transfected mammalian cell
line. A number of vectors suitable for stable transfection of nl~mm~ n cells
are available to the public (e.g., see Pouwels e~ al., Cloning Vectors: A
Laboratory Manual, 1985, Supp.1987), as are methods for constructing such
cell lines (see e.g., Ausubel et al., supra). In one example, cDNA encoding the
desired protein (i.e., p28 Bap31) is cloned into an expression vector that
includes the dihydrofolate reductase (DHFR) gene. Integration of the
expression plasmid bearing the protein-encoding nucleic acid into the host cell
chromosome is selected for by inclusion of 0.01-300 !lM methotrexate in the
cell culture medium (as described in, e.g., Ausubel et al., supra). This
dominant selection can be accomplished in most cell types.
Recombinant protein expression can be increased by DHFR-mediated
amplification of the transfected nucleic acid (e.g., nucleic acid encoding p28
Bap31). Methods for selecting cell lines bearing gene amplifications are
described in Ausubel et al. (supra) and generally involve extended culture in
medium cont~ining gradually increasing levels of methotrexate. The most
commonly used DHFR-containing expression vectors are pCVSEII-DHFR and
pAdD26SV(A) (described in Ausubel et al., supra). The host cells described
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above or, preferably, a DHFR-deficient CHO cell line (e.g., CHO DHFR- cells,
ATCC Accession No. CRL 9096) are among those most preferred for DHFR
selection of a stably-transfected cell line or DHFR-mediated gene
amplification.
Another preferred expression system is the baculovirus system using, for
example, the vector pBacPAK9, which is available from Clontech (Palo Alto,
CA). If desired, this system may be used in conjunction with other protein
expression techniques, for example, the myc tag approach described by Evan et
al. (Mol. Cell. Biol. 5: 3610-3616, 1985).
Once the recombinant protein is expressed, it is isolated by, for example,
affinity chromatography. ln one example, an anti-p28 Bap3 1 antibody, which
may be produced by the methods described herein, can be attached to a column
and used to isolate the p28 Bap3 1 polypeptide. Lysis and fractionation of p28
Bap3 l-expressing cells prior to affinity chromatography may be performed by
standard methods (see e.g., Ausubel et al., supra). Once isolatcd, the
recombinant protein may, if desired, be further purified by e.g., by high
performance liquid chromatography (HPLC; e.g., see Fisher, Laboratory
Techniques In Biochcmistry And Molecular Biolo~y, Work and Burdon, eds.,
Elsevier, 1980).
Polypeptides of the invention, particularly short p28 Bap3 1 fragmcnts,
can also be produced by chemical synthesis (e.g., by the methods described in
Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co.,
Rockford, IL). These general techniques of protein expression and purification
can also be used to produce and isolate useful p28 Bap3 1 fragments or analogs,
as described herein.
Those skilled in the art of molecular biology will understand that a wide
variety of expression systems employing a wide variety of host cells may be
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-54-
used to produce the recombinant protein. The p28 Bap31 polypeptides may be
produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g, S.
cerevisiae, insect cells such as Sf9 cells, or ms~rnm~ n cells such as COS-1,
NIH 3T3, or HeLa cells). These cells are publically available, for example,
from the American Type Culture Collection, Rockville, MD; see also Ausubel
et al., supra). The method of transduction and the choice of expression vehicle
will depend on the host system selected. Transformation and transfection
methods are described, e.g., in Ausubel et al. (supra), and expression vehicles
may be chosen from those provided, e.g., in Pouwels et al., supra.
Testing for the Presence of p28 Bap31 Biological Activity
The identified Bcl-2-interacting protein, p28 Bap31, has been shown to
be linked to apoptotic cell death in t~vo ways. First, p28 Bap 31 is cleaved at
the tv.~o ICE/FLICE recognition sequences following induction of apoptosis.
Both cleavages are observed in intact cells and their appearance closely
correlates with pro-enzyme activation of Caspase-3 (CPP 32) and cell death
following infection of KB cells with adenovirus type 5 lacking expression of
E 1 B 19K. Secondly, ectopic expression of the p20 cleavage product of p28
induces apoptosis. Without being limited to a particular model, the p20
cleavage product may induce apoptosis by a trans-dominant mechanism that
interferes with normal p28 Bap31 function.
Identification of p28 Bap31 allows the study of p28 Bap31 co-
association with a Bc1-2 protein (e.g., BCI-XL and Bc1-2) and/or pro-FLICE, an
ability to be cleaved to produce the p20 product, and involvement in apoptosis-
associated cellular events. These p28 Bap31 biological activities may also be
evaluated based upon the level of expression of p28 Bap31. For example,
interaction of p28 Bap31 with pro-FLICE and a Bc1-2 protein may be measured
CA 022~2422 1998-10-20
W098139434 PCT~B98/00706
by utilizing the various methods known in the art and described herein for
measuring protein:protein interactions (e.g., the yeast two-hybrid interaction
system assay). Furthermore, ~rlmini~tration of a p28 Bap3 1 protein or
polypeptide fragment thereof, or a p28 Bap3 1-inhibiting compound may be
- used to modulate apoptosis, as may be measured by apoptosis assays known in
the art and described herein. Preferably, such assays are carried out in a cell
capable of undergoing apoptosis (e.g., human KB cells).
Another method for assessing the biological activity of p28 Bap3 I by
measuring the level of expression of p28 Bap3 1 may be accomplished by
measuring the amount of protein expression using p28 Bap3 1 -specific
antibodies (e.g., thc antibodies described above), or by measuring the amount
of p28 Bap3 1 mRNA, using detectably labelled p28 Bap3 l nucleic acid as a
probe. In addition, the proteolytic cleavage of p28 Bap3 1 into the p20 product
may be detected using the assays described above (see, e.g., Figs. SA and SB).
These assays may also be used to assess the ability of a reagent or compound to
inhibit or enhance the biological activity of the p28 Bap3 1 protein (which is, in
this particular example, an ability to be cleaved to produce the p20 product),
and thus modulate apoptosis.
P28 Bap31 Fragments
Polypeptide fragments comprising various portions of p28 Bap3 l are
useful in identifying regions of these proteins important for their biological
activities (e.g, their abilities to modulate apoptosis). Methods for generating
such fragments are well known in the art (see, for example, Ausubel et al.,
supra) using the nucleotide sequences encoding the full length p28 Bap3 l
polypeptides. For example, a p28 Bap3 1 fragment may be generated by PCR
amplifying the desired fragment using oligonucleotide primers designed based
, ......... . ..
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-56-
upon the p28 Bap3 1 nucleic acid sequence. Preferably the oligonucleotide
primers comprise unique restriction enzyme sites which facilitate insertion of
the fragment into the cloning site of an expression vector. This vector may
then be introduced into a cell by artifice by the various techniques known in the
art and described herein, resulting in the production of p28 Bap3 1 polypeptide
~agment.
In an alternative approach, polypeptide fragments comprising various
portions of p28 Bap3 1 are useful in modulating p28 Bap3 1 mediated apoptosis,
respectively, as may be assessed in the various apoptosis assays known in the
art and described herein. P28 Bap3 1 polypeptide fragments (e.g., a fragment
corresponding to the p20 product) may be used to induce apoptosis in a cell.
P28 Bap31-specific Antibodies
In order to prepare polyclonal antibodies, p28 Bap3 1 polypeptides,
fragments thereof, or fusion proteins containing defined portions or the entire
p28 Bap3 1 polypeptide can be synthesized in bacteria by expression of
corresponding DNA sequences in a suitable cloning vehicle. A common source
of antigen for producing antibodies are fusion proteins. Two widely used
expression systems for E. coli are lacZ fusions using the pUR series of vectors
and t~pE fusions using the pATH vectors. The proteins may be purified, and
then may be coupled to a carrier protein, mixed with Freund's adjuvant (to help
stimulate the antigenic response by the animal), and injected into laboratory
anim~ls of choice (e.g., rabbits). Alternatively, p28 Bap31 proteins can be
isolated from expressing cultured cells. Following booster injections at bi-
weekly intervals, the immunized rabbits are bled and the sera isolated. The
sera may be used directly or may be purified prior to use by various methods
including affinity chromatography employing reagents such as Protein A-
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Sepharose, Antigen Sepharose, and Anti-rabbit-Ig-Sepharose. The sera may be
used to probe tissue extracted proteins which have been resolved on a
polyacrylamide gel to identify the p28 Bap31 polypeptides. Alternatively,
synthetic peptides can be made that correspond to the antigenic portions of the
protein and used to immunize the ~nim~
In order to generate polypeptide fragments or full-length protein for use
in making p28 Bap31-specific antibodies, the coding sequences can be
expressed as a C-t~rminal fusion with glutathione S-transferase (GST; Smith et
al., Gene 67: 31-40, 1988). The GST fusion proteins can be purified on
glutathione-Sepharose beads, eluted with glutathione, and cleaved with
thrombin (at the engineered cleavage site), and purified to the degree required
to successfully immunizc rabbits. Primary immunizations can be carried out
with Freund's complete adiuvant and subsequent immunizations performed
with Freund's incomplete adjuvant. Antibody titers are monitored by Western
blot and immunoprecipitation analyses using the thrombin-cleaved p28 Bap31
fragment of the GST fusion protein. lmmllne sera are affinity purified using
p28 Bap31 proteins coupled to CNBr-Sepharose. Antiserum specificity is
determined using an unrelated GST protein which may be generated by PCR
using known sequences.
The skilled artisan will understand that p28 Bap31 -specific murine
monoclonal antibodies may be prepared using the p28 Bap31 proteins
described above and standard hybridoma technology (see, e.g., Kohler et al.,
Nature 256: 495-497, 1975; Kohler et al., Eur. J. Immunol. 6: 511-519, 1976;
Kohler et al., Eur. J. Immunol. 6: 292-295, 1976; Hammerling et al., In
Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York, NY,
1981; Ausubel et al., supra). To produce p28 Bap31-specific murine
monoclonal antibodies, for example, mice may be immunized with
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recombinant p28 Bap3 1 proteins and fusion proteins described above, as well
as p28 Bap3 1 isolated from cells which normally express p28 Bap3 1 or p28
Bap3 1 isolated from tissues. Cellular extracts or recombinant protein extracts
containing the p28 Bap3 1, may, for example, be injected with Freund's
adjuvant into mice. Following primary injection and subsequent boosters,
serum samples may be collected and assessed for an ability to bind p28 Bap3 1.
Spleens from mice whose sera are p28 Bap3 1 reactive are removed and minced
to attain a suspension of isolated spleen cells. The spleen cells serve as a
source of B Iymphocytes, some of which are producing antibody of the
a~pro~uliate specificity. The spleen cells are fused with a permanently growing
myeloma partner cells, and the products of the fusion are plated into 96 well
plates in culture media containing a selective agent such as hypoxanthine,
aminopterine, and thymidine (HAT). The wells are then screened by ELISA to
identify those containing cells producing antibody capable of binding p28
Bap3 1 protein or polypeptide fragments or mutants thereof. The cells
producing p28 Bap3 1-reactive antibodies are returned to the selective culture
media and cloned by limiting dilution into 96 well plates. After a period of
growth, wells are again screened to identify p28 Bap3 1 specific antibody-
producing cells. Sevcral cloning procedures are carried out until over 90% of
the wells contain single clones which are positive for p28 Bap3 1 specific
antibody production. From this procedure a stable line of hybridoma clones is
established, each of which produces a monoclonal antibody capable of
specifically binding to the p28 Bap3 1. Ascites containing large amounts of p28
Bap3 1 specific monoclonal antibody can be generated by injecting the p28
Bap3 1 specific monoclonal antibody secreting hybridoma cells into a mouse
having the same MHC haplotype as the mouse whose spleen cells were used to
make the hybridoma cell clone.
... , . , . . . .. . , . . ~ . . _
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The p28 Bap31-reactive monoclonal antibodies can be further purified
by affinity chromatography using Protein A Sepharose, ion-exchange
chromatography, as well as variations and combinations of these techniques.
Truncated versions of monoclonal antibodies may also be produced by
recombinant methods in which plasmids are generated which express the
desired monoclonal antibody fragment(s) in a suitable host.
As an alternate or adjunct immunogen to GST fusion proteins, peptides
corresponding to relatively unique hydrophilic regions of p28 Bap31 may be
generated and coupled to keyhole limpet hemocyanin (KLH) through an
introduced C-terminal Iysine. Antiserum to each of these peptides is similarly
affinity purified on peptides conjugated to CNBr-Sepharose, and specificity
may bc tcsted by ELISA and Western blotting using peptide conjugates, and by
Western blotting and immunoprecipitation using p28 Bap31 expressed as GST
fusion proteins. Methods describing these analysis techniques are well known
in the art (see, for example, Ausubel et al., supra; Hammerling et al., supra;
and Sambrook, e~ al., Molecular Cloning: A Laboratory Manual (2d ed.), CSH
Press, 1989).
Antibodies that specifically recognize p28 Bap31, or fragments thereof
arc considered useful in the invention. They may, for example, be used in an
immunoassay to monitor p28 Bap31 protein exprcssion levels or to determine
the subcellular location of the polypeptides or fragments produced by a
m~mm~l. P28 Bap31 fragments may also be used to inhibit binding of the p28
Bap31 to a Bc1-2 protein and/or to pro-FLICE. Antibodies that inhibit p28
Bap31 interactions described herein may be especially useful in preventing
apoptosis in cells undergoing undesirable cell death.
Preferably, antibodies of the invention are produced using p28 Bap31
protein amino acid sequences that do not reside within highly conserved
.
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-60-
regions, and that appear likely to be antigenic, as analyzed by criteria such asthose provided by the Peptide Structure Program (Genetics Computer Group
Sequence Analysis Package, Program Manual for the GCG Package, Version 7,
1991) using the algorithm of Jameson and Wolf (CABIOS 4: 181, 1988).
These fragments can be generated by standard techniques, e.g., by the PCR,
and cloned into the pGEX expression vector (Ausubel et al., supra). GST
fusion proteins are expressed in E. coli and purified using a glutathione agarose
affinity matrix as described in Ausubel et al. (supra). To generate rabbit
polyclonal antibodies, and to minimize the potential for obtaining antisera thatis non-specific, or exhibits low-affinity binding to p28 Bap31, two or three
fùsions are generated for each protein, and each fusion is injected into at least
two rabbits. Antisera are raised by injections in series, preferably including at
least three booster injections.
In addition to intact monoclonal and polyclonal p28 Bap31 -specific
antibodies, the invention features various genetically engineered antibodies,
humanized antibodies, and antibody fragments, including F(ab')2, Fab', Fab,
Fv, and sFv fragments. Methods for making hllm~ni7ed antibodies are known
in the art, e.g., monoclonal antibodies with a desired binding spccificity can be
commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,
CA). Fully human antibodies, such as those expressed in transgenic animals,
are also features of the invention (Green et al., Nature Genetics 7: l 3-21,
1994).
In addition, Ladner (U.S. Patent 4,946,778 and 4,704,692) describes
methods for preparing single polypeptide chain antibodies. Ward et al. (Nature
341: 544-546, 1989) describe the l)rc~aldlion of heavy chain variable domains,
which they term "single domain antibodies," which have high antigen-binding
affinities. McCafferty et al. (Nature 348: 552-554, 1990) show that complete
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antibody V domains can be displayed on the surface of fd bacteriophage, that
the phage bind specifically to antigen, and that rare phage (one in a million) can
be isolated after affinity chromatography. Boss et al. (U.s. Patent 4,816,397)
describe various methods for producing immunoglobulins, and
immunologically functional fragments thereof, which include at least the
variable domains of the heavy and light chain in a single host cell. Cabilly et
al. (U.s. Patent 4,816,567) describe methods for preparing chimeric antibodies.
These p28 Bap31 -specific reagents are also features of the instant invention.
Use of p28 Bap31-specific Antibodies
Antibodies that specifically bind to p38 Bap31 or fragments thereof may
be used, as noted above, to detect protein expression and modulate the
biological activity of the protein. ln addition, the antibodies may be coupled to
compounds for diagnostic and/or therapeutic uses such as radionucleotides for
imaging and therapy and liposomes for the targeting of compounds to a specific
tissue location.
Detection of p28 Bap31 Gene Expression
As noted, the antibodies described above may be used to monitor p28
Bap31 expression in cells or tissues using Westem blot or immunoprecipitation
analysis (according to methods described herein and in the art.
Another method which may be used to detect the expression of p28
Bap31 genes is in situ hybridization, which relies upon the hybridization of a
specifically labelled nucleic acid probe to the cellular RNA in individual cellsor tissues. Therefore, it allows the identification of mRNA within intact
tissues, such as the brain. In this method, oligonucleotides or cloned nucleic
acid (RNA or DNA) fragments corresponding to unique portions of the desired
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-62-
gene (e.g, p28 Bap3 1) are used to detect specific mRNA species, e.g, in a
neuron.
Identification of Compounds that Modulate p28 Bap31
Molecules that are found, by the methods described herein, to effectively
modulate p28 Bap3 l gene expression or biological activity may be tested
further in animal models. If they continue to function successfully in an in vivo
setting, they may be used as therapeutics to either inhibit or enhance apoptosis,
as a~plopllate.
A) Compounds that Modu}ate p28 Bap3 l Biolo~ical Activity
P28 Bap3 1 -encoding cDNAs may be used to facilitate the identification
of compounds that increase or decrease expression of these proteins. In one
approach for detecting a compound that modulates p28 Bap3 1 expression,
candidate compounds are added, in varying concentrations, to the culture
medium of cells expressing p28 Bap3 1 mRNA. P28 Bap3 1 expression is then
measured, for example, by Northern blot analysis (Ausubel et al., supra) using
a p28 Bap3 1 DNA, or cDNA or RNA fragment, as a hybridization probe. The
level of p28 Bap3 1 expression in the presence of the candidate compound is
compared to the p28 Bap3 1 expression level in the absence of the candidate
compound, all other factors (e.g., cell type and culturc conditions) being equal.
The effect of candidate compounds on p28 Bap3 1 expression may,
instead, be measured at the level of translation by using the general approach
described above with standard protein detection techniques, such as Western
blotting or immunoprecipitation with p28 Bap3 1-specific antibodies (for
example, the antibodies described herein).
In an alternative approach to detecting compounds which modulate p28
Bap3 1 at the level of transcription, candidate compounds may be tested for an
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-63-
ability to regulate a reporter gene whose expression is directed by the p28
Bap3 1 promoter. For example, to detect a compound that can modulate p28
Bap3 1 expression, candidate compounds may then be added, in varying
concentrations, to the culture medium of cells transfected with a expression
plasmid comprising a luciferase reporter gene operably linked to the p28 Bap3 1
promoter. Luciferase expression levels may then be measured on a
luminometer by subjecting the compound-treated transfected cells to standard
luciferase assays known in the art, such as the luciferase assay system kit
commercially available from Promega (Madison, Wl). Luciferase expression
in the presence of the candidate compound is compared to the level of
luciferase expression in the absence of the candidate compound, all other
factors (e.g., cell type and culture conditions) being equal.
Compounds that modulate the level of p28 Bap3 1 expression may be
purified, or substantially purified, or may be one component of a mixture of
compounds such as an extract or supernatant obtained from cells (Ausubel et
al., supra). In an assay of a mixture of compounds, p28 Bap3 1 expression is
tested against progressively smaller subsets of the compound pool (e.g.,
produced by standard purification techniques such as HPLC or FPLC) until a
single compound or minim~l number of effective compounds is demonstrated
to modulate p28 Bap3 1 expression levels.
Compounds may also be screened for an ability to modulate other
biological activities of p28 Bap3 1 . In this approach, the degree of p28 Bap3 1biological activity in the presence of a candidate compound is compared to the
degree of p28 Bap3 I biological activity in its absence, under equivalent
conditions. The biological activity of p28 Bap3 1 may be assayed by its role in
apoptosis. The biological activity of p28 Bap3 1 may additionally be assayed
by its ability to be cleaved into the p20 product. Again, the screen may begin
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with a pool of candidate compounds, from which one or more useful p28
Bap31 biological activity-modulating compounds is isolated in a step-wise
fashion.
A compound that induces an increase in expression and/or biological
activity of p28 Bap31 or the p20 Bap31 product is considered particularly
useful in the invention; such a compound may be used, for example, as a
therapeutic to increase cellular levels of p28 Bap31 or the p20 Bap31 product
and/or the biological activities of these proteins, and thereby exploit their
abilities to promote apoptosis in a cell (e.g., a cancer cell) which has a reduced
level of apoptosis.
A compound that inhibits the levels of p28 Bap31 or the p20 Bap31
product and/or biological activities of these proteins may be used to increase
cellular proliferation in cells which show an undesirably high level of
apoptosis. This would be advantageous in the treatrnent of degenerative
diseases, such as neurodegenerative diseases (e.g., Alzheimer's disease,
Huntington's disease) or other tissue-specific degenerative diseases (e.g.,
cirrhosis of the liver, T-lymphocyte depletion in AIDS).
One method for detecting compounds that modulate the biological
activity of p28 Rap31 is to screen for compounds that alter the physical
interaction of p28 Bap31 with either a Bc1-2 protein (e.g., Bc1-2 or BCI-XL) or
pro-FLICE. These compounds are detected by adapting yeast two-hybrid
expression systems known in the art. Yeast two-hybrid systems detect protein
interactions using a transcriptional activation assay and are generally described
by Gyuris et al. (Cell 75: 791-803, 1993) and Field et al. (Nature 340: 245-246,1989). Reagents for yeast two-hybrid systems are also are commercially
available from Clontech (Palo Alto, CA). Once an interaction between p28
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Bap31 and either a Bc1-2 protein or pro-FLICE is detectable in yeast,
compounds may be screened for an ability to alter that interaction.
In one example, if the Clontech Matchmaker Two-Hybrid System
(Catalog number K1605-1) is used, p28 Bap31, Bc1-2, or BC1-XL polypeptides
fused to either the GAL4 DNA binding domain or activator domain can be
made by cloning the nucleic acid sequences encoding full length polypeptides
or fragments thereof into the a~plo~liate vectors available in the kit. A vector,
for example, a DNA binding domain vector, encoding a p28 Bap31 protein (or
fragment thereof) fusion is then co-transformed with a vector, for example an
activation domain vector, encoding a Bc1-2 protein (or fragment thereof) fusion
into the yeast strains included in the kit. Polypeptides of p28 Bap31 which
form strong interactions with a Bc1-2 polypeptide will result in blue colonies on
X-gal containing media. A candidate compound or combinations thereof which
are being screened for an ability to alter interactions between p28 Bap31 and a
Bc1-2 protein can be a-lmini.~tered to the yeast which form blue colonies on X-
gal plates. A compound which can alter the color of the colony on X-gal plates
as compared to an untreated colony is a compound that alters the interaction
between p28 Bap31 and the protein ( Bc1-2) which ws co-expressed as an
activation fusion protein in thc compound administered blue yeast colony.
By disrupting thc association of p28 Bap31 to either a Bc1-2 protein or
pro-FLlCE, such a compound may function as modulator of p28 Bap31 -
associated apoptotic cell death and may include peptide and non-peptide
molecules such as those present in cell extracts, m~mm~ n serum, or growth
medium in which m~mm~lian cells have been cultured. The effect of
interaction-disrupting compounds on p28 Bap31 apoptosis activity may be
measured by any standard assay, for example, those described herein.
. ..... .
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Another method for detecting compounds that modulate the biological
activity of p28 Bap31 is to screen for compounds that affect the in vitro
association of p28 Bap31 with Bc1-2. Compounds may be assessed for an
ability to affect the binding of p28 Bap31 to Bc1-2 by adding a candidate
compound to p28 Bap31 protein immunoprecipitates from cells co-transfected
with p28 Bap31 and Bc1-2 protein-encoding nucleic acids. Following Western
blotting analysis with an anti-Bcl-2 antibody (such as clones 100 or 4C 11
commercially available from Santa Cruz Biotech., lnc., Santa Cruz, CA),
whether or not a compound affects the binding of p28 Bap31 to Bc1-2 may be
assessed by col~lpalillg the amount of anti-Bcl-2 immunoreactive protein in a
compound-treated reaction versus an untreated reaction. The amount of Bc1-2
may be assessed, for example, by quantitation on a Phorphorimager.
ln a method to detect a compound that affects the cleavage of p28 Bap31
to p20, a candidate compound may be added to the 35S-labelled transcription-
translation product of p28 cDNA, which is then incubated with increasing
concentrations of ICE (caspase- 1). Following resolution by SDS-PAGE, the
products can compared to a reaction carried out in absence of the candidate
compound (control). A compound that affects the cleavage of p28 Bap31 as
compared to the control reaction is a compound that modulates the biological
activity of p28 Bap31.
By either affecting the association of p28 Bap31 with a Bc1-2 protein or
with pro-FLICE, or by affecting the cleavage of p28 Bap31 to p20, a compound
may function as modulator of p28 Bap31-associated apoptotic cell death. Such
a compound may include peptide and non-peptide molecules such as those
present in cell extracts, m~mm~ n serum, or growth medium in which
m~mm~ n cells have been cultured.
B) Compounds that Modulate p20 Biolo~ical Activity
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A compound that affects p20 biological activity may be assessed by
treating cells with a candidate compound prior to intracellular expression of
p20. Apoptosis may be assessed by a variety of assays described herein
including, without limitation, DNA fragmentation, membrane blebbing, and
Annexin V reactivity at the cell surface. A compound found to either enhance
or inhibit the ability of p20 to induce apoptosis in a p20 expressing cell relative
to a p20 expressing cell untreated with the compound is a compound that
modulates p20 biological activity. Such a compound is useful to cither induce
cell death in hyperplasia cells (e.g., cancerous cell), or inhibit cell death in cells
that undergo inal~plo~liately high levels of apoptosis (e.g., T cells in HIV
infected individuals).
High through-put screens may be employed to identify the protease
responsible for the cleavage of p28 Bap3 1 into p20. Since p20 expression will
cause apoptosis, a compound that produces p20 may be readily identified using
the various apoptosis assays known in the art and described herein. For
example, a cell may be stably transfected with a high expressing plasmid
encoding for p28 Bap3 1. The stable p28 Bap3 1 cells may then be transiently
transfected in groups with pools of plasmids from a cDNA expression library,
and thc groups of cells examined for apoptosis. Once a group of cells is
identified as undergoing apoptosis at a frequency higher than is normally
observed in p28 Bap3 I stably expressing cells, the plasmid DNA used to
transfect the cells is sub-divided and each division transfected into groups of
~ p28 Bap3 1 stably expressing cells. Following repetition of this
transfect/subdivision, an cDNA encoding a protein capable of cleaving p28
Bap3 1 to produce p20 may be isolated and subjected to DNA sequence
analysis. Once identified, the protein (z.e., a protease) capable of cleaving p28
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Bap3 1 to produce p20 may be subjected to the manipulations described herein
for p28 Bap3 1.
Genes Related to p28 Bap31
Standard techniques, such as the polymerase chain reaction (PCR) and
DNA hybridization, may be used to clone additional p28 Bap3 1 homologues in
other species. For example, Southern blot analysis may be made of genomic
DNA of various organisms (e.g., C. elegans or mice) with nucleic acid probes
generated from the nucleic acid sequences encoding p28 Bap3 1. Hybridization
at low stringency may reveal bands that correspond to p28 Bap3 1, and/or
related family members. P28 Bap3 1 probes may be based on the codon
preference displayed by the organism, or may be degenerate probes based upon
all possible codon combinations, or a combination of codon preference and
codon degeneracy. This probc may then be used to screen either genomic or
cDNA libraries for sequences which hybridize to the probe. Th~ls, additional
p28 Bap3 1 may be identified using low stringency hybridization. Furthermore,
p28 Bap3 1 probes may be used as primers to clone additional p28 Bap3 1
related genes by RT-PCR.
Therapies
Therapies may be designed to prevent or treat a p28 Bap3 I gene defect
or an inadequate or excessive amount of p28 Bap3 1 gene expression, and thus
modulate apoptosis. In considering various therapies, it is understood that suchtherapies may be targeted at any tissues demonstrated to express the p28 Bap3 1
protein. In particular, therapies to enhance p28 Bap3 1 gene expression are
useful in promoting apoptosis in neoplastic cells. Apoptosis-inducing p28
.. . .. ~ , .
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Bap3 1 reagents may include, without limitation, full length or fragment p28
Bap3 1 polypeptides, p28 Bap3 1 mRNA, or any compound which increases p28
Bap3 1 expression and apoptosis-inducing activity.
a) Protein Therapy
Treatrnent or prevention of an ina~plo~ ate amount of apoptosis may be
accomplished by correcting dysfunction caused by mutant, deficient, or surplus
p28 Bap3 I protein with normal protein, by modulating the function of mutant
protein, or by delivering normal p28 Bap3 1 proteins to the ap~)lo~,iate cells.
The pathophysiological pathway (e.g., the apoptosis signalling pathway) in
which the protein participates may also be modified in order to correct the
physiological defect.
To ~1~ini~ter p28 Bap3 1 proteins to cells which either no longer
express sufficient amounts of either of these proteins, or produce mutant
dysfunctional protein, it is necessary to obtain largc amounts of pure p28
Bap3 I from cultured cell systems which can express the proteins. Delivcry of
the proteins to the affected tissues and/or cells (e.g., cancerous cells) can then
be accomplished using a~rol liate packaging or ~-lmini~trating systems.
Alternatively, compounds which act as p28 Bap3 1 agonists may be
administered to thc affected cells and/or tissues and in this manner produce thedesired physiological effect. Methods for finding such compounds are
provided herein.
b) Gene Therapy
Gene therapy is another potential therapeutic approach for treating cells
which lack a normal level of expression of a functional p28 Bap3 1. In one
approach, copies of the functional p28 Bap3 1 gene are introduced into selected
tissues to encode for the normal level of expression of the functional protein in
affected cell types (e.g., cancer cells which show reduced levels of apoptosis or
... . . .. .. .. .... . .
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degenerative cells which show an increased level of apoptosis). The gene must
be delivered to those cells in a form in which it can be taken up and encode forsufficient protein to provide effective function.
Transducing retroviral vectors are particularly useful for somatic cell
gene therapy because of their high efficiency of infection and stable integration
and expression. The targeted cells, however, must be able to divide and require
high levels of normal protein expression. For example, to treat a deficiency
involving p28 Bap3 1, the full length p28 Bap3 1 gene, or portions thereof, may
be cloned into a retroviral vector with its expression directed by its endogenous
promoter or by the retroviral long terminal repeat or by a promoter specific forthe target cell type of interest (such as a neuron). Other viral vectors which can
be used include adeno-associated virus, vaccinia virus, bovine papilloma virus,
or a herpes virus.
Gene transfer could also be achieved using non-viral means requiring in
vitro transfection of affected cells with p28 Bap3 1 genes or fragrnents thereof.
This in vitro transfection methods include calcium phosphate, DEAE dextran,
electroporation, and protoplast fusion. Liposomes may also be potentially
beneficial for delivery of DNA into a cell. Although these and other methods
known in the art are available, many of these are of low transfection efficiency.
Strategies based upon antisense RNA may be employed to explore p28
Bap3 1 gene function and as a basis for therapeutic drug design. Antisense
RNA based strategies are particularly useful in controlling p28 Bap3 1
overexpression in cells or tissues undergoing an increased level of apoptosis
(e.g., neurons in a patient with a neurodegenerative disorder). The principle isbased upon the hypothesis that sequence-specific suppression of gene
expression can be achieved by intracellular hybridization between mRNA and a
complementary antisense species. The formation of a hybrid RNA duplex may
_. .
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then interfere with the processing, transport, translation, and/or stability of the
target p28 Bap31 mRNA. Antisense strategies may use a variety of approaches
including, without limitation, the use of antisense oligonucleotides and
injection of antisense p28 Bap31 RNA to a cell or tissue that is expected to
undergo undesired apoptosis. The antisense p28 Bap31 RNA may be produced
and isolated by any standard technique, but is most readily produced by in vitrotranscription using an antisense cDNA under the control of a high efficiency
promoter (e.g., the T7 promoter). Administration of antisense p28 Bap31 RNA
to cells can be carried out by any of the methods for direct nucleic acid
administration described above.
Introduction of normal p28 Bap31 genes into the affected cells of a
patient may also be useful therapy. In this procedure, normal p28 Bap31-
encoding nucleic acid is transfected into a cultivatable cell type, which may ormay not be derived from the patient, such that the nucleic acid is either
incorporated into the chromosome, or exists and replicates episomally.
Preferably, the cell has the same MHC haplotype as the affected patient. The
transfected cells are then injected serotologically into the targeted tissue(s) of
the patient.
Retroviral, adenoviral, adenovirus-associated viral vcctors, or other viral
vectors with the appropriate tropism for cclls likely to bc involved in apoptosis
(for examplc, lymphocytes) may be uscd as gene transfer delivery systems for a
therapeutic p28 Bap31 gene construct. Numerous vectors useful for this
purpose are generally known (Miller, A. D., Human Gene Ther. 1: 5-14, 1990;
Friedmann T., Science 244: 1275-1281, 1989; Eglitis and Anderson,
BioTechniques 6: 608-614, 1988; Tolstoshev and Anderson, Curr. Opin. in
Biotech. 1: 55-61, 1990;Cornettaetal., Prog.NucleicAcidRes.Mol.Biol.
36: 311-322, 1989; Anderson, W. F., Science 226: 401-409, 1984; Moen, R.C.,
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W O 98/39434 PCT~B98/00706
Blood Cells 17: 407-416, 1991; Miller et al., BioTechniques 7: 980-990, 1989;
Le Gal La Salle et al., Science 259: 988-990, 1993; and Johnson, L. G., Chest
lû7: 77S-83S, 1995). Retroviral vectors are particularly well developed and
have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:
570-578, 1990; Anderson et al., U.s. Patent No. 5,399,346). Non-viral
approaches may also be employed for the introduction of therapeutic DNA into
cells otherwise predicted to undergo apoptosis. For example, p28 Bap31
proteins may be introduced into a neuron or a T cell by lipofection (Felgner et
al., Proc. Natl. Acad. Sci. USA 84: 7413-7417, 1987; Ono et al., Neurosci.
Lett. 117: 259-2G3, 1990; Brigham et al., Am. J. Mcd. Sci. 298: 278-281, 1989;
Straubinger et al., Methods Enzymol. 101: 512-527, 1983), asialorosonucoid-
polylysine conjugation (Wu et al., J. Biol. Chem. 263: 14621-14624, 1988; Wu
et al., J. Biol. Chem. 264: 16985-16987, l 989); or, less preferably, micro-
injection under surgical conditions (Wolff et al., Science 247: 1465-1468,
1990).
For any of the methods of application described above, the therapeutic
p28 Bap31 DNA construct is preferably applied (for example, by injection) to
the site of cells or tissues affected by either abnormally high or low levels ofapoptosis. However, it may also be applied to tissue in the vicinity of the
affected site or to a blood vessel supplying the cells (e.g., cancerous cells) at
the affected site.
In the constructs described, p28 Bap31 gene expression can be directed
by any suitable promoter (e.g., the human cytomegalovirus (CMV), simian
virus 40 (SV40), or metallothionein promoters), and regulated by any
a~ ;ate m~mm~ n regulatory element. For example, if desired, enhancers
known to preferentially direct gene expression in neural cells, Iymphocytes, or
muscle cells may be used to direct p28 Bap31 gene expression. Alternatively,
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if a p28 Bap3 1 genomic clone is used as a therapeutic construct, regulation maybe mediated by the cognate regulatory sequences or, if desired, by regulatory
sequences derived from a heterologous source, including any of the promoters
or regulatory elements described above.
Ideally, the amount of p28 Bap3 1 protein produced by any gene therapy
approach will result in cellular levels of protein that are at least equivalent to
the normal cellular level of protein in an unaffected cell. Treatment by any p28Bap3 1- mediated gene therapy approach may be combined with more
traditional therapies.
Another therapeutic approach within the invention involves
~lmini.~tration of recombinant p28 Bap3 I protein, either directly to the site of a
cells or tissues affected by increased or decreased levels of apoptosis (for
example, by injection) or systemically (for example, by any conventional
recombinant protein ~1ministration technique). The dosage of p28 Bap3 1
depends on a number of factors, including the size and health of the individual
patient.
Administration of p28 Bap31 Polypeptides, Genes, or Modulators of p28
Bap31 Synthesis or Function
A p28 Bap3 1 protein, gene, or modulator may be ~lministered within a
pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage forrn.
Conventional pharmaceutical practice may be employed to provide suitable
formulations or compositions to ~(lmini.~ter neutralizing p28 Bap3 1 antibodies
or p28 Bap3 1-inhibiting compounds (e.g., a compound that prevents the
cleavage of p28 Bap3 1 to p20) to patients suffering from a disease (e.g., a
degenerative disease) that is caused by excessive apoptosis. Likewise,
formulations or compositions may be provided to ~(lminister compounds which
..... . . ..
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increase p28 Bap3 1 expression (e.g, a compound that induces p28 Bap3 1
expression and/or activity or recombinant p28 Bap3 1 protein) to patients
suffering from a disease (e.g., cancer) that is caused by a reduced level of
apoptosis. Such ~1mini~kation may be limited to the affected tissue or cell.
Administration may also begin before the patient is symptomatic. Any
a~pro,o~iate route of ~lmini~tration may be employed, for example,
~lmini~tration may be parentel~l, intravenous, intra-arterial, subcutaneous,
intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,
intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal,
aerosol, by suppositories, or oral administration. Therapeutic formulations may
be in the form of liquid solutions or suspensions; for oral a-lmini~tration,
formulations may be in the forrn of tablets or capsules; and for intranasal
formulations, in the form of powders, nasal drops, or acrosols.
Methods for m~king formulations are well known in the art and may be
found, for example, in Remington's Pharmaceutical Sciences, (18th edition), ed.
A. Gennaro, 1990, Mack Publishing Company, Easton, PA. Formulations for
parenteral administration may, for example, contain excipients, sterile water, or
saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, or hydrogenated napthalenes. Biocompatiblc, biodegradablc lactide
polymer, lactide/glycolidc copolymer, or polyoxyethylene-polyoxypropylene
copolymers may be used to control thc release of the compounds. Other
potentially useful parenteral delivery systems for p28 Bap3 1 modulatory
compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Forrnulations for inh~l~tion may
contain excipients, for example, lactose, or may be aqueous solutions
cont~inin~, for example, polyoxyethylene-9-lauryl ether, glycocholate and
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W 098139434 PCT~B98100706
deoxycholate, or may be oily solutions for a~lmini~tration in the form of nasal
drops, or as a gel.
If desired, treatment with a p28 Bap3 1 polypeptide or gene, or a p28
~ Bap3 1 modulatory compound may be combined with more traditional therapies
for the disease such as surgery, chemotherapy, or radiation therapy for cancer,
and antiviral therapy for AIDS.
Detection of Conditions Involving Altered Apoptosis
P28 Bap3 1 polypeptides and nucleic acid sequences may be used
diagnostically to detect or monitor conditions involving aberrant levels of
apoptosis. For example, a decreased expression of p28 Bap3 1 is found to be
correlated with decreased apoptosis in humans. Similarly, overexpression of
p28 Bap3 1 is found to be associated with increased apoptosis. Accordingly, a
decrease or increase in the level of p28 Bap3 1 production or expression may
provide an indication of a deleterious condition. Levels of p28 Bap3 1
expression may be assayed by any standard technique. For example, p28
Bap3 1 expression in a biological sample (e.g., a biopsy) may be monitored by
standard Northern blot analysis or may be aided by PCR (see, e.g., Ausubel et
al., supra; PCR Technolo~y: Principles and Applications for DNA
Amplification, H.A. Ehrlich, Ed. Stockton Press, NY; Yap et al., Nucl. Acids
Rcs.19:4294,1991).
Alternatively, a biological sample obtained from a patient may be
analyzed for one or more mutations in the p28 Bap3 1 nucleic acid sequences
using a mismatch detection approach. Generally, these techniques involve PCR
amplification of nucleic acid from the patient sample, followed by
identification of the mutation (i.e., mi~m~tch) by either altered hybridization,aberrant electrophoretic gel migration, binding or cleavage mediated by
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mismatch binding proteills, or direct nucleic acid sequencing. Any of these
techniques may be used to facilitate mutant p28 Bap31 detection, and a}l are
well known in the art; examples of particular techniques are described, without
limitation, in Orita et al., Proc. Natl. Acad. Sci. USA 86: 2766-2770, 1989;
Sheffield et al., Proc. Natl. Acad. Sci. USA 86: 232-236, 1989; Ausubel et al.,
supra).
In yet another approach, immunoassays may be used to detect or
monitor p28 Bap31 expression in a biological sample. P28 Bap31 - specific
polyclonal or monoclonal antibodies (produced as described above) may be
used in any standard immunoassay format (e.g., ELJSA, Western blot, or RIA)
to measure expression levels of p28 Bap31. These levels would be compared
to p28 Bap31 levels in a unaffected biological sample of the same source and
kind, with a decrease in p28 Bap31 production indicating a condition involving
decreased apoptosis. Examples of immunoassays are described, e.g., in
Ausubel et al., supra. Immunohistochemical techniques may also be utilized
for p28 Bap31 detection. For example, a tissue sample may be obtained from a
patient, sectioned, and stained for the presence of p28 Bap31 using an anti-p28
Bap31 antibody and any standard detection system (e.g., one which includes a
secondary antibody conjugated to horseradish peroxidase). General guidance
regarding such techniques may be found in the art (sec, for cxample, Bancroft
and Stevens (Theory and Practice of Histological Technigues, Churchill
Livingstone, 1982, and Ausubel et al., supra).
In one preferred example, a combined diagnostic method may be
employed that begins with an evaluation of p28 Bap31 protein production (for
example, by immunological techniques or the protein truncation test
(Hogel,o~l et al., Nature Genetics 10: 208-212,1995) and also includes a
nucleic acid-based detection technique designed to identify more subtle p28
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Bap3 l mutations (for example, point mutations). As described above, anumber of mismatch detection assays are available to those skilled in the art,
and any preferred technique may be used. Mutations in p28 Bap31 may be
detected that either result in loss of a normal level of p28 Bap3 l expression or
loss of normal p28 Bap3 l biological activity. In a variation of this combined
diagnostic method, p28 Bap3 l biological activity is measured as apoptotic-
inducing activity using any apyrop..atc apoptosis assay system, or as a Bc1-2
interacting activity using a protein:protein interaction assay (e.g., the yeast two-
hybrid system described above).
Mismatch detection assays also providc an opportunity to diagnose a
p28 Bap3 l-mediated predisposition to diseases caused by an inappropriate
amount of apoptosis. For example, a patient heterozygous for a p28 Bap31
mutation that induces a p28 Bap31 overexpression may show no clinical
symptoms and yet possess a higher than normal probability of developing one
or more types of neurodegcnerative or myelodysplastic disorders, or having
severe sequelae to an ischemic event. Likewise, an a-symptomatic patient
found to be heterozygous for a p28 Bap31 mutation that induces a reduced
level of p28 Bap31 or expression may have an increased risk of developing
cancer. Given such a dia~nosis, a patient may take precautions to minimi7.e his
cxposure to adverse environmental factors (for example, W cxposure or
chemical mutagens) and to carefully monitor his medical condition (for
example, through frequent physical e~min~tions). These types of p28 Bap31
diagnostic approaches may also be used to detect p28 Bap31 mutations in
prenatal screens. The p28 Bap3 l diagnostic assays described above may be
carried out using any biological sample (for example, any biopsy sample or
other tissue) in which p28 Bap3 l is normally expressed. Identification of a
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W O 98/39434 PCTAnB98/00706
mutant p28 Bap3 1 gene may also be assayed using these sources for test
samples.
A mutation in either p28 Bap3 1, particularly as part of a diagnosis for
predisposition to p28 Bap3 l-associated disease, may also be tested using a
nucleic acid sample from any cell, for example, by mismatch detection
techniques. Preferably, the nucleic acid sample is subjected to PCR
amplification prior to analysis.
Preventative Anti-Apoptotic Therapy
In a patient diagnosed to be heterozygous for a p28 Bap3 1 mutation that
increases p28 Bap3 1 expression and/or biological activity or to be susceptible
to such a mutation, or a patient diagnosed with a degenerative disease (e.g.,
neurodegenerative disorders such as Huntington's or ALS diseases), or
diagnosed as HIV positive, any of the above therapies may be administered
prior to the occurrence of the disease phenotype. For example, the therapies
may be provided to a patient who is HIV positive but does not yet show a
~1imini.~hed T cell count or other overt signs of AIDS. In particular, the HIV
positive patient may be treated with compounds shown to decrease p28 Bap3 1
expression or decrease p28 Bap3 1 biological activity which may be
administered by any standard dosage and route of ~lmini~tration (see above).
Alternatively, gene therapy using an antisense p28 Bap3 1 mRNA expression
construct may be undertaken to reverse or prevent the T cell defect prior to thedevelopment of AIDS.
The methods of the instant invention may be used to reduce or diagnose
the disorders described herein in any m~mm:~l, for example, humans, domestic
pets, or livestock. Where a non-human m~mm~l is treated or diagnosed, the
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-79~
p28 Bap3 1 polypeptide, nucleic acid, or antibody employed is preferably
specific for that species.
Characterization of p28 Bap31 Biological Activity and Intracellular
Localization Studies
The ability of p28 Bap3 1 to modulate apoptosis can be defined in in
vitro systems in which alterations of apoptosis can be detected. ~ tnm~lian
expression constructs carrying p28 Bap3 1 cDNAs, which are either full-length
or truncated, may be introduced into cell lines such as KB, CHO, NIH 3T3,
HL60, Rat-1, or Jurkat cells. In addition, SF9 insect cells may be used, in
which case the p28 Bap3 1 genes are preferentially expressed using an insect
baculovirus expression system. Following introduction of expression
constructs carrying p28 Bap3 1 cDNAs encoding either full-length or truncated
polypeptides, cells can be induced to undergo apoptosis by standard methods,
which include serum withdrawal, or application of staurosporine, menadione
(which induces apoptosis via free radical formation), or treatment with anti-Fasor anti-TNF-R1 antibodies. Preferably, human KB cells are transfected by
calcium phosphate precipitation and induced to undergo apoptosis by infection
with adenovirus type 5 lacking expression of ElB l9K (pml716/2072). K B
cells thus infected but either not transfccted, or transfected with a vector that
lacks a p28 Bap3 1 insert are used as a control. The ability of each p28 Bap3 1
construct to induce or inhibit apoptosis upon expression can be quantified by
calculating the survival index ofthe cells, i.e., the ratio of surviving transfected
cells to surviving control cells. These experiments can confirm the presence of
apoptosis inducing activity of the full length p28 Bap3 1 protein and, as
discussed below, can also be used to determine the functional regions of p28
Bap3 1. These assays may also be performed in combination with the
.. . ~, ..... ~ ... ...
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application of additional compounds in order to identify compounds that
modulate apoptosis via p28 Bap3 1.
Other Embodiments
In other embodiments, the invention includes any protein which is
substantially identical to a m~mm~ n p28 Bap3 1 polypeptides; such
homologues include other substantially pure naturally-occurring m~mm~ ,n
p28 Bap3 1 proteins as well as allelic variants; natural mutants; induced
mutants; nucleic acid sequences which encode proteins and also hybridize to
the p28 Bap3 1 nucleic acid sequences under high stringency conditions (e.g.,
washing at 1 X SSC at 65~C with a probe length of at least 20 nuclcotides) or,
less preferably, under low stringency conditions (e.g., washing at 2X SSC at
40~C with a probe length of at least 40 nucleotides); and proteins specifically
bound by antisera directed to a p28 Bap3 1 polypeptide. The term also includes
chimeric polypeptides that include a portion from p28 Bap3 1.
The invention further includes analogs of any naturally-occurring p28
Bap3 I polypeptides. Analogs can differ from the naturally-occurring p28
Bap3 1 protein by amino acid sequence differences, by post-translational
modifications, or by both. Analogs of the invention will generally exhibit at
least 85%, morc preferably 90~/O, and most preferably 95% or even 99%
identity with all or part of a naturally occurring p28 Bap3 1 amino acid
sequence. The length of sequence comparison is at least 15 amino acid
residues, preferably at least 25 amino acid residues, and more preferably more
than 35 amino acid residues. Modifications include in vivo and in vitro
chemical derivatization of polypeptides, e.g., acetylation, carboxylation,
phosphorylation, or glycosylation; such modifications may occur during
polypeptide synthesis or processing or following treatment with isolated
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modifying enzymes. Analogs can also differ from the naturally-occurring p28
Bap3 1 polypeptide by alterations in the primary amino acid sequence. These
include genetic variants, both natural and induced (for example, resulting from
random mutagenesis by irradiation or exposure to ethanemethylsulfate (EMS)
or by site-specific mutagcnesis as described in Sambrook, et al., sup~a or
Ausubel et al., supra). Also included are cyclized peptides, molecules, and
analogs which contain residues other than L-amino acids, e.g., D-amino acids
or non-naturally occurring or synthetic amino acids. In addition to full-length
polypeptides, thc invention also includes p28 Bap3 1 polypeptide fragments. As
used herein, thc terrn "fragment," means at least 20 contiguous amino acids,
preferably at least 30 contiguous amino acids, more prefcrably at least 50
contiguous amino acids, and most preferably at lcast 60 to 80 or more
contiguous amino acids. Fragments of p28 Bap3 1 polypeptides can be
generated by methods known to those skilled in the art or may result from
normal protein processing (e.g., removal of amino acids from the nascent
polypeptide that are not required for biological activity or removal of amino
acids by alternative mRNA splicing or alternative protein processing events).
Preferable fragmcnts or analogs according to the invention are those
which facilitate specific detection of a p28 Bap3 I nuclcic acid or amino acid
sequences in a sample to be diagnosed. Particularly useful p28 Bap3 1
fragments for this purpose include, without limitation, the amino acid
fragments which bind Bc1-2 or BC1 XL, which are retained in p20, which arc
cleaved to create the p20 product, or which are located in the death domain.
All publications and patent applications mentioned in this specification
are herein incorporated by reference to the same extent as if each independent
publication or patent application was specifically and individually indicated tobe incorporated by reference.
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SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: McGill University
~ii) TITLE OF THE INVENTION: METHODS AND REAGENTS FOR
MODULATING APOPTOSIS
~iii) NUMBER OF SEQUENCES: 32
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Clark & Elbing LLP
(B) STREET: 176 Federal Street
(C) CITY: Boston
(D) STATE: MA
(E) COUNTRY: USA
(F) ZIP: 02110
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM ComDatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 03-MAR-98
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 2,198,988 Canada
(B) FILING DATE: 3-Mar-97
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bieker-Brady, Kristina
(B) REGISTRATION NUMBER: 39,109
(C) REFERENCE/DOCKET NUMBER: 50013/004WO1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617-428-0200
(B) TELEFAX: 617-428-7045
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 246 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUBNCE DESCRIPTION: SEQ ID NO:1:
Met Thr Leu Gln Trp Thr Ala Val Ala Thr Phe Leu Tyr Ala Glu Val
I 5 10 15
Phe Val Val Leu Leu Leu Cys Ile Pro Phe Ile Ser Pro Lys Arg Trp
Gln Lys Ile Phe Lys Ser Arg ~eu Val Glu Leu Leu Val Ser Tyr Gly
Asn Thr Phe Phe Val Val Leu Ile Val Ile Leu Val Leu Leu Val Ile
Asp Ala Val Arg Glu Ile Arg Lys Tyr Asp Asp Val Thr Glu Lys Val
80'
Asn Leu Gln Asn Asn Pro Gly Ala Met Glu His Phe His Met Lys Leu
Phe Arg Ala Gln Arg Asn Leu Tyr Ile Ala Gly Phe Ser Leu Leu Leu
100 105 110
Ser Phe Leu Leu Arg Arg Leu Val Thr Leu Ile Ser Gln Gln Ala Thr
115 120 125
Leu Leu Ala Ser Asn Glu Ala Phe Lys Lys Gln Ala Glu Ser Ala Ser
130 135 140
Glu Ala Ala Lys Lys Tyr Met Glu Glu Asn Asp Gln Leu Lys Lys Gly
145 150 155 160
Ala Ala Val Asp Gly Gly Lys Leu Asp Val Gly Asn Ala Glu Val Lys
165 170 175
Leu Glu Glu Glu Asn Arg Ser Leu Lys Ala Asp Leu Gln Lys Leu Lys
180 1~5 190
Asp Glu Leu Ala Ser Thr Lys Gln Lys Leu Glu Lys Ala Glu Asn Glu
195 200 205
Val Leu Ala Met Arg Lys Gln Ser Glu Gly Leu Thr Lys Glu Tyr Asp
210 215 220
Arg Leu Leu Glu Glu His Ala Lys Leu Gln Ala Ala Val Asp Gly Pro
225 230 235 240
Met Asp Lys Lys Glu Glu
245
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
~ (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
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Ala Ala Val Asp Gly
l 5
(2) INFORMATION FOR SEQ ID NO:3:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: C-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Lys Lys Xaa Xaa
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) hENGTH: 45 amino aclds
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Ser Gln Gln Ala Thr Leu Leu Ala Ser Asn Glu Ala Phe Lys Lys Gln
l 5 l0 15
Ala Glu Ser Ala Ser Glu Ala Ala Lys Lys Tyr Met Glu Glu Asn Asp
20 25 30
Gln Leu Lys Lys Gly Ala Ala Val Asp Gly Gly Lys Leu
35 40 45
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ser Gln Gln Ala Thr Leu Leu Ala Ser Asn Glu Ala Phe Lys Lys Gln
l 5 l0 15
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Ala Glu Ser Ala Ser Glu Ala Ala Lys Lys Tyr Met Glu Glu Asn Asp
20 25 30
Gln Leu Lys Lys Gly Ala Ala Glu Asp Gly Asp Lys Leu
35 40 45
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Asp Val Gly Asn Ala Glu Val Lys Leu Glu Glu Glu Asn Arg Ser Leu
1 5 10 15
Lys Ala Asp Leu Gln Lys Leu Lys Asp Glu Leu Ala Ser Thr Lys Gln
Lys Leu Glu Lys Ala Glu Asn Glu Val Leu Ala Met Arg Lys Gln Ser
Glu Gly Leu Thr Lys Glu Tyr Asp Arg Leu Leu Glu Glu His Ala
50 55 60
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Asp Ile Gly Asn Thr Glu Met Lys Leu Glu Glu Asn Lys Ser Leu Lys
1 5 10 15
Asn Asp Leu Arg Lys Leu Lys Asp Glu Leu Ala Ser Thr Lys Lys Lys
Leu Glu Lys Ala Glu Asn Glu Ala Leu Ala Met Gln Lys Gln Ser Glu
Gly Leu Thr Lys Glu Tyr Asp Arg Leu Leu Glu Glu His Ala
50 55 60
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Val Glu Tyr Gly Thr Leu Phe Gln Asp Leu Thr Asn Asn Ile Thr
1 5 10 15
Leu Glu Asp Leu Glu Gln Leu Lys Ser Ala Cys Lys Glu Asp Ile Pro
Ser Glu Lys Ser Glu Glu Ile Thr Thr Gly Ser Ala Trp Phe Ser Phe
Leu Glu Ser His Asn Lys Leu Asp Lys Asp Asn Leu Ser Ile Ile Glu
50 55 60
His Ile Phe
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Ala Glu Tyr Gly Thr Leu Leu Gln Asp Leu Thr Asn Asn Ile Thr
1 5 10 15~eu Glu Asp Leu Glu Gln Leu Lys Ser Ala Cys Lys Glu Asp Ile Pro
Ser Glu Lys Ser Glu Glu Ile Thr Thr Gly Ser Ala Trp Phe Ser Phe
Leu Glu Ser His Asn Lys Leu Asp Lys Asp Asn Leu Ser Tyr Ile Glu
His Ile Phe
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
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Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser
1 5 10 15
Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Gly
Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met
Leu Leu Glu Gln Asn Asp Leu Glu Pro Gly His Thr Glu Leu Leu Arg
50 55 60
Glu Leu Leu
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Met Asp Pro Phe Leu Val Leu Leu His Ser Leu Ser Gly Ser Leu Ser
1 5 10 15~ly Asn Asp Leu Met Glu Leu Lys Phe Leu Cys Arg Glu Arg Val Ser
Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Thr Val
Leu Leu Glu Gln Asn Asp Leu Glu Arg Gly His Thr Gly Leu Leu Arg
50 55 60
Glu Leu Leu
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser
1 5 10 15~lu Asp Leu Ala Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln
30~rg Lys Gln Glu Pro Ile Lys Asp Ala Leu Met Leu Phe Gln Arg Leu
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Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu
50 55 60
Leu Leu
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val
1 5 10 15
Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile
Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile
Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu
50 55 60
Lys Arg Val Cys
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Lys Leu Gln Ala Ala Val Asp Gly Pro Met Asp Lys Lys Glu Glu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Lys Leu Gln Ala Ser Val Arg Gly Pro Ser Val Lys Lys Glu Glu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
~ (A) LENGT~: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Met Asp Tyr Lys Asp Asp Asp Asp Lys Ala
1 5 10
(2) INFORMATION FOR SEQ ID NO:l9:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
CTAGCGCCCG CCGCGCCTCT GTGGAATTCT GAA
33
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
AGCTTTCAGA ATTCCACAGA GGCGCGGCGG GCG
33
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
TCTCTAGAAC AAACAGAAGT ACTGGA
26
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GATCTAGACA TCTTCCTGTG GGAA
24
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GCGGATCCAT GAGTCTGCAG TGGACT
26
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GCGAATTCTT ACTCTTCCTT CTTGTC
26
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GCGGATCCAT GAGTCTGCAG TGGACT
26
(2) INFORMATION FOR SEQ ID NO:26:
~ ... .. . _ .. . .
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GCGAATTCAG TCAACAGCAG CTCCCTT
27
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: sin~le
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GCGCGGATCC CTCATTTCGC AGCAGGCC
28
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
GCGAATTCAG TCAACAGCAG CTCCCTT
27
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MO~ECULE TYPE: Other
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
GCGGATCCGG AGGCAAGTTG GATGTC
26
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
GCGAATTCTT ACTCTTCCTT CTTGTC
26
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Arg Val Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Ser Glu Gly Ile
1 5 10 15
Asp Ser Glu Asn Leu Lys Asp Met Ile Phe Leu Leu Lys Asp Ser Leu
Pro Lys Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Phe Leu Glu Lys
Gln Gly Lys Ile Asp Glu Asp Asn Leu Thr Cys Leu Glu Asp Leu Cys
50 55 60
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 68 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
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Cys Lys Val Ser Phe Arg Glu hys Leu Leu Ile Ile Asp Ser Asn heu
l 5 lO 15~ly Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cys Ile Gly Leu Val
Pro Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Ser Asp Val Phe Glu
His heu heu Ala Glu Asp Leu heu Ser Glu Glu Asp Pro Phe Phe heu
Ala Glu Leu Leu
