Note: Descriptions are shown in the official language in which they were submitted.
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Programmed Cell Death and Caspase-12
Background of the Invention
Statement as to Rights to Inventions Made Under
Federally-Sponsored Research and Development
Part of the work performed during development of this invention utilized
U.S. Government funds. The U.S. Government has certain rights in this
im~ention.
Field of the Invention
This invention is generally in the field of molecular biology. This
invention relates to the control of programmed cell death.
Related Art
Programmed Cell Death
Apoptosis, also referred to as programmed cell death or regulated cell
death, is a process by which organisms eliminate unwanted cells. Such cell
death
occurs as a normal aspect of animal development as well as in tissue
homeostasis,
such as in tissue remodeling and the establishment of immune self tolerance,
during aging and in disease (gaff, M.C., Nature 36:397-400 (1992);
Glucksmann, A., Biol. Rev. Cambridge Fhilos. Soc. 26:59-86 ( 1950); Ellis et
al.,
Dev. 112:591-603 (1991); Vaux et al., Cel17G:777-779 (1994); Thompson, C.B.,
Science 267:1456-1462 (1995)). Programmed cell death can also act to regulate
cell number, to facilitate morphogenesis, to remove harmful or otherwise
abnormal cells and to eliminate cells that have already performed their
function.
Additionally, programmed cell death is believed to occur in response to
various
physiological stresses such as hypoxia or ischemia. The morphological
characteristics of apoptosis include plasma membrane blebbing, condensation of
nucleoplasm and cytoplasm and degradation of chromosomal DNA at
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inter-nucleosomal intervals. (Wyllie, A_ H., in Cell Death in Biology and
Pathology, Bowen and Lockshin, eds., Chapman and Hall ( 19$1 ), pp. 9-34).
Apoptosis is achieved through an endogenous mechanism of cellular
suicide (Wyllie, A. H., in Cell Death in Biology and Pathology, Bowen and
Lockshin, eds., Chapman and Hall ( 1981 ), pp. 9-34) and occurs when a cell
activates its internally encoded suicide program as a result of either
internal or
external signals. The suicide program is executed through the activation of a
carefully regulated genetic program (Wylie, A.H., et al., Int. Rev. Cyt. 68:
251
( 1980); Eilis, R.E., et a1_, Ann. Rev. Cell Bio. 7: 663 ( 1991 ); Yuan, Y.
Curr. Op.
Cell. Biol. 7:211-214 (1995)). In many cases, gene expression appears to be
required for apoptosis, since cell death can be prevented by inhibitors of RNA
or
protein synthesis (Cohen et al.. J. Immunol. 32:38-42 (1984); Stanisic et al.,
Invest. Urol. 16:19-22 (1978); Martin et al., J. Cell Biol. 106:829-844
(1988).
Acute and chronic disregulation of cell death is believed to lead to a
number of major human diseases (Barn et al. Biotech. 12:487-493 (1995);
Thompson C.B., Science 267:14561462 (1995)). These diseases include but are
not limited to malignant and pre-malignant conditions, neurological and
neurodegenerative disorders, heart disease, immune system disorders,
intestinal
disorders, kidney disease, aging, viral infections and AIDS.
Malignant and pre-malignant conditions may include solid tumors, B cell
lymphomas, chronic lymphocytic leukemia, prostate hypertrophy, preneoplastic
liver foci and resistance to chemotherapy. Neurological disorders may include
stroke, Alzheimer's disease, amyotrophic lateral sclerosis, prion-associated
disorder and ataxia telangiectasia. Heart disease may include ischemic cardiac
2S damage and chemotherapy-induced myocardial damage. Immune system
disorders may include AIDS. type I diabetes, lupus erythematosus, Sjogren's
syndrome and glomerulonephritis. Intestinal disorder may include dysentery,
inflammatory bowel disease and radiation- and H1V-induced diarrhea. Kidney
disease may include polycystic kidney disease and anemialerythropoiesis.
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Specific references to many of these pathophysiological conditions as
involving
disregulated apoptosis can be found in Barr et al. Id- Table 1.
Caspases
Caspases are a family of proteins, previously referred to as ICE or ced-3
proteins, involved in the regulation of apoptosis. A genetic pathway of
programmed cell death has been identified in the nematode Caenorhabditis
elegans (Ellis, R.E., et al.,Annual Review ofCell Bioloy 7:663-698 (1991).) In
this genetic pathway, the genes ced-3 and ced 4 are required for cell death,
while
another gene, ced-9, is a negative regulator of cell death. The ced-3 gene
encodes
a cysteine . protease (Yuan, J., et al. , Cell 75:641-fi52 ( 1993)) which
shares
sequence similarity to the mammalian interleukin-ll3-converting enzyme (ICE),
also called caspase-1, which catalyzes the processing of pro-interleukin-113
to its
biologically active form (Thornberry, N.A., et al., Nature 3~b:768-774
(1992)).
The active centers of ICE and Ced-3 are remarkably conserved with an identical
pentapeptide QACRG (SEQ ID N0:3) in which the cysteine is the catalytic
residue (Yuan, J., etal., Cell 75:641-652 (1993); Thornberry, N.A., etal.,
Nature
356:768-774 (1992)). ICE and Ced-3 exhibit unique requirement for Asp at P1
position for cleavage with different preference for P2-P4 positions in their
substrates (Thornberry> N.A., et al. , Nature 356:768-774 ( 1992); Xue, D., et
al.,
Genes and Development 10:1073-1083 (1996)). The ICE/Ced-3 family of
cysteine proteases have recently been renamed as caspases (AInemri, E.S., et
al.,
Cell 87:171 (1996)), which stands for cysteine aspartic acid specific
proteases.
There have been at least ten human caspases identified (Stennicke, H.R. and
Salvesen, G.S., J. Biol. Chem. 272(41):25719-25723 (1997)).
Overexpression of ICE in a rat fibroblast cell line results in apoptosis,
which can be blocked by mammalian Bcl-2. a homolog of the nematode cell death
suppressor, Ced-9 (Miura, M., et al., Cell 75:653-660 (1993)). While Ced-3 is
the only caspase identified in C. elegans, at least twelve caspases have been
identified in mammals (Kumar, S., et al., Genes and Development 8:1613-1626
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(1994); Wang, L., et al., Cell 78:739-750 (1994); Fernandez-Alnemri, T., et
al.,
J. Biol. Chem. 269:30761-30764 (1994); Tewari, M., Cell 81:801-809 (1995);
Faucheu, C., et al., The EMBOJournal 14:1914-1922 (1995); Munday, N.A., et
al., J. Biol. Chem. ?70:15870-15876 (1995); Duan, H., et al., J. Biol. Chem.
271:1621-1625 (1996); Wang, S., et al.,J. Biol. Chem. 271:20580-20587 ( 1996);
Van de Craen, M., et al. , FEBS Letters 403:61-69 ( 1997)).
The members of the caspase family are expressed in multiple tissues and
cell types during development and adult life. Thus, unlike that in C elegans
where a single ced-3 gene controls alt programmed cell death, multiple
homologs
of CED-3 regulate mammalian apoptosis. Interesting results have come from
gene knockout experiments where mice that are mutant for ICE and caspase-3
have been generated (Li, P., et al., Cell 80:401-411 (1995); Kuida, K., et
al.,
Science 267:2000-2003 (1995); Kuida, K., et al., Nature 384:368-372 (1996)).
The results emerging from analyses of these mutant mice support the hypothesis
1 S that these homologs act in both parallel and sequential fashion to
regulate
apoptosis (Enari, M., et al., Nature 380:723-725 ( 1996)). Ice-I- embryonic
fibroblasts and B lymphoblasts are resistant to granzyme B-induced apoptosis
(Shi, L., et al., Proc. Natl. Acacf Sci. US A 93:11002-11007 (1996)).
Caspase-3-/- mutant mice are defective in brain development, exhibiting a
variety
of hyperplasias and disorganizations which may be due to the absence of
apoptosis, although their thymocytes respond normally to a variety of
apoptotic
agents (Kuida, K., et al., Nature 384:368-372 (1996)). These results suggest
that
either ICE or caspase-3 is critical for certain types of apoptosis in certain
cells
while their functions in other cell types are redundant.
Inhibitors of the caspase family prevent apoptosis induced by a variety of
signals such as trophic factor deprivation, Fas and TNF, suggesting that the
members of the caspase family are critical regulators of mammalian apoptosis
(Miura, M., et al., Cell 75:653-660 {1993); GagIiardini, V., et al., Science
263:97-100 (1994); Tewari, M., and Dixit, V.M., J. Biol. Chem. 270:3255-3260
(1995)). Since cells have been found to express multiple caspases (Wang, L.,
et
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al., Cell 78:739-750 (1994)), it is proposed that caspases may act in a
proteolytic
cascade to carry out the execution of cells (Enari, M., et al., Nature 380:723-
725
( 1996)). Caspases are synthesized as proforms which are to be activated
through
cleavages after specific Asp residues during activation process. For example,
active ICE is generated from its 45 kDa precursor protein through specific
cleavages at four Asp residues, which give rise to the 20 kDa (p20} and 10 kDa
(p 10) subunits.
The unique specificity of the caspase family suggests that either
autoprocessing or cleavage by other homoiogs is responsible for the activation
of
the praforms. In vitro, pro-ICE can be activated by active caspase-4 (Faucheu,
C., et al., The ENfBO Journal 14:1914-19'?2 (1995)}, pro-caspase-3 by active
ICE
(Tewari, M., Cell 81:801-809 (1995); Nicholson, D.W., et al., Nature 376:37-43
(1995}} and pro-caspase-3 and pro-caspase-7 by active caspase-10
(Fernandes-Alnemri. T., etal., Proc. Natl. Acad. Sci. USA 93:7464-7469
(1996)).
In apoptosis induced by anti-Fas antibody, a transient increase in ICE-like
activity
was detected prior to an elevation in caspase-3-Iike activity, supporting the
idea
that the caspase family of proteases act in a proteolytic cascade (Enari, M.,
et al.,
Nature 380:723-725 (1996)).
summary of the Invention
An object of the present invention is to provide a Programmed Cell Death
and Caspase-I2. In accordance with an aspect of the present invention, these
is
provided an isolated nucleic acid molecule at least 90% identical to a nucleic
acid
molecule selected from the group consisting of:
(a) a nucleic acid molecule comprising a nucleotide sequence
encoding the amino acid sequence of easpase-12S as shown in Figure 9 (SEQ ID
N0:14);
(b) a nucleic acid molecule comprising a nucleotide sequence
encoding the amino acid sequence of caspase-12S as encoded by the eDNA clone
contained in ATCC Deposit No. 209710;
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5a
(c) a nucleic acid molecule comprising the nucleotide
sequence of caspase-12S as shown in Figure 9 {SEQ ID N0:13};
(d) a nucleic acid molecule comprising a nucleotide sequence
encoding caspase-12~ (amino acid residues 95 to 419 of SEQ ID N0:2);
(e) a nucleic acid molecule comprising a nucleotide sequence
encoding caspase-122 (amino acid residues 145 to 419 of SEQ ID N0:2); and
(f) a nucleic acid molecule comprising a nucleotide sequence
complementary to (a), (b), (c), (d) or (e).
In accordance with another aspect of the invention, there is provided an
isolated nucleic acid molecule encoding a caspase-12S polypeptide, said
nucleic
acid molecule prepared by a process comprising:
(a) hybridizing a population of nucleic acid molecules to a
nucleic acid molecule comprising the sense or antisense nucleotide sequence of
SEQ ID N0:13, wherein the hybridization is performed under stringent
hybridization conditions;
(b) selecting those nucleic acid molecules of said population
that hybridize to said nucleic acid molecule comprising the sense or antisense
nucleotide sequence of SEQ ID N0:13; and
(c) selecting nucleic acid molecules of (b) that encode
caspase-125.
In accordance with another aspect of the invention, there is provided a
nucleic acid construct comprising a nucleic acid molecule encoding a caspase-
12S
polypeptide operably linked to a heterologous promoter.
In accordance with another aspect of the invention, there is provided an
isolated polypeptide at least 90% identical to a polypeptide selected from the
group
consisting of:
(a) an isolated polypeptide comprising the amino acid
sequence of caspase-12S as shown in Figure 9 (SEQ ID N0:14);
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(b) an isolated poiypeptide comprising the amino acid
sequence of caspase-12S as encoded by the cDNA clone contained in ATCC
Deposit No. 209710;
(c) an isolated polypeptide comprising the amino acid
sequence of caspase-12~ (amino acid residues 95 to 419 of SEQ iD N0:2); and
(d) an isolated polypeptide comprising the amino acid
sequence of caspase-1202 (amino acid residues 145 to 4I9 of SEQ ID N0:2).
In the present invention, the gene encoding caspase-12, {casp-12
previously referred to as Ich-4), was isolated through degenerate polymerise
chain reaction and sequenced. Casp-12 has at least two alternative splicing
products, casp-12S and casp-12L. Casp-12L contains an open reading frame of
419 amino acid residues. Casp-12S contains an open reading frame of 349 amino
acid residues. Fragments of caspase-12 were also obtained: rasp-1201,
consisting
of the open reading frame for amino acid residues 95-419 of carp-12L; and casp-
12d2, consisting of the open reading frame for amino acid residues 145-419 of
Gasp-12L.
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This invention is thus directed, inter- alia, to isolated nucleic acid
molecules comprising a nucleotide sequence encoding the amino acid sequence
of caspase-12S as shown in Figure 9 (SEQ ID NO:14) or as encoded by the
cDNA clone contained in ATCC Deposit No. 209710. 'the invention is also
directed to isolated nucleic acid molecules comprising the nucleotide sequence
of caspase-12S as shown in Figure 9 (SEQ ID N0:13); nucleic acid molecules
comprising a nucleotide sequence encoding caspase-12~ (amino acid residues 95
to 419 of SEQ ID N0:2); and nucleic acid molecules comprising a nucleotide
sequence encoding caspase-122 (amino acid residues 145 to 419 of SEQ ID
N0:2). The invention is also directed to nucleic acid molecules comprising a
nucleotide sequence complementary to the above-described sequences.
Also provided for are nucleic acid molecules at least 80%, preferably 85%
or 90%, still more preferably 95%, 96%, 97%, 98%, or 99% identical to any of
the above-described nueieic acid molecules. Also provided for are nucleic acid
molecules which hybridize under stringent conditions to any of the above-
described nucleic acid molecules.
The present invention also provides for recombinant vectors comprising
an above-described nucleic acid molecule, and host cells transformed with such
vectors.
Also provided are isolated polypeptides comprising the amino acid
sequence of caspase-12S as shown in Figure 9 (SEQ ID N0:14) or as encoded by
the cDNA clone contained in ATCC DepositNo. 209710, as well as polypeptides
comprising the amino acid sequence of caspase-I 20 (amino acid residues 95 to
419 of SEQ ID N0:2) and polypeptides comprising the amino acid sequence of
2~ caspase-1202 {amino acid residues 145 to 419 of SEQ ID N0:2). Also provided
are polypeptides at least 80%, more preferably 85% or 90%, still more
preferably
95%, 96%, 97%, 98%, ar 99% identical to any of the above-described
polypeptides.
Also provided are methods for inducing programmed cell death in a cell
comprising contacting the cell with an above-described polypeptide.
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Overexpression of cusp-12L, cusp-12S, and casp-12~ in rat fibroblast
cells caused apoptosis. The apoptotic activity of caspase-12 was not
inhibitabte
by CrmA, a serpin encoded by cowpox virus which is a specific inhibitor of
ICE,
by Bcl-2, a mammalian cell death suppressor. 'fhe pro-caspase-12 protein was
cleaved effectively by several other members of the caspase family in vitro,
while
recombinant caspase-12 protein and fragments thereof poorly cleaved other
caspases except pro-caspase-12 itself. Therefore, the invention provides for a
method of modulating apoptosis comprising contacting a cell with the protein
encoded for by carp-12L, case-12S, casp-12~ or casp-1202.
Brief Description of the Bigures
Figures IA-1B. caspase-12 cDNA. Figure 1 A shows the cDNA sequence
(SEQ ID NO: l ) and deduced amino acid sequence (SEQ ID N0:2) of caspase-12.
Horizontal arrows indicate the position of degenerate PCR primers used for the
initial screening of mouse thymus eDNA library. The alternative splicing sites
are indicated by vertical arrows. Diamonds indicate the position of the
termination codon. Figure 1 B is a diagram of the alternative splicing and
protein
structure of caspase-12. The exons and introns between them are shown as bars
and lines, respectively (top). Nucleotides at the exon/intron boundaries are
indicated. Numbers above the bars correspond to nucleotide numbers in the
caspase-12L cDNA sequence. A schematic comparison of caspase-12L (419
residues) and caspase-12S (349 residues) proteins is shown at the bottom.
Potential cleavage sites for maturation of the precursor proteins are
indicated
below the bars with the residues at the sites.
Figure 2A-2B. Sequence alignment of caspase-12. Figure 2A shows the
sequence alignment of caspase-12 protein with other members of the caspase
family: caspase-5 (SEQ TD N0:4), hICE (SEQ ID NO:S), mICE (SEQ ID N0:6),
caspase-l I (SEQ ID N0:7), caspase-4 (SEQ ID N0:8), caspase-5 (SEQ ID
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N0:9), caspase-2 (SEQ ID NO:10), caspasc-3 (SEQ ID NO:1 I ) and CED-3 (SEQ
ID N0:12). Seven amino acid residues well conserved among the caspase family
are marked with either asterisks (catalytic residues) or filled diamonds (P1
binding). Open diamonds indicate the residues for S2-S4 subsites. Figure 2B
shows the alignment of amino acid residues involved in substrate binding.
Amino acid residues of each member of the caspase family are described by
one-letter codes: ICE (SEQ ID N0:31 ), caspase-I 2 (SEQ ID N0:32); caspase-1 I
(SEQ ID N0:33), caspase-2 (SEQ ID N0:34), caspase-3 (SEQ ID N0:35) and
CED-3 (SEQ ID N0:36). Numbering above the residues corresponds to the
positions in human ICE. Seven residues which form the P 1 active pocket of ICE
are grouped into those involved in catalysis (His, Gly, Cys) and those for
binding
(Arg, Gln, Arg, Ser).
Figure 3. RT PCR analysis of caspase-12 in mouse adult tissues. cDNA
samples were reverse transcribed from mRNA isolated from brain, thymus, heart,
1 ~ lung, liver, kidney, spleen, and intestine. Lane 12L, PCR products (617
bp) by
using cusp-12L as a positive control. Lane 12S , PCR products (407 bp) by
using
cusp-12S as a positive control. Arrows indicate these Gasp-12L and casp-12S
specific fragments. M, lambda phage DNA HindIII digest as molecular weight
standard. N, negative control as no DNA templates were used for PCR.
Figure 4A-4C. Cell death induced by overexpression of caspase-1Z.
Figure 4A shows Rat-1 cells that were transiently transfected with the
casp-12L-IacZ construct, fixed 24 hr later, and stained with anti-(3-
galactosidase
antibody (left panels) or by Hoechst dye 33258 (right panels). The round cells
expressing the casp-12L-lacZ fusion proteins show condensed and fragmented
2~ nuclei. Figure 4B shows Rat-1 cells that were transiently transfected with
the
control lacZ vector and treated as in A. The nuclear morphology in
13-galactosidase-positive cells is nornlal and noncondensed (indicated by
arrows).
Figure 4C shows Rat-1 cells and rat embryonic fibroblast cells that were
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transfected with the expression constructs, fixed 24 hr later. and stained
with
X-Gal solution for 3 hr. The data shown are the percentage of round blue cells
among total number of blue cells counted. In each transfection, over 200 blue
cells were randomly chosen and counted. The data of Rat-I cells collected from
at least three independent experiments. T'he constructs used were: casp-12L-
lacZ,
casp-125-lacZ, Gasp-12~-lacZ, ICE-lacZ, lac2. Rat embryonic fibroblast cells
(REF) were also transfected with either Gasp-12L-lacZ or IacZ.
Figure SA-SB. Effect of cell death suppressors on tl:e killing activity of
caspase-12. Figure 5A shows a bcl-2 expressing stable transfectant, Rat-1lbcl-
2,
that was transiently transfected with carp-12L-lacZ. Cell death was assayed as
described in Figure 4C. Figure SB shows a crmA expressing transfectant,
Rat-1/crmA, that was transient)}' transfected with casp-12L-lacZ. The data
from
transfection of the parental cell Line, Rat-1 (Figure 4C), are included in the
figures
for comparison. The lacZ construct was also used for transfection as a
control.
Figure 6. The cell type spec(city of caspase-12-induced cell killing
activity. NG108-15, HeLaand COS-1 cells were transfected with Gasp-12L-lacZ,
casp-12S-LacZ, lacZ or ICE-lacZ. Staining of transfectants were done and the
data are presented as in Figure 4C.
Figure 7. In vitro cleavage of caspase-I2 by other members of the
caspase fan:ily. [35S]-labeled caspase-12, either casp-12L or casp-12~, was
incubated with E. coli lysate expressing other caspases (l, ICE; 2, easpase-2;
3,
caspase-3) for 3 hr at 30°C. Control samples (-) were incubated in the
absence
of E. coli Iysate.
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Figure 8A-8C. Protease activity of caspase-12d2. Figure 8A shows
SDS-polyacrylamide gel electrophoresis of partially purified proteins. D,
casp-12L1 (amino acid residues 95-419 of SEQ ID N0:2); O?, easp-122 (amino
acid residues 145-419 of SEQ ID NO:2). Proteins were run on 15% gel and
detected by Coomassie brilliant blue staining. Arrowheads indicate
polypeptides
derived from caspase-12 proteins. Figure 8B is a diagram of processing sites
on
caspase-120 and caspase-1202. Figure 8C shows [355)-labeled proteins that were
incubated with (+) or without (-) partially purified caspase- I 2112 for 1 hr
at 37°C.
Reaction was stopped by boiling with the addition of sample buffer for
SDS-polyacrylamide gel electrophoresis. The molecular sizes in kilodaltons are
indicated at the left.
Figure 9. Sequence of Gasp-12,5 cDNA sequence (SEQ ID N0:13) and
deduced amino acid sequence (SEQ ID N0:14) of~ caspase-125.
Detailed Description of the Preferred Embodiments
In the description that follows, a variety of technical terms are used.
These terms shall have their ordinary well-recognized meaning in the art,
unless
the context indicates otherwise. In order to provide clearer and more
consistent
understanding of the specification and the claims, the following definitions
are
provided.
Caspase. This is the current designation for members of the ICE/Ced-3
family of apoptosis proteins.
Caspase-12 activity. A potypeptide having caspase-12 activity should be
understood to be a polypeptide having "caspase-12 activiy" similar, but not
necessarily identical activity to the caspase-12 protein measured in a
particular
biological assay. For example, caspase-12 is able to induce programmed cell
death in rat embryonic fibroblast cells. Therefore. a polypeptide which is
able to
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induce programmed cell death in rat embryonic fibroblast cells is said to have
''caspase-12 activity."
Cell Extract. A cell extract that contains the nucleic acid or polypeptide
of interest should be understood to mean a preparation such as a homogenate or
cell-free preparation obtained from cells that express the polypeptide or
contain
the nucleic acid of interest. The term "cell extract" is intended to include
culture
media, especially spent culture media from which the cells have been removed.
Fragment. A fragment of a molecule should be understood as referring
to a nucleic acid molecule or polypeptide that contains a shortened nucleotide
or
amino acid sequence compared to a reference nucleic acid molecule or
polypeptide. The fragment may or may not retain one or more desired chemical
or biological properties of the full-length nucleic acid molecule or
polypeptide.
Examples of caspase-12 fragments are casp-12~ or casp-122.
Functional Derivative. A functional derivative of caspase-12 should be
I 5 understood as referring to a polypeptide, or nucleic acid encoding a
polypeptide,
that possesses a biological activity that is substantially similar to the
biological
activity of caspase-12. A functional derivative may or may not contain post-
translational modifications such as covalently linked carbohydrates, depending
on the necessity of such modifications for the performance of a specific
function.
The term "functional derivative" is intended to include the "fragments,"
"variants," "analogues," or "chemical derivatives" of a molecule. The
derivative
retains at least one of the naturally-occurring functions of the parent gene
or
protein. The function can be any of the regulatory gene functions or any of
the
function of the finally processed protein. The degree of activity of the
function
need not be quantitatively identical to the caspase-12 activity as long as the
qualitative function is substantially similar.
Host. As used herein, a "host" or a "host cell" is a cell into which a
recombinant nucleic acid has been introduced. A heterologous host is a host
that
normally does not express the gene or protein of interest.
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Isolated. As used herein, an "isolated" molecule refers to a molecule that
has been removed from its native environment. Isolated nucleic acid molecules
include nucleic acid molecules contained in a recombinant vector; purified
nucleic acid molecules in solution, and nucleic acid molecules produced in
S heterologous host cells. Isolated polypeptides include recombinantly
produced
polypeptides expressed in host cells; native or recombinant polypeptides which
have been essentially or partially purif ed by any suitable technique known in
the
art; and polypeptides produced synthetically.
Modulating programmed cell death. Modulating programmed cell
death should be understood as referring to either an increase or a decrease in
programmed cell death following manipulation of a cell. Such manipulation can
be the result of transfection or transformation of cells with particular carp-
12
constructs, e.g., casp-12S or casp-12L. ,Alternatively, one may contact a cell
with
a polypeptide of interest.
I S Mutation. A "mutation" should be understood as referring to a
detectable change in the genetic material which may be transmitted to daughter
cells and possibly even to succeeding generations giving rise to mutant cells
or
mutant organisms. If the descendants of a mutant cell give rise only to
somatic
cells in muIticellular organisms, a mutant spot or area of cells arises.
Mutations
in the germ line of sexually reproducing organisms may be transmitted by the
gametes to the next generation resulting in an individual with the new mutant
condition in both its somatic and germ cells.
A mutation may be any (or a combination of) detectable change affecting
the chemical or physical constitution, mutability, replication, phenotypic
function,
or recombination of one or more nucleotides. In a mutation, nucleotides may be
added, deleted, substituted, inverted or transposed to new positions with and
without inversion. Mutations may occur spontaneously or can be induced
experimentally by application of mutagens. A mutant variation of a nucleic
acid
molecule results from a mutation. A mutant polypeptide may result from a
mutant nucleic acid molecule.
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Nucleotide sequence. As used herein "nucleotide sequence" refers to a
series of deoxyribonucleotides, in the case of DNA, or of ribonucleotides, in
the
case of RNA. Nucleotide sequences are herein presented as a sequence of
deoxyribonucleotides, abbreviated as follows: A for adenine, C for cytosine, G
for guanine, and T for thymine. However, the nucleotide sequences presented
herein are intended to also represent RNA sequences, wherein each
deoxyribonucleotide thymine is replaced by a ribonucleotide uracil.
Operably linked. Two nucleotide sequences are said to be "operably
linked" if induction ofpromoter function results in the transcription ofthe
coding
sequence and if the nature of the linkage between the two DNA sequences does
not: ( 1 ) result in the introduction of a frame-shift mutation; (2) interfere
with the
ability of regulatory sequences to direct the expression of the coding
sequence;
or (3) interfere with the ability of the coding sequence template to be
transcribed
by the promoter region sequence. Thus, a promoter region would be operably
I S linked to a nucleotide sequence if the promoter were capable of effecting
transcription of that nucleotide sequence.
identical. As used herein. a first nucleotide sequence is said to be, for
instance, "95% identical" to the nucleotide sequence of a second reference
nucleic
acid if the first nucleotide sequence is identical to the complete length of
the
reference sequence, except that the nucleotide sequence of the first nucleic
acid
may include up to 5 substitutions per each 100 nucleotides of the second,
reference sequence. This includes any combination of deletions, insertions, or
single nucleotide substitutions up to five per each 1 U0 nucleotides of the
reference
sequence. Likewise, a sequence is "85% identical" to a reference sequence if
the
nucleotide sequence of the nucleic acid is identical to the reference sequence
except that the nucleotide sequence may include up to I 5 substitutions per
each
100 nucleotides of reference sequence. This applies equally to amino acid
sequences, therefore one amino acid sequence may be, for example, 95%
identical
to a second amino acid sequence by having 95 out of 100 of the same amino
acids
as the reference amino acid sequence.
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As a practical matter, whether any particular nucleic acid molecule is at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for instance,
the nucleotide sequence shown in Figure 1 {SEQ ID NO:1 ) or to the nucleotide
sequence of the deposited cDNA clone can be determined conventionally using
known computer programs such as the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group, University
Research Park, 575 Science Drive, Madison, WI 53711 ). Bestfit uses the local
homology algorithm of Smith and Waterman (Advances in Applied Mathematics
2:482-489 ( 1991 )) to find the best segment of homology between two
sequences.
When using Bestfit or any other sequence alignment program to determine
whether a particular sequence is, for instance, 95% identical to a reference
sequence according to the present invention, the parameters are set, of
course,
such that the percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5% of the
total
number of nucleotides in the reference sequence are allowed.
Population. A "population of nucleic acid molecules" is a mixture of
nucleic acid molecules, in which at least some nucleic acid molecules are non-
identical to the other nucleic acid molecules in the mixture. This includes,
inter
alia, cDNA libraries made from one cell type; extractions from heterologous
cells, which include both DNA and RNA, or which include DNA or RNA only;
and extractions from homologous cells, which include both DNA and RNA, or
which include DNA or RNA only.
PuriFed. Preparations made fiom biological cells or hosts that are said
to be "purif ed" are any cell extract containing the indicated nucleic acid or
polypeptide including a crude extract of the nucleic acid or polypeptide of
interest. Such preparations include molecules removed from their native
environment. For example, in the case of a polypeptide, a purified preparation
can be obtained following an individual technique or a series of preparative
or
biochemical techniques, and the polypeptide of interest can be present at
various
degrees of purity in these preparations. The procedures include, but are not
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limited to, ammonium sulfate fractionation, gel filtration, ion exchange
chromatography, affinity chromatography, density gradient centrifugation,
electrophoresis, and other techniques known to those of skill in the art. See,
e.g.,
Current Protocols in Molecular Biology, John Wiley and Sons (I996).
A preparation of nucleic acid or polypeptide that is "pure" should be
understood to mean a preparation free from naturally occurnng materials with
which such nucleic acid or polypeptide is normally associated in nature.
''Essentially pure" should be understood to mean a ''highly" purified
preparation
that contains at least 95% of the nucleic acid or protein of interest.
Preparations
of nucleic acids or polypeptides of interest that are ''partially pure" are
less pure
than preparations that are essentially pure, and include crudely purified
nucleic
acids or polypeptides.
Stringent Hybridization. Stringent hybridization conditions should be
understood to be those conditions normally used by one of skill in the art to
establish at least a 90% homology between complementary pieces of nucleic
acids. Lesser homologies, such as at least 70% homology or preferably at least
80% homology, may also be desired and obtained by varying the hybridization
conditions.
There are only two required conditions for hybridization to a denatured
strand of nucleic acid to occur: (1) there must be complementary single
strands
in the sample; and (2) the ionic strength of the solution of single-stranded
nucleic
acid must be fairly high so that the bases can approach each other;
operationally,
this is greater than 0.2 M. A third condition affects the rate of
hybridization: the
nucleic acid concentration mush be high enough for intermolecular collisions
to
occur at a reasonable frequency.
Conditions routinely used by those of skill in the art are set out in readily
available procedure texts, e.g., Current Protocols in Molecular Biolo~ry, Vol.
I,
Chap. 2.10, John Wiley 8c Sons, Publishers ( 1994) or Sambrook et al.,
Molecular
Cloning, Cold Spring Harbor ( 1989), incorporated herein by reference. As
would
be known by one of skill in the art, the ultimate hybridization stringency
reflects
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both the actual hybridization conditions as well as the washing conditions
following the hybridization. One of skill in the art would know the
appropriate
manner in which to change these conditions to obtain a desired result.
For example, a prehybridization solution should contain sufficient salt and
nonspecific nucleic acid to allow for hybridization to non-specific sites on
the
solid matrix, at the desired temperature and in the desired prehybridization
time.
For example, for stringent hybridization, such prehybridization solution could
contain 6x sodium chloride/sodium citrate (IxSSC is 0.15 M NaCI, 0.015 M Na
citrate; pH 7.0), Sx Denhardt's solution, 0.05% sodium pyrophosphate and 100
pg
per ml of herring sperm DNA. An appropriate stringent hybridization mixture
might then contain 6x SSC, 1x Denhardt's solution, 100 pg per ml of yeast tRNA
and 0.05% sodium pyrophosphate.
Alternative conditions for nucleic acid hybrid analysis could entail the
following:
1 ) prehybridization at room temperature and hybridization at 68 °C;
2) washing with 0.2x SSC/0.1% SDS at room temperature;
3) as desired, additional washes at 0.2x SSC/0.1% SDS at 42°C
(moderate-stringency wash); or
4) as desired, additional washes at O.lx SSC/0.1% SDS at 68°C
(high stringency).
Known hybridization mixtures, e.g., that of Church and Gilbert, Proc.
Natl. Acad.. Sci. USA 81:1991-1995 (1984), comprising the following
composition may also be used: I % crystalline grade bovine serum albumin/ 1 mM
EDTA/O.SM NaHP04, pH 7.2/7% SDS. Additional, alternative but similar
reaction conditions can also be found in Sambrook et al.. Molecular Cloning,
Cold Spring Harbor (1989). Formamide may also be included in
prehybridization/hybridization solutions as desired.
It should be understood that these conditions are not meant to be definitive
or limiting and may be adjusted as required by those of ordinary skill in the
art to
accomplish the desired objective.
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Vector. As used herein, a vector is a vehicle which provides an
appropriate nucleic acid environment for a transfer of a nucleic acid of
interest
into a host cell. Such a vector may be used for cloning or expressing a
desired
sequence, such as a nucleotide sequence c>f a caspase-12 encoding nucleic acid
of the invention, in an apprapriate host. An expression vector may contain the
nucleic acid of interest operably linked to ahomologous or heterologous
promoter
or regulatory sequences.
In one embodiment of the present invention, isolated nucleic acid
molecules having a nucleotide sequence encoding an amino acid sequence
corresponding to caspase-I2 are provided. In one preferred embodiment, the
isolated nucleic acid molecule has a nucleotide sequence encoding the amino
acid
sequence of casp-I2S as shown in Figure 9(SEQ ID N0:14). In another preferred
embodiment, the isolated nucleic acid molecule has a nucleotide sequence
encoding the amino acid sequence encoded by the cDNA clone deposited as
ATCC Deposit No. 209710 on March 18, 1998 at the American Type Culture
Collection (ATCC), 10801 University Boulevard, Manassas, VA, 20110-2209.
In another preferred embodiment, the isolated nucleic acid molecule has a
nucleotide sequence encoding the amino acid sequence of case-12D (amino acids
95 to 419 of SEQ ID N0:2). In yet another preferred embodiment, the isolated
nucleic acid molecule has a nucleotide sequence encoding the amino acid
sequence of casp-12Q2 (amino acids 145 to 419 of SEQ ID N0:2).
Additionally, nucleic acid molecules of the invention may include only the
coding region of the sequence, with or without leader sequences.
Isolated nucleic acid molecules of the present invention may be in the
form of RNA, for example, mRNA, or in the form of DNA, for example, cDNA.
The DNA may be double-stranded or single-stranded. Single-stranded DNA or
RN A
may by the coding strand, which is also known as the sense strand, or it may
be
the non-coding strand, which is also known as the anti-sense strand.
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The nucleic acid molecules of the present invention may be chemically
synthesized or they may be produced using standard cloning and screening
procedures which are well known in the art. See, e.g., Current Protocols in
Molecular Biology, John Wiley and Sons (1996). Provided in Example 1 is a
description of one preferred method for cloning and screening for the nucleic
acid
molecules of the present invention.
As can be appreciated by one of ordinary ski 1l in the art, there are nucleic
acid molecules other than those specifically depicted here, which due to the
degeneracy of the genetic code, will encode the amino acid sequence of cusp-
12S
as shown in Figure 9 (SEQ ID N0:14) or as encoded by the cDNA clone
contained in ATCC Deposit No. 209710. All degenerate variants of the claimed
sequences are also encompassed by the invention. The genetic code is well
known in the art. Thus, it would be routine for one skilled in the art to
generate
nucleic acids, other than those specifically depicted here, that still encode
the
I 5 amino acid sequence of the invention.
In another embodiment of the present invention, there are provided
nucleic acid molecules having a nucleotide sequence that is at least 80%, and
preferably at least 85% or 90%, still more preferably at least 95%, 96%, 97%,
98%, or 99%, identical to the nucleotide sequences described above. The
present
invention is directed to nucleic acid molecules having a nucleotide sequence
at
least 80% identical to the nucleotide sequence of the above-recited nucleic
acid
molecules irrespective of whether or not they encode a polypeptide having
caspase-I2 activity. This is because, even where a particular nucleic acid
molecule does not encode a polypeptide having caspase-12 activity, one of
skill
in the art would still know how to use the nucleic acid molecule, for instance
as
a probe. Uses of the nucleic acid molecules of the present invention that do
not
encode a polypeptide having caspase-t2 activity include, inter alia, isolating
the
caspase-12 gene or allelic variants thereof in a cDNA library: flourescent in
situ
hybridization ("FISH") to metaphase chromosomal spreads to provide precise
chromosomal location of the caspase-12 gene as described in Verma et al.,
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Human Chromosomes: a Manual of Basic Technigues, Pergamon Press, New
York (1988); and Northern Blot analysis for detecting caspase-I2 mRNA
expression in specific tissues.
Preferred, however, are nucleic acid molecules having a nucleotide
sequence at least 80%, more preferably 85% or 90%, still more preferably at
least
9~%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence of the above-
described nucleic acid molecules which in fact encode a polypeptide having a
caspase-12 activity. One of ordinary skill in the art would immediately
recognize
that, due to the degeneracy of the genetic code, a large number of nucleic
acid
molecules having a nucleotide sequence at least 80%, more preferably 85% or
90%, still more preferably at least 95%, 96%, 97%, 98%, or 99% identical to
the
nucleotide sequence of the above-described nucleic acid molecules will encode
a polypeptide of the present invention. Since the degenerate variants of the
claimed molecules all encode the same polypeptide as the original sequence, it
would be clear to the skilled artisan that degenerate the variants will encode
a
polypeptide having activity similar to that of the original sequence, even
without
performing screening assays, e.g. for caspase-12 activity. Uses of nucleic
acid
molecules which encode a polypeptide having caspase-I2 activity include, inter
alia, modulating programmed cell death by inserting the nucleic acid molecule
into a cell, and production of polypeptides with caspase-12 activity by
inserting
the nucleic acid molecule into a vector, transforming a host cell with the
vector,
and inducing expression of the polypeptide.
It will be further recognized by those skilled in the art that, even for such
nucleic acid molecules that are not degenerate variants, a reasonable number
will
also encode a polypeptide having caspase-12 activity. This is because the
skilled
artisan is aware of possible amino acid substitution that are less likely or
not
likely to significantly affect protein function.
For example, "conservative" amino acid substitutions generally have little
effect on activity of a polypeptide. Typically seen as conservative amino acid
substitutions are: exchange of aromatic residues Phe and Try; exchange of the
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basic residues Lys and Arg; exchange of the amide residues Asn and Gln;
exchange of the acidic residues Asp and Glu; exchange of the hydroxyl residues
Ser and Thr; and substitution among the aliphatic residues AIa, Val, Leu, and
Ile.
Further guidance concerning how to make phenotypically silent amino acid
substitution is provided, for example, in Bowie et al., Science 247:1306-1310
( 1990).
A further embodiment of the present invention provides for a vector
comprising a nucleic acid molecule described above. Vectors useful in the
present invention include chromosomal-, episomal- and virus-derived vectors,
for
example, vectors derived from bacterial plasmids, yeast episomes, yeast
chromosomal elements, viruses such as bacteriophage, baculoviruses, papova
viruses, herpes viruses, vaccinia viruses, adenoviruses, fowl pox viruses;
pseudorabies viruses and retroviruses, and vectors derived from combinations
thereof, such as cosmids and phagemids.
If desired, a recombinant vector encoding a fusion product of a
polypeptide of the invention may be constructed. For example, the nucleotide
sequence encoding a polypeptide of the invention may be linked to a signal
sequence which allows secretion of the polypeptide from, or the
compartmentalization of the polypeptide in, a particular host. Such signal
sequences may be designed with or without specific protease sites such that
the
signal peptide sequence is amenable to subsequent removal. Alternatively, the
polypeptide may be fused to a sequence which enabled easier purification of
the
polypeptide, for example, a Histidine tag.
For cloning into a vector, a nucleic acid molecule of the present invention
is randomly sheared or enzymatically cleaved, and ligated into an appropriate
vector. Methods for cloning nucleic acids into vectors are well known in the
art.
See, for example, Sambrook et al., ~Ylolecular C.'loning, Cold Spring Harbor
( 1989).
The nucleic acid molecules may be joined to a vector containing a
selectable marker suitable for use in identifying cells transformed with the
vector.
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Examples of selectable markers include dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture and tetracycline or ampicillin
resistance
genes for culturing in bacteria.
Certain preferred vectors provide for specific expression of the nucleic
acid molecule, which may be inducible and/or cell-type specific. Particularly
preferred among such vectors are those inducible by environmental factors that
are easily manipulated, such as temperature or nutrient additives. In this
regard,
the nucleic acid insert should be operably linked to an appropriate promoter,
for
example, the phage lambda PL promoter, the E. coli 1ac promoter, the SV40
early
and late promoters, the CMV immediate early promoter, the HSV thymidine
kinase promoter, the promoters of retroviral LTRs, and metallotionein
promoters.
Still another embodiment of the present invention is a host transformed
with a vector described above. The sequence may be incorporated into the
genome of the host cell, or it may be maintained extrachromasomally.
Recombinant vectors may be introduced into host cells using well known
techniques such as infection, transduction, transfection, transvection,
electroporation, and transformation.
Examples of appropriate hosts include, but are not limited to, bacterial
cells, such as E. coli and Salmonella typhimurium cells; fungal cells, such as
yeast
cells; insect cells, such as Drosophila S2 and Spodoptera Sf~ cells; animal
cells,
such as CHO, COS cells, NIH 3T3 cells, human 293 cells, 293T cells, and HeLa
cells; and plant cells. Appropriate culture conditions for the above-described
host
cells are known in the art.
Expression of the polypeptides of the invention from recombinant vectors
2~ in hosts may require the presence of regulatory regions functional in such
hosts.
The precise nature of the regulatory regions needed for gene expression may
vary
between species or cell types, but shall in general include, as necessary, 5'
non-
transcribed and/or non-translated and/or 3' non-transcribed and/or non-
translated
(non-coding) sequences involved with the regulation of transcription and
translation, such as the TATA box, capping sequence, CAAT sequence,
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sequences for transcriptional termination, and the like. Such 5' non-
transcribed
control sequences may include the region which contains a promoter for
transcriptional control of the operably linked gene.
In eukaryotic hosts, where transcription is not linked to translation, control
regions may or may not provide an initiator methionine (ATG) codon, depending
on whether the cloned sequence contains such a methionine. Such regions will,
in general, include a promoter region sufficient to direct the initiation of
RNA
synthesis in the host cell. The promoter region may be a homologous or
heterologous promoter. A heterologous promoter is one that is not normally
associated with the nucleic acid being expressed. For example, the use of a
cytomegalovirus promoter with a mouse ICE gene on or an acting promoter with
a mouse ICE gene are uses of a heteroIogous promoter. The homologous
promoter for carp-12 is the promoter with which the casp-12 gene is operably
linked naturally.
Further embodiments of this invention relate to caspase-12 polypeptides.
In one preferred embodiment, there is provided an isolated polypeptide having
an
amino acid sequence of caspase-12S as shown in Figure 9 (SEQ ID N0:14). In
another preferred embodiment, there is provided an isolated polypeptide having
an amino acid sequence of caspase-12S as encoded by the eDNA clone contained
in ATCC Deposit No. 209710. In another preferred embodiment, there is
provided an isolated polypeptide having an amino acid sequence of caspase-12d
(amino acid residues 95-419 of SEQ ID N0:2). In yet another preferred
embodiment, there is provided an isolated polypeptide having an amino acid
sequence of caspase-1202 (amino acid residues 145-419 of SEQ ID N0:2).
Polypeptides of the present invention include, but are not limited to,
naturally purified products, chemically synthesized polypeptides, and
polypeptides produced by recombinant techniques. Expression of polypeptides
by recombinant techniques may result in different post-translational
modifications, dependent on the host cell. These modified forms of the
polypeptides are also encompassed by the claimed invention.
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It would be readily recognized by one of skill in the art that some amino
acid residues of caspase-12S could be varied without significant effect on the
structure or function of the protein. Such variations include deletions,
insertions,
inversions, repeals, arid type substitutions. Guidance concerning which amino
acid changes are likely to be phenotypically silent can be found in Bowie et
al.,
Science 247:306-1310 (1990).
Thus, another embodiment of the present invention are polypeptides
which are 80%, more preferably 85% or 90%, still more preferably at least 95%,
96%, 97%, 98%, or 99% identical to the above-described polypeptides.
Preferably, these polypeptides will display caspase-12 activity. A skilled
artisan
is fully aware of possible amino acid substitution that are less likely or not
likely
to significantly affect protein function. Guidance concerning how to make
phenotypically silent amino acid substitution is provided, for example, in
Bowie
et al., Science 247:1306-1310 (1990).
The polypeptides of the invention may be used for the purpose of
generating polyclonal or monoclonal antibodies using standard techniques known
in the art (Klein, J., Immunology: The Science of Cell-Noncell Discrimination,
John Wiley & Sons, N.Y. (1982); Kennett et al., Monoclonal Antibodies,
Hybridoma: A New Dimension in Biological Analyses, Plenum Press, N.Y.
( 1980); Campbell, A., "Monoclonal Antibody Technology," In: Laboratory
Technigues in Biochemistry and Molecular Biology 13, Burdon et al. eds.,
Elseiver, Amsterdam (1984); Harlow and Lane, Antibodies, A Laboratory
Manual, Cold Spring Harbor Laboratory, N.Y. (1988)). Such antibodies may be
used in assays for determining gene expression and for screening expression
libraries. Purified protein would serve as the standard in such assays.
The present inventors have shown that caspase-12 induces apoptosis in
cells. Thus, another embodiment of the present invention is a method of
inducing programmed cell death in a cell comprising contacting the cell with a
polypeptide described above. For the purpose of inducing programmed cell death
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in a cell, the polypeptides of the present invention can be administered to a
cell
in vitro or in vivo.
The polypeptides may be administered to the cell exogenously. The
polypeptides may also be administered through recombinant expression. For
example, homologous recombination can be used to express the polypeptides of
the invention in cells. Extrachromosomal nucleic acids with the appropriate
nucleotide sequence can also be introduced into cells.
Induction of apoptosis can be used to treat, inter alia, malignant and pre-
malignant conditions, and autoimmune disorders. Malignant and pre-malignant
conditions may include solid tumors, B cell lymphomas, chronic Iymphocytic
leukemia, prostate hypertrophy, preneoplastic liver foci and resistance to
chemotherapy.
The following are presented as representative, but non-limiting, examples
of the present invention. Other suitable modifications are within the scope of
the
present invention and will be apparent to one of ordinary skill in the art.
Example 1
Caspase-12 is a Member of the Caspase Family
To isolate additional members of the caspase family, two degenerate
primers based upon sequences of two conserved regions among the members of
the caspase family were designed: the pentapeptide motif QACRG (SEQ ID
N0:3) around the active cysteine residue and a hexapeptide sequence FYLFPG
(SEQ ID N0:17) at the C-terminus. This pair of degenerate primers were used
to amplify cDNA from mouse thymus which generated a 400 by fragment as
expected for caspases. This fragment was cloned and subjected to restriction
enzyme and sequence analyses, which showed that there are several different
cDNA species in this 400 by band. One of them, named caspase-12, was a novel
member of the caspase family with a deduced amino acid sequence sharing about
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35% identity with mouse ICE. Southern blot analysis of mouse genomic DNA
showed that caspase-12 is a single copy gene. Using this cDNA clone as a
probe,
nine clones from mouse thymus cDNA library were isolated. These nine positive
clones can be classified into two groups: Long forni of the caspase-12 cDNA
(plasmid pNB6, casp-12L) and short form (plasmid pNB7, casp-12S ).
Methods
PCR amplification of caspase-12 cDNA
Standard techniques of molecular cloning were used as described
(Sambrook, J., et al., Molecular cloning: a laboratory manual (second
edition),
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)), unless
otherwise stated. A partial cDNA clone for caspase-12 was amplified from
murine thymus cDNA by polymerase chain reaction (PCR) using degenerate
oligonucleotide primers: upstream, 5'- TG(GATC) CC(GATC) GGG AA(GATC)
AGG TAG AA-3' (SEQ ID NO:15); downstream, 5'-ATC AT(ATC) ATC CAG
GC(GATC) TGC AG(AG) GG-3' (SEQ ID N0:16) (bases in parentheses indicate
degenerate nucleotides). The following conditions were used for the PCR
reactions: 1 x reaction buffer (Promega), 1.5 mM MgCl2, 200 mM dNTP, 2 mM
each primer, I unit of Taq DNA polymerase (Promega) in a total volume of 50
~cl. cDNA was denatured for 4 min at 94°C prior to 25 cycles of
94°C for 1 min,
46°C for I min, 72°C for 2 min. A 0.4 kb PCR product was cloned
into the
EcoRV site of pBluescript II SK(-) plasmid vector (Stratagene). E. coli cells
were
transformed with the plasmid DNA and individual colonies were isolated and
prepared for DNA sequencing. A novel caspase cDNA was identified based upon
DNA sequence and was named caspase-12.
The 0.4 kb cDNA fragment was labeled with [32P) dATP (Amersham) and
used to screen a murine thymus cDNA library (Stratagene) under high stringent
conditions. Nine positive clones were identified from about 10' phage clones.
The plasmid DNAs were excised from phage DNAs by an in vivo excision
protocol (Stratagene). Eight out of the nine clones contain identical
sequences
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with a 419 amino acid residue open reading frame and was designated as
caspase-12L (Figure 1A). The ninth clone is 210 by shorter than caspase-12L
because it does not contain the bases 94 through 303 of caspase-12L (Figure 1
B).
The missing sequence starts fiom a dinucleotide sequence, GT, which is a
S probable mammalian splice donor site. Analysis of the caspase-12 genomic
clones showed that the base 303/304 is located exactly at a splice junction,
and
the base 304 is preceded by a dinucleotide sequence AG in the intronic
sequence,
conforming to the GT/AG rule for splice donor/acceptor site (Figure 1B). Thus,
the shorter clone was likely derived from an alternatively spliced mRNA. The
short form of mRNA was named caspase-12S which contains an open reading
frame of 349 amino acid residues.
A potential processing site in pro-caspase-12L is found at Asp94/G1y95
(Asp24/GIy25 in caspase-12S). The 210 by sequence which is missing in
caspase-12S (codons 17-87) probably encodes the sequence within the
pro-domain of the caspase-12 (Figure 1B), which could be removed during
proteolytic processing of precursor form to generate a mature protein. Both
caspase-12L and caspase-12S proteins have another possible processing site
(Asp318/Thr319 in caspase-12L, Asp248/Thr249 in caspase-12S), corresponding
to the cleavage site between p20 and p 10 fragments of ICE. Thus, the
difference
between caspase-12L and caspase-12S is in their pro-domains and processing of
caspase-12L and caspase-12S would give rise to a mature enzyme of identical
subunit composition and sequences.
The caspase family can be divided into three subfamilies according to
their amino acid sequence similarity: the ICE subfamily, the caspase-2
subfamily,
and the caspase-3 subfamily (Duan, H., et al., J. Biol. Chenz 271:1621-1625
( I 996)). The caspase-12L protein shares 41 % sequence identity with human
ICE
(Thornberry, N.A., et al., Nature 356:768-774 ( 1992)), 41 % sequence identity
with mouse ICE (Miura, M., et al., Cell 75:653-660 (1993); Nett, M.A., et al.,
J.
Immunol. 149:3254-3258 (1992), 42% sequence identity with mouse caspase-11
(Wang, S., et al., J. Biol. Chem. 271:20580-20587 (1996)), 49% with human
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caspase-4 (Faucheu, C., et al., The EMBD.Iournal 14:1914-1922 (1995)), 46%
with human caspase-5 (Munday, N.A., et al., J. Biol. Chem. 270:15870-15876
( 1995)), 21 % with human caspase-2L (Wang, L., et al., Cell 78:739-750 {
1994)),
20% with human caspase-3 (I~ernandez-Alnemri, 't., et al., J. Biol. Chem.
269:30761-30764 (1994)), and 18% with the C. elegans Ced-3 (Yuan, J., et al.,
Cell 75:641-652 { 1993)) (Figure 2A, Table 1 ). The comparison of the caspase-
I2
amino acid sequence with those of other caspase family members suggests that
caspase-12 belongs to the ICE subfamily, which includes ICE, caspase-4, -5 and
1 o Table 1
Sequence Similarity Among Caspase Family (% Identity)
( ~ hICEcaspase-llcaspase-4caspase-5cas~pase-2caspase-3CED-3
~ ~ ( ( ( J
caspase-1241 42 49 46 21 20 18
NICE 46 49 49 26 32 28
caspase-1l 60 54 30 32 26
1 J caspase-4 73 28 32 27
caspase-5 25 30 25
caspase-2 28 28
caspase-3 34
The X-ray crystallography of the ICE protein has revealed that the p10
20 subunit apparently makes a direct contact with P2-P4 amino acid residues of
a
substrate (Walker, N.P.C., et al., Cell 78:343-352 (1994}; Wilson, K.P., et
al.,
Nature 370:270-275 (1994)). Three groups of amino acid residues in ICE are
assumed to be the subsites (S2-S4) which recognize and bind to the P2-P4
substrate determinants (Figure 2B). The first group (Va1338 and Trp340 in
25 human ICE) is best conserved among the caspase family. The second one
(His342 and Pro343) is least conserved among the ICE subfamily {ICE,
caspase-12 and caspase-11), whereas the other three members (caspase-2,
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caspase-3 and CED-3) show similar composition (Asn and Thr/Ser). The
variation in the amino acid composition of the subsites probably reflects
different
substrate specificity.
Sequence alignment of caspase-12 with ICE reveals that the three residues
S involved in catalysis of ICE {His237, G1y238 and Cys285, (Walker, N.P.C.,
etal.,
Cell 78:343-352 (/994); Wilson, K.P., et al., Nature 370:270-27~ {1994)) are
conserved in caspase-12 (His250, G1y251 and Cys298. Figs. 2A and 2B) as well
as the residues that are part of the P1 Asp binding pocket in ICE (Arg179,
G1n283, Arg341 and Ser347) which correspond to Arg192. G1n286, Lys356,
Ser362 in caspase-12 with the only substitution of Arg with Lys at residue
356.
This is the first example that a member of the caspase family shows such a
substitution at this position. The residues in ICE that make up the groove for
binding P2-P4 residues of the substrate (Va1338, Trp340. His342, Pro343,
Arg383 and GIn385) are less conserved in caspase-12, in which only Trp and Gln
are conserved with the corresponding amino acid residues, IIe353, Trp355,
Va1357, G1y368, Leu398, and GIn400 (Figure 2B). These results suggest that
caspase-12 is a member of the caspase family with P1 Asp specificity but
likely
to have different P2-P4 preference in its substrates in comparison to that of
ICE.
Example 2
Expression Pattern of Casp-12
To examine the expression pattern of both casp-l~'L and Gasp-12S,
fragments of casp-12L and casp-12S messages were amplified by RT-PCR.
Methods
RT-PCR
2~ mRNA was isolated from mouse brain, thymus, lung. heart, liver, kidney,
spleen, and intestine using the MicroFast mRNA isolation kit (Invitrogen). One
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microgram of mRNA was used for reverse transcription with random primers and
Moloney murine leukemia virus (MoMLV) reverse transcriptase {lnvitrogen).
The caspase-12 cDNA was amplified with PCR. The conditions of PCR were as
follows: 94°C, 1 min; 50°C, 2 min; 72°C, 2 min, 30
cycles. The primers used
were: upstream, 5'-GAGAT'CCAATC'TACAAGATC-3' (SEQ ID N0:18);
downstream, 5'-CACCACAGAGTATCCAAG-3' (SEQ ID N0:19). These two
specific primers span the 210 by region that is deleted in casp-12S and were
designed using sequences from separate exons so that the possibility of
amplification from contaminating genomic DNA could be eliminated.
In the PCR reaction, both case-12L and case-12S can be amplified
simultaneously to produce DNA fragments of 617 by and 407 bp, respectively.
Both fragments were amplified from all the tissues examined which include
brain,
thymus, lung, heart, liver, kidney, spleen and intestine (Figure 3). These
results
indicate that both caspase-12L and caspase-125 are ubiquitously expressed in
adult tissues. Since ubiquitous expression of several members of the caspase
family has been found in adult tissues (Kumar, S_, et al., Genes and
Development
8:1613-1626 (1994); Wang, L., et al., Cell 78:739-750 (1994);
Fernandez-Alnemri, T., et al., J. Biol. Chem. 269:30761-30764 (1994); Tewari,
M., Cell 81:801-809 (1995); Faucheu, C., et al., The EMBO Journal
14:1914-1922 (1995); Munday, N.A., et al., J. Biol. Chem. 270:15870-15876
(1995); Duan, H., et al , J Biol. Chem. 271:1621-1625 (1996); Wang, S., et
al.,
J. Biol. Chem. 271:20580-20587 (1996); Van de Craen, M., etal., FEBSLetters
403:61-69 (/997)), the caspase-12 protein is likely coexpressed with other
members of the caspase family in a single cell.
There was another intense band of 900 by in each lane of the RT-PCR
products (Figure 3). Cloning and sequencing of this fragment showed that it
contains no similarity to carp-12, and it did not hybridize to the casp-12
genomic
clones, indicating that this band was amplified from a different genetic
locus.
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Example 3
Overexpression of casp-12 cDNA Induces Apoptosis in Mammalian Cells
To examine if expression of caspase-12 induces apoptosis, a transient
expression system was used to overexpress caspase-12 in mammalian cells.
S Expression constructs of ICE, cusp-2 and casp-11 fused with E. coli lacZ
have
been successfully used to demonstrate that expression of these ICE family
members induces apoptosis (Miura, M., et al., Cell 75:653-660 ( 1993); Wang,
L.,
et al., Cell 78:739-750 (1994); Wang, S., et al., J. Biol. Chem. 271:20580-
20587
(1996)). Transfected cells are easily detected by staining with X-gal because
of
the i3-galactosidase activity of IacZ. Expression constructs of case-12L and
casp-125 fused with lacZ gene in pcDNA3 vector, which contains the
cytomegalovirus (CMV) promoter, were generated. These expression constructs
were introduced into Rat-1 cells by transfection, and the expression of the
genes
were examined 24 hr after transfection using X-gal reaction.
Methods
Plasmid construction
The long form of caspase-12 cDNA (casp-12L) within pNB6 was
amplified by using Vent DNA polymerase (New England Biolabs) and the
following primers: upstream, S'-CTC GAA TTC ATG GCG GCC AGG AGG
ACA CAT G-3' (SEQ ID N0:20); and downstream, S'-CTC GGA TCC TTC
CCG GGA AAA GGT AG-3' (SEQ ID N0:21 ). The amplified fragments were
digested with EcoRI and BamHI and then cloned into pBluescript II SK(-)
(pBSI9S). pBI9SZ was made by inserting BamHI fragment of lacZ (Miura, M.,
et al., Cell 75:653-660 (1993)) into pBSI9S. The cusp-12-IacZ fusion gene was
cut out from pBI9SZ by using KpnI and XbaI and inserted into peDNA3 vector
(Invitrogen) digested with Kpnl and XbaI to generate pNB 15. The expression
construct pNB 16, a fusion of the short form of caspase-12 cDNA (casp-12S) and
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lacZ in pcDNA3 vector, was generated through the same strategy as described
above, using pNB7 instead of pNB6 as a template for PCR.
For the expression construct of caspase-12D (amino acid residues 95-419
of SEQ ID N0:2), a pan of Gasp-12L was amplified by PCR with following
primers: upstream, 5'-CTC GG'I~ ACC ATG GGA CCT CAG AAG ATA TGT
AC-3' (SEQ ID N0:22); and downstream, 5'-CTC GTC GAC CCA TTC CCG
GGA AAA AGG TAG-3' (SEQ ID N0:23). The upstream primer contains an
ATG for an artificial initiation codon. The amplified fragments were digested
with KpnI/SaII and fused with IacZ in pBluescript II SK(-) vector. The
rasp-12~-lacZ fusion gene was excised from the pBluescript-based plasmid using
Kpnl and XbaI and inserted into KpnI/Xbal sites of peDNA3 vector.
Cell culture and transient transfection
Rat-1, rat embryonic fibroblast cells, NG108-1 S, HeLa and COS-1 were
maintained in culture at 37°C with 5% CO~ in Dulbecco's modified
Eagle's
medium containing 10% (v/v) fetal bovine serum (BioWhittaker) and 50 units/ml
penicillin and 50 ~g/ml streptomycin (Sigma). The day before transfection,
cells
were seeded at a density of 1.3 x 104 cells/ cmz. Expression constructs were
transferred to cells with either calcium phosphate or lipofectamine (GIBCO
BRL)
as previously described (Miura, M., et al., Cell 7~:6~3-660 (1993); Kumar, S.,
et
al., Genes and Development 8:1613-1626 (1994); Wang, L., et al., Cell
78:739-750 ( 1994)). The expression of chimeric genes in cells was detected by
staining of cells with 5-bromo-4-chloro-3-indolyl t3- D-galactoside (X-gal) as
previously described (Miura, M., etal., Ce1175:6~3-660 (1993); Kumar, S.,
etal.,
Genes and Development 8:1613-1626 (1994); Wang, L., et al., Cell 78:739-750
( 1994)). Immunostaining of transfected cells was done as previously described
using mouse monoclonal anti-lacZ antibody (Miura, M., et al., Cell 75:653-660
(1993); Kumar, S., et al., Genes and Development 8:1613-1626 (1994); Wang,
L., et al., Cell 78:739-750 (1994)).
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More than halfofX-gal positive cells transfected with casp-12-lacZ fusion
construct are round and smaller than healthy X-gal positive cells transfected
with
lacZ alone. Such reduction of cell size is a characteristic feature of
apoptotic cells
(Miura, M., et al., Cell 75:653-660 (1993); Jacobson, M.D., et al., Nature
361:365-369 (1993)). To confirm that the casp-12 overexpression causes
apoptosis, the nuclear morphology of the cell death induced by casp-I2
expression was examined by staining the casp-12L-lacZ transfected Rat-1 cells
with anti-13-galactosidase antibody and Hoechst dye 33258.
13-galactosidase-positive round cells which had been transfeeted with
casp-12L-lacZ had condensed and fragmented nuclei, characteristic of cells
undergoing apoptosis (Figures 4A and 4B).
The percentage of cell death induced by caspase-12L and caspase-125 in
Rat-1 cells are about 60% (Figure 4C). A similar result was obtained using
primary culture of rat embryonic fibroblasts. About 60% of transfected rat
embryonic fibroblasts were round and small (Figure 4C). Thus, overexpression
of casp-12 causes cell death of both an established cell line and primary
cells.
The apoptotic activity of casp-Z 2, however, is weaker than that of ICE and
carp-2,
since overexpression of ICE and casp-2 caused over 90% of cells to die (Miura,
M., et al., Cell 75:653-660 (1993); Wang, L., et al., Cell 78:739-750 (1994)).
In
a parallel experiment (Figure 4C), the same culture of Rat-1 cells were
efficiently
killed by overexpression of ICE (Miura, M., et al., Cell 7.1:653-660 (1993)).
The N-terminal pro-domains of the caspase family may have a role in
regulating proteolytic activation of the precursor proteins and removal of the
pro-domain often results in higher activity (Yuan, J., et al., Cell 75:641-652
(1993); Thornberry, N.A., et u1., Nature 356:768-774 (1992); Duan, H., et al.,
J.
Biol. Chem. 271:1621-1625 (1996)). Based upon the sequence homology of
caspase-12 with other members of the caspase family, Asp94-G1y95 of
caspase-12L was identified as a possible cleavage site (Figure 1B). To examine
the possibility that the pro-domain of caspase-12 may have inhibitory function
which reduces cell-killing activity of caspase-12, an expression construct was
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made which lacked the first 94 amino acids of caspase-12L with remaining
GIy95-Asn419 (caspase-120) fused to the IacZ sequence. This casp-I2L1-IacZ
fusion construct was transfected into Rat-I cells and the efficiency of cell-
killing
was examined by X-gal staining. The truncated construct exhibited a cell-
killing
activity comparable to that of caspase-12L and caspase-12S (60% dead cells,
Figure 4C). Thus, unlike the pro-domains of other members of the caspase
family
{Duan, H., et al., J. Biol. C.'hena. 271:1621-1625 (199b)), the pro-domain of
caspase-12 does not appear to have an inhibitory function to the apoptotic
activity
of caspase-12. The relatively lower cell-killing activity of caspase-12 is
likely
intrinsic to the caspase-12 protein, rather than due to the inhibition of the
pro-domain. This result also shows that the G1y95-Asn419 portion of the
caspase-12 protein is sufficient to exhibit its killing activity.
Although the percentages of apoptotic cells induced by overexpression of
either full-length caspase-12 or a truncated caspase-120 eDNA are comparable
IS to each other, the full-length caspase-12 protein is not active in vitro.
It is thus
likely that the maturation of caspase-12 rn vivo requires other caspases.
Example 4
Effects of Cell Death Suppressors oh Caspase-12 Cytotoxicil3~
Cell death induced by overexpression of ICE, casp-2 and case-I 1 can be
effectively inhibited by bcl-2 (Miura, M., et al., Cell 7:653-660 (1993);
Wang,
L., et al., Cell 78:739-750 (1994); Wang, S., et al., J. Biol. Chem.
271:20580-20587 (1996)). bcl-2 is a mammalian homolog of the C. elegans
ced-9 gene, which is a cell death suppressor in the worm (Elks, R.E., et al.,
Annual Review of Cell Biology 7:663-698 ( 1991 )). CrtnA is a serpin encoded
by
cowpox virus which is a discriminatory inhibitor of the caspase family with
high
affinity for ICE and low affinity for caspase-2 and caspase-3 (Ray, C.A., et
al.,
Cell 69:597-604 (1992); Komiyama,T., et al., J. Biol. Chem. 269:19331-19337
(1994)). Apoptosis induced by ICE and easpase-11 but not caspase-2 can be
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suppressed by CrmA (Miura, M., et al., C.'ell 75:653-660 ( 1993); Wang, L., et
al.,
Cell 78:739-750 (1994); Wang, S., et al., J. Biol. Chem. 271:20580-20587
( 1996)). Expression of either bcl-2 or crmA prevents apoptosis of many cell
types induced by different stimuli (Miura, M., et al., Cell 75:653-660 (1993);
Gagliardini, V., et al., Science 263:97-100 (1994); Tewari, M., and Dixit,
V.M.,
J. Biol. Chem. 270:3255-3260 (1995); Tewari, M., et al., J. Biol. Chem.
270:22605-22708 (1995); Enari, M., et al., Nature 375:78-81 ( 1995); Los, M.,
et
crl. , Nature 375:81-83 ( 1995); Miura, M., et al. , Proc. Natl. Acad. 5c1.
USA
92:8318-8322 (1995)).
To determine if expression of bcl-2 and crmA can inhibit apoptosis
induced by case-12 overexpression, casp-12L-lacZ fusion construct was
transfected into stable cell lines of Rat-I that overexpresses either bcl-2 or
crmA
(Miura, M., et al., Cell 75:653-660 (1993)). Cell death was assayed as
described
above (Example 3). The results showed that the cell death induced by
overexpression of casp-12 can be only very weakly inhibited by Bcl-2 (Figure
5A)
but not by CrmA (Figure 5B). The magnitude of the bcl-2 suppression against
toxicity of Gasp-12 overexpression was about 10% (60% for Rat-1, 50% for
Rat-1/bcl-2) which is much smaller than that observed for ICE and casp-2
(Figure SA; Wang, L., et al., Cell 78:739-750 (1994)). Statistical analysis
suggests that the suppression of caspase-12 by Bcl-2 is not significant with
the
t-value of 0.35 (P < 0.05). Although the ICE-induced apoptosis was suppressed
by 50% in Rat-I/crmA cells (Wang, L., et al., Cell 78:739-750 (1994)),
caspase-12-induced apoptosis is not suppressed in the same stable cell line
(Figure 5B).
CrmA is known to be a suicide substrate, which forms an equimolar
complex with ICE (Komiyama,T., et al., J. Biol. Chem. 269:19331-19337
( 1994)). The results thus indicate that although caspase-12 may belong to the
ICE
subfamily, its substrate specificity is quite different from that of ICE. It
has been
found that another virus inhibitor for the caspase family, baculovirus p35,
could
not eff cientIy suppress the cell-killing activity of caspase-12 (N. Morishima
et
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al., unpublished data). As p35 is an effective inhibitor of ICE, caspase-2 and
caspase-3 (Xue, D., and Horvitz, H.R., Nature 3 77:248-251 ( I 995)), the
substrate
specificity of caspase-12 appears to be unique among the members of the
caspase
family.
Unlike ICE, casp-2 and casp-1 l, cytotoxicity of Gasp-12 overexpression
was not effectively suppressed by Bel-2. Thus, easpase-12 may control
apoptotic
pathways that are not sensitive to the inhibition of Bcl-2. Although Bcl-2 is
a
potent inhibitor of cell death under many circumstances, some types of cell
death
have been shown to be resistant to BcI-2 action. For example, Fas-induced
apoptosis is not inhabitable by Bcl-2 in several cell types examined (Memon,
S.A., et al., J Immunol. 15.5_4644-4652 (1995); Strasser, A., et al., The EMBO
Journal 14:6136-6147 (1995)). Caspase-12 could be involved in such Bcl-2
resistant cell death pathway. Alternatively, caspase-12 may control a step
which
is downstream from the point of Bcl-2 inhibition in the apoptosis pathway.
This
hypothesis will predict that ICE, caspase-2 and caspase-I I activate apoptosis
before or at the step inhabitable by Bcl-2 whereas caspase-12 activates
apoptosis
after that step. Armstrong et al. (Armstrong, R.C., et al., J. Biol. Chem.
271:16850-16855 (1996)) have shown that Bc1-2 prevents pro-caspase-3
processing. It is thus probable that downstream effectors activated by caspase-
3
are insensitive to Bcl-2. The evidence shows that pro-caspase-12 can be
cleaved
by caspase-3 while caspase-12 cannot efficiently cleave other caspases tested.
Example S
Cell Type Specificity of Caspase-12 Cytotoxicity
The cytotoxic effects of caspase-12 exhibited cell type specificity. The
casp-I2L-LacZ constructs were transfected into NG108-15, HeLa and COS-1
cells and cell killing effects were assayed to see if caspase-12 kills tumor
cell
lines as well as Rat-I fibroblast cells. The results showed that these tumor
cell
lines are resistant to apoptosis induced by casp-12L expression (Figure 6).
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COS-1 cells were established by transformation of monkey kidney cells with
SV40 (Gluzman, Y., C.'ell 23:175-1$2 ( 1981 )) and are resistant to apoptosis
induced by overexpression of ICE and casp-2 (Wang, L., et al., Cell 78:739-750
(1994)). The neuroblastomalglioma hybrid cell line NG108-15 and HeLa cells
are sensitive, however, to apoptosis induced by overexpression of ICE and casp-
2
(Wang, L., et al., Cell 78:739-750 (1994)). Control experiments using Ice-lacZ
construct showed that overexpression of ICE induced apoptosis of both tumor
cell
lines effectively under the same experimental conditions (about 70% dead cells
as to NG108-15 and 90% for HeLa). These results suggest that the apoptosis
inducing activity of caspase-12 may be more sensitive to transformation status
of
cells than that of ICE and caspase-2.
Caspase-12 is unique among the caspases because of its cell type
specifccity; fibroblast cells are sensitive to its overexpression, while tumor
cell
lines examined are resistant. Therefore, one use of casp-12 may be to
specifically
1 S kill fibroblast cells. The spectrum of caspase-12 killing activity could
be related
to regulatory mechanism of cell death. One of the possibilities is that the
action
spectrum that caspase-12 shows is somehow relevant to difference in terms of
cell
transformation. Tumor cells may lack either substrates or activators of
caspase-12, or tumor cells could have specific inhibitors of caspase-12. An
aspect of tumorigenesis now well recognized is that cells lose their ability
to
undergo apoptosis during tumor progression (Symonds, H., et al., Cell
78:703-7I1 (1994); White, E.. Nature 371:21-22 (1994); Fisher, D.E., Cell
78:539-542 ( I 994)). Identification of the proteins which interact with
caspase-12
is thus important and could provide further insight into both apoptosis and
tumorigenesis.
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Example 6
In vitro Cleavage ojCaspase-12
Recent data suggest an ordered activation of caspases (Enari, M., et al.,
Nature 380:723-725 ( 1996)). To examine if the caspase-12 protein is a
substrate
for other members of the caspase family, [35S]-labeled caspase-12 precursor
was
incubated with Escherichia coli lysates containing the caspase family members.
Methods
Plasmid construction
Casp-12L was amplified through PCR with the following primers:
upstream, 5'-CTG GAT CCG TAT GGC GGC CAG GAG GAC ACA TGA
AAG AGA TCC-3' (SEQ ID N0:24); and downstream, 5'-CTC GTC GAC CCA
TTC CCG GGA AAA AGG TAG-3' (SEQ ID NO:25). Casp-I20 was amplified
through PCR with the following primers: upstream, 5'-CTC GGT ACC ATG
GGA CCT CAG AAG ATA TGT AC-3' (SEQ ID N0:22); and downstream,
5'-CTC CTC GAG CTA ATT CCC GGG AAA AAG G-3' (SEQ ID N0:26).
The amplified fragments were cleaved by either BamHI/SaII or KpnI/XhoI, and
cloned into pcDNA3.
In vitro cleavage
ICE, casp-2 and Gasp-3 were cloned into a pET-15b vector (Novagen) and
their expression was induced in the presence of 0.2-0.3 mM IPTG (Wang, S., et
u1., J. Biol. Chem. 271.20580-20587 ( 1996); Cryns, V.L., et al., J. Biol.
Chem.
271:31277-31282 (1996)). Bacterial lysates containing the ICE-like proteases
were prepared as described (Wang, S., et al., J. Biol. Chem. 271:20580-20587
(1996); Cryns, V.L., et al., J Biol. Chem. 271:31277-31282 (1996)).
2 ~ [35S]-labeled proteins were prepared by in vitro transcription and
translation using
a TNT coupled reticulocyte lysate system (Promega) and ['SS]methionine
{Amersham). Labeled proteins were incubated at 37°C with purified
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caspase-1202 or at 30°C with E. coli lysates containing the caspase
family
members. The reaction mixture contained 25 mM Hepes (pH 7.5) with 5 mM
EDTA, S mM dithiothreitoi, 10% sucrose, 10 ug/ml leupeptin and 250 ,uM
phenylmethylsulfonyl fluoride. Amount of proteins used for cleavage reaction
S were: 0. I ~cg for partially purified caspase-1202, 25-70 E.cg for E. coli
lysate. To
inactivate caspase-12,x.2 with thiol reagents, purified caspase-122 was
preincubated with either 1 mM 5, S'-dithio-bis(2-nitrobenzoic acid) (DTNB) at
25°C for 60 min or 2 mM iodoaceamide at 0°C for 60 rnin in the
absence of
reducing reagents such as dithiothreitol, and subsequently the enzyme solution
was used for reaction with [355]-labeled proteins. For the inhibition
experiments
using DTNB, dithiothreitol was omitted from reaction mixture. Peptide
inhibitors, YVAD-CHO (Thornberry, N.A., et al., Nature 356:768-774 (I992))
and DEVD-CHO (Nicholson, D.W., et al., Nature 376:37-43 (1995)) were
purchased from Peptide Institute Inc. (Osaka, Japan). Cleavage products were
analyzed by either 10- or I S% SDS-polyacrylamide gel electrophoresis:
Detection ofproteins were done by fluorography using ENLIGHTNING solution
(New England Nuclear) as previously described (Cryns, V.L., et al., J. Biol.
Chem. 271:31277-31282 (1996)).
It was found that caspase-2 and caspase-3 can cleave pro-caspase-12
protein, generating smaller fragments of about 40 kDa (Figure 7). Thus, the
precursor form of caspase-I2 may be a substrate of several caspases. Among
these proteases, caspase-3 was focused on because the probable processing site
in pro-caspase-12 (Asp9,Gluq,Asp93Asp94/G1y95) matches the consensus sequence
for caspase-3 cleavage, DXXD (SEQ ID N0:30); Nicholson, D.W., et al., Nature
3 7b:37-43 ( 1995)). Cleavage of the caspase-12 precursor at this site by
caspase-3
would generate a fragment of 38 kDa. whose size is comparable to that of the
fragment observed in cleavage experiments with caspase-3. Caspase-2 has a
preference for the DXXD (SEQ ID N0:30) sequence, while its cleavage
efficiency is also dependent on a PS residue. which makes this enzyme unique
among the caspase family (Van de Craen, M., et al., FEBS Letters 403:61-69
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( I 997)). Cleavage of pro-caspase-12 by caspase-2 generated a fragment of
about
40 kDa at a lower cleavage efficiency, compared to caspase-3 digestion.
To examine if caspase-3 cleaves at the peptide bond between Asp94 and
G1y95, in vitro cleavage of caspase-12D was tried. Casp-12~ was cloned into
pcDNA3 vector with an artificial initiation codon. Figure 7 shows that the
truncated form is identical in size to the fragment generated from pro-caspase-
12
by caspase-3 digestion. Caspase-3 treatment of the truncated form did not
produce any smaller fragments. These results suggest that the truncated
protein
does not contain the cleavage site for caspase-3, and that caspase-3 likely
cleaves
between Asp94 and G1y95.
Caspase-12 can be cleaved by other members of the caspase family
(caspase-2, caspase-3}. Removal of the N-terminal portion of caspase-12 (Metl
through Asp94} resulted in autoprocessing of the truncated protein when it was
expressed in E. coli. These results suggest the possibility that caspase-12
can be
1 S activated by caspase-3 (or caspase-3-like protease) in vivo through
removal of the
N-terminal pro-domain. The 38 kDa fragment of caspase-12 generated by the
action of caspase-3, however, did not get processed further. Autoprocessing of
ICE occurs under particular conditions (Ramage, P., et al., J. l3iol. Chem.
270:9378-9383 (1995)).
Example 7
Protease Activity of Recombinant Caspase-12
To study the enzymatic properties of caspase-12, caspase-I2 cDNA was
expressed in E. coli for protein production.
Methods
Plasmid construction
For production of histidine-tagged proteins, casp-12L1 was inserted into
a prokaryotic expression vector, pRSET-A (lnvitrogen). Primers used were:
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upstream, 5'-CTC GGA TCC GGA CCT CAG AAG ATA TGT AC-3' (SEQ ID
N0:27); and downstream, 5'-CTC GGA'rCC CTA ATT CCC GGG AAA AAG
GTA G-3' (SEQ ID N0:28). The amplified fragments were digested with BamHI
and inserted into the BamHI site of pRSET-A, which encodes an N-terminal
histidine tag. Similarly, bacterial expression plasmid for caspase-12L12
(amino
acid residues 145-419 of SEQ ID NO:2) was constructed by using another
upstream primer, 5'-CTC GGA TCC ACA CTG AAG CTT TGT CCA CG-3'
(SEQ ID N0:29).
Production of recombinant Caspase-12 protein
casp-12D and easp-1202 were cloned into a pRSET-A vector (Invitrogen),
and the resultant plasmid was introduced into E. coli BL21(DE3) pLys.
Production of the histidine tagged proteins were induced by 0.1 mM
isopropyl-I-thio-13- D-galactopyranoside (IPTG), and the protein was purified
with a pET system (Novagen) according to the manufacturer's protocol. Purified
protein was stored at -80°C in 50 mM Tris-HCl (pH7.S) containing 100 mM
NaCI and 50% glycerol. N-terminal sequencing was done according to the
method of Matsudaira (LeGendre, N., and Matsudaira, P., BioTechniques
6:154-159 (1988)) with an Applied Biosystems 473A Protein Sequences".
Full-length caspase-12 protein expressed and purified from E. coli was
stable without any processing and protease activity in vitro. Thus, a
truncated
caspase-12 cDNA (caspase-120 corresponding to Gly9~-Asn419) was expressed
in E. coli after removal of N-terminal pro-domain coding region and tagged its
N-terminal with a His rich sequence. The His-tagged caspase-1261 protein
(approximately 42 kDa including the tag portion of 4.2 kDa) from E. coli was
partially purified. The purified proteins consisted of three polypeptides of
42
kDa, 17 kDa and 10 kDa (Figure 8A). Microsequencing of these polypeptide
chains revealed that the 42 kDa polypeptide contains the N-terminal His-tag,
suggesting that it is an intact form of caspase-120. The 10 kDa polypeptides
were a mixture of two polypeptides (roughly equal quantities), one of which
has
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the N-terminal His-tag. The other polypeptide of 10 kDa is the predicted small
subunit of caspase-120 whose sequence started right after Asp318 in caspase-12
sequence. The 17 kDa poiypeptide turned out to be a contaminating E. toll His
rich piotein, the ferric uptake regulation protein. Since there is no reported
protease of bacterial origin which shows Asp specificity, it is very likely
that the
autoprocessing of the caspase-12 protein took place at the position of Asp318
either before or during the purification of the truncated protein. It is also
probable
that the smaller subunit was obtained by the Ni column chromatography because
of its physical association with the intact form of caspase-120.
The presence of the smaller tagged protein ( 10 kDa) suggests that another
autoproeessing occurred at an Asp residue which is approximately SO residues
apart from G1y95 (about 90 residues including the tag). It was suspected that
the
mature p20 subunit of caspase-12 was lost because the N-terminal His tag was
removed through autoproeessing (Figure 8B). To recover the mature form of
caspase-12 (p20 and pI0), another version of truncated caspase-12
(caspase-12x2) which lacked a region of Metl through Asp144 was expressed.
Figure 8A shows that purified caspase-12112 consists of two major fragments
(p24 and p10). p24 had the His tag and the N-terminal of p10 had the sequence
starting from Thr319, as revealed by microsequencing. The sequence of p10
indicates that autoprocessing at the Asp318 again occurred in caspase-12112 as
well as caspase-12~ (Figure 8B). The subunit composition (two polypeptides)
and sizes (the 20 kDa subunit tagged with the 4.2 kDa His-rich sequence and
the
other subunit of 10 kDa) of the purified protein is similar to the mature form
of
ICE, which consists of 20 kDa and 10 kDa subunits.
The His-tagged caspase-12 recombinant protein was incubated with other
members of the caspase family in vitro to determine if caspase-12 can cleave
these pro-enzymes. Among the caspase family members examined, caspase-12L12
can efficiently process pro-caspase-12 (48 kDa) into fragments. With the
disappearance of the intact pro-caspase-12, fragments of about 35 kDa and 13
kDa by caspase-12112 were generated (Figure 8C). When [35SJ-labeled
CA 02402599 2002-10-15
WO 99/52925 PCT/US99/08064
-42-
caspase-120 was incubated with active caspase-1202. the 38 kDa polypeptide
was cleaved into 26 kDa and 13 kDa fragments (Figure 8C). Therefore the
truncated polypeptide contains the major cleavage site which divides
caspase-122 into the N-terminal 26 kDa and the C-terminal 13 kDa.
Other members of the family including pro-ICE, pro-caspase-2 and
pro-caspase-3, however, were resistant to the recombinant caspase-12112 as
only
faint bands of cleavage products were visible after incubation with caspase-
122
for the same (Figure 8C) or longer time period (3 hr). Resistance of ICE,
caspase-2 and caspase-3 to caspase-1202 cleavage suggests that these proteases
are unlikely to be the substrate for caspase-12 in vivv.
The cleavage of pro-caspase-I2 by caspase-12L12 was inhibited by thiol
reagents. Pretreatment of caspase-12L12 with either 5, 5'-dithio-bis(2-
nitroben2oic
acid) or iodoaeetamide completely inhibited its proteolytic activity. This
result
demonstrates that caspase-12 is a cysteine protease. On the other hand,
caspase-12 cleavage activity cannot be inhibited by up to I O uM YVAD-CHO,
the peptide inhibitor with a preference for ICE (Thornberry, N.A., et al.,
Nature
356:768-774 {1992)), or up to 10 uM DEVD-CHO, a peptide inhibitor with a
preference for CPP32 (Nicholson, D.W., et al., Nature 376:37-43 (1995)).
Consistent with this result is that purified caspase-122 showed little
activity for
cleavage of pro-IL-1 (3 and poly(ADP-ribose) polymerase, which are the
preferred
substrates for ICE and caspase-3, respectively. 'these data suggest that the
substrate specificity of caspase-12 mature protein is unique among the known
caspases. Therefore, one use of caspase-12 and shortened forms of case-12,
such
as casp-1212, may be to specifically cleave proteins for polypeptide mapping.
CA 02402599 2002-10-15
WO 99/52925 PCTlUS99/08064
--43-
Example 8
Cloning DNA Encoding caspase-12
A DNA molecule encoding the caspase-12 protein is cloned by
hybridizing a desired DNA molecule to the sequence or antisense sequence of,
for
S example, DNA SEQ ID NO:1 or DNA SEQ ID N0:13 under stringent
hybridization conditions. Those DNA molecules hybridizing to the probe
sequences are selected and transformed into a host cell. The transformants
that
express caspase-12 are selected and cloned.
One possible set of hybridization conditions for the cloning of the DNA
encoding caspase-12 protein is as follows:
1 ) prehybridizing far I hour;
2) hybridizing overnight at 65 °C in the hybridization buffer; and
3) washing once for I 5 minutes in 2xSSX at room temperature, then
two times for 30 minutes in 0.1 xSSC and 0. I % SDS at 60 ° C.
I S Example 9
Molecular Weigl:t Markers
The ca.spase-12 proteins produced recombinantly are purified by routine
methods in the art (Current Protocols in Molecular Biology, Vol. 2, Chap. 10,
John Wiley & Sons, Publishers ( 1994)). Because the deduced amino acid
sequence is known, the molecular weight of these proteins can be precisely
determined, and the proteins can be used as molecular weight markers for gel
electrophoresis. The calculated molecular weight of the full length caspase-12
protein based on the deduced amino acid sequence is 48kDa.
CA 02402599 2002-10-15
WO 99/52925 PCT/US99/08064
__
Example 10
Treatment of cells with caspase-12
Since caspase-12 can induce programmed cell death (see, Example 3},
caspase-12 is used to modulate cell death in a cell. This is accomplished by
contacting a cell with a caspase-12 poIypeptide. Caspase-12L, caspase 12S,
caspase-1211, or caspase-1202 is used for this purpose.
All art mentioned herein is incorporated by reference into the disclosure.
Having now fully described the invention by way of illustration and example
for
purposes of clarity and understanding, it will be apparent to those of
ordinary skill
in the art that certain changes and modifications may be made in the disclosed
embodiments and such modifications are intended to be within the scope of the
present invention.
CA 02402599 2002-10-15
WO 99/529Z5 PCT/US99/08064 -
-44 .1-
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
OR OTHER BIOLOGICAL MATERIAL
(PCT Rule l3bis)
A. The indications made below
relate to the deposited microorganism
or other biological material
referred to in the
description on page 6 , fine
4
B. IDENTIFICATION OF DEPOSIT
Further deposits arc identified
on an additional sheet ~
Name of depositary institution
American Type Culture Collection
Address of depositary institution
(including postal code and country)
10801 University Boulevard
Manassas, Virginia 20110-2209
United States of America
Date of deposit Accession Number
March 18, 1998 ATCC 209710
C. ADDITIONAL INDICATIONS !leave
blank if not opplicable) This
information is continued on
an additions! sheet CI
Murine caspase-12s (Plasmid pNB7)
D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE (iftlxindtcationsarenotjoralldesiguuedStates)
E. SEPARATE FURNISHING OF INDICATIONS
rrr~~Yr~mk ilnnrapphcahlr)
The indications listed below
wilt be submitted to the international
Bureau later (specify the general
nature ajthe indications e.g.,
"Accession Number of Deposit')
Far receiving Office use only For International Bureau use only
f~'This sheet was received withd This sheet was received by the International
the imernational application Bureau on:
Authori ,~~ ;..~,~>~ .~ ~"~ Authorized officer
T.r-~-r~1 a~
~ .~.~~N
;
;:
fir
-
... ~
,
Form PCTIROIl34 (July 1998) 440PC134.inscrtapp
CA 02402599 2002-10-15
-1-
SEQUENCE LTSTING
<110> The General Hospital Corporation
<120> Programmed Cell Death and Caspase-12
<130> 184-325
<140> 2,327,516
<1 41 > 1 999-04-1 4
<150> US 60/081,962
<151> 1998-04-16
<160> 38
<170> PatentIn Ver_ 2.0
<21 0> 1
<211> 1402
<212> DNA
<213> Artificial Sequence
<220>
<221> CDS
<222> (45)..(1301)
<220>
<223> Description of Artificial Sequence. cDNA
<400> 1
cttttttttt tttttttttt tatgtcctgg agtcctgcac agcc atg gcg gcc agg 56
Met Ala Ala Arg
1
agg aca cat gaa aga gat cca atc tac aag atc aaa ggt ttg gcc aag 104
Arg Thr His Glu Arg Asp Pro Ile Tyr Lys I1e Lys Gly Leu Ala Lys
10 15 20
gac atg ctg gat ggg gtt ttt gat gac ctg gtg gag aag aat gtt tta 152
Asp Met Leu Asp Gly Val Phe Asp Asp Leu Val Glu Lys Asn Val Leu
25 30 35
aat gga gat gag tta ctc aaa ata ggg gaa agt gcg agt ttc atc ctg 200
Asn Gly Asp Glu Leu Leu Lys Ile Gly Glu Ser Ala Ser Phe Ile Leu
40 45 50
aac aag get gag aat ctg gtt gag aac ttc tta gag aaa aca gac atg 248
Asn Lys Ala Glu Asn Leu Val Glu Asn Phe Leu Glu Lys Thr Asp Met
55 60 65
CA 02402599 2002-10-15
-2-
gca gga aaa ata ttt get ggc cac att gcc aat tcc cag gaa cag ctg 296
Ala Gly Lys Ile Phe Ala Gly His Ile Ala Asn Ser Gln Glu Gln Leu
70 75 80
agt tta caa ttt tct aat gat gag gat gat gga cct cag aag ata tgt 344
Ser Leu Gln Phe Ser Asn Asp Glu Asp .Asp Gly Pro Gln Lys Ile Cys
85 90 9S 100
aca cct tct tct cca tca gaa tcc aag aga aaa gta gag gat gat gaa 392
Thr Pro Ser Ser Pro Ser Glu Ser Lys Arg Lys Val Glu Asp Asp Glu
105 110 115
atg gag gta aat get gga ttg gcc cat gaa tca cat cta atg ctg aca 440
Met Glu Val Asn Ala Gly Leu Ala His Glu Ser His Leu Met Leu Thr
120 125 130
get cct cat gga ctc cag agc tca gaa gtc caa gat aca ctg aag ctt 488
Ala Pro His Gly Leu Gln Ser Ser Glu Val Gln Asp Thr Leu Lys Leu
135 140 145
tgt cca cgt gat cag ttt tgt aag ata aag aca gaa agg gca aaa gag 536
Cys Pro Arg Asp Gln Phe Cys Lys Ile Lys Thr Glu Arg Ala Lys Glu
150 155 160
ata tat eca gtg atg gag aag gag gga cga aca egt ctg get ctc ate 584
Ile Tyr Pro Val Met Glu Lys Glu Gly Arg Thr Arg Leu Ala Leu Ile
165 170 175 180
atc tgc aac aaa aag ttt gac tac ctt ttt gat aga gat aat get gat 632
Ile Cys Asn Lys Lys Phe Asp Tyr Leu Phe Asp Arg Asp Asn Ala Asp
185 190 195
act gac att ttg aac atg caa gaa cta ctt gaa aat ctt gga tac tct 680
Thr Asp Ile Leu Asn Met Gln Glu Leu Leu Glu Asn Leu Gly Tyr Ser
200 205 210
gtg gtg tta aaa gaa aac ctt aca get cag gaa atg gag aca gag tta 728
Val Val Leu Lys Glu Asn Leu Thr Ala Gln Glu Met Glu Thr Glu Leu
215 220 225
atg cag ttt get ggc cgt cca gag cac cag tcc tca gac agc aca ttc 776
Met Gln Phe Ala Gly Arg Pro Glu His Gln Ser Ser Asp Ser Thr Phe
230 235 240
ctg gtg ttt atg tcc cat ggc atc ctg gaa gga atc tgt ggg gtg aag 824
Leu Val Phe Met Ser His Gly Ile Leu Glu Gly Ile Cys Gly Val Lys
245 250 255 260
cac cga aac aaa aag cca gat gtt ctt cat gat gac act atc ttc aaa 872
His Arg Asn Lys Lys Pro Asp Val Leu His Asp Asp Thr Ile Phe Lys
265 270 275
CA 02402599 2002-10-15
-3-
att ttc aac aac tct aac tgt cgg agt ctg aga aac aaa ccc aag att 920
Ile Phe Asn Asn Ser Asn Cys Arg Ser Leu Arg Asn Lys Pro Lys Ile
280 285 290
ctc atc atg cag gcc tgc aga ggc aga tat aat gga act att tgg gta 968
Leu Ile Met Gln Ala Cys Arg Gly Arg Tyr Asn Gly Thr Ile Trp Val
295 300 305
tcc aca aac aaa ggg ata gcc act get gat aca gat gag gaa cgt gtg 1016
Ser Thr Asn Lys Gly Ile Ala Thr Ala Asp Thr Asp Glu Glu Arg Val
31 0 31 5 320
ttg agc tgt aaa tgg aat aat agt ata aca aag gcc cat gtg gag aca 1064
Leu Ser Cys Lys Trp Asn Asn Ser Ile Thr Lys Ala His Val Glu Thr
325 330 335 340
gat ttc att get ttc aaa tct tct acc eca cat aat att tct tgg aag 1112
Asp Phe Ile Ala Phe Lys Ser Ser Thr Pro His Asn Ile Ser Trp Lys
345 350 355
gta ggc aag act ggt tcc ctc ttc att tcc aaa ctc att gac tgc ttc 1160
Val Gly Lys Thr Gly Ser Leu Phe Ile Ser Lys Leu Ile Asp Cys Phe
360 365 370
aaa aag tac tgt tgg tgt tat cat ttg gag gaa att ttt cga aag gtt 1208
Lys Lys Tyr Cys Trp Cys Tyr His Leu Glu Glu Ile Phe Arg Lys Val
375 380 385
caa cac tca ttt gag gtc cca ggt gaa ctg acc cag atg ccc act att 1256
Gln His Ser Phe Glu Val Pro Gly Glu Leu Thr Gln Met Pro Thr Ile
390 395 400
gag aga gta tcc atg aca cgc tat ttc tac ctt ttt ccc ggg aat 1301
Glu Arg Val Ser Met Thr Arg Tyr Phe Tyr Leu Phe Pro Gly Asn
405 41 0 41 5
tagcacaggc aactctcatg cagttcacag tcaagtattg ctgtagctga gaagaaaaga 1361
aaattccaag atcccaggat ttttaaatgt gtaaaacttt t 1402
<210> 2
<211> 419
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Deduced amino acid of Caspase-12
<400> 2
Met Ala Ala Arg Arg Thr His Glu Arg Asp Pro Ile Tyr Lys Tle Lys
1 5 10 15
CA 02402599 2002-10-15
-4-
Gly Leu Ala Lys Asp Met Leu Asp Gly Val Phe Asp Asp Leu Val Glu
20 25 30
Lys Asn Val Leu Asn Gly Asp Glu Leu Leu Lys Ile Gly Glu Ser Ala
35 40 45
Ser Phe Ile Leu Asn Lys Ala Glu Asn Leu Val Glu Asn Phe Leu Glu
50 55 60
Lys Thr Asp Met Ala Gly Lys Ile Phe Ala Gly His Ile Ala Asn Ser
65 70 75 80
Gln Glu Gln Leu Ser Leu Gln Phe Ser Asn Asp Glu Asp Asp Gly Pro
85 90 95
Gln Lys Ile Cys Thr Pro Ser Ser Pro Ser Glu Ser Lys Arg Lys Val
100 105 110
Glu Asp Asp Glu Met Glu Val Asn Ala Gly Leu Ala His Glu Ser His
115 120 125
Leu Met Leu Thr Ala Pro His Gly Leu Gln Ser Ser Glu Val Gln Asp
130 135 140
Thr Leu Lys Leu Cys Pro Arg Asp Gln Phe Cys Lys Ile Lys Thr Glu
145 150 155 160
Arg Ala Lys Glu Ile Tyr Pro Val Met Glu Lys Glu Gly Arg Thr Arg
165 170 175
Leu Ala Leu Ile Ile Cys Asn Lys Lys Phe Asp Tyr Leu Phe Asp Arg
180 185 190
Asp Asn Ala Asp Thr Asp Ile Leu Asn Met Gln Glu Leu Leu Glu Asn
195 200 205
Leu Gly Tyr Ser Val Val Leu Lys Glu Asn Leu Thr Ala Gln Glu Met
21 0 21 5 220
Glu Thr Glu Leu Met Gln Phe Ala Gly Arg Pro Glu His Gln Ser Ser
225 230 235 240
Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Leu Glu Gly Ile
245 250 255
Cys Gly Val Lys His Arg Asn Lys Lys Pro Asp Val Leu His Asp Asp
260 265 270
Thr Ile Phe Lys Ile Phe Asn Asn Ser Asn Cys Arg Ser Leu Arg Asn
275 280 285
Lys Pro Lys Ile Leu Ile Met Gln Ala Cys Arg Gly Arg Tyr Asn Gly
CA 02402599 2002-10-15
-5-
290 295 300
Thr Ile Trp Val Ser Thr Asn Lys Gly Ile Ala Thr Ala Asp Thr Asp
305 310 315 320
Glu Glu Arg Val Leu Ser Cys Lys Trp Asn Asn Ser Ile Thr Lys Ala
325 330 335
His Val Glu Thr Asp Phe Ile Ala Phe Lys Ser 5er Thr Pro His Asn
340 345 350
Ile Ser Trp Lys Val Gly Lys Thr Gly Ser Leu Phe Ile Ser Lys Leu
355 360 365
Ile Asp Cys Phe Lys Lys Tyr Cys Trp Cys Tyr His Leu Glu Glu Ile
370 375 380
Phe Arg Lys Val Gln His Ser Phe Glu Val Pro Gly Glu Leu Thr Gln
385 390 395 400
Met Pro Thr Ile Glu Arg Val Ser Met Thr Arg Tyr Phe Tyr Leu Phe
405 410 415
Pro Gly Asn
<210> 3
<211> 5
<212> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: conserved
pentapeptide
<400> 3
Gln Ala Cys Arg Gly
1 5
<21 0> 4
<211> 418
<21 2> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: Caspase-5
<400> 4
Met Phe Lys Gly Ile Leu Gln Ser Gly Leu Asp Asn Phe Val Ile Asn
1 5 10 15
CA 02402599 2002-10-15
-6-
His Met Leu Lys Asn Asn Val Ala GIy Gln Thr Ser Ile Gln Thr Leu
20 25 30
Val Pro Asn Thr Asp Gln Lys Ser Thr Ser Val Lys Lys Asp Asn His
35 40 45
Lys Lys Lys Thr Val Lys Met Leu Glu Tyr Leu Gly Lys Asp Val Leu
50 55 60
His Gly Val Phe Met Tyr Leu Ala Lys His Asp Val Leu Thr Leu Lys
65 70 75 80
Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Ala Lys Thr Glu Asp Lys Ala
85 90 95
Leu Ile Leu Val Asp Ser Leu Arg Lys Asn Arg Val Ala His Gln Met
100 105 110
Phe Thr Gln Thr Leu Leu Asn Met Asp Gln Lys Ile Thr Ser Val Lys
115 120 125
Pro Leu Leu Gln Ile Asp Ala Gly Pro Pro Glu Ser AIa Glu Ser Thr
130 135 140
Asn Ile Leu Lys Leu Cys Pro Arg Glu Glu Phe Leu Arg Leu Cys Lys
145 150 155 160
Lys Asn His Asp Glu Ile Tyr Pro Ile Lys Lys Arg Glu Asp Arg Arg
165 170 17S
Arg Leu Ala Leu Ile Ile Cys Asn Thr Lys Phe Asp His Leu Pro Ala
180 185 190
Arg Asn Gly Ala His Tyr Asp Ile Val Gly Met Lys Arg Leu Leu Gln
195 200 205
Gly Leu Gly Tyr Thr Val Val Asp Glu Lys Asn Leu Thr Ala Arg Asp
210 21 5 220
Met Glu Ser Val Leu Arg Ala Phe AIa Ala Arg Pro Glu His Lys Ser
225 230 235 240
Ser Asp Ser Thr Phe Leu Val Leu Met Ser His Gly Ile Leu Glu Gly
245 250 255
Ile Cys Gly Thr Ala His Lys Lys Lys Lys Pro Asp Val Leu Leu Tyr
260 265 270
Asp Thr Ile Phe Gln Ile Phe Asn Asn Arg Asn Cys Leu Ser Leu Lys
275 280 285
Asp Lys Pro Lys Val Ile Tle Val Gln Ala Cys Arg Gly GIu Lys His
CA 02402599 2002-10-15
290 295 300
Gly Glu Leu Met Val Arg Asp Ser Pro Ala Ser Leu Ala Val Ile Ser
305 310 315 320
Ser Gln Ser Ser Glu Asn Leu Glu Ala Asp Ser Val Cys Lys Ile His
325 330 335
Glu Lys Lys Asp Phe Ile Ala Phe Cys Ser Ser Ser Pro His Asn Val
340 345 350
Ser Trp Arg Asp Arg Thr Arg Gly Ser Ile Phe Ile Thr Glu Leu Ile
355 360 365
Thr Cys Phe Gln Lys Tyr Ser Cys Cys Cys His Leu Met Glu Ile Phe
370 375 380
Arg Lys Val Gln Lys Ser Phe Glu Val Pro Gln Ala Lys Ala Gln Met
385 390 395 400
Pro Thr Ile Glu Arg Ala Thr Leu Thr Arg Asp Phe Tyr Leu Phe Pro
405 410 415
Gly Asn
<210> 5
<211> 404
<212> PRT
<213> Homo
Sapiens
<400> 5
Met Ala Lys Leu Lys LysArg Lys Leu IleArg
Asp Val Glu Phe Ser
1 5 10 15
Met Gly Gly Ile Asn LeuLeu Asp Glu LeuGln
Glu Thr Gly Leu Thr
20 25 30
Arg Val Asn Glu Glu GluLys Val Lys GluAsn
Leu Lys Met Arg Ala
35 40 45
Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp Ser Val Ile Pro Lys
50 55 60
Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Tyr Ile Cys Glu Glu Asp
65 70 75 80
Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala Asp Gln Thr Ser Gly
85 90 95
Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Val Leu Ser Ser Phe Pro
100 105 110
CA 02402599 2002-10-15
_g_
Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly
115 120 125
Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile
130 135 140
Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser
145 150 155 160
Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile
165 170 175
Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu
180 185 190
Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala
195 200 205
Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His
21 0 21 5 220
Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg
225 230 235 240
Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu
245 250 255
Gln Leu Met Ala Ile Phe Asn Met Leu Asn Thr Lys Met Cys Pro Ser
260 265 270
Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp
275 280 285
Ser Pro Gly Val Val Trp Phe Lys Asp Ser Val Gly Val Ser Gly Asn
290 295 300
Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys
305 310 315 320
Ala His Ile Lys Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp
325 330 335
Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg
340 345 350
Leu Ile Gly His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu
355 360 365
Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala
370 375 380
Gln Met Pro Thr Thr Glu Arg Val Thr Leu Thr Arg Cys Phe Tyr Leu
CA 02402599 2002-10-15
-9-
385 390 395 400
Phe Pro Gly His
<210> 6
<211> 402
<21 2> PRT
<213> Mus musculus
<400> 6
Met Ala Asp Lys Ile Leu Arg Ala Lys Arg Lys Gln Phe Ile Asn Ser
1 5 10 15
Val Ser Ile Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Glu Lys
20 25 30
Arg Val Leu Asn Gln Glu Glu Met Asp Lys Ile Lys Leu Ala Asn Ile
3S 40 45
Thr Ala Met Asp Lys Ala Arg Asp Leu Cys Asp His Val Ser Lys Lys
50 55 60
Gly Ala Pro Ala Ser Gln Ile Phe Ile Thr Tyr Ile Cys Asn Glu Asp
65 70 75 80
Cys Tyr Leu Ala Gly Ile Leu Glu Leu Gln Ser Ala Pro Ser Ala Glu
85 90 95
Thr Phe Val Ala Thr Glu Asp Ser Lys Gly Gly His Pro Ser Ser Ser
100 105 110
Glu Thr Lys Glu Glu Gln Asn Lys Glu Asp Gly Thr Phe Pro Gly Leu
115 120 125
Thr Gly Thr Leu Lys Phe Cys Pro Leu Glu Lys Ala Gln Lys Leu Trp
130 135 140
Lys Glu Asn Pro Ser Glu Ile Tyr Pro Ile Met Asn Thr Thr Thr Arg
145 150 155 160
Thr Arg Leu Ala Leu Ile Ile Cys Asn Thr Glu Phe Gln His Leu Ser
165 170 175
Pro Arg Val Gly Ala Gln Val Asp Leu Arg Glu Met Lys Leu Leu Leu
180 185 190
Glu Asp Leu Gly Tyr Thr Val Lys Val Lys Glu Asn Leu Thr Ala Leu
195 200 205
Glu Met Val Lys Glu Val Lys Glu Phe Ala Ala Cys Pro Glu His Lys
21 0 21 5 220
CA 02402599 2002-10-15
Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Gln Glu
225 230 235 240
Gly Ile Cys Gly Thr Thr Tyr Ser Asn Glu Val Ser Asp Tle Leu Lys
245 250 255
Val Asp Thr Ile Phe Gln Met Met Asn Thr Leu Lys Cys Pro Ser Leu
260 265 270
Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Glu Lys
275 280 285
Gln Gly Val Val Leu Leu Lys Asp Ser Val Arg Asp Ser Glu Glu Asp
290 295 300
Phe Leu Thr Asp Ala Ile Phe Glu Asp Asp Gly Ile Lys Lys Ala His
305 310 315 320
Ile Lys Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp Asn Val
325 330 335
Ser Trp Arg His Pro Val Arg Gly Ser Leu Phe Ile Glu Ser Leu Ile
340 345 350
Lys His Met Lys Glu Tyr Ala Trp Ser Cys Asp Leu Glu Asp Ile Phe
355 360 365
Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Lys Glu Arg Leu Gln Met
370 375 380
Pro Thr Ala Asp Arg Val Thr Leu Thr Lys Arg Phe Tyr Leu Phe Pro
385 390 395 400
Gly His
<210> 7
<211> 373
<212> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: Caspase-11
<400> 7
Met Ala Glu Asn Lys His Pro Asp Lys Pro Leu Lys Val Leu Glu Gln
1 5 10 15
Leu Gly Lys Glu Val Leu Thr Glu Tyr Leu Lys Lys Leu Val Gln Ser
20 25 30
Asn Val Leu Lys Leu Lys Glu Glu Asp Lys GIn Lys Phe Asn Trp Ala
CA 02402599 2002-10-15
-11-
35 40 45
Glu Arg Ser Asp Lys Arg Met Val Phe Val Asp Ala Met Lys Lys Lys
50 55 60
His Ser Lys Val Gly Glu Met Leu Leu Gln Thr Phe Phe Ser Val Asp
65 70 75 80
Pro Gly Ser His His Gly Glu Ala Asn Leu Glu Met Glu GIu Pro Lys
85 90 95
Lys Ser Leu Met Thr Leu Lys Leu Cys Ser Pro Glu Glu Phe Thr Arg
100 105 110
Leu Cys Arg Glu Lys Thr Gln Glu Ile Tyr Pro Ile Lys Glu Ala Asn
115 120 125
Gly Arg Thr Arg Lys Ala Leu Ile Ile Cys Asn Thr Glu Phe Lys His
130 135 140
Leu Ser Leu Arg Tyr Gly Ala Lys Phe Asp Ile Ile Gly Met Lys Gly
145 150 155 160
Leu Leu Glu Asp Leu Gly Tyr Asp Val Val Val Lys Glu Asn Leu Thr
165 170 175
Ala Glu Gly Met Glu Ser Glu Met Lys Asp Phe Ala Ala Leu Ser Glu
180 185 190
Lys Gln Thr Ser Asp Ser Thr Phe Leu Val Leu Met Ser His Gly Thr
195 200 205
Leu His Gly Ile Cys Gly Thr Met His Ser Glu Lys Thr Pro Asp Val
21 0 21 5 220
Leu Gln Tyr Asp Thr Ile Tyr Gln Ile Phe Asn Asn Cys His Cys Pro
225 230 235 240
Gly Leu Arg Asp Lys Pro Lys Val Ile Ile Val Gln Ala Cys Arg Gly
245 250 255
Gly Met Ser Gly Glu Met His Ile Arg Glu Ser Ser Lys Pro Gln Leu
260 265 270
Cys Arg Gly Val Asp Leu Pro Arg Asn Met Glu Ala Asp Ala Val Lys
275 280 285
Leu Ser His Val Lys Lys Asp Phe Ile Aia Phe Tyr Ser Thr Thr Pro
290 295 300
His His Leu Ser Tyr Arg Asp Lys Thr Gly Gly Ser Tyr Phe Ile Thr
305 310 315 320
CA 02402599 2002-10-15
-12-
Arg Leu Ile Ser Cys Phe Arg Lys His Ala Cys Ser Cys His Leu Phe
325 :330 335
Asp Ile Phe Leu Lys Val Gln Gln Ser Phe Glu Lys Ala Ser Ile His
340 345 350
Ser Gln Met Pro Thr Ile Asp Arg Ala Thr Leu Thr Arg Tyr Phe Tyr
355 360 365
Leu Phe Pro Gly Asn
370
<210> 8
<211> 379
<212> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism:Caspase-4
<400> 8
Met Ala Glu Gly Asn His Arg Lys Lys Pro Leu Lys Val Leu Glu Ser
1 5 10 15
Leu Gly Lys Asp Phe Leu Thr Gly Val Leu Asp Asn Leu Glu Val Gln
20 25 30
Asn Val Leu Met Trp Lys Lys Glu Glu Lys Lys Lys Tyr Tyr Asp Ala
35 40 45
Lys Thr Glu Asp Lys Val Arg Ala Met Ala Asp Ser Met Gln Glu Lys
50 55 60
Gln Arg Met Ala Gly Gln Met Leu Leu Gln Thr Phe Phe Asn Ile Asp
65 70 75 80
Gln Ile Ser Pro Asn Lys Lys Ala His Pro Asn Met Glu Ala Gly Pro
85 90 95
Pro Glu Ser Gly Glu Ser Thr Asp Ala Leu Lys Leu Cys Pro His Glu
100 105 110
Glu Phe Leu Arg Leu Cys Lys Glu Arg Ala Glu Glu Ile Tyr Pro Ile
115 120 125
Lys Glu Arg Asn Asn Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Thr
130 135 140
Glu Phe Asp His Leu Pro Pro Arg Asn Gly Ala Asp Phe Asp Ile Thr
145 150 155 160
CA 02402599 2002-10-15
-13-
Gly Met Lys Glu Leu Leu Leu Leu Glu Gly Leu Asp Tyr Ser Val Asp
165 170 175
Val Glu Glu Asn Leu Thr Ala Arg Asp Met Glu Ser Ala Leu Arg Ala
180 185 190
Phe Ala Thr Arg Pro Glu His L,ys Ser Ser Asp Ser Thr Phe Leu Val
195 200 205
Leu Met Ser His Gly Ile Leu Glu Gly Ile Cys Gly Thr Val His Asp
21 0 21 5 220
Glu Lys Lys Pro Asp Val Leu Leu Tyr Asp Thr Ile Phe Gln Ile Phe
225 230 235 240
Asn Asn Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro Lys Val Ile Ile
245 250 255
Val Gln Ala Cys Arg Gly Ala Met Arg Gly Glu Leu Met Val Arg Asp
260 265 270
Ser Pro Ala Ser Leu Glu Val Ala Ser Ser Gln Ser Ser GIu Asn Leu
275 280 285
Glu Glu Asp Ala Val Tyr Lys Thr His Val Glu Lys Asp Phe Ile Ala
290 295 300
Phe Cys Ser Ser Thr Pro His Asn Val Ser Trp Arg Asp Ser Thr Met
305 310 315 320
Gly Ser IIe Phe Ile Thr Gln Leu Ile Thr Cys Phe Gln Lys Tyr Ser
325 330 335
Trp Cys Cys His Leu Glu Glu Val Phe Arg Lys Val Gln Gln Ser Phe
340 345 350
Glu Thr Pro Arg Ala Lys Ala Gln Met Pro Thr Ile Glu Arg Leu Ser
355 360 365
Met Thr Arg Tyr Phe Tyr Leu Phe Pro Gly Asn
370 375
<21 0> 9
<211> 13
<212> PRT
<213> Caenorhabditis elegans
<400> 9
His Gly Cys Arg Gln Arg Ser Val Trp Asn Ser Ser Gly
1 5 10
CA 02402599 2002-10-15
-14-
<210> 10
<211> 435
<212> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: Caspase-2
<400> 10
Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gly Val Cys Gly Met His
1 5 10 15
Fro His His Gln Glu Thr Leu Lys Lys Asn Arg Val Val Leu Ala Lys
20 25 30
Gln Leu Leu Leu Ser Glu Leu Leu Glu His Leu Leu Glu Lys Asp Ile
35 40 45
Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Ala Lys Val Gly Ser Phe
50 55 b0
Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pro Lys Arg Gly Pro Gln
65 70 75 80
Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Glu Thr Lys Gln GIy His
85 90 95
Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gly Leu Gln His Val Leu
100 105 110
Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro Val Cys
115 120 125
Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp Thr Val
130 135 140
Glu His Ser Leu Asp Asn Lys Asp Gly Pro Val Cys Leu Gln Val Lys
145 150 155 160
Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala Tyr Arg
165 170 17S
Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val His
180 185 190
Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp Val Asp
195 200 205
His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp Val His
210 215 220
CA 02402599 2002-10-15
-15-
Val Leu Cys Asp Gln Thr Ala Gln Val Met Gln Glu Lys Leu Gln Asn
225 230 235 240
Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp Ser Cys Ile Val Ala
245 250 255
Leu Leu Ser His Gly Val Glu Gly Ala Ile Tyr Gly Val Asp Gly Lys
260 265 270
Leu Leu Gln Leu Gln Glu Val Phe Gln Leu Phe Asp Asn Ala Asn Cys
275 280 285
Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Phe Ile Gln Ala Cys Arg
290 295 300
Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gln Asp Gly Lys Asn His
305 310 315 320
Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Ala Gly Lys Glu Lys Leu
325 330 335
Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Met Ile Cys Gly Tyr Ala
340 345 350
Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser Trp
355 360 3b5
Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys Asp Met
370 375 380
His Val Ala Asp Met Leu Val Lys Lys Asn Ala Leu Ile Lys Asp Arg
385 390 395 400
Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu Met Ser
405 41 0 41 5
Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro Gly His
420 425 430
Pro Pro Thr
435
<210> 11
<211> 277
<212> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: Caspase-3
<400> 11
CA 02402599 2002-10-15
-16-
Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu
1 5 10 15
Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser
20 25 30
Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile
35 40 45
Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Asn Thr Ser Arg
50 55 60
Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn
65 70 75 80
Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Lys Glu Ile
85 90 95
Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser
100 105 110
Ser Phe Val Cys Val Leu Leu Ser His Gly Lys Glu Gly Ile Ile Phe
115 120 125
Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg
130 135 140
Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile
145 150 155 160
Gln Ala Cys Arg Gly Thr Lys Leu Asp Cys Gly Ile Glu Thr Asp Ser
165 170 175
Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp
180 185 190
Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn
195 200 205
Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys
21 0 21 5 220
Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn
225 230 235 240
Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe
245 250 255
His Ala Lys Lys Gln Ile Pro Cys Il.e Val Ser Met Leu Thr Lys Glu
260 265 270
Leu Tyr Phe Tyr His
CA 02402599 2002-10-15
-17-
275
<210> 12
<211> 503
<212> PRT
<213> Caenorhabditis elegans
<400> 12
Met Met Arg Gln Asp Arg Arg Ser Leu Leu Glu Arg Asn Ile Met Met
1 5 10 15
Phe Ser Ser His Leu Lys Val Asp Glu Ile Leu Glu Val Leu Ile Ala
20 25 30
Lys Gln Val Leu Asn Ser Asp Asn Gly Asp Asn Ile Asn Ser Cys Gly
35 40 45
Thr Val Arg Glu Lys Arg Arg Glu Ile Val Lys Ala Val Gln Arg Arg
50 55 60
Gly Asp Val Ala Phe Asp Ala Phe Tyr Asp Ala Leu Arg Ser Thr Gly
65 70 75 80
His Glu Gly Leu Ala Glu Val Leu Glu Pro Leu Ala Arg Ser Val Asp
85 90 95
Ser Asn Ala Val Glu Phe Glu Cys Pro Met Ser Pro Ala Ser His Arg
100 105 110
Arg Ser Arg Ala Leu Ser Pro Ala Gly Tyr Thr Ser Pro Thr Arg Val
115 120 125
His Arg Asp Ser Val Ser Ser Val 5er Ser Phe Thr Ser Tyr G1n Asp
130 135 140
Ile Tyr Ser Arg Ala Arg Ser Arg Ser Arg Ser Arg Ala Leu His Ser
145 150 155 160
Ser Asp Arg His Asn Tyr Ser Ser Prc> Pro Val Asn Ala Phe Pro Ser
165 170 175
Gln Pro Ser Ser Ala Asn Ser Ser Phe Thr Gly Cys Ser Ser Leu Gly
180 185 190
Tyr Ser Ser Ser Arg Asn Arg Ser Phe Ser Lys Ala Ser Gly Pro Thr
195 200 205
Gln Tyr Tle Phe His Glu Glu Asp Met Asn Phe Val Asp Ala Pro Thr
210 21 5 220
Ile Ser Arg Val Phe Asp Glu Lys Thr Met Tyr Arg Asn Phe Ser Ser
CA 02402599 2002-10-15
-18-
225 230 235 240
Pro Arg Gly Met Cys Leu Ile Ile Asn Asn Glu His Phe Glu Gln Met
245 250 255
Pro Thr Arg Asn Gly Thr Lys Ala Asp Lys Asp Asn Leu Thr Asn Leu
260 265 270
Phe Arg Cys Met Gly Tyr Thr Val Ile Cys Lys Asp Asn Leu Thr Gly
275 280 285
Arg Gly Met Leu Leu Thr Ile Arg Asp Phe Ala Lys His Glu Ser His
290 295 300
Gly Asp Ser Ala Ile Leu Val Ile Leu Ser His Gly Lys Glu Asn Val
305 310 315 320
Ile Tle Gly Val Asp Asp Ile Pro Ile Ser Thr His Glu Ile Tyr Asp
325 330 335
Leu Leu Asn Ala Ala Asn Ala Pro Arg Leu Ala Asn Lys Pro Lys Ile
340 34S 350
Val Phe Val Gln Ala Cys Arg Gly Glu Arg Arg Asp Asn Gly Phe Pro
355 360 365
Val Leu Asp Ser Val Asp Gly Val Pro Ala Phe Leu Arg Arg Gly Trp
370 375 380
Asp Asn Arg Asp Gly Pro Leu Phe Asn Phe Leu Gly Cys Val Arg Pro
385 390 395 400
Gln Val Gln Gln Val Trp Arg Lys Lys Pro Ser Gln Ala Asp Ile Leu
405 410 415
Ile Ala Tyr Ala Thr Thr Ala Gly Ile Val Ser Trp Arg Asn Ser Ala
420 425 430
Arg Gly Ser Trp Phe Ile Gln Ala Val Cys Glu Val Phe Ser Thr His
435 440 445
Ala Lys Asp Met Asp Val Val Glu Leu Leu Thr Glu Val Asn Lys Lys
450 455 460
Val Ala Cys Gly Phe Gln Thr Ser Gln Gly Ser Asn Ile Leu Lys Gln
465 470 475 480
Met Pro Glu Met Thr Ser Arg Leu Leu Lys Lys Phe Tyr Pro Trp Pro
485 490 495
Glu Ala Arg Asn Ser Ala Val
500
CA 02402599 2002-10-15
-19-
<210> 13
<211> 1050
<21 2> DNA
<213> Artificial Sequence
<220>
<221 > CDS
<222> (1)..(1047)
<220>
<223> Description of Artificial Sequence: cDNA
<400> 13
atg gcg gcc agg agg aca cat gaa aga gat cca atc tac aag atc aaa 48
Met Ala Ala Arg Arg Thr His Glu Arg Asp Pro Ile Tyr Lys Ile Lys
1 5 10 15
gaa ttt tct aat gat gag gat gat gga cct cag aag ata tgt aca cct 96
Glu Phe Ser Asn Asp Glu Asp Asp Gly Pro Gln Lys Ile Cys Thr Pro
20 25 30
tct tct cca tca gaa tcc aag aga aaa gta gag gat gat gaa atg gag 144
Ser Sex Pro Ser Glu Ser Lys Arg Lys Val Glu Asp Asp Glu Met Glu
35 40 45
gta aat get gga ttg gcc cat gaa tca cat cta atg ctg aca get cct 192
Val Asn Ala Gly Leu Ala His Glu Ser His Leu Met Leu Thr Ala Pro
50 55 60
cat gga ctc cag agc tca gaa gtc caa gat aca ctg aag ctt tgt cca 240
His Gly Leu Gln Ser Ser Glu Val Gln Asp Thr Leu Lys Leu Cys Pro
65 70 75 80
cgt gat cag ttt tgt aag ata aag aca gaa agg gca aaa gag ata tat 288
Arg Asp Gln Phe Cys Lys Ile Lys Thr Glu Arg Ala Lys Glu Ile Tyr
85 90 95
cca gtg atg gag aag gag gga cga aca cgt ctg get ctc atc atc tgc 336
Pro Val Met Glu Lys Glu Gly Arg Thr Arg Leu Ala Leu Ile Ile Cys
100 105 110
aac aaa aag ttt gac tac ctt ttt gat aga gat aat get gat act gac 384
Asn Lys Lys Phe Asp Tyr Leu Phe Asp Arg Asp Asn Ala Asp Thr Asp
115 120 12S
att ttg aac atg caa gaa cta ctt gaa aat ctt gga tac tct gtg gtg 432
IIe Leu Asn Met Gln Glu Leu Leu Glu Asn Leu Gly Tyr Ser Val Val
130 135 140
tta aaa gaa aac ctt aca get cag gaa atg gag aca gag tta atg cag 480
CA 02402599 2002-10-15
-20-
Leu Lys Glu Asn Leu Thr Ala Gln Glu Met Glu Thr Glu Leu Met Gln
145 150 155 160
ttt get gge cgt eea gag cac cag tcc tca gac agc aca ttc ctg gtg 528
Phe Ala Gly Arg Pro Glu His Gln Ser Ser Asp Ser Thr Phe Leu Val
165 170 175
ttt atg tcc cat ggc atc ctg gaa gga atc tgt ggg gtg aag cac cga 576
Phe Met Ser His Gly Ile Leu Glu Gly Ile Cys Gly Val Lys His Arg
180 185 190
aac aaa aag cca gat gtt ctt cat gat gac act atc ttc aaa att ttc 624
Asn Lys Lys Pro Asp Val Leu His Asp Asp Thr Ile Phe Lys Ile Phe
195 200 205
aac aac tct aac tgt cgg agt ctg aga aac aaa ccc aag att ctc atc 672
Asn Asn Ser Asn Cys Arg Ser Leu Arg Asn Lys Pro Lys Ile Leu Ile
210 215 220
atg cag gcc tgc aga ggc aga tat aat gga act att tgg gta tcc aca 720
Met Gln Ala Cys Arg Gly Arg Tyr Asn Gly Thr Ile Trp Val Ser Thr
225 230 235 240
aac aaa ggg ata gce act get gat aca gat gag gaa cgt gtg ttg age 768
Asn Lys Gly Ile Ala Thr Ala Asp Thr Asp Glu Glu Arg Val Leu Ser
245 250 255
tgt aaa tgg aat aat agt ata aca aag gcc cat gtg gag aca gat ttc 816
Cys Lys Trp Asn Asn Ser Ile Thr Lys Ala His Val Glu Thr Asp Phe
260 265 270
att get ttc aaa tct tct acc eca cat aat att tct tgg aag gta ggc 864
Ile Ala Phe Lys Ser Ser Thr Pro His Asn Ile Ser Trp Lys Val Gly
275 280 285
aag act ggt tcc ctc ttc att tcc aaa ctc att gac tgc ttc aaa aag 912
Lys Thr Gly Ser Leu Phe Ile Ser Lys Leu Ile Asp Cys Phe Lys Lys
290 295 300
tac tgt tgg tgt tat cat ttg gag gaa att ttt cga aag gtt caa cac 960
Tyr Cys Trp Cys Tyr His Leu Glu Glu Ile Phe Arg Lys Val Gln His
305 310 315 320
tca ttt gag gtc cca ggt gaa ctg acc cag atg ccc act att gag aga 1008
Ser Phe Glu Val Pro Gly Glu Leu Thr Gln Met Pro Thr Ile Glu Arg
325 330 335
gta tcc atg aca cgc tat ttc tac ctt ttt ccc ggg aat tag 1050
Val Ser Met Thr Arg Tyr Phe Tyr Leu Phe Pro Gly Asn
340 345
CA 02402599 2002-10-15
-21-
<210> 14
<211> 349
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Deduced amino acid of caspase-12S
<400> 14
Met Ala Ala Arg Arg Thr His Glu Arg Asp Pro Ile Tyr Lys Ile Lys
1 5 10 15
Glu Phe Ser Asn Asp Glu Asp Asp Gly Pro Gln Lys Ile Cys Thr Pro
20 25 30
Sex Ser Pro Ser Glu Ser Lys Arg Lys Val Glu Asp Asp Glu Met Glu
35 40 45
Val Asn Ala Gly Leu Ala His Glu Ser His Leu Met Leu Thr Ala Pro
50 55 60
His Gly Leu Gln Ser Ser Glu Val Gln Asp Thr Leu Lys Leu Cys Pro
65 70 75 80
Arg Asp Gln Phe Cys Lys Ile Lys Thr Glu Arg Ala Lys Glu Ile Tyr
85 90 95
Pro Val Met Glu Lys Glu Gly Arg Thr Arg Leu Ala Leu Ile Ile Cys
100 105 110
Asn Lys Lys Phe Asp Tyr Leu Phe Asp Arg Asp Asn Ala Asp Thr Asp
115 120 125
Ile Leu Asn Met Gln Glu Leu Leu Glu Asn Leu Gly Tyr Ser Val Val
130 135 140
Leu Lys Glu Asn Leu Thr Ala Gln Glu Met Glu Thr Glu Leu Met Gln
145 150 155 160
Phe Ala Gly Arg Pro Glu His Gln Ser Ser Asp Ser Thr Phe Leu Val
i65 170 175
Phe Met Ser His Gly Ile Leu Glu Gly Ile Cys Gly Val Lys His Arg
180 185 190
Asn Lys Lys Pro Asp Val Leu His Asp Asp Thr Ile Phe Lys Ile Phe
195 200 205
Asn Asn Ser Asn Cys Arg Ser Leu Arg Asn Lys Pro Lys Ile Leu Ile
210 21 5 220
Met Gln Ala Cys Arg Gly Arg Tyr Asn Gly Thr Ile Trp Val Ser Thr
CA 02402599 2002-10-15
-22-
225 230 235 240
Asn Lys Gly Ile Ala Thr Ala Asp Thr Asp Glu Glu Arg Val Leu Ser
245 250 255
Cys Lys Trp Asn Asn Ser Ile Thr Lys Ala His Val Glu Thr Asp Phe
260 265 270
Ile Ala Phe Lys Ser Ser Thr Pro His Asn Ile Ser Trp Lys Val Gly
275 280 285
Lys Thr Gly Ser Leu Phe Ile Ser Lys Leu Ile Asp Cys Phe Lys Lys
290 295 300
Tyr Cys Trp Cys Tyr His Leu Glu Glu Ile Phe Arg Lys Val Gln His
305 310 315 320
Ser Phe Glu Val Pro Gly Glu Leu Thr Gln Met Pro Thr Ile Glu Arg
325 330 335
Val Ser Met Thr Arg Tyr Phe Tyr Leu Phe Pro Gly Asn
340 345
<21 0> 1 5
<211> 20
< 21 2 > DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<220>
<221> unsure
<222> (3)
<223> May be any nucleic acid
<220>
<221> unsure
<222> (6)
<223> May be any nucleic acid
<220>
<221> unsure
<222> (12)
<223> May be any nucleic acid
<400> 15
tgnccnggga anaggtagaa 20
<210> 16
CA 02402599 2002-10-15
-23-
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<221> unsure
<222> (6)
<223> May be adenine, thymine or cytosine
<220>
<223> Description of Artificial Sequence: DNA Primer
<220>
<221> unsure
<222> (15)
<223> May be any nucleic acid
<400> 16
atcatnatcc aggcntgcag rgg 23
<210> 17
<211> 6
<21 2> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 17
Phe Tyr Leu Phe Pro Gly
1 5
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 18
gagatccaat ctacaagatc 20
<210> 19
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
CA 02402599 2002-10-15
-24-
<223> Description of Artificial Sequence: DNA Primer
<400> 19
caccacagag tatccaag 18
<210> 20
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 20
ctcgaattca tggcggccag gaggacacat g 31
<210> 21
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 21
ctcggatcct tcccgggaaa aggtag 26
<210> 22
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 22
ctcggtacca tgggacctca gaagatatgt ac 32
<210> 23
<211> 30
<21 2> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 23
ctcgtcgacc cattcccggg aaaaaggtag 30
CA 02402599 2002-10-15
-25-
<210> 24
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 24
ctggatccgt atggcggcca ggaggacaCa tgaaagagat cc 42
<210> 25
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 25
ctcgtcgacc cattcccggg aaaaaggtag 30
<210> 26
<211> 28
< 21 2 > DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 26
ctcctcgagc taattcccgg gaaaaagg 28
<210> 27
<211> 29
<21 2> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 27
ctcggatccg gacctcagaa gatatgtac 29
<210> 28
<211> 31
CA 02402599 2002-10-15
-26-
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 28
ctcggatccc taattcccgg gaaaaaggta g 31
<210> 29
<211> 29
< 21 2 > DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: DNA Primer
<400> 29
ctcggatcca cactgaagct ttgtccacg 29
<210> 30
<211> 4
<212> PRT
<213> Unknown
<220>
<221> UNSURE
<222> (2) .. (3)
<223> May be any amino acid
<400> 30
Asp Xaa Xaa Asp
1
<210> 31
<211> 13
<212> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: ICE
<400> 31
His Gly Cys Arg Gln Arg Ser Val Trp His Pro Arg Gln
1 5 10
<210> 32
<211> 13
CA 02402599 2002-10-15
-27-
<212> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: Caspase-12
<400> 32
His Gly Cys Arg Gln Lys Ser Ile Trp Val Gly Leu Gln
1 5 10
<210> 33
<211> 13
< 21 2 > PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: Caspase-11
<400> 33
His Gly Cys Arg Gln Arg Ser Leu Tyr Asp Lys His Gln
1 5 10
<210> 34
<211> 13
<212> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: Caspase-2
<400> 34
His Gly Cys Arg Gln Arg Ser Ala Met Asn Thr Tyr Pro
1 5 10
<210> 35
<211> 13
<21 2> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: Caspase-3
<400> 35
His Gly Cys Arg Gln Arg Ser Tyr Trp Asn Ser Phe Phe
1 5 10
<210> 36
<211> 4
CA 02402599 2002-10-15
-28-
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Peptide
Inhibitor
<400> 36
Tyr Val Ala Asp
1
<210> 37
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Peptide
Inhibitor
<400> 37
Asp Glu Val Asp
1
<210> 38
<211> 5
<212> PRT
<213> Unknown
<220>
<223> Description of Unknown Organism: Pro-caspase-12
<400> 38
Asp Glu Asp Asp Gly
1 5
