JP2003533997A - CARD domain containing polypeptides, encoding nucleic acids and methods of use - Google Patents

Abstract

(57) SUMMARY The present invention provides caspase recruitment domain (CARD) containing polypeptides, CARD, NB-ARC, ANGIO-R, LRR and SAM domains derived therefrom, as well as encoding nucleic acid molecules and specific antibodies. The invention also includes related screening, diagnostic and therapeutic methods.

Description

Detailed Description of the Invention

This invention was made in part with Government support under the NIH Grant GM61694 awarded by the National Institutes of Health. The US Government has certain rights in this research.

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to molecular biology and medicine, and more specifically to the identification of proteins involved in programmed cell death, cytokine processing and receptor signaling, and the association of these proteins. Regarding

BACKGROUND INFORMATION Programmed cell death is a physiological process that causes homeostasis to be maintained during cell production and cell turnover in virtually all self-renewing tissues. Often, a characteristic morphological change called "apoptosis" occurs in dying cells. Since similar changes occur in different types of dying cells, cell death appears to proceed by a common pathway in different cell types.

In addition to maintaining tissue homeostasis, apoptosis also includes growth factor deficiency, alternating calcium levels, free radicals, cytotoxic lymphokines, some viral infections, radiation and most chemotherapeutic agents. It occurs in response to various external stimuli. Therefore, apoptosis is an inducible event that is subject to similar regulatory mechanisms such as those that occur in metabolic pathways. In this respect,
Dysregulation of apoptosis also occurs, for example in some cancer cells that survive longer than the corresponding normal cells, and in neurodegenerative diseases in which neurons die before maturation. In viral infections, the induction of apoptosis may be prominent in the pathophysiology of the disease process, as the immunity underlying the eradication of viral infections depends on the loss of virus-producing host cells due to the immune cell attack that causes apoptosis. is there.

Some proteins involved in programmed cell death have been identified and the association of some of these proteins has been described. However, further apoptosis that regulates proteins remains undiscovered and the mechanisms by which these proteins mediate their activity have not been described. Identification of proteins involved in cell death and understanding of the association between these proteins selectively regulates the means of manipulating intracellular apoptosis and thus the relative lifespan of cells or their relative resistance to cell death stimuli. Means are provided.

The major effectors of apoptosis are a family of intracellular proteases known as caspases, which are abbreviations for cysteine aspartyl proteases. Caspases are inactive zymogens in virtually all animal cells. During apoptosis, caspases are activated by proteolytic processing at specific aspartate residues and usually assemble as an active protease consisting of a heterotetramer containing two large subunits and two small subunits. As a result. The phenomenon of apoptosis occurs directly or indirectly by activation of caspases in cells, resulting in proteolytic cleavage of specific substrate proteins. Furthermore, in many cases, caspases cleave and activate themselves and each other, creating a cascade of protease activation and a mechanism of "self" activation. Therefore, knowledge of proteins that interact with and regulate caspases is important for devising strategies to manipulate cell life and death in therapeutically useful ways. In addition, because caspases can also be involved in cytokine activation and other processes, knowledge of proteins that interact with caspases can be important for manipulating immune responses and other biochemical processes in a useful way.

One of the mechanisms that regulates caspase activation involves protein-protein interactions mediated by a family of protein domains known as caspase recruitment domains (CARDs). Therefore, CARD
The identification of proteins containing domains and the elucidation of proteins with which they interact can underlie strategies designed to alter apoptosis, cytokine production, cytokine receptor signaling and other cellular processes. Therefore, there is a need to identify proteins that contain the CARD domain. The present invention meets this need and provides other advantages as well.

SUMMARY OF THE INVENTION The present invention provides Caspase recruitment domain (CARD) containing polypeptides and CARD, NB-ARC, ANGIO-R, LRR and SAM domains derived therefrom. Also provided are chimeric polypeptides that include CARD, NB-ARC, ANGIO-R, LRR, and SAM domains of CARD-containing polypeptides. Also provided are methods of producing a CARD-containing polypeptide, and compositions comprising the CARD-containing polypeptide and a pharmaceutically acceptable carrier.

The present invention further provides a CARD-containing polypeptide and its CARD, NB-ARC, ANGIO-R, L
Nucleic acid molecules encoding the RR and SAM domains are provided. Antibodies to such polypeptides are also provided.

The present invention also provides a method of identifying a nucleic acid molecule encoding a CARD-containing polypeptide and a method of detecting the presence of a CARD-containing polypeptide in a sample.

Further provided are methods for identifying CARD associated polypeptides (CAP), and for identifying effective agents that alter the binding of CARD-containing polypeptides to CAP. The invention also provides methods for identifying effective agents that modulate the activity of the NB-ARC domain of a CARD-containing polypeptide.

The present invention also provides a method of altering the level of a biochemical process regulated by a CARD-containing polypeptide.

The invention further provides a method of treating a medical condition characterized by abnormal cell proliferation, abnormal cell death or inflammation.

Also provided is a method of diagnosing or predicting the clinical prognosis of a condition characterized by elevated or decreased levels of a CARD-containing polypeptide in a subject.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel polypeptides involved in programmed cell death, or apoptosis. The major effectors of apoptosis are a family of intracellular cysteine aspartyl proteases known as caspases. Intracellular caspase activity is regulated by protein-protein interactions. Similarly, protein-protein interactions affect the activity of other proteins involved in apoptosis. Multiple protein interaction domains are involved in the interactions between some apoptosis-regulating proteins. Among these is the caspase recruitment domain, a CARD-containing polypeptide, and is so named for its ability to bind caspases. In addition to their ability to bind caspases, many CARD-containing polypeptides contain other proteins,
In particular it binds to other CARD containing polypeptides. Furthermore, the CARD-containing polypeptide is
It affects various cellular and biochemical processes other than apoptosis, including cell adhesion, inflammation and cytokine receptor signaling.

According to the present invention, SEQ ID NOs: 12, 168, 188, 170, 172, 174, 176, 97, 99, 101
, 103, 178, 180, 182, 184, 86 and 90, and an isolated CARD-containing polypeptide or functional fragment thereof comprising an amino acid sequence substantially the same as that described in any of

The sequence identifiers shown above correspond to the molecules described herein as shown in Table 1.

[0018]

[Table 1]

As used herein, the term “CARD containing protein” or “CARD containing polypeptide” means a protein or polypeptide containing a CARD domain.
As used herein, the term "CARD domain" means a caspase recruitment domain. The CARD domain is described, for example, by Hofmann et al., Trends Bioc
hem. Sci. 22: 155-156 (1997), a well-known protein domain of about 80 amino acids with a characteristic sequence conservation. The CARD domain is found in some members of the caspase family of cell death proteases. Caspase-1, 2, 4, 5, 9 and 11 contain a CARD domain near its NH2-terminus. These CARD domains mediate the interaction of the zymogen inactive forms of caspases with other proteins that either activate or inhibit activation of these enzymes.

For example, the CARD domain of pro-caspase-9 binds to the CARD domain of a caspase activating protein called Apaf-1 (apoptotic protease activator-1). Similarly, the CARD domain of pro-caspase-1 allows it to interact with another CARD protein known as Cardiac (also called RIP2 and RICK), resulting in activation of the caspase-1 protease (Tome). (
Thome) et al., Curr. Biol. 16: 885-888 (1998)). In addition, Pro-Caspase-2
It binds to the CARD protein Raidd (also known as Cradd), which allows recruitment of pro-caspase-2 to the tumor necrosis factor (TNF) receptor complex, resulting in activation of caspase-2 protease ( Cancer Re, Ahmad et al.
s.57: 615-619 (1997)). The CARD domain is also involved in homotypic interactions with itself, resulting in self-association of polypeptides containing these protein interaction domains, producing dimers, and even oligomeric complexes.

The CARD domain binds to other types of functional domains within one polypeptide and brings the functional domains into close proximity or contact with the target protein by CARD: CARD association involving two CARD-containing polypeptides. A mechanism can be provided. For example, the Caenorhabiditis elegans cell death gene ced-4 encodes a protein containing an ATP-binding oligomerization domain called the CARD domain and the NB-ARC domain (van der Biezen and Jones). ), Curr. Biol. 8: R226-R227). The CARD domain of the CED-4 protein is CED
It interacts with the CARD domain of pro-caspase called -3. The NB-ARC domain causes CED-4 to self-associate, thereby forming an oligomeric complex, which brings the associated pro-CED-3 molecules into close proximity to each other. Since most pro-caspases have at least a small amount of protease, even in the proform, the assembly of complexes lining the alternative forms of caspase results in trans- and proteolytic processing of the zymogen. Active caspases are produced. Thus, CED-4 uses the CARD domain to bind pro-caspase and the NB-ARC domain for self-oligomerization, resulting in caspase clustering,
Process and activate proteolysis.

In addition to its role in caspase activation, the CARD domain is also involved in other cellular processes. Some CARD-containing polypeptides, for example, induce activation of the transcription factor NF-kB. NF-kB activation is induced by many cytokines and plays an important role in the cytokine receptor signal transduction mechanism (DiDonato et al., Nature 388: 548-554 (1997)). In addition, the CARD domain is, for example, an IAP (apoptosis protein inhibitor) family member cIAP1 and cIAP2 (Rothe et al., Cell 83: 1243-1252 (1995)) and an oncogenic mutant of the Bcl-10 protein (Willis Willis) et al., Cell 96: 35-45 (1999)), which is found in some proteins that inhibit rather than activate caspases. Also, CARD
Caspase activation resulting from domain interactions is often involved in inducing apoptosis, while other caspases are primarily proteolytic and activate inflammatory cytokines such as pro-IL-lb and pro-IL-18. Get involved in. Thus, CARD-containing polypeptides are also involved in cytokine receptor signaling and cytokine production, and thus may be involved in the regulation of immune and inflammatory responses.

In light of the function of the CARD domain within the CARD-containing polypeptide of the invention or a functional fragment thereof, the polypeptide of the invention is herein referred to as apoptosis, NF-kB induction, cytokine processing, cytokine receptor. Use in methods that alter biochemical processes such as signal transduction, caspase-mediated proteolysis, and thus have regulatory effects on cell life and death (ie apoptosis), inflammation, cell adhesion, and other cellular and biochemical processes I am considering doing.

The CARD-containing polypeptide of the present invention or a functional fragment thereof (including a CARD domain)
Also alters apoptosis, NF-kB induction, cytokine processing, cytokine receptor signaling, caspase-mediated proteolysis and thus cell life and death (ie apoptosis), inflammation, cell adhesion, other cellular and biochemical In a method to identify CARD binding agents and CARD associated polypeptides (CAPs), which have a modulatory effect on biological processes.

It is also contemplated herein that CARD-containing polypeptides of the invention associate with other CARD-containing polypeptides to form hetero-oligomers or homo-oligomers of the invention, such as heterodimers or homodimers. In particular, the CARD domain of the polypeptide of the present invention and Apaf-1, CED-4, caspase-1, 2, 9, 11, cIAP-1 and 2, CARDIAK, Raidd, Dark, CLAN and other invention CARD-containing Association with other CARD-containing polypeptides, including homo-oligomerization, such as polypeptides, is sufficiently specific such that the binding complex is formed intracellularly in vivo or under suitable conditions in vitro. . Thus, likewise, CARD-containing polypeptides of the invention can be used with other CARDs.
It is possible to associate the containing polypeptide with a CARD: CARD formed heterooligomer or homooligomer of the invention, such as a heterodimer or homodimer.

In accordance with the present invention, the sequences of novel CARD-containing polypeptides have been determined. Therefore, the present invention provides CARD2X, CARD3X, CLAN A, CLAN B, CLAN C, CLAN D, COP-1.
And a novel CARD-containing polypeptide, including the newly identified CARD-containing polypeptide, designated as COP-2 (described in SEQ ID NOs: 12, 188, 97, 99, 101, 103, 86 and 90) To do.

In addition to the CARD domain, the polypeptides of the present invention may contain one or more additional domains. The positions within the reference sequence of the domains described herein are shown in Table 2.

[0028]

[Table 2]

CARD3X (SEQ ID NO: 88) has at least four different domains: CARD-A and C.
It contains two CARD domains called ARD-B, the NB-ARC domain and the angio-R domain. An open reading frame in a second frame, beginning after the stop codon, encodes a domain with several leucine-rich repeats (LRRs) (SEQ ID NO: 189) (see Examples). Accordingly, a CARD3X polypeptide of the invention comprises an amino acid sequence designated SEQ ID NO: 188 and an amino acid sequence designated SEQ ID NO: 189; including SEQ ID NO: 188 but not SEQ ID NO: 189; or SEQ ID NO: 189, but not SEQ ID NO: 188. The murine CARD3X polypeptide is a human CARD3X AN with or without one or more additional CARD3X domains.
It comprises an amino acid sequence called SEQ ID NO: 193, which is homologous to part of the GIO-R domain.

CLAN exists in four isoforms, each containing a CARD domain (see Examples). The longest isoform, CLAN-A, also contains the NB-ARC (NACHT) domain, the LRR domain and the SAM domain. CLAN becomes a new member of the CED-4-related protein family. Recently, numerous CED-4-related proteins have been identified. These proteins belong to the CED-4 family of proteins, CED-4 (Yuan and Horvitz, Development 116: 309-320 (1992)), Ap.
af-1, (Zou et al., Cell 90: 405-413 (1997)), Dark (Rodriguez (Ro
driguez) et al., Nature Cell Biol. 1: 272-279 (1999)), and CARD4 / Nod1.
(Bertin et al., J. Biol. Chem. 274: 12955-12958 (1999) and Inohara et al., J. Biol. Chem. 274: 14560-14567 (1999)). As used herein, a “CED-4 family” member or “CED-4 protein family” member, also referred to herein as a “NAC” polypeptide, is NB-ARC.
A polypeptide comprising a domain and a CARD domain.

The human and rodent CED-4 homolog, called Apaf-1, is (i) a CARD domain,
Includes multiple copies of (ii) NB-ARC domain and (iii) WD repeat domain. In contrast to CED-4, which is capable of spontaneous oligomerization, the mammalian Apaf-1 protein
It is an inactive monomer until the oligomerization is induced by the binding of the cofactor protein cytochrome c (Li et al., Cell 91: 479-489 (1
997)). In Apaf-1, the WD repeat domain prevents oligomerization of the Apaf-1 protein until it comes into contact with cytochrome c. Therefore, the WD-repeated sequence is oligomeric Apaf- by the release of cytochrome c from damaged mitochondria.
It functions as a negative regulatory domain that keeps Apaf-1 in a latent state until assembly of one complex is induced (Saleh, J. Biol. Chem. 274: 17941-17945 (1999)).
. When pro-caspase-9 is bound by its CARD domain, the Apaf-1 oligomeric complex brings the proto-enzymes of caspase-9 into close proximity, cleaving them together and proteolytically processing the active caspase-9 protease. (Zou et al., J. Biol. Chem. 274: 11549-11556 (1999)).

Another feature of the CARD-containing polypeptides of the present invention is that they are capable of associating with pro-caspase, caspase or caspase associated proteins, thereby altering the proteolytic activity of caspases. The proteolytic activity of caspases is associated with cell apoptosis, as well as cytokine production. Thus, the CARD-containing polypeptides of the present invention can alter apoptosis or cytokine production by altering the proteolytic activity of caspases. As used herein, "caspase" is any member of the cysteine aspartyl protease. Caspases are usually able to associate with CARD-containing polypeptides of the invention, such as NAC polypeptides. Similarly, "pro-caspases" are inactive or less active precursor forms of caspases, which are commonly further impaired by proteolytic events, often after protein: protein interactions such as CARD: CARD interactions. Converted to highly active caspase.

As described in the examples, COP-1 is also associated with the pro-domain of pro-caspase-1, and RIP2, a protein previously demonstrated to bind to the pro-domain of pro-caspase-1. Interact. COP-1 competes with RIP2 for binding to pro-caspase-1, thereby inhibiting RIP2-mediated oligomerization of caspase-1. As a result, COP-1 induces mature IL-1 with RIP2 induced by caspase-1.
interfere with the ability to secrete β. Therefore, COP-1 is likely to play a role in controlling IL-1β production, thereby impeding IL-1β-induced inflammation. IL-1β plays an important role in septic shock, currently the most common cause of death of patients treated in an intensive care setting. Therefore, COP-1 is likely to play a role in IL-1β homeostasis in preventing systemic inflammatory responses when attacked by Gram-negative bacteria and other causes of inflammatory stroke.

As also described in the Examples, the isoforms of CLAN (A, B, C and D) interact with a wide variety of other CARD proteins, resulting in apoptosis, cytokine processing or NF-kB. Highly likely to affect activity. CLAN and Pro-
Caspase-1 interactions likely represent a role for CLAN as an IL-1β regulator. In this regard, various isoforms of CLAN appear to have the effect of interfering with pro-caspase-1 activation. For example, the longest isoform, CLAN-A, induces pro-caspase-1 activation by an "inducible proximity" mechanism as a result of its NB-ARC (NACHT) domain-mediated oligomerization. You can In contrast, the shorter isoform of CLAN lacking this self-oligomerization is similar to ICEBERG, a CARD-containing protein that competes with CARDIAK (RIP2 / RICK) for binding to pro-caspase-1, a pro- It is possible to act as a competitive antagonist of caspase-1. NAC also interacts with CLAN and NAC.
Suggests that this protein can influence Apaf-1-mediated apoptosis by enhancing its ability to bind Apaf-1 and activate caspase-9 in response to cytochrome c . Finally, CLAN association with NF-kB regulators such as Bcl-10 and Nod2 suggests that at least some of the CLAN isoforms affect the activity of this transcription factor.

In addition to the ability to bind caspases, the CARD-containing polypeptides of the invention can include protease domains such as those found in caspases. The caspase protease domain hydrolyzes amide bonds, especially amide bonds in the peptide or polypeptide backbone. Generally, the caspase protease domain comprises a P20 / P10 domain in the active site region of the caspase protease domain. Therefore, the caspase protease domain has proteolytic activity.

CARD-containing polypeptides are also known to induce activation of the transcription factor NF-kB. Thus, the CARD-containing polypeptides of the invention can also alter transcription, such as by modulating NF-kB activity.

NB-ARC of NAC polypeptides of the invention, such as CLAN and CARD3X (see Examples)
The (NACHT) domain associates with other polypeptides, especially those containing the NB-ARC domain. Thus, the functional NB-ARC domain associates with the NB-ARC domain-containing polypeptide by the NB-ARC: NB-ARC association. As used herein, the term "associate" or "association" means that a CARD-containing polypeptide, such as a NAC polypeptide, binds to the polypeptide relatively specifically, resulting in the formation of a binding complex. It means that you can do it. For example, the association of a CARD domain of a CARD-containing polypeptide of the invention with another CARD-containing polypeptide, or the NB-ARC domain of NAC and another NARD.
The association with the B-ARC domain-containing polypeptide is sufficiently specific that binding complexes can be generated in vivo in cells or in vitro under suitable conditions.

In addition, the NB-ARC domain exhibits both nucleotide binding (eg ATP binding) and hydrolytic activity, which is generally required for the ability to associate with NB-ARC domain-containing polypeptides. Therefore, the NB-ARC domain of NAC of the present invention is 1
It contains one or more nucleotide binding sites. As used herein, a nucleotide binding site is the portion of the polypeptide that specifically binds a nucleotide, such as ADP, ATP. Generally, the nucleotide binding site of NB-ARC comprises the NAC P-loop, Kinase 2 or Kinase 3a motif of the invention (these motifs are eg van der Biezen and Jones). ), As defined above). Preferably, the nucleotide binding site of NB-ARC comprises the NAC P-loop of the invention. Therefore, the NB-ARC domain of the CARD-containing polypeptides of the invention can be used in homo- or hetero-oligomerization
Can associate with the NB-ARC domain. In addition, the NB-ARC domain is characterized by nucleotide hydrolytic activity that can affect the ability of NB-ARC to associate with other NB-ARC domains.

Therefore, the NAC of the present invention is capable of CARD: CARD association and / or NB-ARC: NB-ARC association, and is a polyfunctional compound capable of one or more specific associations with other polypeptides. Produce a sex polypeptide. The NAC of the present invention can change cellular processes such as apoptosis and cytokine production. For example, it is contemplated herein that the NAC polypeptides of the invention can increase the level of intracellular apoptosis. It is also considered herein that the NACs of the invention may reduce the level of intracellular apoptosis. For example, does not induce apoptosis
NAC forms heterooligomers with apoptotic NAC and thus inhibits NAC's apoptotic activity.

In another embodiment of the invention, CLAN (SEQ ID NOs: 96, 98, 100 and 102) and
CARD-containing polypeptides of the invention, such as the CARD3X isoforms (including SEQ ID NO: 189), also contain leucine-rich repeat (LRR) domains. LRR domains are known in the art and, in some embodiments, of CARD-containing polypeptides of the invention.
The LRR domain has substantially the same sequence as LRR, described in another CARD-containing polypeptide known as Nod1 (Inohara et al., J. Biol. Chem. 274).
: 14560-14567 (1999)). The function of the LRR domain is to mediate specific interactions with other polypeptides.

In another aspect of the invention, a CARD-containing polypeptide comprising an NB-ARC domain and a CARD domain is provided. NAC polypeptide sequences disclosed herein, such as CARD4 / 5X (CLAN), regulate various biochemical processes such as apoptosis. NA
C polypeptides can also have other domains that regulate biochemical processes, such as the LRR domain and the WD domain.

Those skilled in the art will appreciate that many residues in the above sequences will be similar to other chemically, sterically and / or electronically without substantially altering the biological activity of the resulting CARD-containing polypeptide species. It will be recognized that it can be substituted with the residue of. In addition, SEQ ID NO:
12, 168, 188, 170, 172, 174, 176, 97, 99, 101, 103, 178, 180, 182, 184,
Larger polypeptide sequences, which include substantially the same sequences as the amino acids set forth at 86 and 90, are considered within the scope of the invention.

As used herein, a “substantially identical amino acid sequence” has about 70% or 75% identity with a reference amino acid sequence and is similar to a polypeptide defined by the reference amino acid sequence. By an amino acid sequence that retains the properties of function and biological activity. Preferably, the polypeptides having "substantially the same amino acid sequence" are at least about 80%, 82%, 84%, 86% or 88%, more preferably 90%, 91%, 92% with respect to the reference amino acid sequence. , 93% or 94% amino acid identity; amino acid sequence identities greater than about 95%, 96%, 97%, 98% or 99% are particularly preferred. However, polypeptides or nucleic acids that occur as splice variants or have been modified by conservative amino acid substitutions or have been modified by substitution of degenerate codons and that have less than the above-identified sequence identities also fall within the scope of the invention. It is recognized that it is included in.

In accordance with the present invention, within the definition of substantially the same amino acid sequence is specifically included the major amino acid sequences of the particular inventive CARD-containing polypeptides or domains disclosed herein. The predominant amino acid sequence is the most commonly occurring naturally occurring amino acid sequence in the species population. The predominant polypeptide with multiple isoforms has the most commonly expressed amino acid sequence for each isoform. The predominant CARD-containing polypeptides of the invention are amino acids that have greater sequence identity to the amino acid sequences disclosed herein than the amino acid sequences of any other naturally occurring protein of a particular species (eg, human). Means an array.

By disclosing the position and the nucleic acid or amino acid sequence corresponding to the CARD-containing polypeptide of the present invention, those skilled in the art can easily identify the nucleic acid or amino acid sequence associated with the CARD-containing polypeptide of the present invention. And can be modified if necessary. For example, the sequence can be confirmed by using PCR or other known methods to probe a cDNA library with a nucleic acid probe corresponding to the nucleic acid of the invention. further,
Suitable bacterial artificial chromosomes containing a genomic region encoding a CARD-containing polypeptide of the invention are commercially available and can be probed using PCR, restriction mapping, sequencing and other known methods.

The term “biologically active” or “functional”, as used herein as a modifier of the CARD-containing polypeptide of the invention, or a polypeptide fragment thereof, is a CARD-containing polypeptide of the invention. A polypeptide having the same functional properties as described above. CA
The biological activity of the RD-containing polypeptide is, for example, preferably in vivo, binds to or homo-oligomerizes to nucleotides, CARD-associated polypeptides, NB-ARC-containing polypeptides, or alters protease activation, especially caspase activation. , Or catalyze reactions such as proteolysis or nucleotide hydrolysis,
Or including the ability to alter NF-kB activity, or alter apoptosis, cytokine processing, cytokine receptor signaling, inflammation, immune response and other biological activities described herein.

The ability of CARD-containing polypeptides to bind to other polypeptides, such as CARD-associated polypeptides, has been demonstrated in, for example, yeast two-hybrid assays, co-immunoprecipitation, GST-fusion co-purification, and Sambrook, supra, And Ausube
l) et al., can be assayed using other methods described in standard technical manuals such as those listed above. Another biological activity of CARD-containing polypeptides is the ability to act as an immunogen to generate polyclonal and monoclonal antibodies that specifically bind the CARD-containing polypeptides of the invention. Accordingly, the nucleic acids of the invention encoding a CARD-containing polypeptide have SEQ ID NOs: 12,168,188,170,172,174,176,97,99.
, 103, 178, 180, 182, 184, 86, and 90 (preferably human) that also specifically recognizes a CARD-containing polypeptide, which encodes a polypeptide that is specifically recognized by an antibody Can be converted. Such immunological activity can be assayed by any method known to those of skill in the art. For example, a test polypeptide can be used to raise an antibody and then assay its ability to bind a polypeptide of the invention. A polypeptide retains the necessary immunobiological activity if the antibody binds to the test and reference polypeptides with substantially the same affinity.

As used herein, the term “substantially purified” refers to contaminating lipids,
By a polypeptide, a nucleic acid or a form of polypeptide which is relatively free of other cellular substances, which is normally associated with the polypeptide within the cell. Substantially purified CARD-containing polypeptides can be obtained by a variety of methods known in the art, such as recombinant expression systems described herein, chemical synthesis or purification from natural sources. Purification methods can include, for example, precipitation, gel filtration, ion exchange, reverse phase and affinity chromatography and the like. Another known method is the Deutscher (Deutsc
her) et al., “Guide to Protein Purification” Meth
ods in Enzymology Vol. 182 (Academic Press, (1990)).
Alternatively, an isolated polypeptide of the invention can be isolated, for example, from Sambrook et al.
Obtained using known recombination methods described in (1989) supra, as well as Ausubel et al., Supra (2000). Methods and conditions for biochemical purification of the polypeptides of the present invention can be selected by those of skill in the art, purification may be for example immunoassay,
Monitored by binding or functional assays.

In addition to the ability of the CARD-containing polypeptides of the invention, or functional fragments thereof, to interact with other heterologous proteins (eg, CARD-containing polypeptides),
CARD-containing polypeptides have the ability to self-associate to form homo-oligomers of the invention, such as homo-dimers. This self-association is possible by interactions between CARD domains and also by interactions between NB-ARC domains. Furthermore, self-association can also occur as a result of interactions between LRR domains.

In accordance with the invention, there are also provided mutations and fragments of CARD-containing polypeptides that have an activity that differs from the activity of the predominant naturally-occurring CARD-containing polypeptide. As used herein, a "mutation" is a deletion, insertion or change of one or more amino acids in a major naturally occurring protein sequence (eg wild type), and a "fragment" is It is a truncated form of either the carboxy terminus or the amino terminus of the major naturally occurring protein, or both. Preferably, the different activities of the mutations or fragments are the result of mutant polypeptides or fragments that retain some, but not all, of the activity of each major naturally occurring CARD-containing polypeptide.

For example, a functional fragment of a polypeptide of the invention may comprise or consist of one or more of the following: CARD domain, NB-ARC domain, L
RR domain, SAM domain, or angio-R domain. In the concrete example, CLAN
Fragments of CARD-containing polypeptides, such as, may contain CARD and LRR domains, but lack the functional NB-ARC domain. Such fragments are predominantly naturally occurring
It retains some of the CLAN activity (eg CARD domain functionality) but not all such activity (eg lacks the active NB-ARC domain).
. Thus, the resulting fragment has an activity that differs from the predominant naturally occurring CLAN activity. In another example, the CLAN polypeptide has only the NB-ARC domain and may interact with other NB-ARC domain proteins during the formation of homo-oligomers or hetero-oligomers. In one embodiment, the activity of the fragment is "dominant inhibition". Due to its dominant inhibitory activity, the fragment is one of the major naturally occurring CARD-containing polypeptides.
The activity of one or more isoforms can be reduced or inactivated.
Other functional fragments can include the angio-R domain (see Examples), or any of the domains disclosed herein (see, eg, Table 2).

Isoforms of CARD-containing polypeptides resulting from alternative mRNA splicing are also provided and may alter or modify the interaction of CARD-containing polypeptides with other polypeptides. For example, the four isoforms of CLAN and the three isoforms of CARD3X are disclosed herein. SEQ ID NO: 12, 188, 97, 99, 101,
Other isoforms of CARD-containing polypeptides, referred to as 103, 86 and 90, are discussed herein and are therefore included within the scope of CARD-containing polypeptides of the invention.

Methods for identifying polypeptides, including functional fragments of CARD-containing polypeptides of the invention, are known in the art and disclosed herein. For example, genomic or cDNA libraries, including universal cDNA libraries, can be probed according to the methods disclosed herein or other methods known in the art. Full length polypeptides encoding nucleic acids such as full length cDNA can be obtained by various methods known in the art. For example, for the 5'and 3'RACE, methods are known in the art and described in Ausubel et al., Supra.

In another aspect of the invention there is provided a chimeric polypeptide comprising a CARD containing polypeptide or functional fragment thereof fused to another protein or functional fragment thereof. Functional fragments of CARD-containing polypeptides are eg NB-ARC (NACHT), C
ARD, LRR, and ANGIO-R domains, or other fragments that retain the biological activity of the CARD-containing polypeptides of the invention. Polypeptides to which the CARD-containing polypeptide or functional fragment thereof is fused include, for example, glutathione-S-transferase, antibodies, or other proteins that facilitate recovery of chimeras, or functional fragments thereof. In addition, the polypeptide to which the CARD-containing polypeptide or functional fragment thereof is fused includes, for example, luciferase, green fluorescent protein, antibodies, or other proteins that facilitate the identification of chimeras, or functional fragments thereof. Still further, a CARD-containing polypeptide or a polypeptide to which a functional fragment thereof is fused may be, for example, a LexA DNA binding domain, lysine, alphasarcin, an antibody or fragment thereof, or any other therapeutic activity or other biological activity. Polypeptides are included.

Further, a chimeric polypeptide of the invention contemplated herein is a chimeric polypeptide in which a functional fragment of a CARD-containing polypeptide is fused to a catalytic domain or protein interaction domain of a heterologous polypeptide. Is. For example, the NB-ARC domain of CLAN as disclosed herein can be replaced by the NB-ARC domain of other CARD polypeptides such as CARD3X. Another example of such a chimera is CLAN
Is a polypeptide in which the CARD domain of is replaced with a CARD domain such as CARD2X or CARD3X. In another example, the NB-ARC domain can be fused with a caspase catalytic P20 domain to form a novel chimera with caspase activity. One of skill in the art will recognize that a large number of chimeric polypeptides can be readily obtained by linking the domains of two or more CARD-containing polypeptides of the invention. Further, the chimeric polypeptide may comprise a functional fragment of the CARD-containing polypeptide of the invention fused to a domain of a protein known in the art such as CED-4, Apaf-1, caspase-1.

In another embodiment of the invention, the polypeptide consisting of 10 or more consecutive amino acids selected from the group consisting of SEQ ID NOs: 12, 188, 97, 99, 101, 103, 86 and 90. Provided.

As used herein, the term “polypeptide” when used in reference to a CARD-containing polypeptide or fragment shall mean a peptide or polypeptide of 2 or more amino acids. The term "polypeptide analog" is substantially the same as a sequence specifically described herein, in which one or more residues are conservatively substituted with functionally similar residues. Demonstrating the ability to functionally mimic a CARD-containing polypeptide described herein having an amino acid residue sequence that is
Includes all polypeptides. "Modification" of the polypeptide of the present invention also includes conservative substitution of the amino acid sequence of the polypeptide of the present invention. Conservative substitutions of encoded amino acids include, for example, amino acids belonging to the following groups: (1) non-polar amino acids (Gly,
(Ala, Val, Leu and Ile); (2) Polar neutral amino acids (Cys, Met, Ser, Thr, Asn)
And Gln); (3) polar acidic amino acids (Asp and Glu); (4) polar basic amino acids (Lys, Arg and His) and (5) aromatic amino acids (Phe, Trp, Tyr and H).
is). Other classes of amino acids are, for example, incorporated herein by reference,
Found in Taylar, J. Theor. Biol. 119: 205-218 (1986). Other minor modifications are included in the polypeptides of the invention as long as the polypeptide retains some or all of the functions described herein.

The amino acid length of the functional fragments or polypeptide analogs of the invention can range from about 5 amino acids to the full length protein sequence of the CARD-containing polypeptide of the invention. In some embodiments, the amino acid length is, for example, at least about 10 amino acids, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least. about
55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about
100, at least about 125, at least about 150, at least about 175, at least about 200
, At least about 250, or up to the full length CARD-containing polypeptide sequence. A functional fragment is a contiguous amino acid sequence of a polypeptide of the invention,
SEQ ID NOs: 12, 188, 97, 99, 101, 103, 86 and 90 contiguous amino acid sequences. Peptides of at least about 10 amino acids can be used, for example, as immunogens to raise antibodies specific for CARD-containing polypeptides of the invention.

Modifications of the polypeptide can also include derivatives, analogs and functional mimetics of such a polypeptide, provided that such polypeptide exhibits the biological activity of the CARD-containing polypeptide. For example, the derivative can include chemical modifications of the polypeptide, such as alkylation, acylation, carbamylation, iodination, or any modification that derivatizes the polypeptide. Such derivatized molecules include, for example, amine hydrochloride derivatized with free amino groups, p-toluenesulfonyl groups, carbobenzoxy groups, t-
Includes molecules that form butyloxycarbonyl, chloroacetyl, or formyl groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters and other types of esters, or hydrazides. Free hydroxyl groups can be derivatized to form o-acyl or o-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. Derivatives or analogs also include polypeptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids, such as 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine, ornithine or carboxyglutamate. Amino acids that are included and are not linked by peptide bonds can also be included. Polypeptides of the present invention are all polypeptides that have one or more additions and / or deletions of residues with respect to the sequence of the polypeptides shown herein, while maintaining CARD-containing polypeptide activity. Including as long as

Modifications of the polypeptides of the invention include functional mimics thereof. Mimetics include chemicals that include chemical moieties that mimic the function of a polypeptide. For example, if a polypeptide contains two charged chemical moieties that have functional activity, the mimetic will place the two charged chemical moieties in a certain spatial arrangement and constrained such that the charged chemical functions are maintained in three-dimensional space. Place in the structure. Thus, mimetics that orient functional groups that provide the function of a CARD-containing polypeptide are included within the meaning of CARD-containing polypeptide derivatives. All of these modifications are included in the term "polypeptide" as long as the polypeptide functional fragment of the invention retains its function. Examples of mimetics also include peptidomimetics, peptoids, or other peptide-like polymers such as poly (b-amino acids), and non-polymeric compounds in which functional groups that mimic a peptide are located.

Another aspect of the invention includes a CARD-containing polypeptide or functional fragment thereof fused to a moiety to form a conjugate. As used herein, a "moiety" is a physical, chemical or biological entity that serves the functionality of a CARD-containing polypeptide or functional fragment thereof. Functionality contributed by a moiety includes the ability to facilitate therapeutic or other biological activity, or identification or recovery of a CARD-containing polypeptide. Thus, the moieties include molecules known to those of skill in the art to be useful in detecting conjugates, for example by fluorescence, magnetic imaging, detection of radioactive emissions. The moieties are also useful for recovering conjugates, eg, His-tags or other known tags used for protein isolation and / or purification, or physical substances such as beads. The moiety can also be a therapeutic compound such as, for example, a cytotoxic agent useful in affecting biological changes within the cell in which the conjugate is localized.

Examples of means for preparing the polypeptide or polypeptides of the present invention include bacterial cells, yeast cells, amphoteric cells such as oocytes, or mammalian cells, such as mammalian cells, using methods known in the art. Expression of the nucleic acid encoding the CARD-containing polypeptide in a suitable host cell and recovery of the expressed polypeptide using known purification methods. The polypeptides of the present invention can be isolated directly from cells transformed with expression vectors known in the art. The recombinantly expressed polypeptides of the invention can also be expressed as a fusion protein with the appropriate affinity tag, such as glutathione S-transferase (GST) or polyHis, and affinity purified. Polypeptides, biologically functional fragments and functional equivalents thereof of the present invention can be isolated from in vitro transcription / art known in the art using, for example, reticulocyte lysates used in the TNT system (Promega). It can also be generated by the translation method. The polypeptides of the invention, biologically functional fragments, and functional equivalents thereof, can also be produced by chemical synthesis. Synthetic polypeptides, for example, are produced using the manufacturer-supplied chemicals using an Applied Biosystems, Inc. Model 430A or 431A automated peptide synthesizer (Foster City, Calif.). Can be generated.

Another aspect of the invention provides an isolated nucleic acid encoding a CARD-containing polypeptide or functional fragment thereof. The isolated nucleic acid is as follows: (a) SEQ ID NO: 12, 168, 188, 170, 172, 174, 176, 97, 99, 101, 103, 178
, 180, 182, 184, 86 and 90, or a (b) a DNA encoding a polypeptide comprising the amino acid sequence described above, or (b) when the DNA encodes a bioactive CARD-containing polypeptide under moderately stringent conditions. , (A) a DNA that hybridizes to the DNA, or (c) when the DNA encodes a bioactive CARD-containing polypeptide, it is selected from the degeneracy of DNA for (b).

The nucleic acid molecules described herein are useful for producing the polypeptides of the invention when such nucleic acids are incorporated into various protein expression systems known to those of skill in the art. In addition, such nucleic acid molecules or fragments thereof are labeled with readily detectable substituents to assay for the presence and / or amount of CARD-containing genes or mRNA transcripts of the invention in a given sample. It can be used as a hybridization probe. The nucleic acid molecules and fragments thereof described herein are also useful as primers and / or templates in PCR reactions that amplify the genes encoding the polypeptides of the invention described herein.

The term “nucleic acid” (also referred to as polynucleotide) includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), probes, oligonucleotides and primers, and is single-stranded or double-stranded. DNA is complementary DNA (cDNA) or, for example,
Either genomic DNA, such as a CARD-encoding gene, which can be the sense strand, the antisense strand, or both. Examples of nucleic acids are RNA, cDNA, or isolated genomic DNA encoding a CARD-containing polypeptide. One means of isolating CARD-encoding nucleic acids is to probe a mammalian genomic or cDNA library with naturally occurring or artificially designed DNA probes using methods well known in the art. DNA probes derived from the CARD-encoding gene are particularly useful for this purpose. DNA and cDNA molecules encoding CARD-containing polypeptides can be used to obtain complementary genomic DNA, cDNA or RNA from a mammal (eg, human, mouse, rat, rabbit, pig, etc.) or other animal source, or Related cDNA or genomic clones can be isolated by screening a cDNA or genomic library using the methods described in more detail in. Such nucleic acids include, but are not limited to, SEQ ID NOs: 11,167,187,169,171,17.
Nucleic acids containing substantially the same nucleotide sequences as described in 3, 175, 96, 98, 100, 102, 177, 179, 181, 183, 85 and 89 are included. In general, the genomic sequence of the invention is
It contains control regions such as promoters, enhancers, and introns that are outside the exons that encode the CARD-containing polypeptide, but not the base gene that does not encode the CARD-containing polypeptide.

Thus, a CARD encoding nucleic acid as used herein refers to a nucleic acid encoding a CARD containing polypeptide of the invention, or a functional fragment thereof.

As used herein and in the claims, “isolated” and / or “purified”
And / or using the term "substantially purified" as a modifier of DNA, RNA, polypeptide or protein, DNA, RNA, as so called.
It means that the polypeptide or protein is purified by human hands to such a form and thus isolated from the native in vivo cellular environment and is substantially free of nucleic acids or proteins of other species. As a result of this human intervention, the recombinant of the present invention
The DNA, RNA, polypeptide or protein is of no use in native DNA, RNA, polypeptide or protein, but in the methods described herein,
It is useful.

The nucleic acid of the present invention encoding the CARD-containing polypeptide and the CARD-containing polypeptide of the present invention can be used in organisms of any species, such as prokaryotes, eukaryotes, plants, fungi, vertebrates and invertebrates. Can also be obtained from The particular species is a mammal. As used herein, "mammal" refers to a subset of the species from which the CARD-encoding nucleic acids of the invention are derived, such as human, rat, mouse, rabbit, monkey, baboon, cow, pig, sheep, dog, cat. And so on. Preferred CARD encoding nucleic acids herein are:
It is a human CARD-encoding nucleic acid.

In one embodiment of the invention, the cDNA encoding the CARD-containing polypeptide of the invention disclosed herein is SEQ ID NO: 11,167,187,169,171,173,175,96.
, 98, 100, 102, 177, 179, 181, 183, 85, and 89, and substantially the same nucleotide sequence as the coding region.

As used herein, the term “substantially the same nucleotide sequence” refers to
A nucleic acid molecule (DNA or RNA) having sufficient identity to a reference polynucleotide so that it hybridizes to the reference nucleotide under moderately or highly stringent hybridization conditions. In one embodiment,
Nucleic acid molecules having substantially the same nucleic acid sequence as the reference nucleotide have the reference numbers: 12, 16
It encodes an amino acid sequence substantially the same as that described in any of 8, 188, 170, 172, 174, 176, 97, 99, 101, 103, 178, 180, 182, 184, 86 and 90. In other embodiments, the nucleic acid molecule having "substantially the same nucleotide sequence" as the reference nucleotide sequence is at least 70%, 72%, 74%, 76% relative to the reference nucleotide sequence.
, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%
, 96%, 97%, 98% or 99% identity, with at least 60%, or at least 65% identity to the reference nucleotide sequence.

According to the invention, the predominant nucleotide sequences of the CARD-containing polypeptides described herein, in particular of the invention, are specifically included in the definition of substantially the same nucleotide sequence. A predominant nucleotide sequence means the most commonly occurring naturally occurring nucleotide sequence in a species population. The predominant CARD-encoding nucleic acids of the invention have sequence identity to a nucleotide sequence disclosed herein (eg, human) that is greater than the nucleotide sequence of any naturally occurring nucleotide of a particular species, Means a nucleotide sequence.

In one embodiment, the nucleic acid molecule having a nucleotide sequence that is substantially the same as the reference sequence is a modification of the reference sequence. As used herein, a “modification” of a nucleic acid can include the addition, deletion or substitution of one or more nucleotides with respect to the reference sequence. Modifications of nucleic acids can include substitutions that do not alter the encoded amino acid sequence due to the degeneracy of the genetic code. Such modifications correspond to deliberate mutations or mutations that occur as mutations during nucleic acid replication.

Examples of re-referenced nucleotide sequence modifications include mice, primates including monkeys and baboons, rat, rabbit, cow, pig, sheep, dog, mammalian species including cats, or other animal species. Included are sequences corresponding to homologs of other species, including. The corresponding nucleotide sequence of the non-human species can be determined by methods known in the art, such as by PCR or by screening genomic, cDNA or expression libraries.

Other examples of modifications of the CARD-encoding nucleic acids or CARD-containing polypeptides of the invention include CA
It may correspond to a splice variant of the RD-encoding nucleotide sequence. In addition, nucleotide sequence modifications include, for example, modifications at the base, sugar, or phosphate moieties,
Alternatively, one or more non-native nucleotides containing modified phosphodiester bonds can be included. Such modifications are advantageous in improving the stability of the nucleic acid molecule.

In addition, modifications of the nucleotide sequence can include, for example, radiolabels, fluorochromes, ferromagnetic substances, luminescent tags or detectable binding agents such as biotin. Such modifications are advantageous in applications where detection of CARD-encoding nucleic acids is desired.

In another embodiment, a nucleic acid molecule having a nucleotide sequence substantially the same as the reference sequence is a functionally equivalent nucleic acid, indicating that it is phenotypically the same as the reference nucleic acid. As used herein, the phrase “functionally equivalent nucleic acid” refers to a small, functionally functional compound that produces the same polypeptide product as the nucleic acids disclosed herein. To nucleic acids characterized by contiguous or non-contiguous sequence changes. In particular, a functionally equivalent nucleic acid encodes a polypeptide that is the same as the polypeptide encoded by the nucleic acids disclosed herein, or that has conservative amino acid changes as described above. These changes include those recognized by those of skill in the art such that they do not substantially change the tertiary structure of the protein.

Furthermore, the degeneracy of the genetic code also provides CARD-containing polypeptides that do not necessarily hybridize to the nucleic acids of the invention under designated hybridization conditions. Preferred nucleic acids encoding the CARD-containing polypeptides of the invention are SEQ ID NOs: 12,168,188,170,172,174,176,97,99,101,103,178,180,182,184.
, 86 and 90, which encodes substantially the same amino acid sequence.

Hybridization means the binding of complementary strands of nucleic acids to each other by hydrogen bonding (ie sense: antisense strand or probe: target DNA), which naturally occurs in chromosomal DNA. The degree of stringency used to hybridize a given probe with target DNA can be readily varied by one of ordinary skill in the art.

The term “stringent hybridization” as used herein refers to conditions under which a polynucleic acid hybrid is stable. As known to those of skill in the art, the stability of a hybrid is reflected in the melting temperature (Tm) of the hybrid. Hybrid stability is generally a function of sodium ion concentration and temperature. Generally, hybridization reactions are performed under conditions of lower stringency, then washed under conditions of different but higher stringency. Reference to the stringency of a hybridization reaction refers to such wash conditions.

As used herein, the phrase “moderately stringent hybridization” refers to conditions that allow a target nucleic acid to bind a complementary nucleic acid. The hybridized nucleic acids typically have at least about 75% identity, such as at least about 60% identity, at least about 85% identity; or at least about 90% identity. Moderately stringent conditions are hybridization at 42 ° C in 50% formamide, 5x Denhardt's solution, 5xSSPE, 0.2% SDS, followed by 0.2xSSPE, 0.2% SDS at 42 ° C. It is equivalent to washing.

The phrase “high stringency hybridization” refers to 0.01 ° C. at 65 ° C.
In 8M NaCl, it means the conditions that allow hybridization of only nucleic acid sequences that form stable hybrids. For example, the hybrid is 0.018MN at 65 ℃
If it is not stable in aCl, it will not be stable under conditions of high stringency as considered herein. High stringency conditions are eg 50 at 42 ° C.
% Formamide, 5x Denhardt's solution, 5xSSPE, 0.2% SDS, followed by a wash with 0.1xSSPE, 0.1% SDS at 65 ° C.

The phrase “low stringency hybridization” refers to 10 at 22 ° C.
% Formamide, 5 × Denhardt's solution, 6 × SSPE, hybridization in 0.2% SDS, followed by washing at 37 ° C. with 1 × SSPE, 0.2% SDS. Denhardt's solution is 1% Ficoll, 1% polyvinylpyrrolidone, and 1%
Contains% bovine serum albumin (BSA). 20 × SSPE (sodium chloride, sodium phosphate, ethylenediamide tetraacetic acid (EDTA)) contains 3M sodium chloride, 0.2M sodium phosphate, and 0.025M (EDTA). Other suitable, moderate stringency, and high stringency buffers and conditions are known to those of skill in the art, eg, Sambrook et al., Supra (1989);
subel) et al., supra (2000). A nucleic acid that encodes a polypeptide is, under moderately stringent or highly stringent conditions,
Substantially the entire sequence, or a substantial portion, eg, usually SEQ ID NOs: 11,167.
, 187, 169, 171, 173, 175, 96, 98, 100, 102, 177, 179, 181, 183, 85 and 89 hybridize to at least 15 to 30 nucleotides of the nucleic acid sequence.

As used herein, the term “degenerate” refers to a reference nucleic acid, eg, SEQ ID NO: 11,167,187,169,171,173,175,96,98,100,102, 177, 179, 18
By codons that differ by at least one nucleotide from 1, 183, 85 and 89, but encode the same amino acids as the reference nucleic acid. For example, the triplet "UCU",
The codons designated by "UCC", "UCA", and "UCG" are degenerate with respect to each other because all four codons encode the amino acid serine.

The invention provides for CARD-encoding nucleic acid molecules, such as SEQ ID NOs: 11,167,187,169,171,173,175,96,98,100,102,177, under moderately stringent conditions. 1
Also provided is a modification of the nucleic acid sequence that hybridizes to the nucleic acid molecule referenced as any of 79, 181, 183, 85 and 89. A modification of the nucleotide sequence is also provided if the modification has at least 60% identity to the CARD encoding nucleotide sequence. The invention provides at least 65% identity, at least 70% identity, at least 72% identity, at least 74% identity, at least 76% identity, at least 78% identity, at least 80% identity. Identity, at least 82% identity, at least 84% identity, at least 86% identity, at least 88% identity, at least 90% identity, at least 91% identity, at least 92% identity Gender, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity. Also provided is a modification of the CARD-encoding nucleotide sequence.

The identity of any two nucleic acid or amino acid sequences can be determined by one of ordinary skill in the art using deformation parameters, for example based on BLAST 2.0 computer alignments. BLAST 2.0 searches are known in the art and can be found at Tatiana
) Et al., FEMS Microbiol Lett. 174: 247-250 (1999); Altschu (Altschu).
l) et al., Nucleic Acid Res., 25: 3389-3402 (1997),
Publicly available at http: //www.ncbi.nlm.nih.gov/BLAST.

One means of isolating nucleic acids encoding CARD-containing polypeptides is to probe a cDNA or genomic library with naturally or artificially designed nucleic acid probes using methods known in the art. That is. For this purpose, CA
Nucleic acid probes derived from the RD-encoding gene are particularly useful. DNA and cDNA molecules encoding CARD-containing polypeptides have been used to form complementary genomic DNA, cDN, from mammals such as humans, mice, rats, rabbits, pigs, or from other animal sources.
Relevant cDNA or genomic clones can be isolated by obtaining A or RNA or screening the cDNA or genomic libraries by methods known in the art (eg, the Examples described below; and See Sambrook et al., Supra, 1989; Ausubel et al., Supra, 2000).
.

Another useful method of producing the CARD-encoding nucleic acids of the present invention is the purification of the resulting products by PCR and amplification of nucleic acid molecules using the oligonucleotides of the present invention, optionally by gel electrophoresis. Including. PCR or RT-PCR can be used to generate CARD-encoding nucleic acid molecules with any desired nucleic acid boundaries as described in the examples. The desired modification to the nucleic acid sequence can also be introduced by selecting the appropriate oligonucleotide primer with one or more additions, deletions or substitutions. Such nucleic acid molecules can be exponentially amplified starting from any cell, tissue or species of interest from as few as one gene or mRNA replication.

The present invention further provides SEQ ID NOs: 168, 170 under conditions of high stringency.
, 172 and 178, SEQ ID NO: 11, 187, 96, 98, 100, 102, 85
And a nucleic acid that hybridizes to the CARD encoding portion of any of 89.
The present invention is SEQ ID NO: 11, 167, 187, 169, 171, 173, 175, 96, 98, 100, 102,
A nucleic acid having substantially the same nucleic acid sequence as that of any of 177, 179, 181, 183, 85 and 89 is provided.

The present invention involves contacting a sample containing nucleic acid with one or more nucleic acid molecules or oligonucleotides of the present invention, contacting under highly stringent conditions to identify nucleic acids that hybridize to the oligonucleotide. Mammal CA by
Also provided are methods of identifying a nucleic acid encoding an RD-containing polypeptide. The invention further provides a method of detecting a CARD-encoding nucleic acid molecule in a sample by contacting the sample with two or more oligonucleotides of the invention, amplifying the nucleic acid molecule and detecting the amplification. . Amplification can be performed, for example, using PCR. The invention further provides oligonucleotides that function as single-stranded nucleic acid primers for amplification of CARD-encoding nucleic acids, wherein the primers are SEQ ID NOs: 11, 187, 96, 98, 1
Includes nucleic acid sequences derived from the nucleic acid sequences set forth in 00, 102, 85 and 89.

According to a further aspect of the invention, the selectively labeled CARD-encoding cDNA or a fragment thereof is used for the main nucleic acid sequence or additional nucleic acid sequence encoding a novel CARD-containing polypeptide, cDNA, genomic , One or more libraries such as BAC can be investigated. Suitable mammalian cDNA, including human cDNA library
Library construction and screening is performed, for example, by Ausubel et al.
It is known in the art as shown above. The screening of such a cDNA library is initially carried out under low stringency conditions consisting of temperatures below about 42 ° C., formamide concentrations below about 50% and moderate to low salt concentrations.

Probe-based screening conditions include a temperature of about 37 ° C., a concentration of formamide of about 20%, about 5X standard citrate saline (SSC; 20 × SSC contains 3M sodium chloride, 0.3M sodium citrate, pH 7). .0). Such conditions allow the identification of sequences that have a substantial degree of similarity to the probe sequence without the need for complete homology. The expression "substantial similarity" means sequences that share at least 50% homology. Hybridization conditions are selected which allow the identification of sequences having at least 70% homology to the probe, while discriminating against sequences that have a low degree of similarity to the probe. As a result, SEQ ID NOs: 11, 167, 187, 169, 171
, 173, 175, 96, 98, 100, 102, 177, 179, 181, 183, 85 and 89 are obtained.

As used herein, nucleic acid “probes” include SEQ ID NOs: 11, 187, 96, 9
At least 15, at least 20, at least 50, at least 100, at least 200, at least 300, at least 400 or at least substantially the same as, or a complement of, any of the contiguous bases described in 8, 100, 102, 85 and 89. A single-stranded nucleic acid or an analogue thereof having a nucleotide sequence containing 500 consecutive bases. In addition, the
Entire cDNA coding region of CARD-containing polypeptide, or SEQ ID NOs: 11, 187, 96
, 98, 100, 102, 85 and 89 can be used as probes in their entirety. The probe is labeled by methods known in the art as described below,
It can be used in various diagnostic kits.

The invention further provides a nucleotide consisting of 15-300 contiguous nucleotides of any of SEQ ID NOs: 11, 187, 96, 98, 100, 102, 85 and 89, or an antisense strand thereof. As used herein, the term "oligonucleotide" means a nucleic acid molecule that comprises at least 15 contiguous nucleotides from a reference nucleotide sequence and has at least 16, 17, 18, 19, 20, or at least 25 contiguous nucleotides. From the reference nucleotide sequence at least 30, 40, 50
, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, max
Often contains 350 contiguous nucleotides. The reference nucleotide can be the sense strand or the antisense strand.

An oligonucleotide of the invention containing at least 15 contiguous nucleotides of a reference CARD-containing nucleotide sequence is capable of hybridizing to a CARD-encoding nucleotide sequence under moderately stringent hybridization conditions, and , As a probe for detecting CARD-encoding DNA or RNA in a sample and for detecting splice variants thereof; as a sequencing or PCR primer; an antisense reagent that inhibits transcription of CARD-encoding RNA in cells As; or hybridization to a CARD-encoding nucleic acid molecule is desirable,
It can be used to advantage in other applications known to those skilled in the art.

According to another aspect of the invention, there is provided a method of identifying a nucleic acid encoding a CARD-containing polypeptide. The method comprises contacting a sample containing nucleic acid with a probe of the invention or an oligonucleotide of the invention under highly stringent hybridization conditions and identifying the nucleic acid to be hybridized. Methods for identifying nucleic acids encoding CARD-containing polypeptides are disclosed herein and exemplified in the Examples.

According to the present invention, SEQ ID NOs: 11, 167, 187, 169, 171, 173, 175, 96, 98, 10
Identifying a candidate CARD-encoding nucleotide sequence using a CARD-encoding nucleotide sequence selected from 0, 102, 177, 179, 181, 183, 85 and 89;
Candidate CAs by aligning candidate sequences with CARD-encoding nucleotide sequences
A CARD-encoding nucleotide that includes the step of validating the RD-encoding nucleotide, wherein a conserved CARD domain sequence resembling a known CARD domain three-dimensional structure or a predicted three-dimensional polypeptide structure confirms a candidate sequence as a CARD-encoding sequence. Also provided is a method of identifying a sequence. Methods for identifying CARD coding sequences are provided herein (see Examples).

The CARD-encoding nucleic acid molecule of the present invention, as used herein, can be found at http: /// www.
nr, dbest, dbsts, gss and htgs, searchable at ncbi.nlm.nih.gov/blast/
Expression sequence tags (ESTs) deposited in public databases such as databases,
CARD-encoding nucleotide sequences (SEQ ID NO: 11, 167, 187, 169, 171, 173, 175, 96, 98, 100, 102, 177, 179, such as sequence tag sites (STS) and genomic fragments.
, 181, 183, 85 and 89), which specifically excludes previously known nucleic acid molecules consisting of nucleotide sequences having identities.

In particular, the CARD-encoding nucleic acid molecule of the present invention is a GenBank (gb), E, described below.
Exclude exact, specific and complete nucleic acid molecule sequences that match any nucleotide sequence with an MBL (emb) or DDBJ (dbj) accession number. Accession numbers that are specifically excluded are GI: 6165147 (Phase-1), AC007728 (
Phase-1), NT-002476 (Phase-1), AC010968 (Phase-1), AP001153
, AC022468 (Phase-1), GI: 6253000 (Phase-1), AC0097959 (Phase)
-1), GI: 6497652 (Phase-1) (contig: 23086: 40635), G
I: 6497652 (Phase-1) (Contig: 41136: 57024), AC023068 (Phase
-1), W58453, AA257158, AA046000, AW085161, AI189838, AA418021, AA046105
, W58488, AA418193, AA257066, AI217611, AW295205, AI023795, AL389934, AA
070591, AA070591, AC027011, AP002787, AQ889169, AV719179, AI263294, AV65
6315, AW337918, BF207840, AW418826, BK903662, AI023795, H25984, AL121653
And NT_005194.1 is included. The human contig, referred to as GenBank Accession No. AC007608, is also specifically excluded from CARD-encoding nucleic acid molecules. The genomic contigs referred to as GenBank Accession Nos. GI 5001450, GI 8575872 and GI 9795562 are also specifically excluded from the nucleic acid molecules of the invention. Those of ordinary skill in the art will recognize that the above-described excluded sequences will be modified at a later date, and therefore, those of skill in the art will recognize that the above-described sequences will be excluded if they are based on the priority date of the present application.

The isolated nucleic acid molecules of the invention can be used in various diagnostic and therapeutic applications. For example, the isolated nucleic acid molecule of the present invention may be used as a probe, as described above; as a template for recombinant expression of a CARD-containing polypeptide; Can be used in the screening assay.

Accordingly, the invention provides a method of detecting CARD encoding nucleic acid in a sample. The method of detecting CARD-encoding nucleic acid in a sample can be qualitative or quantitative, as desired. For example, the presence, abundance, integrity or structure of the CARD-encoding nucleic acid can be determined as desired by the assay format and the probe used for the hybridization or primer pair selected for the application.

Useful assays for detecting CARD-containing nucleic acids based on specific hybridization with isolated oligonucleotides of the invention are known in the art, eg, nucleic acid molecules depending on the assay format used. In situ hybridization, which can be used to detect changes in chromosomal location, changes in gene copy number and RNA abundance. Other hybridization assays include, for example, Northern blots and RNAse protection assays that can be used to determine the abundance and integrity of various RNA splice variants, and Southern blots that can be used to determine DNA copy number and integrity. Including. Hybridization probes can be detected by radioisotopes, fluorescent dyes, chemiluminescent markers, biotin, analytical methods,
Labeling can be with any suitable detection moiety, such as other detection moieties known in the art.

As used herein, “label” and “indicator”, in their various grammatical forms, mean a single atom and molecule that is directly or indirectly involved in producing a detectable signal. To do. Labels or indicating means can be attached to the nucleic acid probes, expressed proteins, polypeptide fragments or antibody molecules of the invention. These atoms or molecules can be used alone or in combination with additional reagents. Such labels per se are known in clinical diagnostic chemistry.

Useful assays for detecting CARD-encoding nucleic acids in a sample based on amplification of CARD-encoding nucleic acids using two or more oligonucleotides of the invention are also known in the art, For example, qualitative or quantitative polymerase chain reaction (PC
R): Reverse transcription-PCR (RT-PCR); easily identify single point mutations in DNA based on differences in secondary structure of single-stranded DNA that alter electrophoretic mobility during non-denaturing gel electrophoresis Yes, single-stranded conformational polymorphism (SSCP) analysis; includes transcription and translation assays, such as bound PCR, protein cleavage tests where mutations in DNA are determined by alterations in protein production on electrophoretic gels . In addition, the amplified CARD-encoding nucleic acids can be sequenced to detect mutations and mutation hotspots, and specific assays for large-scale screening of samples to identify such mutations. Can also be developed.

Also provided is an antisense nucleic acid having a sequence capable of specifically binding to the full length of an mRNA encoding a CARD-containing polypeptide or any portion thereof to prevent translation of the mRNA. The antisense nucleic acid can have a sequence that can specifically bind to any portion of the sequence of the cDNA encoding the CARD-containing polypeptide. As used herein, the phrase "specifically binds" refers to the ability of a nucleic acid sequence to recognize complementary nucleic acids and, in addition, to form double helix segments by hydrogen bond formation between complementary base pairs. including. Examples of antisense nucleic acids are antisense nucleic acids containing chemical analogs of nucleotides.

The present invention provides synthetic antisense nucleic acid compositions (hereinafter,
The use of SANC) provides a means of altering the expression level of CARD-containing polypeptides. The chemical structure of a synthetic oligonucleotide, or other antisense nucleic acid, designed to recognize and selectively bind mRNA is SEQ ID NO: 11,
187, 96, 98, 100, 102, 85 and 89 containing substantially the same nucleotide sequence, C
It is designed to be complementary to the full length or part of the ARD encoding strand.

SANCs are designed to be stable in the bloodstream or in laboratory cell culture conditions when administered to a subject by injection. SANC is designed to pass through the cell membrane to enter the cytoplasm of cells due to the physical and chemical properties of SANC. For example, by designing small hydrophobic SANC chemical structures, or SANC
SA is recognized by a specific intracellular transport system that recognizes and transports SANC into cells.
Allows the NC to cross the cell membrane. In addition, SANCs target certain SANCs by targeting them in such a way that they are recognized by a specific cellular uptake mechanism that binds only to SANCs in the selected cell population and absorbs only SANCs. It can be designed to be administered to only. In some embodiments, SANC is an antisense oligonucleotide.

For example, SANCs can be designed to bind to receptors found only on certain cell types, as described above. SANC recognizes and selectively binds to a target mRNA sequence that may be a sequence contained within the sequences set forth in SEQ ID NOs: 11, 187, 96, 98, 100, 102, 85 and 89. Is also designed. SANC binds to a target mRNA sequence, induces mRNA degradation, for example by digestion with RNAse I, interferes with translational regulators or ribosomes, degrades or chemically modifies ribozyme sequences or target mRNA It is designed to inactivate the target mRNA sequence by inhibiting translation of the mRNA target sequence by the inclusion of other chemical structures such as reactive chemical groups. SANCs have been shown to be capable of such properties when directed against mRNA targets (Cohen et al., TIPS, 10: 435 (1989) and Weintraub, Sci. .American, January (1990), pp.40).

The invention further provides a method of altering the level of a biochemical process regulated by a CARD-containing polypeptide by introducing an antisense nucleotide sequence into the cell. Here, the antisense nucleotide sequence specifically hybridizes to the CARD-encoding nucleic acid molecule, and the hybridization reduces or inhibits expression of the CARD-containing polypeptide in the cell. The use of recombinant antisense nucleic acids or antisense nucleic acids, including SANC, can be advantageously used to inhibit cell death.

[0109] An amount of the antisense nucleic acid of the invention that is effective to reduce expression of CARD-containing polypeptides by entering cells to prevent translation and specifically binding to CARD-encoding mRNA is provided. Also provided herein are compositions that include and acceptable hydrophobic carriers that can cross cell membranes. Suitable hydrophobic carriers are eg US Pat. No. 5,334,76.
No. 1; No. 4,889,953; No. 4,897,335. Acceptable hydrophobic carriers that are able to cross cell membranes may also contain structures that bind to receptors specific for the selected cell type and are thus absorbed by cells of the selected cell type. For example, the structure may be part of a protein known to bind to cell type specific receptors such as tumors.

Antisense nucleic acid compositions are useful for inhibiting translation of mRNA encoding a polypeptide of the invention. Synthetic oligonucleotides or other antisense chemical structures designed to bind to a CARD-encoding mRNA and inhibit translation of the mRNA, and encode a CARD-encoding gene or CARD-associated polypeptide in a tissue sample or subject. It is useful as a composition that inhibits gene expression.

The invention also provides a vector comprising the CARD-encoding nucleic acid of the invention. Suitable expression vectors are known in the art and include vectors that are capable of expressing nucleic acids that are operably linked to regulatory sequences or elements, such as promoter or enhancer regions, that can control the expression of such nucleic acids. Suitable expression vectors include those that are replicable in eukaryotic cells and / or prokaryotic cells, those that remain episomal, or those that integrate into the host cell genome.

The promoter or enhancer may be constitutive, depending on the nature of the control,
Can be controlled. The control sequences or elements are operably linked to the nucleic acids of the invention such that the physical and functional relationship between the nucleic acid and the control sequences allows transcription of the nucleic acid.

Vectors suitable for expression in prokaryotic and eukaryotic cells are known to those of skill in the art (see, eg, Ausubel et al., Supra, 2000). Vectors useful for expression in eukaryotic cells include, for example, the SV40 early promoter, cytomegalovirus (
It may include regulatory elements including CMV) promoter, mouse mammary tumor virus (MMTV), steroid-inducible promoter, Moloney murine leukemia virus (MMLV) promoter and the like. The vectors of the present invention are useful for subcloning and amplifying CARD-encoding nucleic acid molecules and for recombinant expression of CARD-containing polypeptides. Vectors of the invention include, for example, viral vectors such as bacteriophage, baculovirus or retroviruses; including cosmids or plasmids; especially for cloning large nucleic acid molecules, bacterial artificial chromosome vectors (BAC) and yeast artificial chromosome vectors (YAC). Is. Such vectors are commercially available and their use is known in the art. Those skilled in the art know and can easily determine a suitable promoter for expression in a particular host cell.

The invention further provides a recombinant cell comprising a CARD encoding nucleic acid of the invention. Recombinant cells are produced by introducing a vector containing a CARD-encoding nucleic acid molecule into a host cell. Recombinant cells are transduced, transfected or otherwise genetically modified. Examples of host cells that can be used to express recombinant CARD molecules include mammalian primary cells; established mammalian cell lines such as COS, CHO, HeLa, NIH3T3, HEK293 and PC12 cells; Xenopus embryo and oocytes. Includes amphibian cells and other vertebrate cells. Examples of host cells are insect cells such as Drosophila,
Saccharomyces cerevisiae, Saccharomyces
Pombe (Saccharomyces pombe) or Pichia pastoris
It also includes yeast cells such as and prokaryotic cells such as Escherichia coli. Other host cells are eg ATCC
(Manassas, Virginia).

In one embodiment, CARD-encoding nucleic acids can be delivered into mammalian cells either in vivo or in vitro using suitable vectors known in the art. Suitable vectors for delivering CARD-containing polypeptides or functional fragments thereof to mammalian cells include retroviral vectors, adenoviruses, adeno-associated viruses, lentiviruses, as well as non-viral vectors such as plasmid vectors.
Includes viral vectors such as herpes virus. Such vectors are useful in providing therapeutic amounts of CARD-containing polypeptides (see, eg, US Pat. No. 5,399,346, issued Mar. 21, 1995). Therapeutic delivery of CARD polypeptides or nucleic acids is particularly useful when targeted to tumor cells, thereby inducing apoptosis in the tumor cells. In addition, introduction of the antisense strand of the nucleic acids of the invention is considered when it is desirable to limit or reduce in vivo expression of CARD-containing polypeptides.

Virus-based systems offer the advantage that relatively high levels of heterologous nucleic acid can be introduced into a variety of cells. Viral vectors suitable for introducing the CARD-encoding nucleic acids of the invention into mammalian cells are known in the art. These viral vectors are, for example, simple perpesvirus vectors (Geller et al., Scienc
e, 241-1667-1669 (1988); vaccinia virus vector (Piccini (Pi
ccini) et al., Meth. Enzymology, 153: 545-563 (1987); cytomegalovirus vector (Mocarski) et al., "Viral Vector (Viral Vector).
s) ”, edited by Y. Gluzman and SH Hughes, Cold.
Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988, pp.78-84))
Moloney murine leukemia virus vector (Danos et al., Proc.Natl.Aca
d.Sci.USA, 85: 6460-6464 (1988); Blaese et al., Science, 270: 4.
75-479 (1995); Onodera et al., J. Virol, 72: 1769-1774 (1998).
); Adenovirus vector (Berkner, Biotechniques, 6: 616)
~ 626 (1988); Cotton et al., Proc. Natl. Acad. Sci. USA, 89: 6094-6.
098 (1992); Graham et al., Meth. Mol. Biol., 7: 109-127 (1991).
Li et al., Human Gene Therapy, 4: 403-409 (1993); Zabne (Zabne).
r) et al., Nature Genetics, 6: 75-83 (1994)); adeno-associated virus vector (Goldman et al., Human Gene Therapy, 10: 2261-2268 (1997)).
Greelish et al., Nature Med., 5: 439-443 (1999);
Wang) et al., Proc. Natl. Acad. Sci. USA, 96: 3906-3910 (1999); Snider (Sn
yder) et al., Nature Med., 5: 64-70 (1999); Herzog et al., Nature.
Med., 5: 56-63 (1999); Retroviral vector (Donahue
) Et al., Nature Med., 4: 181-186 (1998); Shackleford.
Proc. Natl. Acad. Sci. USA, 85: 9655-9659 (1988); U.S. Pat. No. 4,405,712.
Nos. 4,650,764 and 5,252,479, and WO92 / 07573, 90.
/ 06997, 89/05345, 92/05266 and 92/14829, and a lentiviral vector (Kafri et al., Nature Genetics, 17: 314-317 (1997).
)including.

For example, in one embodiment of the invention, the adenovirus transferrin / polylysine-DNA (TfAdpl-DNA) vector complex (Wagner et al., Proc. N.
atl.Acad.Sci., USA, 89: 6099-6103 (1992); Curiel et al., Hum.Ge.
ne.Ther., 3: 147-154 (1992); Gao et al., Hum.Gene.Ther., 4: 14-24.
(1993)) are used to transduce mammalian cells with heterologous CARD-encoding nucleic acids. All of the plasmid expression vectors described herein can be used in the TfAdpl-DNA complex.

Vectors useful for therapeutic administration of CARD-encoding nucleic acids can include control elements that provide tissue-specific or inducible expression of the operably linked nucleic acid. One of skill in the art can readily determine an appropriate tissue-specific promoter or enhancer that allows expression of the CARD polypeptide or nucleic acid in the desired tissue. Any of a variety of inducible promoters or enhancers can also be included in the vector for controllable expression of the CARD polypeptide or nucleic acid. Such induction systems include, for example, the tetracycline induction system (Gossen and Bizard (Bi
zard), Proc.Natl.Acad.Sci.USA, 89: 5547-5551 (1992); Gossen.
) Et al., Science, 268: 1766-1769 (1995); Clontech, Palo Alto, Calif.); Heavy metal-inducible metallothionein promoters; ecdysone-responsive insect steroid hormones or related steroids such as muristerone. No et al., Proc. Natl. Acad. Sci. USA.93: 3346-3351.
(1996); Yao et al., Nature, 366: 476-479 (1993); Invitrogen, Carlsbad, CA); mouse mammary tumor virus induced by steroids such as glucocortocoid and estrogen (
MMTV) (Lee et al., Nature, 294: 228-232 (1981); and heat shock promoters induced by temperature changes.

Inducible systems that are particularly useful for therapeutic administration deliver a level of therapeutic product in response to a given level of agent administered to an individual and, in the absence of the agent, therapeutic product. Inducible promoters are utilized that can be regulated to have little or no expression. One such system utilizes Gal4 fusions induced by antiprogestins such as mifepristone in a modified adenovirus vector (Burien et al., Proc.Natl.Acad.Sci.USA, 96: 355 ~ 3
60 (1999). In other such induction systems, the drug rapamycin is used to induce reconstitution of transcriptional activators containing the rapamycin binding domain of FKBP12 and FRAP in adeno-associated viral vectors (Ye et al., Science, 283: 88 ~ 91 (19
99)). It is understood that any combination of inducible systems can be combined in any suitable vector, including the vectors disclosed herein. Such controllable inducible systems allow therapeutic expression levels to be controlled by the amount of drug administered to an individual or, if desired, by stopping administration of the drug. This is advantageous because the expression of the product can be terminated.

The present invention also provides a method of expressing a CARD-containing polypeptide by culturing cells containing the CARD-encoding nucleic acid under conditions suitable for expression of the CARD-containing polypeptide. Accordingly, methods of recombinant production of CARD-containing polypeptides of the invention by expressing the CARD-encoding nucleic acid sequences in a suitable host cell are also provided. Recombinant DNA expression systems suitable for producing the CARD-containing polypeptides described herein are known in the art (see, eg, Ausubel et al., Supra, 2000).
. For example, the nucleotide sequences described above can be included in a vector for further manipulation. Vector, as used herein, means a recombinant DNA or RNA plasmid, or a virus that contains independent elements used to introduce heterologous DNA into a cell and direct its expression or replication. To do.

The invention further provides an isolated anti-CARD antibody that has specific reactivity with a CARD-containing polypeptide of the invention. The anti-CARD antibody can be a monoclonal antibody or a polyclonal antibody. The present invention further provides a cell line producing a monoclonal antibody that has specific reactivity with the CARD-containing protein of the present invention.

The present invention therefore provides antibodies that specifically bind a CARD-containing polypeptide. As used herein, the term "antibody" is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. With respect to the anti-CARD antibodies of the invention, the term "antigen" means a native or synthetic CARD-containing polypeptide or fragment thereof.
The anti-CARD antibody, or antigen-binding fragment of such an antibody, is characterized by having a specific binding activity of at least about 1 × 10 ⁵ M ⁻¹ for the CARD polypeptide or peptide portion thereof. Thus, Fab, F (ab ′) ₂ , Fd and Fv fragments of anti-CARD antibodies that have specific binding activity for CARD-containing polypeptides are included in the definition of antibody. CA
The specific binding activity of an RD-containing polypeptide can be readily determined by those skilled in the art, for example, by comparing the binding activity of an anti-CARD antibody to a CARD-containing polypeptide and a reference polypeptide that is not a CARD-containing polypeptide. Methods of preparing polyclonal or monoclonal antibodies are known to those of skill in the art (eg, Harl
ow) and Lane, “Antibodies: Experimental Manual (Antibodies: A Laborato
ry Manual) ", Cold Spring Harbor Laboratory Press (1988)).

Furthermore, the term “antibody” as used herein refers to naturally occurring antibodies, such as single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as their antibody binding. It also includes non-naturally occurring antibodies, such as fragments. Such non-naturally occurring antibodies can be made using solid phase peptide synthesis, can be produced recombinantly, or as described, for example, in Huse et al., Science 246: 1275-1281 (1989). It is obtained by screening a combinatorial library consisting of various heavy chains and various light chains. These methods and for example chimera,
Other methods of making humanized, CDR-grafted, single chain and bifunctional antibodies are known to those of skill in the art (Winter and Harris, Immunol. Today).
14: 243-246 (1993); Ward et al., Nature 341: 544-546 (1989).
Harlow and Lane, supra, (1988); Hily
ard) et al., “Protein Engineering: A Antibody Eng.
ineering) ", 2nd edition (Oxford University Press 1995)).

Anti-CARD antibodies can be prepared from natural sources or recombinantly produced,
A CARD immunogen, such as an isolated CARD-containing polypeptide, or a peptide portion of a CARD-containing polypeptide having an amino acid sequence substantially identical to SEQ ID NOs: 12, 188, 97, 99, 101, 103, 86 and 90, or a fragment thereof. Can be produced using. Such a peptide portion of the CARD-containing polypeptide is a functional antigenic fragment if it can be used to generate CARD-specific antibodies. Making a non-immunogenic or weakly immunogenic CARD-containing polypeptide or portion thereof immunogenic by linking the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). You can Various other carrier molecules and methods for attaching haptens to carrier molecules are known in the art (eg, Halow (Ha
rlow) and Lane, supra, 1988). The immunogenic CARD-containing polypeptide fragment can also be produced by expressing the peptide as a fusion protein in, for example, glutathione S transferase (GST), polyHis and the like. Methods of expressing peptide fusions are known to those of skill in the art (Ausubel (Au
Subel) et al., supra (2000)).

The invention further provides a method of detecting the presence of a human CARD-containing polypeptide in a sample by contacting the sample with a CARD-specific antibody, and detecting the presence of specific binding of the antibody to the sample, Thereby provide a method of detecting the presence of human CARD-containing polypeptide in a sample. CARD-specific antibodies can be used in diagnostic methods and systems to detect the level of CARD-containing polypeptide present in a sample. As used herein, the term "sample" includes CARD nucleic acids or polypeptides,
Or, it is intended to mean any biological fluid, cell, tissue, organ or part thereof that potentially comprises. The term includes samples obtained from and derived from an individual, as well as samples present in the individual. For example, the sample can be a tissue section of a sample obtained by biopsy, or can be a cell that has undergone tissue culture or is suitable for tissue culture.
The sample can also be a subcellular fraction or extract, or an unpurified or substantially pure nucleic acid or polypeptide preparation.

CARD-specific antibodies can be used for immunoaffinity or affinity chromatographic purification of CARD-containing polypeptides of the invention. In addition, contacting the cells with an antibody that specifically binds to the CARD-containing polypeptide under conditions that allow the antibody to bind to the CARD-containing polypeptide and detecting the presence of the antibody that binds to the CARD-containing polypeptide Also contemplated herein are methods of detecting the presence of a CARD-containing polypeptide of the invention in a cell, comprising: and thereby detecting the presence of a polypeptide of the invention in the cell. For detection of such polypeptides, the antibodies can be used in in vitro diagnostic or in vivo imaging methods.

Immunological procedures useful for the in vitro detection of target CARD-containing polypeptides in a sample include immunoassays using detectable antibodies. Such immunoassays include, for example, immunohistochemistry, immunofluorescence, ELISA assays, radioimmunoassays, FACs.
Includes S-analysis, immunoprecipitation, immunoblot analysis, Pandex microfluorescence quantitation assay, agglutination assay, flow cytometry and serodiagnosis assay, which are known in the art (Harlow and Lane, supra). See 1988; Harlow and Lane, "Antibody Used: Experimental Manual (Using Anti.
bodies: A Laboratory Manual) ", Cold Spring Harbor Press (1999)).

Antibodies can be detected by various methods well known in the art. For example, the detectable marker can be attached directly to the antibody or indirectly, for example using a second intermediary that recognizes the CARD-specific antibody. Useful markers include, for example, radionucleotides, enzymes, binding proteins such as biotin, fluorogens, chromogens and chemiluminescent labels.

Antibodies can also be detected by, for example, a fluorescent labeling agent that chemically binds to the antibody or antigen without denaturation to form a fluorescent dye (dye) useful as an immunofluorescent tracer. A description of immunofluorescence analysis techniques is incorporated herein by reference, "Antibody As a Tool, Marc Saronis (Marc
halonis) et al., John Wiley & Sons, Lt.
d), pp.189-231 (1982), DeLuca, "Immunofluorescence analysis (Immnoflu
orescence Analysis) ”.

In one embodiment, the indicator group is an enzyme such as horseradish peroxidase (HRP), glucose oxidase. In other embodiments, radioactive elements are used as labeling agents. Binding of labels to substrates, ie labeling of nucleic acid probes, antibodies, polypeptides and proteins, is known in the art. For example, the antibodies of the invention can be labeled by the metabolic inclusion of radiolabeled amino acids given in culture. See, for example, Galfre et al., Meth. Enzymol., 73: 3-46 (1981). Conventional means of protein conjugation or coupling by activated functional groups are especially available. For example, Aurameas et al., Scand.J.Immunol., Volume 8, Addendums 7: 7-23 (1978), Rodwell et al., Biotech., 3: 889-894 (
1984), and U.S. Pat. No. 4,493,795.

In addition to detecting the presence of a CARD-containing polypeptide, the anti-CARD antibodies of the invention alter the activity of the CARD-containing polypeptide in living animals, humans or biological tissues or fluids isolated therefrom. To be used herein. "
The term "alter" increases or decreases the biological activity modulated by a CARD-containing polypeptide, CARD-encoding nucleic acid, drug or other compound by acting as an agonist or antagonist of that compound. Means ability. Thus, a carrier and a certain amount of an antibody with specificity for a CARD-containing polypeptide that effectively inhibits the binding of naturally occurring ligands or other CARD-associated polypeptides to the CARD-containing polypeptides of the invention. Compositions that include are considered herein. For example, an epitope-directed monoclonal antibody directed against a CARD-containing polypeptide of the invention comprising an amino acid sequence substantially the same as SEQ ID NOs: 12, 188, 97, 99, 101, 103, 86 and 90 may be used for this purpose. It is useful.

The invention further provides a transgenic non-human mammal capable of expressing an exogenous nucleic acid encoding a CARD-containing polypeptide. As used herein, an "exogenous nucleic acid" is a nucleic acid sequence that is not native to the host, or a nucleic acid that is present in the host outside the native environment, eg, as part of a genetically modified DNA construct. Means
In addition to naturally occurring CARD-containing polypeptide levels, the CARD-containing polypeptides of the invention can be over- or under-expressed in transgenic mammals, such as under-expression in knockout animals.

Also provided is a transgenic non-human mammal capable of expressing a CARD-encoding nucleic acid for mutating such that its normal activity cannot be used. Therefore, the transgenic animal does not express the native CARD-containing polypeptide or reduces the expression of the native CARD-containing polypeptide. The present invention is complementary to a CARD-encoding nucleic acid that is arranged to be transcribed into an antisense mRNA that is complementary to the CARD-encoding mRNA, which hybridizes to the mRNA and thereby reduces its translation. Transgenic non-human mammals having a genome comprising a sex antisense nucleic acid are also provided. Nucleic acids further include inducible promoters and / or tissue-specific regulatory elements so that expression is induced or restricted to specific cell types.

Model animal systems are also provided to elucidate the physiological and behavioral roles of CARD-containing polypeptides, created by forming transgenic animals that alter the expression of CARD-containing polypeptides using a variety of techniques. . Examples of such techniques include inserting a normal or mutated nucleic acid encoding a CARD-containing polypeptide into a properly fertilized embryo by microinjection, retroviral infection or other means known to those of skill in the art. , Including creating transgenic animals. For example, Hogan et al., "Manipulating the Mouse Embryo: A Laborator.
y Manual) "(Cold Spring Harbor Laboratory (1986)). The transgenic model animal system is useful for in vivo screening of compounds to identify specific ligands such as agents or antagonists that activate or inhibit biological activity.

Herein, a native locus in transgenic animals for altering expression control or structure of CARD-containing polypeptides by replacing a foreign gene with a recombinant or mutated CARD-encoding gene is provided. Mutant or normal CARD
Also consider the use of homologous recombination of the encoded gene. Methods of making transgenic non-human mammals, including gene knockout non-human mammals, are well known to those of skill in the art (Capecchi et al., Science 244: 1288 (1989); Zimmer (Z
immer) et al., Nature 338: 150 (1989); Shastry, Experentia.
51: 1028-1039 (1995); Shastry, Mol.Cell.Biochem., 18
1: 163-179 (1998); and U.S. Pat. No. 5,616,491 issued April 1, 1997.
Issue and No. 5,981,830 issued November 9, 1999).

In addition to homologous recombination, other methods of adding the gene to the host genome without removing the host gene, such as microinjection, can also be used. Microinjection can create transgenic animals capable of expressing both endogenous and exogenous CARD-containing polypeptides. An inducible promoter can be attached to the coding region of the nucleic acid to provide a means for controlling the expression of the transgene. Attachment of a tissue-specific regulatory element to the coding region allows for tissue-specific expression of the transgene. The transgenic animal model system is CA
It is useful for in vivo screening of compounds to identify specific ligands, agonists and antagonists, that activate or suppress RD-containing polypeptide responses.

Another aspect of the invention provides a method of identifying a CARD associated polypeptide (CAP). The method involves contacting a CARD-containing polypeptide of the invention with a candidate CAP to generate C
It is performed by detecting the association of the ARD-containing polypeptide with CAP.

As used herein, a “CARD associated polypeptide” or “CAP” is specific for a CARD-containing polypeptide of the invention, or for any functional fragment of a CARD-containing polypeptide of the invention. Means a polypeptide capable of binding to. Since the CARD-containing polypeptide of the present invention contains a domain capable of self-association, the CARD-containing polypeptide is CA
Included in the word P. An example of CAP is the NB-ARC (NA
CHT), CARD, LRR or ANGIO-R domain is a protein or polypeptide portion of a protein. For example, CAP is, for example, Ausbel
), Et al., Supra, using in vitro protein binding assays similar to those described in 2000, by in vivo methods including methods such as the yeast two-hybrid assay, or other protein interaction assays known in the art. And can be identified by the method.

The normal association of CARD-containing and CAP polypeptides in cells can be altered by, for example, intracellular expression of the mutant CAP or CARD-containing polypeptide, respectively, either of which It is possible to compete with the normal binding function of the CARD-containing polypeptide and thus reduce the association of CAP and CARD-containing polypeptide within the cell. The term “variant” is generally used herein
Used to mean a polypeptide that differs from the CAP- or CARD-containing polypeptide normally found in a particular cell type. Thus, variants may include muteins or naturally occurring proteins, such as isoforms not normally found in particular cell types.

The CARD-containing and CARD-associated polypeptides of the invention are characterized, for example, for use in in vitro binding assays or yeast two-hybrid systems. In vivo transcription activation assays such as the yeast two-hybrid system are particularly useful for identifying and manipulating protein association. In addition, the results obtained with such an assay reflect events that occur naturally within the cell. Thus, the results obtained in such an in vivo assay may be predictive of the results that may occur in cells of a subject, such as a human subject.

Transcriptional activation assays, such as the yeast two-hybrid system, use functionally separable DNs.
It is based on the modular nature of transcription factors, which consist of an A-binding domain and a transcriptional activation domain. When expressed as independent proteins, these two domains are unable to mediate gene transcription. However, the transcriptional activation activity can be restored by cross-linking the DNA binding domain and the transcriptional activation domain together, eg by the association of two proteins. The DNA binding domain and the transcriptional activation domain can be cross-linked, for example, by expressing the DNA binding domain and the transcriptional activation domain as a fusion protein (hybrid), provided that the proteins fused to the domain can associate with each other. The non-covalent cross-linking of the two hybrids joins the DNA binding domain and the transcription activation domain to create a transcriptionally competent complex. Protein association is determined by observing transcriptional activation of the reporter gene.

The yeast two-hybrid system exemplified herein uses various strains of S. cerevisiae as host cells for vectors expressing hybrid proteins. The transcription activation assay can also be performed using, for example, mammalian cells. However, the yeast two-hybrid system is particularly useful due to the ease of handling the yeast and the speed with which the assay can be performed. For example, using a yeast host cell containing a lacZ reporter gene linked to a LexA operator sequence, it can be demonstrated that the CARD domain of a CARD-containing polypeptide of the invention can interact with itself or with other CARD-containing polypeptides. . For example, the DNA-binding domain is L
It consists of a LexA DNA binding domain that binds the ex promoter, fused to the CARD domain of a CARD-containing polypeptide of the invention, and the transcriptional activation domain is individually fused to multiple cDNA sequences encoding known CARD-containing polypeptides. B42 can consist of acidic regions. Non-covalently cross-linking the LexA domain to the transcriptional activation region fused to the CARD-containing polypeptide can activate transcription of the reporter gene by association.

CAP, such as CARD-containing polypeptides, NB-ARC-containing polypeptides or LRR-containing polypeptides, can be prepared using well-known in vitro assays such as, for example, glutathione-S-transferase (GST) fusion protein-based assays. Can also be identified. Such in vitro assays provide a simple, rapid and inexpensive method for identifying and isolating CAP. Such in vitro assays include
It is particularly useful for confirming the results obtained in vivo and can be used to characterize the specific binding domain of CAP. For example, GST can be expressed and purified by fusing to a CARD-containing polypeptide of the invention and binding to an affinity matrix containing immobilized glutathione. CAP or CAP if desired
The sample containing the active fragment of E. coli is passed through an affinity column containing the bound GST / CARD to obtain CAP that binds to the CARD-containing polypeptide. In addition, GST / CARD can be used to screen cDNA expression libraries. In that case, GST /
Binding of the CARD fusion protein to the clone indicates that the clone contains a cDNA encoding CAP.

Accordingly, one of skill in the art will recognize that CAP can be identified using a variety of methods such as the CARD-containing polypeptides, protein purification, protein interaction cloning or protein mass spectrometry described herein. To be

Although the term “CAP” is commonly used, it should be recognized that the CAP identified with the novel polypeptides described herein can be a fragment of a protein. Thus, as used herein, CAP also includes a polypeptide that specifically associates with the portion of the CARD-containing polypeptide of the invention that does not contain a CARD domain. For example, the CAP can associate with the NB-ARC domain of CLAN or CARD3X. As used herein, "candidate CAP" means a polypeptide comprising a polypeptide sequence known or suspected of binding one or more CARD-containing polypeptides of the invention. Therefore, CAP may be an alternative to the full length protein or its CARD associated fragment. As the CAP polypeptide can be a full-length protein or a CARD-associated fragment thereof, one of skill in the art will recognize that CAP-encoding nucleic acids such as genomic sequences, mRNA sequences or cDNA sequences need not encode the full-length protein. Thus, the cDNA can encode a polypeptide that is nevertheless a fragment of full-length CAP that binds one or more CARD-encoding polypeptides of the invention. For example, due to steric inhibition of the binding site, full-length CAP without post-translational modification
It is also within the scope of the invention that it can adopt a conformation that binds the CARD-containing polypeptide of the invention. Thus, a CAP may be a protein or a polypeptide portion of a protein capable of binding one of the CARD-containing polypeptides of the invention. CAPs can also be identified by using the smallest polypeptide derived from the sequences of the CARD-containing polypeptides of the invention, and it is not necessary to use the full length molecule to identify such CAPs. You need to be aware of that.

Since CARD-containing polypeptides can be involved in apoptosis, association of CAP with CARD-containing polypeptides affects the susceptibility or resistance of cells to apoptosis or induces apoptosis induced by external or internal stimuli. Or it can be inhibited. CAR with the identification of various CAPs using known methods
It is possible to determine the function of these CAPs in the controlled cell death or signaling pathways of D-containing polypeptides, useful in identifying agents that effectively alter the association of CAPs with CARD-containing polypeptides. Assay development is expected. Such agents may be useful for effectively treating the symptoms caused, at least in part, by cancer, autoimmune disease or certain viral infections. Such agents are also useful in effectively treating diseases known to cause excessive apoptosis, such as stroke, heart failure or AIDS.

The assays of the invention can be used to identify agents that alter the self-association of CARD-containing polypeptides of the invention. Therefore, using the method of the invention (SEQ ID NO:
CARD2X, CARD3X, CLAN A, C described in 12, 188, 97, 99, 101, 103, 86 and 90)
LAN B, CLAN C, CLAN D, COP-1 and COP-2 self-association, its CARD domain, N
Agents that are altered by the B-ARC domain, LRR domain or other domains within these polypeptides can be identified.

ATP binding and hydrolysis of the NB-ARC domain is important for the function of NAC polypeptides, for example by altering NAC oligomerization. Therefore, C
NB-A of NAC polypeptide of the invention, such as LAN (SEQ ID NO: 97, 99, 101 or 103)
Agents that interfere with or enhance ATP or nucleotide binding and / or hydrolysis by the RC domain may also be useful in altering the activity of these polypeptides within cells.

A further aspect of the invention is a method of identifying an agent capable of effectively altering CARD-containing polypeptide activity, eg, the ability of a CARD-containing polypeptide to associate with one or more heterologous proteins. provide. Therefore, the present invention is a CA
Provided are screening assays useful in identifying effective agents that can alter the association of RD containing polypeptides with CARD associated polypeptides (CAP), such as heterologous CARD containing polypeptides. Since CARD-containing polypeptides are involved in biochemical processes such as apoptosis, the identification of such effective agents is
For example, it may be effective in altering the level of biochemical processes, such as apoptosis, in cells of a subject having a condition characterized by elevated or decreased levels of apoptosis.

In addition, effective agents may be useful in altering other biochemical processes regulated by the CARD-containing polypeptides of the invention. Other biochemical processes regulated by CARD-containing polypeptides include, for example, NF-kB induction, cytokine processing, cytokine receptor signaling, cJUN N-terminal kinase induction, and caspase-mediated proteolytic activation / inhibition, transcription, Including inflammation and cell adhesion.

As used herein, the term “agent” alters the association of a single or complex organic molecule, peptide, peptidomimetic, polypeptide, CARD-containing polypeptide with a heterologous protein. Means a chemical or biological molecule, such as a protein or oligonucleotide, that has the potential to alter, or alter the ability of a CARD-containing polypeptide to self-associate, or alter the ligand binding or catalytic activity of a CARD-containing polypeptide. Examples of ligand binding activities are nucleotide binding activities such as ADP or ATP binding activities; examples of catalytic activities are nucleotide hydrolytic and proteolytic activities. In addition, the term "effective agent" is used herein to alter the association of a CARD-containing polypeptide with a heterologous protein, or CARD.
Used to mean an agent that has been identified with the ability to alter the ability of a containing polypeptide to self-associate, or alter the ligand binding or catalytic activity of a CARD containing polypeptide. For example, the effective agent can be an anti-CARD antibody, a CARD-associated polypeptide, a caspase inhibitor and the like.

As used herein, the term “altering association” means that the association between two specifically interacting polypeptides is increased or decreased by the presence of an effective agent. Means to do. As a result of the altered association of the CARD-containing polypeptide with other polypeptides in the cell, the activity of the CARD-containing polypeptide or CAP may be increased or decreased, which may result in biochemical processes such as intracellular The level of apoptosis changes. As used herein, the term "altering activity" means that an agent increases or decreases the activity of a CARD-containing polypeptide within a cell, thereby causing an intracellular biochemical process, such as a cell. Means altering the level of apoptosis within. Similarly, the term "altering" a biochemical process regulated by a CARD-containing polypeptide means an increase or decrease in the biochemical process that occurs when altering the activity of the CARD-containing polypeptide. For example, an effective agent can increase or decrease the CARD: CARD association activity of a CARD-containing polypeptide, which results in decreased apoptosis. In another example, modification of ATP hydrolysis activity results in NB-of CARD-containing polypeptides.
The ability of the ARC domain to modulate the association with other NB-ARC containing polypeptides such as Apaf-1 may be affected by such association between CARD containing and NB-ARC containing polypeptides. The process can also be changed.

Effective agents act by inhibiting the ability of CARD-containing polypeptides to associate with other polypeptides, or CARD from complexes with CARD-containing polypeptides.
Can act by causing dissociation of the containing polypeptide,
In that case, the ratio of bound CARD-containing polypeptide to free CARD-containing polypeptide is related to the level of biochemical processes such as intracellular apoptosis. Binding of a ligand to CAP, for example, allows CAP to then bind to a specific CARD-containing polypeptide because all of the specific CARD-containing polypeptides bind to CAP, resulting in reduced apoptosis. it can. For example, the association of a CARD-containing polypeptide with a CARD-containing polypeptide results in the NB-A of the CARD-containing polypeptide.
RC: Can result in activation or inhibition of NB-ARC association activity. In the presence of effective agents, it is possible to alter the association of CARD-containing polypeptides with CAP, which can, for example, alter intracellular activation of caspases. As a result of altered caspase activation, intracellular apoptosis levels can be increased or decreased. Therefore, by identifying effective agents that alter the association of CARD-containing polypeptides with other polypeptides, it is possible to use effective agents to increase or decrease the level of biochemical processes such as apoptosis. Can be considered.

Effective agents may be useful, for example, in increasing intracellular apoptotic levels, such as cancer cells, which are characterized by reduced apoptotic levels relative to corresponding normal cells. Effective agents can also be used in T subjects of subjects with viral diseases, such as acquired immunodeficiency syndrome, characterized by increased levels of apoptosis in infected T cells compared to normal T cells. It may also be effective in reducing the level of apoptosis in cells such as lymphocytes. Thus, an effective agent may be useful as a drug to alter the level of apoptosis in a subject having a condition characterized by increased or decreased apoptosis. In addition, effective agents can be used, for example, to reduce apoptosis levels and thus increase the survival time of cells such as hybridoma cells in culture. The use of agents effective to prolong cell survival in vitro can significantly improve the bioproduction yield in industrial tissue culture applications.

Lacking the ability to bind the NB-ARC or LRR domain to another polypeptide, but retaining the ability of its CARD domain to self-associate or bind to another CARD-containing polypeptide CARD-containing polypeptides are examples of effective agents. This is because intracellular non-NB-ARC associated or non-catalytically active CARD-containing polypeptides can alter the association of endogenous CARD-containing polypeptides with themselves or with CAP.

Thus, mutations in CARD-containing polypeptides can be effective agents, eg, depending on the normal levels of CARD-containing and CARD-associated polypeptides occurring in a particular cell type. In addition, an active fragment of a CARD-containing polypeptide can be an effective agent if the active fragment can alter the association of the CARD-containing polypeptide with other polypeptides in the cell. Such active fragments, which can be peptides as small as about 5 amino acids, can be isolated, for example, by screening peptide libraries to identify peptides capable of binding CARD-associated polypeptides (see, eg, Ladner et al., US Pat. See No. 5,223,409).

Similarly, fragments of CARD associated polypeptides are also effective agents. Fragments of CARD-associated polypeptides may be useful, for example, to reduce intracellular association of CARD-containing polypeptides with CAP by competing for binding to CARD-containing polypeptides. Non-natural peptidomimetics may also be useful as effective agents. Such peptidomimetics can include, for example, peptoids, which are peptidic sequences containing N-substituted glycines, or oligocarbamates. Peptoid mimetics, for example, are particularly useful as effective agents due to their increased stability against enzymatic degradation in vivo.

According to another aspect of the invention, the following steps provide a method of identifying an effective agent that alters the association of a CARD-containing polypeptide of the invention with a CARD associated polypeptide (CAP): (A) a CARD-containing polypeptide is prepared by using an agent believed to be capable of changing the association between the CARD-containing polypeptide and the CAP polypeptide under the conditions for associating the CARD-containing polypeptide with the CAP polypeptide. Contacting the CAP polypeptide; (b) detecting an altered association of the CARD-containing polypeptide with the CAP polypeptide when identifying an agent for which the altered association is effective.

The bioassays described herein use CARD-containing polypeptides and CAP to identify agents as agonists or antagonists of CARD-containing polypeptides.
Methods well known in the art for detecting altered polypeptide association can be used, eg, measurement of protein: protein binding, proteolytic or apoptotic activity. As described herein, CARD-containing polypeptides are capable of self-association. Thus, methods of identifying agents that alter the association of CARD-containing polypeptides with CAP are useful for identifying effective agents that alter the ability of CARD-containing polypeptides to self-associate.

As used herein, “conditions for associating the CARD-containing polypeptide and the CAP polypeptide” means the environmental conditions under which the CARD-containing polypeptide and CAP specifically associate. Such conditions will generally be aqueous conditions with a pH between 3.0 and 11.0 and temperatures below 100 ° C. The conditions are preferably such that the salt concentration is equal to or less than 1M NaCl and the pH is 5.0.
~ 9.0, the temperature is 0 ℃ ~ 50 ℃ in the aqueous conditions. Most preferably, the conditions fall within the range of normal physiological conditions of yeast or mammalian cells, or preferred conditions for in vitro assays such as immunoprecipitation and GST protein: protein association assays.

In another aspect of the invention, methods of identifying an agent that modulates ligand binding or catalytic activity of a CARD-containing polypeptide of the invention are provided. The method is of the invention
Contacting the CARD-containing polypeptide with an agent that is believed to modulate the ligand-binding or catalytic activity of the CARD-containing polypeptide, and measuring the ligand-binding or catalytic activity of the CARD-containing polypeptide, wherein the ligand-binding Alternatively, an agent is identified as an agent that alters ligand binding or catalytic activity of a CARD-containing polypeptide by catalytic activity.

As used herein with respect to ligand binding or catalytic activity, “modulate” means an increase or decrease in ligand binding or catalytic activity. Therefore,
Modulation includes activating or increasing ligand binding or catalytic activity as well as inhibition of ligand binding or catalytic activity. Examples of ligand binding activity include nucleotide binding activity. Examples of catalytic binding activities include nucleotide hydrolysis and proteolytic activity.

Methods of ligand binding or catalytic activity are known in the art as disclosed herein. For example, an agent known or suspected of modulating ligand binding or catalytic activity can be contacted with a CARD-containing polypeptide of the invention in vivo or in vitro and ligand binding Alternatively, catalytic activity can be measured using known methods. For example, enzymatic activity can be measured using a cleavable reporter if the cleavable reporter produces or alters a measurable signal such as absorption, fluorescence or radioactive decay. Examples of agents capable of modulating ligand binding or catalytic activity include peptides, peptidomimetics and other peptide analogs, non-peptide organic molecules such as naturally occurring protease inhibitors or their derivatives, nucleotides and nucleotide analogs, etc. Including. Such inhibitors can be either reversible or irreversible, as is well known in the art.

Agents that modulate the ligand binding or catalytic activity of CARD-containing polypeptides identified using the methods of the invention can be used to modulate the activity of CARD-containing polypeptides. For example, the agent can modulate the nucleotide binding or nucleotide hydrolytic activity of the NB-ARC domain of the CARD-containing polypeptide. In other examples, the agent can modulate the catalytic activity of a protease domain, such as the caspase domain. The method of modulating ligand binding or catalytic activity of a CARD-containing protein of the invention can be used in a method of altering a biochemical process regulated by a CARD-containing protein, such as a biochemical process disclosed herein. .

In yet another aspect of the invention, there is provided a method of altering the ligand binding or catalytic activity of a CARD-containing polypeptide of the invention, the method comprising: activating the CARD-containing polypeptide as described herein. Contacting with an effective amount of the agent identified by the assay.

The present invention also provides in vitro screening assays. Such screening assays are particularly useful in that they can be automated and alter the association of CARD-containing polypeptides and CAP or the catalytic or ligand binding activity of CARD-containing polypeptides, thereby altering CARD, such as apoptosis. To identify those agents that alter the biochemical processes regulated by the containing polypeptide, of agents such as agents, peptidomimetics or peptides, eg randomly or rationally designed agents High throughput screening is considered. In vitro screening assays can utilize CARD-containing polypeptides, including, for example, CARD-containing fusion proteins such as CARD-glutathione-S-transferase fusion proteins. CARD for use in in vitro screening assays
The containing polypeptide must have the ability to associate with the CARD associated polypeptide as well as its affinity for solid substrates. For example, when a CARD containing polypeptide is used in the assay, the solid substrate can include a covalently bound anti-CARD antibody. Alternatively, the GST / CARD fusion protein can be used in the assay,
The solid substrate can include covalently bound glutathione bound by the GST component of the GST / CARD fusion protein. Similarly, CARD associated polypeptides, or GST / NB-ARC containing polypeptide fusion proteins are known in the art,
Any of the various in vitro enzymes or in vitro binding assays described in the text such as Ausubel et al., Supra, 2000 can be used.

In vitro screening assays can be performed, for example, by attaching the CARD-containing polypeptide to a solid support and then adding the CARD-associated polypeptide and the agent to be tested. Reference reactions without agents can be run in parallel. For example, in the absence of an agent after incubation under suitable conditions, including suitable buffer concentrations and pH and time and temperature that allow binding of a particular CARD-containing polypeptide to a CARD-associated polypeptide, and The amount of protein associated with the agent can be determined. Association of the CARD-associated polypeptide with the CARD-containing polypeptide is detected, for example, by attaching a detectable moiety such as a radionuclide or a fluorescent label to the CARD-associated polypeptide and measuring the amount of label associated with the solid support. The amount of label that is possible and detected is indicative of the amount of association of the CARD-associated polypeptide with the CARD-containing polypeptide. Effective agents are determined by comparing the amount of specific binding in the presence of the agent to a reference level of binding, where the effective agent alters the association of the CARD containing polypeptide with the CARD associated polypeptide. Such an assay is particularly useful for screening a range of agents, such as peptide libraries, to detect effective agents.

Various binding assays to identify cellular proteins that interact with protein binding domains are known in the art, eg, yeast two-hybrid screening assays (eg, US Pat. Nos. 5,283,173, 5,468,614 and 5,667,
Issue 973; Ausubel et al., Supra, 2000; Luban et al., Curr.Opi.
n. Biotechnol. 6: 59-64 (1995)) and affinity column chromatography methods using cell extracts. The CARD binding interface can be readily identified by synthesis or expression of polypeptide fragments containing various CARD associated sequences or deletions.

Other assay methods for screening agents that alter the activity of CARD-containing polypeptides are based on altering the yeast phenotype by expressing CARD-containing polypeptides. In one embodiment, expression of the CARD-containing polypeptide is inducible (Tao et al., J. Biol. Chem. 273: 23704-23708 (19
98)), compounds can be screened if CARD-containing polypeptide expression is induced. The CARD-containing polypeptides of the invention can also be co-expressed in yeast with a CAP polypeptide used to screen compounds that antagonize the activity of the CARD-containing polypeptide.

Assays for identifying agents that alter the expression of CARD-containing polypeptides are also provided by the present invention. A method of determining the expression of a CARD-containing polypeptide can include detecting a change in the amount of the CARD-containing polypeptide in response to contacting the cell with an agent that modulates the expression of the CARD-containing polypeptide. Assays that detect altered expression of a polypeptide include, for example, immunoassays with CARD-specific antibodies, such as immunoblotting, immunofluorescence, immunohistochemistry and immunoprecipitation assays, as described herein.

As will be appreciated by those in the art, assay methods for identifying agents that alter the activity of a CARD-containing polypeptide generally require comparison with a reference. One type of "reference" is a cell or a cell that is treated in substantially the same manner as a test cell or test culture exposed to an agent, except that the "reference" cell or culture is not exposed to the agent. It is a culture. The other type of "reference" cell or culture can be the same cell or culture as the test cell, except that the "reference" cell or culture does not express the CARD-containing polypeptide. Thus, the response of a transfected cell to an agent is compared to the response of a “reference” cell or culture to the same agent under the same reaction conditions, or lack thereof.

Including simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies and the like,
Methods for producing numerous agents for use in screening compounds that alter the activity of CARD-containing polypeptides are known in the art and include, for example, Huse (Hus
e), U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem. Biol.
: 422-428 (1998); Tietze et al., Curr. Biol. 2: 363-371 (1998).
); Sofia, Mol. Divers. 3: 75-94 (1998); Eichler
) Et al., Med. Res. Rev. 15: 481-496 (1995). Libraries containing large numbers of natural and synthetic agents are also available from commercial sources. A combinatorial library of molecules can be created using known combinatorial chemistry methods (Gordon et al., J. Med. Chem. 37: 1233-1251 (1994).
); Gordon et al., J. Med. Chem. 37: 1385-1401 (1994); Gordon (G
Ordon) et al., Acc. Chem. Res. 29: 144-154 (1996); Wilson and Czrarnik, eds., "Combinatorial Chemistry: Synthesis and Application," John. Wiley & Sons (New York (1997)).

The invention further provides a method of diagnosing or predicting the clinical prognosis of a subject characterized by elevated or decreased levels of a CARD-containing polypeptide in a subject. The method is (a
) Obtaining a test sample from a subject; Contacting; and (c) comparing the amount of specific binding in the test sample with the amount of specific binding in the reference sample, wherein the increased amount of specific binding in the test sample compared to the reference sample and Including a stage in which the amount of reduction is a diagnosis of symptoms, or a prediction of clinical prognosis of symptoms. The agent is, for example, an anti-CARD antibody, a CARD-associated polypeptide (
CAP) or CARD-encoding nucleic acid.

Examples of conditions for diagnosis or prediction of clinical prognosis are neoplastic conditions (eg cancer),
Includes any condition described herein, such as autoimmune diseases and other conditions associated with abnormal cell proliferation or abnormal cell death (eg, apoptosis) disclosed herein.

The present invention provides that a test sample from a patient is contacted with a CARD-specific antibody by
Also provided are methods of diagnosing cancer or monitoring cancer therapy. The invention further provides a method of assessing the prognosis (eg, predicting the clinical prognosis) of a cancer patient, comprising contacting a test sample from the patient with a CARD-specific antibody.

The invention further provides methods of diagnosing cancer or monitoring cancer therapy by contacting a test sample from a patient with an oligonucleotide that selectively hybridizes to a CARD-encoding nucleic acid molecule. The invention further provides a method of assessing the prognosis (eg, predicting clinical prognosis) of a cancer patient by contacting a test sample from the patient with an oligonucleotide that selectively hybridizes to a CARD-encoding nucleic acid molecule. .

Using the methods of the invention for diagnosing cancer or monitoring cancer therapy using CARD-specific antibodies or oligonucleotides or nucleic acids that selectively hybridize to CARD-encoding nucleic acid molecules, eg, cancer Patients can be divided into high-risk or low-risk groups for diagnosing or predicting risk of metastasis or failure to respond to treatment. Therefore, the method of the present invention is
For example, it can be advantageously used to diagnose the risk of metastasis in a cancer patient, or the risk of an autoimmune disease in a patient, or as a prognostic indicator of survival or disease progression in a cancer patient or an autoimmune disease patient. Those of skill in the art will recognize that the prognostic indicators of survival of stage I cancer patients may differ from those of stage IV cancer patients. For example, the
The prognosis of patients with stage I cancer may be directed to the potential for continued growth and / or metastasis of cancer, whereas the night wear of patients with stage IV cancer may be directed to the potential efficacy of therapeutic treatments for cancer treatment. Can be done. Therefore, the method of the present invention, which is directed to measuring the level of CARD-containing polypeptide or CARD-encoding nucleic acid, or determining their presence, is advantageously used as a prognostic indicator of the presence or progression of cancer or its return to therapy. Can be used.

The invention further provides methods of introducing a CARD-encoding nucleic acid into cells of a subject, eg, for gene therapy. Viruses are specialized infectious agents that can evade host defense mechanisms and infect and propagate in specific cell types. Viral-based systems offer the advantage of being able to introduce relatively high levels of heterologous nucleic acid into a variety of cells. Suitable viral vectors for introducing the CARD-encoding nucleic acids of the invention into mammalian cells (eg, vascular tissue fragments) are well known in the art. These viral vectors include, for example, herpes simplex virus vectors (eg, Geller et al., Science, 241: 1667-1669 (1988)); vaccinia virus vectors (eg, Piccini et al., Meth. In Enzy.
mology, 153: 545-563 (1987); Cytomegalovirus vector (Mocarski et al., "Viral Vectors", Y. Gluzman and SH Hughes, edited by Cold. Spring Harbor La
boratory, Cold Spring Harbor, NY, 1988, pp.78-84)); Moloney murine leukemia virus vector (Danos et al., Proc.Natl.Acad.Sci.USA, 85: 6).
469 (1980)); adenovirus vectors (eg, Logan et al., Pr.
oc.Natl.Acad.Sci., USA, 81: 3655-3659 (1984); Jones et al., C.
ell, 17: 683-689 (1979); Berkner, Biotechniques, 6: 616.
~ 626 (1988); Cotton et al., Proc. Natl. Acad. Sci. USA, 89: 6094-6.
098 (1992); Graham et al., Meth. Mol. Biol., 7: 109-127 (1991)).
Adeno-associated viral vectors, retroviral vectors (see, eg, US Pat. Nos. 4,405,712 and 4,650,764) and the like. Particularly preferred viral vectors are adenovirus and retroviral vectors.

Suitable retroviral vectors for use herein are, for example, US Pat. No. 5,252,479 and WO 9
2/07573, 90/06997, 89/05345, 92/05266 and 92/14829, and using such retroviral vectors to efficiently target nucleic acids to human cells. The method of introducing it is described. Other retroviral vectors include, for example, the mouse mammary tumor virus vector (eg, Shacklford).
Eford) et al., Proc. Natl. Acad. Sci. USA, 85: 9655-9659 (1988)) and the like.

In particular, the specificity of the viral vector for a particular cell type can be exploited to target the intended cell type. Thus, the choice of viral vector will depend in part on the targeted cell type. For treating neurodegenerative diseases, eg, by increasing the level of CARD-containing polypeptides in the nerve cells affected by the disease, viral cells targeting the nerve cells can be used. Vectors derived from herpes simplex virus are an example of viral vectors that target nerve cells (Battleman et al., J. Neurosci, 13
: 941-951 (1993), incorporated herein by reference). Similarly, for treating diseases or conditions of the hematopoietic system, viral vectors specific for particular blood cells or their progenitor cells can be used. Human immunodeficiency virus-based vectors are an example of such viral vectors (Carroll et al., JC.
ell.Biochem.17E: 241 (1993), incorporated herein by reference).
In addition, viral or other vectors can be engineered to express a CARD-encoding nucleic acid in a tissue-specific manner by incorporating a tissue-specific promoter or enhancer within the vector (Dai et al., Proc.Natl.A
cad.Sci.USA 89: 10892-10895 (1992), incorporated herein by reference).

In the case of gene therapy, the vector containing the CARD-encoding nucleic acid or antisense nucleotide sequence can be administered to the subject in a variety of ways. For example, when using a viral vector, administration can take advantage of the vector's target specificity. In such cases, it is not necessary to administer the vector locally to the disease site. However, topical administration can be a particularly effective method of administering CARD-encoding nucleic acids. Administration can also be by intravenous or subcutaneous injection into the subject. After injection, the viral vector circulates until it recognizes a host cell with the appropriate target specificity for injection. Injection of the viral vector into the spinal fluid can be an effective mode of administration, for example in the treatment of neurodegenerative diseases.

To deliver a CARD-encoding nucleic acid molecule intracellularly in a tissue-specific manner using a tissue-specific ligand or antibody that is non-covalently bound to the nucleic acid molecule by a bridging molecule, the receptor Mediated DNA delivery techniques can also be used (Curiel et al., Hum
.Gene.Ther. 3: 147-154 (1992); Wu and Wu, J. Biol. Chem. 262.
: 4429-4432 (1987), each incorporated herein by reference).
Direct injection of naked nucleic acid or nucleic acid encapsulated in, for example, value-voiced liposomes can also be used for stable gene transfer to non-dividing or dividing cells in vivo (Ulmer et al., Science 259). : 1745-1748 (1993), incorporated herein by reference). Furthermore, CARD-encoding nucleic acid molecules can be transplanted into various tissues using particle irradiation (Williams et al., Proc. Natl. Acad. Sci. USA 88: 2726-2730 (1991), as a reference. Incorporated herein). Such nucleic acid molecules can be attached to the appropriate nucleotide sequences required for transcription and translation.

A particularly useful mode of administration of CARD-encoding nucleic acids is by local direct inoculation at the site of the disease or condition. Topical administration is advantageous because it has no effect of dilution, thereby increasing the likelihood that the majority of target cells will come into contact with the nucleic acid. Therefore, local inoculation is
It is possible to reduce the targeting requirements of other modes of administration, and if desired, vectors can be used that infect all cell types in the inoculated area. If expression in only a specific subset of cells of the inoculated portion is desired, a promoter, enhancer or other vocabulary element specific for the desired subset of cells can be attached to the nucleic acid molecule. Vectors containing such nucleic acid molecules and regulatory elements may be viral vectors, viral genomes, plasmids, phagemids and the like. Transfection vehicles such as liposomes can also be used to introduce non-viral vectors into recipient cells. Such excipients are known in the art.

The present invention also provides therapeutic compositions useful in practicing the therapeutic methods described herein. A therapeutic composition of the invention, such as a pharmaceutical composition, is identified by a CARD-containing polypeptide (or functional fragment thereof) of the invention, a CARD-encoding nucleic acid of the invention, a method described herein, A physiologically compatible carrier is included with an agent that alters CARD activity or expression, or an anti-CARD antibody that is dissolved or dispersed in the composition as an active ingredient, as described herein. In a preferred embodiment, the therapeutic composition is not immunogenic when administered therapeutically to a mammalian or human patient.

As used herein, “pharmaceutically acceptable,” “physiologically compatible,” and grammatical variations thereof refer to compositions, carriers, diluents and reagents,
Used interchangeably, means that a substance can be administered to a mammal without causing unwanted physiological effects.

The preparation of physiological compositions containing dissolved or dispersed active ingredients is known in the art. Such compositions are usually prepared for injection as either liquid solutions or suspensions; however, solid forms suitable for solution in, or suspensions in, liquid prior to use can also be prepared. . The preparation can also be emulsified.

The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the amounts of active ingredient suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, as well as combinations of any two or more thereof. If more desirable, the composition enhances the effectiveness of the active ingredients, such as wetting agents, emulsifying agents, pH buffering agents,
Small amounts of auxiliary substances can be included.

Therapeutic compositions of the present invention can include pharmaceutically acceptable salts of the ingredients.
Pharmaceutically acceptable non-toxic salts include, for example, hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, Includes acid addition salts (formed by free amino groups of the polypeptide) formed by inorganic acids such as malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid.

Salts formed by free carboxyl groups also include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide; and mono-, di- and trialkyl and aryl amines (eg triethylamine, diisopropylamine, methylamine, It can be derived from organic bases such as dimethylamine) and optionally substituted ethanolamines (eg ethanolamine, diethanolamine, etc.).

Physiologically tolerable carriers are well known in the art. Examples of liquid carriers are free of any substance other than the active ingredient and water, or at a physiological pH, physiological salt concentration or both, such as phosphate buffered saline, a buffering agent such as sodium phosphate. It is a sterile aqueous solution containing. Still further, the aqueous carrier can include more than one buffer salt, as well as salts such as sodium and potassium chloride, dextrose, polyethylene glycol and other solutes.

Liquid compositions can also include a liquid phase in addition to and to the exclusion of water. Examples of additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.

As described herein, an “effective amount” refers to a CARD to achieve the desired therapeutic effect, ie, alter the protein binding activity of the CARD-containing polypeptide or the catalytic activity of the CARD-containing polypeptide. A predetermined amount calculated to result in the altered biochemical process regulated by the containing polypeptide. The dose required will depend on the particular treatment and the desired duration of treatment; however, it is expected that doses of between about 10 micrograms to about 1 milligram per kg body weight daily will be used for therapeutic treatment. It is particularly advantageous to administer such agents in depot or sustained form, as discussed herein. A therapeutically effective amount is usually about 0.1 μg / ml to about 100 μg / ml, preferably about 0.1 μg / ml to about 50 μg / ml, more preferably about 2 μg when administered as a physiologically acceptable composition. / ml, and generally 5 μg
An amount of an agent identified herein that is sufficient to achieve a plasma concentration of / ml to 10 μg / ml. Therapeutic anti-CARD antibodies of the invention can be administered in proportionately appropriate amounts according to practices known in the art.

Also provided herein is a method of treating a condition characterized by abnormal cell proliferation, abnormal cell death, or inflammation, the method comprising administering an effective amount of a therapeutic composition of the present invention. . Such compositions are usually administered as physiologically compatible compositions.

Examples of aberrant cell proliferative disorders associated with CARD-containing polypeptides, which are considered herein for treatment according to the invention include cancerous conditions, keratinocyte hyperplasia, neoplasia,
Includes keloids, benign prostatic hypertrophy, inflammatory thickening, fibrosis, smooth muscle cell proliferation (restenosis) in arteries after balloon angioplasty. Examples of cancer conditions considered herein for treatment include glioma, carcinoma, line carcinoma, sarcoma, melanoma, hamartoma,
Includes leukemia and lymphoma. Additional diseases associated with CARD-containing polypeptides that are considered herein for treatment in accordance with the present invention include inflammatory and cytopathic diseases. Such diseases include allergies, inflammatory diseases including arthritis, lupus, Schrogen syndrome, Crohn's disease, ulcerative colitis, as well as allograft rejection such as graft-versus-host disease. . CARD
The contained polypeptides prevent diseases associated with abnormal cell death in neurodegenerative disease conditions such as stroke, myocardial infarction, heart failure, Parkinson's disease and Alzheimer's disease, and for immune deficiency related diseases such as HIV infection and HIV related diseases. It is also useful for strategy design.

Methods of treating a medical condition can include methods of modulating the activity of one or more oncogenic proteins that specifically interact with a CARD-containing polypeptide of the invention. A method of modulating the activity of such an oncogenic protein is described in the following:
Contacting the binding fragment). This contact alters the activity of the oncogenic protein, thereby providing a method of treating the condition caused by the oncogenic protein. A further method of modulating the activity of oncogenic proteins is to
Contacting with an agent that alters the interaction between the CARD-containing polypeptide and the oncogenic protein.

Also provided herein are methods of treatment using the pharmaceutical compositions herein for treating pathological disorders in which there is too little cell division, such as myelodysplasia, immunodeficiency due to decreased lymphocyte counts. Consider. Also contemplated herein are methods of treating various inflammatory disorders with the therapeutic compositions of the invention, such as sepsis, fibrosis (eg, scarring), arthritis, graft-versus-host disease, and the like.

The present invention provides biochemical methods for determining whether increased or decreased levels of biochemical processes are due to, for example, increased or decreased expression of CARD-containing polypeptides, or expression of mutant CARD-containing polypeptides. Also provided is a method of diagnosing a medical condition characterized by increased or decreased levels of physical processes. Such biochemical processes, as disclosed herein, include apoptosis, NF-kB induction, cytokine processing, caspase-mediated proteolysis, transcription, inflammation, cell adhesion and the like. Described herein by identifying such conditions by altering the association of CARD-containing polypeptides with CARD-associated polypeptides in cells, or by altering the ligand binding or catalytic activity of CARD-containing polypeptides. Intervention therapy with other effective agents or nucleic acid molecules or antisense nucleotide sequences can be considered. Generally, the test sample can be obtained from a subject who exhibits symptoms characterized by increased or decreased apoptosis, or suspected of being the cells in the test sample, eg, expressing the CARD-encoding gene. Can be compared to a reference sample by a normal subject to determine whether it exhibits an increase or decrease in Intracellular CA
The level of RD-containing polypeptide can be determined by contacting the sample with a reagent such as an anti-CARD antibody or a CARD-associated polypeptide, both of which are capable of specifically binding to a CARD-containing polypeptide. For example, the level of intracellular CARD-containing polypeptides can be determined by well-known immunoassays or immunohistochemistry using anti-CARD antibodies (eg, Reed et al. Anal. Biochem. 205: 70-76 (1992). )
See also Harlow and Lane, supra (1988)). As used herein, the term "reagent" means a chemical or biological molecule capable of specifically binding to a CARD-containing polypeptide or to a bound CARD / CARD associated polypeptide complex. For example, both anti-CARD antibodies or CARD associated polypeptides can be reagents for CARD-containing polypeptides, while both anti-CARD antibodies or anti-CARD associated polypeptide antibodies can be reagents for CARD / CARD associated polypeptide complexes.

As used herein, the term “test sample” means a cell or tissue sample obtained from a subject and tested for expression of the CARD-encoding gene in cells in the sample. . A test sample can be obtained, for example, during surgery or needle biopsy and can be tested using the methods described herein to diagnose a medical condition characterized by increased or decreased apoptosis. Increased or decreased expression of a CARD-encoding gene in cells in a test sample can be determined, for example, by comparing to expected normal levels of CARD-containing polypeptide or CARD-encoding mRNA in a particular cell type. The normal range of levels for CARD-containing polypeptides or CARD-encoding mRNA in various cell types can be determined by sampling a statistically significant number of normal subjects. In addition, the reference sample can be evaluated in parallel with the test sample to determine if the pathology characterized by increased or decreased apoptosis is due to increased or decreased expression of the CARD encoding gene. The test sample can be tested, for example, using the immunohistochemistry methods described above, or the sample can be further processed and tested. For example, whether the CARD-containing polypeptide in the sample can associate with the CARD-associated polypeptide in the same manner as the CARD-containing polypeptide by the reference cell, or, alternatively, whether the mutant CARD-containing polypeptide is expressed intracellularly. An extract of the test sample can be prepared and tested to determine.

According to another aspect of the present invention, at least one inventive CARD-encoding nucleic acid, CARD-containing polypeptide and / or CARD-containing nucleic acid described herein, in suitable packaging material.
Alternatively, a diagnostic system is provided, preferably in kit form, comprising an anti-CARD antibody.
In one embodiment, for example, the diagnostic nucleic acid is SEQ ID NO: 11,187, 96, 98, 100, 1.
Derived from any of 02, 85 and 89. The diagnostic system of the present invention is useful for assaying for the presence or absence of CARD-encoding nucleic acids in genomic DNA or in transcribed CARD-encoding nucleic acids such as mRNA or cDNA.

A suitable diagnostic system comprises at least one CARD-encoding nucleic acid of the invention, a CARD-containing polypeptide and / or an anti-CARD antibody, preferably two or more nucleic acids, proteins and / or antibodies of the invention, Include as an individually packaged chemical reagent an amount sufficient for at least one assay. Instructions for use of the packaged reagents are usually included. One of skill in the art will readily include the nucleic acid probes and / or primers of the invention in a kit format in combination with appropriate buffers and solutions to perform the methods of the invention as described herein. You can

As used herein, the expression “packaging material” means one or more physical structures used to house the contents of the kit, such as the nucleic acid probes or primers of the invention. To do. The packaging material is preferably constructed using well known methods to provide a sterile, contaminant-free environment. The packaging material detects that the nucleic acid of the invention detects the specific CARD coding sequence comprising the nucleic acid sequence set forth in SEQ ID NOs: 11, 187, 96, 98, 100, 102, 85 and 89 or a mutation or deletion. , Thereby having a label indicating that it can be used for diagnosing the presence or predisposition to conditions such as cancer and autoimmune diseases. In addition, the packaging material will also include instructions on how to use the materials in the kit to detect a particular sequence and to diagnose the presence or predisposition to conditions such as cancer or autoimmune disease.

The packaging materials used herein in connection with diagnostic systems are those customarily utilized in nucleic acid-based diagnostic systems. "Packing," as used herein
The term means a solid matrix or a material such as glass, plastic, paper, foil or the like, within which the isolated nucleic acids, oligonucleotides or primers of the invention can be retained within fixed limits. Thus, for example, the package may be a glass vial used to contain a milligram quantity of the considered nucleic acid, oligonucleotide or primer, or a microgram quantity of the considered nucleic acid probe is operably attached. In some cases, microtiter plate wells are used.

The “instruction for use” usually means the concentration of the reagent, or the relative amount of the reagent and the sample to be mixed, the reagent.
/ Includes specific expressions describing at least one assay method parameter such as retention period of sample mixture, temperature, buffer conditions, etc.

Diagnostic assays should include a simple method for detecting the amount of CARD containing polypeptide or CARD encoding nucleic acid in a sample that binds to a reagent. Detection can be carried out by labeling reagents using known methods and detecting the presence of the label (for labeling of antibodies, see, for example, Harlow and Lane, supra, 1988; See Chapter). Reagents can be labeled with a variety of detectable moieties including radioactive labels, enzymes, biotin or fluorescent dyes. The substance that labels the reagent can be included in the diagnostic kit, or a commercially available product can be purchased separately. After contacting the labeled reagent with a test sample, if desired a control sample, the specifically bound reagent can be identified by detecting a particular moiety.

A labeled antibody capable of specifically binding a reagent can be used to identify the specific binding of an unlabeled reagent. For example, if the reagent is an anti-CARD antibody, a second antibody can be used to detect specific binding of the anti-CARD antibody. The second antibody will generally be specific for a particular class of first antibody. For example, if the anti-CARD antibody is of IgG class, the second antibody is anti-I
It becomes a gG antibody. Such a second antibody is easily available as a commercial product. The second antibody can be labeled with a detectable moiety as described above. When labeling a sample with a second antibody, the sample is first contacted with the first antibody and then the sample is contacted with the labeled second antibody, which specifically binds to the first antibody and can get.

According to another aspect of the invention, there is provided a method of determining the prognosis of disease-free or overall survival of a patient suffering from cancer. For example, herein, an abnormal level (high or low) of a CARD-containing polypeptide in primary tumor tissue is highly correlated with either tumor recurrence or an increase or decrease in spread and is therefore disease-free or total. Consideration is given to the likelihood of survival. Thus, the present invention provides a significant advance in cancer management because early identification of patients at risk of tumor recurrence or spread significantly increases the likelihood of survival and enables aggressive early treatment. Bring Methods of predicting the risk of tumor recurrence or spread in individuals with cancerous tumors; methods of screening cancer patients to determine the risk of tumor metastasis; and appropriate therapeutic course for patients suffering from cancer A method of determining is also provided. These methods collect a sample from a patient and determine the expression level of the patient's CARD-encoding gene, a control expression level or a reference level for determining patient population sampling, tissue culture analysis, or disease prognosis. It is carried out by comparison with reference levels of CARD-encoded gene expression defined by other known methods. And the patient's level of CARD-encoding gene expression is classified as either above or below the reference level, where in the case of high level patients, the prognosis of survival or tumor recurrence is the prognosis of low level patients. Is different from.

All US patents and all publications mentioned herein are incorporated by reference in their entirety. The invention will now be described in further detail by reference to the following non-limiting examples.

[0208] Example 1.0 Identification of CARD-containing polypeptides   The process of gene identification and assembly involves the following steps.

A) Identification of New Candidate CARD-Containing Polypeptides A database search was performed using the TBLASTN program, querying the CARD domains of caspase-1 and caspase-12 in the following NCBI database: Rapidly Determining Genome Sequence (HTGS). ), Genomic search sequence (GSS) and expressed sequence tag (E
ST) Database.

B) Verification that New Candidate CARD-Containing Polypeptides are Novel PSI-BLAST is used to query NCBI's annotated non-redundant (NR) database for each new candidate CARD gene. During this study, if the new candidate gene shows significant, but not identical, homology with other known CARD-containing polypeptides, the CARD-containing polypeptides are retained for further analysis.

C) 3-D model construction of new candidate CARD polypeptides: If the sequence homology is low (<25% identity), apply the three-dimensional criterion for characterization of new CARD polypeptides. Candidate CARD fragments were analyzed by a profile-profile sequence comparison method, which aligned candidate CARD domains with a database of sequences of known three-dimensional structure. From this analysis, a sequence alignment was created and a three-dimensional model was constructed according to the known structure of the CARD domain of IAP-1. In most cases, the highest score was obtained using a CARD domain sequence with a known three-dimensional structure. The quality of the three-dimensional model obtained from the alignment confirmed that a novel CARD domain-containing polypeptide was identified.

D) Identification of additional domains in the full length protein The full length protein sequence was obtained using as query the full length caspase homolog closest to the new CARD identified in step B. A TBLASTN search was performed for sequences containing the newly identified CARD domain. Longer sequence fragments or multiple sequence fragments at the accession numbers corresponding to the newly identified CARD-containing polypeptides were shown to be longer proteins.

E) These additional domains were assembled using the following gene construction procedure: Genomic DNA fragments were constructed from incomplete DNA genomic sequences using mouse caspase-12 and human caspase-1 full length proteins as queries. The HTGS database was scanned from NCBI and identified by T-BLAST-N analysis. New fragments homologous to caspase-12 and caspase-1 were further confirmed by psi-blast analysis, scanning the NR database using the TBLASTN genomic DNA homologue fragment as a query. The boundaries of each fragment were identified by the following criteria: Breaking the sequence similarity between the protein alignment of the target fragment and the query; Extending the protein sequence alignment between the query and the target using the ORF finder; Protein sequence overlap between two contiguous fragments in the sequence for; conservation of exon-intron junction between target DNA sequence and query; orientation of ORFs of different genomic DNA fragments; sequence alignment by query on the same contig Presence of proximal fragments based on.

Finally, the reconstructed sequences were aligned by CLUSTALW using the query and exon-intron junctions, which was further refined by repeating the above steps.

2.0 Identification of CARD2X, CARD3X and CLAN Nucleic acids encoding the CARD-containing proteins CARD2X, CARD3X and CLAN are tblast.
All databases of gss, htgs and NCBI were systematically scanned using n and identified from various CARD queries. Further analysis was carried out using the translated genomic fragment containing the CARD domain, which is larger than the CARD domain itself, as a query to identify other domains. Genomic DNA was translated in all open reading frames and other domains were examined using the psi-blast and nr databases.

Cloning and Sequencing of cDNAs Larger than 3.0 For cDNAs larger than 1500 bp, cloning is done by amplification of multiple fragments of the cDNA. Jurkat total RNA is reverse transcribed into complementary DNA using MMLV reverse transcriptase (Stratagene) and random hexanucleotide primers. Overlapping DNA fragments of a CARD-containing polypeptide are Jurkat complementary by Turbo Pfu DNA polymerase (Stratagene) using one oligonucleotide primer set for every 1500 bp of cDNA.
Amplify from DNA. The cDNA fragment amplified here contains a unique restriction site near the end that binds to an adjacent amplified cDNA fragment.

The resulting cDNA fragment is a mammalian expression vector pcDNA-myc (Invitrogen
, Roy et al., Modified as described in EMBO J. 16: 6914-6925 (1997)), flanking adjacent fragments at unique endonuclease sites forming a full-length cDNA. Assemble full-length cDNA by ligating. Assembled full-length cDNA
Can be performed to identify splice isoforms of CARD-containing polypeptides.

4.0 Construction of Plasmids Complementary DNA encoding a CARD-containing polypeptide, or a functional fragment thereof, was Turbo
Amplify from the Jurkat cDNA using Pfu DNA polymerase (Stratagene) and the desired primers as described above. The resulting PCR fragment contains EcoRI and X
It is digested with a restriction enzyme such as ho and ligated into pGEX-4T1 (Pharmacia) and pcDNA-myc vector.

5.0 In Vitro Protein Binding Assay CARD-containing or fragments thereof encoded in pGEX-4T1 were expressed in XL-1 blue E. coli cells (Stratagene) to determine “current protocols in molecular biology (Cu
rrent Protocols in Molecular Biology ”, eds. Ausubel et al., John Wiley and Sons (1999), and affinity purification using glutathione (GSH) -Sepharose according to known methods. For GST pull-down assay, purified CARD-GST fusion protein and GST alone (10 to 15 μl GSH
-0.1-0.5 μg immobilized on Sepharose beads, 100 μl Co-IP buffer (142.
4mM KCl, 5mM MgCl ₂ , 10mM HEPES (pH7.4), 0.5mM EGTA, 0.2% NP-40, 1mM DT
Incubate for 30 minutes at room temperature in 1 mg / ml BSA in T and 1 mM PMSF). The beads were then loaded with 1 μl of rat reticulocyte lysate (TnT-lysate; Promega) containing ³⁵ S-labeled in vitro translated CARD-containing or control protein Skp-1 in 100 μl Co-IP supplemented with 0.5 mg / ml BSA. , Inc.) overnight at 4 ° C. The beads are washed 4 times in 500 μl Co-IP buffer and then boiled in 20 μl Laemmli-SDS sample buffer. The eluted protein is analyzed by SDS-PAGE. Bands on the SDS-PAGE gel are detected by fluorometry.

The resulting oligomerization pattern reveals that CARD: CARD and other protein: protein interactions are caused by CARD-containing polypeptides or fragments thereof.

In vitro translated candidate CARD associations such as Apaf-1 (lacking its WD domain), CED4, using GSH-Sepharose beads conjugated with GST and GST-CARD containing polypeptides as described above. A GST pull-down assay is performed on the polypeptide and control Skp-1. Lanes containing GST-CARD generate a significant signal when incubated with CARD-associated polypeptide, whereas control GST alone and Skp-1 give negligible signal.

6.0 Protein Interaction Studies in Yeast EGY48 yeast cells (Saccharomyces cerevisiae: MATα, trpl, ura3, his, leu
2 :: plexApo6-leu2) is transformed with the pGilda-CARD plasmid (his marker) encoding the LexA DNA binding domain fused to a CARD-containing polypeptide, fragment thereof, or CARD-associated polypeptide. EGY48 is also transformed with the LexA-LacZ reporter plasmid pSH1840 (ura3 marker) as previously described (Durfree et al., 1993; Sato et al., 1995). Cell and plasmid sources are described in US Pat. No. 5,632,994, Zervous et al., Ce.
ll 72: 223-232 (1993); Gruris et al., Cell 75: 791-803 (1993).
Golemis et al., "Current Protocols in Molecular Biology (Current P
rotocols in Molecular Biology) ”Ausubel et al .; Green Publ
.; NY 1994), each incorporated herein by reference. Transformants were prepared as described previously (Sato et al., Gene 140: 291).
~ 292 (1994)) Burkholder's minimal medium supplemented with leucine and 2% glucose (BM
M) Replica plating on. 2% protein-protein interaction
Assessment is by growth of transformants on leucine-deficient BMM plates containing galactose and 1% raffinose.

Protein-protein interactions are also assessed using the β-galactosidase activity assay. Colonies that grow on BMM / Leu / glucose plates are filter lifted onto nitrocellulose membranes and incubated overnight on BMM / Leu / galactose plates. The filter is immersed in liquid nitrogen and thawed at room temperature to lyse the yeast cells. β-galactosidase activity was determined by 3.2 ml Z buffer (60 mM Na ₂ HPO ₄ , 40 mM Na ₂ HPO ₄ , 10 mM KCl, 1 mM MgSO ₄ ) supplemented with 50 μl X-gal solution (20 mg / ml).
Incubate the filter and measure. The level of β-galactosidase activity is estimated by the intensity of the blue color generated in each transformant.

The results of this experiment show colonies on leucine-deficient plates of yeast containing a CARD / LexA fusion with a CARD-associated polypeptide / B42. Furthermore, CARD / LexA: CARD
The associated polypeptide / B42 cells have a sufficient amount of LacZ activity.

7.0 Self-Association of NB-ARC Domain of CARD-Containing Polypeptides In vitro translated ³⁵ S-labeled rat reticulocyte lysates (1 μl) containing NB-ARC or Skp-1 (used as a control) were subjected to GST pull-down assay. For use, it is incubated with purified GST-NB-ARC or GSH-Sepharose beads coupled with GST alone, resolved by SDS-PAGE and visualized by fluorometry as described above. For NB-ARC or Skp-1 as control, add 1 / 10th of the input.

8.0 Protein-Protein Interactions of CARD-Containing Polypeptides Transient transfections of 293T, a human embryonic kidney fibroblast cell line, are performed using SuperFect reagent (Qiagen) according to the manufacturer's instructions. A cDNA fragment encoding full-length CED4, and Apaf-1 containing amino acids 1-420 of human Apaf-1 protein
Truncated form (Apaf-1ΔWD) was amplified by PCR, and EcoRI and Xho in pcDNA3HA
Subcloning at I site. Expression plasmids encoding catalytically inactive forms of caspases such as pro-Casp8 (pro-Casp8 (C / A) were prepared by substituting Ala for Cys 377 using site-directed mutagenesis and Casp9 (Pro-Casp9 (C / A)) has been previously described, Cardone et al., Science 282: 1318-1321 (1998).
). 293T cells were transfected with a plasmid (2 μg
) Presence or absence of HA-labeled human Apaf-1ΔWD, CED4, pro-Casp8 (C / A
) Or C-terminal Flag-tagged pro-Casp9 (C / A) -encoding expression plasmid (2 μg)
Transfect temporarily by. After growing in culture for 24 hours, the transfected cells were harvested and combined with 12.5 mM β-glycerol phosphate, 2 mM NaF, 1 mM Na ₃ VO ₄ , 1 mM PMSF, and 1 × protease inhibitor mix (Boehringer Mannheim).
) Added Co-IP buffer (142.4 mM KCl, 5 mM MgCl ₂ , 10 mM HEPES (pH 7.4),
Lysis in 5 mM EGTA, 0.1% NP-40 and 1 mM DTT). Cell lysates are clarified by microcentrifugation and subjected to immunoprecipitation with either mouse monoclonal antibody to myc (Santa Cruz Biotechnologies, Inc) or control mouse IgG. Immune-complexed proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and anti-HA antibody followed by anti-myc antibody using standard Western blotting procedures and ECL reagents from Amersham-Pharmacia Biotechnologies, Inc. Immunoblot analysis (Krajewski et al., Proc. Natl. Acad.S.
ci.USA 96: 5752-5757 (1999)).

9.0 Cloning and Characterization of CARD2X The CARD2X encoding cDNA was 0.9k from a human skeletal muscle cDNA library (Clontech).
primers for amplifying bcDNA molecules Was obtained by PCR using. The PCR product was then purified by agarose gel electrophoresis and the purified product was subcloned into the pBluescript II SK vector (Stratagene). ABI PRISM Big Dye Term using forward primer
PCR fragments were sequenced directly using the inal Cycle Sequencing Kit according to the manufacturer's instructions (Perkin Elmer). Array based on the obtained sequence A third CARD2X-specific primer with was generated. This third CARD2X-specific primer is used to identify the 3'end of the CARD2X-encoding cDNA. 0.3 kb cD using the method described above with a phage specific primer
The NA molecule was amplified. The 0.3 kb cDNA molecule was cloned and sequenced as described above, and the sequences of the 0.3 and 0.9 kb cDNA molecules were combined to create the 1.0 kb cDNA sequence.

The sequence of CARD2X was confirmed. Other 5'untranslated sequences identified (including 5'untranslated sequences
The nucleotide sequence of CARD2X, SEQ ID NO: 84). The CARD domain spans amino acids 4-78 of SEQ ID NO: 12.

The association of CARD2X with other CARD-containing proteins was determined. H in 6-well plate
EK293T cells can be labeled with Myc or FLAG labeled CARD2X, CARDIAK or NOD1 (2 μg total DNA).
) Pair combination was used to transfect with SuperFect (Qiagen). Twenty-four hours later, the cells were added to 400 μl of lysis buffer (1 x protease inhibitor mix (Roc
he / Boehringer Mannheim) 20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-4
0 and 1 mM EDTA). The cell lysate was clarified by centrifugation and anti-FLAG M2
Immunoprecipitation was performed using agarose beads conjugated with antibody (Sigma). Immune complexes were washed with buffer (20 mM Tris, pH 7.4, 100 mM NaCl, 0.05% NP-40 and 1 mM ED).
Washed 3 times with (TA) and separated on SDS-PAGE gel. The proteins in the gel were transferred to a nitrocellulose membrane, immunoblotted with anti-Myc antibody, and ECL (Amersham-Pharmacia Biote
ch). Epitope-specific antibodies for myc, FLAG or HA tags were obtained from Santa Cruz Biotech, Roche / Boehringer Mannheim and Sigma. The results of these co-immunoprecipitation assays demonstrated that CARD2X specifically associates with both NOD1 and CARDIAK.

Next, the effect of CARDIAK on the phosphorylation of CARD2X was determined. FLAG-CARDIA
HEK 293T cells that transiently express K were lysed and immunoprecipitated with agarose beads conjugated with anti-FLAG M2 antibody. In vitro phosphorylation was performed in immune complexes with or without purified Myc-CARD-2X as a substrate. 10 μl Kinase buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 6 mM MgCl ₂ , 1 mM MnCl, 1 mM EDTA
1) of [γ- ³² P] ATP in) was added to start the kinase reaction. After 20 minutes at 37 ° C., 10 μl of 2 × SDS sample buffer was added to stop the reaction and subjected to SDS-PAGE and autoradiography. The results of these assays showed that CARD2X is not directly phosphorylated by CARDIAK.

A phosphatase assay was also performed to examine the phosphorylation of CARD2X. HEK 293 cells were treated with FLAG-CARDIAK, a kinase deficient mutant of CARDIAK or
Transfection with a plasmid encoding Myc-CARD-2X with or without FLAG-CARDIAK (K47M). Clear lysate with 20 μl reaction buffer (2
It was diluted 1:20 with 5 mM Tris, pH 8.0, 50 mM NaCl, 5 mM MgCl ₂ ) and selectively treated with 2 units of calf intestinal alkaline phosphatase (Gibco BRL) for 30 minutes at 37 ° C. 7
The reaction was terminated by adding μl of 4 × SDS sample buffer and subjected to SDS-PAGE and immunoblotting. The phosphorylated form of CARD2X migrates more slowly than its CARD2X and is not seen after phosphatase treatment. The results of these assays showed that CARD2X is phosphorylated in vivo in the presence of CARDIAK or kinase-deficient CARDIAK, but not in the absence. Taken together with the in vivo phosphorylation results above, these results indicate that CARDIAK is indirectly involved in CARD2X phosphorylation.

The carboxyl-terminal 30-35 residues of CARD2X contain human Alu family sequences and R
It has homology with hoGAP. Thus, this region can have activities similar to those found in human Alu family sequences and RhoGAP.

10.0 Cloning and Characterization of CLAN To amplify cDNA from a human genomic library, CLAN encoding the cDNA was used as a primer. Was obtained by polymerase chain reaction (PCR). 30 cycles of PCR were performed with Turbo Pfu DNA polymerase (Stratagene) at an annealing temperature of 47 ° C and an extension temperature of 72 ° C. The PCR product was then purified by agarose gel electrophoresis and the purified product was subcloned into the pGEM-T vector (Promega).

Using the CARD amino acid sequence of cIAP-1 as a query by the TBLASTn method, CARD
The HTSG database of human genomic DNA sequence data was searched for regions that could encode This search revealed strong homology with a human genomic clone (accession number: AQ889169) that maps to human chromosome 2p21-22. This locus is not recognized in the human genome database and has not previously been annotated. Early studies identified two genes, encoding CARD domain-containing polypeptides, called CARD4X and CARD5X. For further characterization, C
It has been determined that ARD4X (also known as NAC-X or NAC-4) and CARD5X are actually encoded by the same gene and are therefore referred to as CARD4 / 5X. CARD4 / 5X is then encoded by at least one protein encoded by it, CARD, leucine-rich repeat (LRR) and NACH, as described below.
To include T (NB-ARC) domain, it was called CLAN representing "C ARD, L RR a nd (and) N Acht containing protein".

The CLAN locus is frequently mutated in autosomal dominant hereditary spastic paraplegia AAA
It is in close proximity to the gene encoding the protein, Spastin (on chromosome 2p21-22). The CLAN locus is found on the opposite strand of the SPG4 (SPAST) locus, but without overlapping regions. This result suggests that mutations in the CLAN gene potentially occur in patients with this neurodegenerative disorder.

Using GENESCAN for exon prediction, additional regions potentially encoding the NACHT (NB-ARC) domain and the leucine-rich repeat (LRR) domain are also potential CARs.
The D coding sequence was recognized as 3 ', suggesting the presence of a CED4-like gene.

10.1 Cloning of CLAN cDNAs CLAN-specific primers corresponding to sequences within the putative CARD and NACHT (NB-ARC) regions (determined from genomic DNA sequence data) were used with two universal primers to allow nesting. Follow the manufacturer's protocol by PCR (SMART RACE, Clont
ech), CLAN cDNA was isolated from first strand liver and lung cDNA. 5'RACE primer used for amplification Is. The amplification product was purified from an agarose gel and the TA cloning vector (Pr
omega) and sequenced. 4 open reading frames (
ORF) was estimated and multiple clones of each isoform were sequenced to ensure fidelity of PCR products.

The longest transcript, called CLAN-A, is 3.370 kilobase pairs (kbp) in length (SEQ ID NO:
96) and had a predicted SAM domain as well as an open reading frame encoding a 1024 amino acid protein (SEQ ID NO: 97) containing CARD, NACHT (NB-ARC) and LRR domains. The second transcript, called CLAN-B, is 1.374 kbps (SEQ ID NO: 98) in length and encodes a 359 amino acid protein (SEQ ID NO: 99) containing the same CARD spliced directly into the LRR. Had an open reading frame. The isolated third transcript, CLAN-C, is 0.768 kbp (SEQ ID NO: 102) in length and contains a 156 amino acid protein (SEQ ID NO: Code: 103). Finally, the shortest transcript found, CLAN-D, has a length of 0.578 kbp.
(SEQ ID NO: 100) and contained an open reading frame encoding a 92 amino acid protein (SEQ ID NO: 101) containing only a CARD followed by 9 amino acids.

Comparison of these cDNA sequence data with the genomic DNA sequence data found in the HTSG database suggested that the CLAN gene is composed of 12 exons spanning 41.3 kbp spanning chromosome 2p21-22. (Figure 1A). Six differences were found between the CLAN cDNA sequence and the sequence in the public database. Furthermore, nucleotide regions 1-12 and 3372-3396 do not have equivalent fragments in public databases.

Southern blot analysis was also performed. For Southern blot analysis, 10 μg of restriction endonuclease (EcoRI or PstI) -digested genomic DNA was added per lane to obtain CLAN
CARD domain of was used as a probe for hybridization. The probe is cDNA
CLAN A isoform, which is present in genomic DNA but is not present in
It was derived from nucleotides 276 to 507 and 20 nucleotides further upstream (see Figures 1 and 2). CLAN was found to be a single copy gene.

Utilizes two different transcription start sites (corresponding to the beginning of either exon 1 and 2); however, both are spliced to exon 3 at the beginning of the CARD. Exons 6 and 7 contain additional internal splice donor sites used to generate CLAN-G. FIG. 1B shows patterns of mRNA splicing events predicted to produce CLAN-A, CLAN-B, CLAN-C and CLAN-D transcripts and encoded proteins. All exon / intron splice junctions follow the conserved GT / AG consensus rules.

CLAN is a CARD, as predicted by SMART (EMBL, Heidelberg, Germany)
(Amino acids 1-87 of SEQ ID NO: 97). Ψ-BLAST searches of non-redundant databases using CLAN CARD as a query revealed cIAP1 and 2 (58%), caspase-1 and ICEBERG (50%), Nod1, Nod2 and Card8 (~ 38%) and caspases.
Multiple homologous CARDs were identified, including those from -13, Ced3, caspase-9, Bc110 (CIPER) and CARKIAK / RIP2 (~ 30%).

After CARD, the domain containing the consensus sequence of Walker A and B boxes (
Walker et al., EMBO J. 8: 945-951 (1982), added additional properties of a family of NTPases called the NACHT family (Koonin et al., Trends.Bio).
chem.Sci.25: 2230224 (2000)). CLAN by Ψ-BLAST search
NACHT domain ("NB" in Figure 1, amino acids 161-457 of SEQ ID NO: 97) of NAIPT
60%) of the NACHT domains show the highest similarity to Nod1 (49%) and Nod2
(47%) continues.

The leucine-rich repeat (LRR) domain is found near the C-terminus of the A and B isoforms of the protein. The C-terminal end is composed of four repeating LRRs, each containing a predicted β-sheet and α-helix structure, consistent with the prototypical horseshoe structure of LRR (Kobe). Curr. Opin. Struct. Biol. 5: 4
09-416 (1999). LRR 1 (amino acids 760-791 of SEQ ID NO: 97) exhibits non-Kobe and Deisenhofer (non-K / D) LRRs, whereas LRR 2, 3 and 4 (amino acids 817 of SEQ ID NO: 97, respectively). ~ 848; 845-876 and 934-965) are described by Kobe and De.
Follows the isenhofer (K / D) LRR. LRR2 also shares sequence homology with the archetypal ribonuclease inhibitor type A (RI type A). By ψ-BLAST search, LRR shows 49% sequence identity to placental ribonuclease / angiogenin inhibitor (RAI).

The sequence located between the NACHT (NB-ARC) and the LRR domain is a sterility domain, which is a domain composed of five α-helices originally found in proteins involved in many developmental processes. Partial similarity is shown to the α motif (SAM) (amino acids 642 to 696 of SEQ ID NO: 97). The SAM domain has been shown to function as a protein-protein interaction domain, capable of homo-oligomerizing as well as hetero-oligomerizing with other SAMs (Stapleton et al., Nat. Str.
uct.Biol.6: 44-49 (1999)).

10.2 In Vivo Expression of CLAN Northern blot analysis was performed to determine which of the various splice variants of CLAN are expressed in human adult tissue. Hybridization probes that match all four CLAN isoforms, or the common CARD domain of the NACHT and LRR regions, are radiolabeled by random priming with hexanucleotides (Roche) and α- ³² p-dCTP or are commercially available. Digoxigenin labeled using the kit (Roche), incubated with blots containing human poly (A) ⁺ RNA from different human tissues, washed at high stringency and exposed to X-ray film. Positive signals were detected by autoradiography or by immunoblotting with HRP-conjugated anti-DIG antibody and enhanced chemiluminescence (ECL) Amersham).

Northern blots with CLAN CARD showed CLAN in almost all tissues examined.
The expression of a transcript of about 1.5 kbp corresponding to -B was revealed, and the maximum expression was found in lung and spleen. Northern blot analysis using CLAN-A NACHT and LRR as probes revealed expression of approximately 3.5 kbp mRNA corresponding to CLAN-A mainly in the lung.

To further investigate the tissue-specific pattern of expression of CLAN splicing variants, an RT-PCR assay specific for the A, B, C and D isoforms was devised. Similar to blood cells prepared as follows, a series of cDs derived from various human tissues
NA samples (Clontech) were used. Peripheral blood leukocytes were obtained from heparinized venous blood by Ficoll-Paque (Amersham) density gradient centrifugation. Erythrocytes were obtained from granulocytes by brief incubation with hypotonic lysis buffer. Monocytes were isolated by adhering lymphocytes to plastic dishes. Total RNA was isolated from cells using TRIZOL reagent (BRL),
2 μg was used to generate cDNA by reverse transcription reaction with Superscript II (BRL).

PCR was performed on cDNA samples in an Eppendorf thermal cycler using Taq polymerase (BRL) and the following isoform-specific primer pairs:

By RT-PCR analysis, CLAN-B was found in whole human tissues (brain, heart, kidney, liver, lung, pancreas, placenta, skeletal muscle, colon, ovary, leukocyte, prostate, small intestine, spleen, testis, thymus). It was shown to be present in the B. In contrast, CLAN-A was restricted to lung, colon, brain, prostate, spleen and white blood cells, but not in other tissues. Further analysis of the leukocyte subpopulation showed that CLAN-A isoform expression was predominantly in the monocyte cell fraction, less in granulocytes, and not in lymphocytes at all. . No expression of CLAN-C was found in all normal cells tested, whereas expression in the cell line HEK293T is apparent, which indicates that
It is suggested that such transcription may occur in some situations. CLAN-C transcript
Detected in brain only by RT-PCR.

RT-PCR was also performed on the cell lines. RT-PCR was performed using the same CLAN primers used for RT-PCR in normal tissues as described above. RT-PCR was performed on various tumor-derived cell lines: M2, OVCAR3, HEY, HaCaT, 293T, SKOV-3, Jurkat, BG-1, 69.
7, HL-60, PC3, DU145, MDA-MB-231, MCF-7, MDA-MB-4, HS578T, BT-549 and T
-47D. Beta actin primer was used as a control. In contrast to normal tissues, the cell lines tested had few transcripts for CLAN. Cell line 697, MDA
-Weak expression was seen with MB-231, MVF-7, MDA-MB-4, HS578T and T-47D.

10.3 CLAN Protein Interactions The interactions between CARD of CLAN and known CARD domains were tested in vitro and in vivo.

To test the interaction of CLAN with other molecules, an in vitro binding assay was performed. CLAN was translated in vitro without a label (ie cold). Other cellular proteins were labeled with ³⁵ S-Met in vitro: CLAN, caspase 1,
Caspase 2, Caspase 8, Caspase 9, Caspase 10, Apaf1, Apaf1-CARD,
NACa, NAC-CARD, Bc110, ASC, cIAP1, cIAP2, XIAP, Nod1, Ced4, RAIDD and C
ARDIAK. In vitro translated proteins were individually mixed with unlabeled CLAN and co-immunoprecipitated with antibodies to the epitope tag fused to the CARD5X of either myc or hemagglutinin (HA). CLAN-associated proteins were boiled and eluted in Laemmli denaturing buffer and separated by 12% SDS-PAGE. Radioactive bands were visualized by fluorometric analysis.

Caspase 2 and cIAP1 showed weak binding to CLAN, Nod1 and Cardiak
A stronger bond was seen in. The strongest binding was found with Ced4. Caspase-8 binding is probably due to its stickiness. No meeting was seen between CLAN and itself.

To prepare a suitable expression vector for in vivo interaction studies, the cDNA encoding the CLAN domain was labeled with PFU polymerase and specific primers containing BamHI and HindIII sites. Was amplified using. pcDNA3.1 that places the myc-His ₆ tag at the C-terminus of expressed proteins
The resulting PCR product was bound in (-) / Myc-His ₆ A (Invitorgen). PcDNA3 / HA-CLAN (CARD) was created using a similar strategy. DNA sequencing confirmed the authenticity of all vectors.

The CARD of CLAN was expressed as an epitope-tagged protein in HEK293T cells in co-transfection with various other epitope-tagged CARD-containing proteins, and lysates from these cells were used for co-immunoprecipitation assays. . Briefly, HEK293T cells were seeded in 6-well plates (35 mm wells) and after 24 hours Superfec
Transfected with 0.2 mg to 2 mg plasmid DNA using t (Qiagen). After culturing for 1 day, the cells were collected and isotonic lysis buffer (142.4 mM KCl, 5 mM MgCl ₂ , 1
0 mM HEPES (pH7.4), 0.5 mM EGTA, 0.2% NP-40, 12.5 mM b-glycerophosphate, 2
mM NaF, 1 mM Na ₃ VO ₄ , 1 mM PMSF, and 1x Protease Inhibitor Mix (Roche
)). The lysate was clarified by centrifugation and agarose-conjugated anti-c-my.
Immunoprecipitation with antibody c (Santa Cruz) or non-specific control antibody and protein-G-agarose for 2-24 hours at 4 ° C. Immune complexes were washed 4 times with lysis buffer, boiled in Laemmli buffer and separated by 12% -15% PAGE. Immune complexes were then transferred to PVDF membrane and immunoblotted with anti-C-myc (Santa Cruz), anti-HA (Roche) or anti-flag (Sigma). Membranes were washed, incubated with HRP-conjugated secondary antibody and ECL was used to detect reactive proteins.

Co-immunoprecipitation analysis showed that CARD of CLAN bound readily to full-length pro-caspase-1, but poorly to other CARD-containing caspases, caspase-9. Among other CED-4 family members containing CARD with a nucleotide binding domain, CLAN interacted with CARDs of Nod2 and NAC, but not Apaf-1 or N.
It did not interact with od-1. Finally, the CLAN CARD was found to associate with Bcl-10 but not with another adapter protein, RAIDD.

11.0 Cloning and Characterization of CARD3X Based on the analysis of overlapping genomic contigs GI 8575872 and GI 5001450.
Was predicted to be the encoded amino acid sequences designated SEQ ID NOS: 83 and 107.

To identify a novel domain of CARD3X, the tblastn search of the HTGS database used the sequence of the CARD domain of the polypeptide CARD3X as a query and found two overlapping genomic contigs (GI # 5001450 and 8575872). . This contig is a GenScan server (http: //ccr-081.mit.edu/GEN) for the existence of exons.
SCAN.html) (Burge and Karlin,
J. Mol. Biol. 268: 78-94 (1997)). The predicted protein sequences encoded by the exons were analyzed using PSI-BLAST in comparison to the NCBI nr protein sequence database. The predicted protein sequence encoded by the exon is http: /// bi
Profiles from oinformatics.burnham-inst.org/FFAS_apotosis-A profile comparison server was used to analyze and compare databases of proteins with known three-dimensional structures and apoptosis-related domains (Rychlewski et al., Protein Science). 9: 232-241 (2000)).

CARD3X contains two CARD domains, CARD-A and CARD-B domains (see Figure 3). The NB-ARC domain was also observed (see Figure 3). NB-ARC is CLAN and APAF-1
Similar to both the NB-ARC domain and the NB-ARC domain with multiple plant disease resistance proteins (Aravind et al., Trends Biochem. Sci. 24: 47-53 (1999).
); Young, Curr. Opin. Plant Biol. 4: 285-289 (2000).

The angio-R domain was also identified at amino acids 457-839 of SEQ ID NO: 107. "An
"gio-R" is a region of a polypeptide chain that has substantial similarity (eg, 25%, 30%, 40% sequence identity) to a 514-residue protein "angiotensin II / vasopressin receptor". Is a new domain (described in Ruiz-Opazp et al., Nature Med. 1: 1074-1081 (1995)). "
The "angio-R" domain has not been previously described in any protein.

The cDNA was cloned and sequenced to confirm the predicted sequence.
CARD3X cDNA is available from Origene Technologies, Inc.
) Was cloned using the Rapid-Screen ™ Sequence Placenta cDNA Library Panel by Inc.). Library cDNA is preselected for long clones,
It was unidirectionally cloned into the vector pCMV6-XL4 and sequenced in 96 well format. Five
The initial master plate containing 00,000 cDNA clones was the forward primer (SEQ ID NO: 185) and reverse primer (SEQ ID NO: 186) was used to screen by PCR. The set of 5000 clones that were initially positive by PCR was rescreened with the same set of primers. Plating positive clones on LB / Amp plates,
Single colony PCR was performed again to obtain the desired clone.

Nucleotide Sequence Three separate clones were sequenced, each corresponding to SEQ ID NO: 187. The cDNA sequence is based on CARD3X and N predicted from analysis of genomic exons.
Both the and C-termini were different. SEQ ID NO: 187 is followed by a stop codon,
It encodes a polypeptide of 795 amino acids (SEQ ID NO: 188). The second open reading frame begins after the stop codon and encodes a 180 amino acid polypeptide (SEQ ID NO: 189). SEQ ID NO: 189 contains multiple leucine-rich repeats.

Following identification of two polypeptides encoded by SEQ ID NO: 187, one paper reported the cloning of a cloned gene called Nod2 (Ogura et al., J. Biol. Chem. .276: 4812-4818 (2001)). Announced No
The d2 sequence has another N-terminal amino acid corresponding to SEQ ID NO: 188, in place of the stop codon between the residues encoding SEQ ID NO: 188 and SEQ ID NO: 189, and is associated with multiple other leucines. Other cloning sequences exist that encode rich repeats. The published Nod2 sequence is 1040 amino acids.

It has been proposed that SEQ ID NO: 188 is a splice variant of CARD3X / Nod2 that does not contain the LRR domain. Nod2's LRR has been shown to inhibit the ability of proteins to activate NFkB (Ogura et al., Supra (2001)).
Therefore, SEQ ID NO: 188 is likely to be expressed under physiological conditions that require activation of NFkB.

The human CARD3X cDNA sequence was used as a query for BLAST searches of multiple mouse databases. The genomic sequence SEQ ID NO: 190 was identified. SEQ ID NO: 190
Nucleotides 191 to 614 are homologous to the ANGIO-R coding region of human CARD3X. Nucleotide positions 193 to 612 of SEQ ID NO: 191 are predicted to encode SEQ ID NO: 191, which has high homology to amino acids 214 to 341 (SEQ ID NO: 176) of the ANGIO-R domain of human CARD3X. It was

PCR was then performed on mouse genomic DNA obtained from C57B6 and NIH3T3 cell lines using the following primers: Forward primer: (SEQ ID NO: 194), reverse primer: (SEQ ID NO: 195). The PCR product so obtained was sequenced (SEQ ID NO: 192)
, It was shown that there are multiple nucleotide differences compared to the relevant region of SEQ ID NO: 190. The predicted amino acid sequence encoded by SEQ ID NO: 192 (called SEQ ID NO: 193) had one amino acid difference compared to SEQ ID NO: 191.

Both CARD-A and CARD-B domains were individually cloned into pcDNA3 with an epitope tag, such as myc or HA, as described above, and CA2 as described above.
To test the binding of the RD3X CARD domain to other known CARD domains, CARD
Domain binding is tested by co-immunoprecipitation.

To determine if NB-ARC self-associates in an ATP-dependent method / P-loop mutation, the NB-ARC domain is combined with a yeast two-hybrid vector and two selective epitope tags (eg myc). And Flag). NB-ARC
The P-loop that binds the gamma phosphate of ATP at the domain is mutated to remove the conserved Lys in the consensus P-loop sequence GS / TK, where Lys is generally mutated to Met. The NB-ARC domain is associated with other CED-4-like proteins (eg apaf1,
The binding of nod1, nac) to the NB-domain is also tested.

12.0 Characterization of COP-1 The amino acid sequence of the caspase-1 prodomain is shown in public databases.
The CARD of pro-caspase-1 (SEQ ID NO: 87) was used as a BLASTn search query.
) Was identified as a human EST clone (GenBank Accession No. AA070591) containing an ORF encoding a 97 amino acid protein (SEQ ID NO: 86) predicted to provide 92% sequence identity with A. The predicted protein contains a CARD (residues 1-91) followed by 6 amino acids, followed by a stop codon. The CARD region of COP-1 showed 97% identity to the CARD of pro-caspase-1.

To confirm the predicted sequence, cDNA was amplified and sequenced from various human adult tissues. The sequenced COP-1 cDNA (SEQ ID NO: 85) had the same nucleotide sequence as the original EST.

The start codon that initiates the ORF within the COP-1 cDNA clone is present in favor of translation and is preceded by an in-frame stop codon. The 3'untranslated region contains the TAAA and TATA motifs, which are representative of short-lived mRNAs that are post-translationally regulated, and a candidate polyadenylation signal sequence (AATAAA). Therefore, this protein is substantially
Since it contains only CARD, it is named CARD-only protein (COP-1).

In order to determine the genomic organization of the COP-1 gene, the COP-1 cDNA nucleotide sequence was used to search the High Speed Determination Genome Sequence (HTGS) database to obtain 3 genomic clones containing the COP-1 gene. Identified as a result (GenBank accession number AC
027011, AP001153 and AP002787). Comparison of the COP-1 cDNA and genomic DNA sequences suggests three exon structures, where only the first two amino acids are so that most coding regions (including the entire CARD) are derived from exon 2. Is encoded in exon 1 and only the last 5 residues are encoded in exon 3. Sylintrons separating exons 1, 2 and 3 are 631 bp and 844 bp in length, respectively, and have consensus sinucleotide splice donor (GT) motifs and splice acceptors (GT).
AG) motif included.

COP-1 genomic clones identified in the HTSG database were pro-caspase-4-,
Similar to pro-caspase-5 and ICEBERG, it was mapped to human chromosome 11q22 to which the pro-caspase-1 gene belongs. To address the genomic localization of COPs, pro-caspase-4, pro-caspase-5 and ICEBERG, search the Human Genome Project Working Draft (www.genome.cse.ucsc.edu) to extract telomeres from centromeres. The order of these genes up to was determined to be pro-caspase-4, pro-caspase-5, pro-caspase-1, COP and ICEBERG. This result suggests that COP-1 is an independent gene, probably resulting from replication of other homologous genes within this locus.

14.1 Expression of COP-1 To study expression of COP-1, RNA and ³² P-labels from multiple human adult tissues were studied.
Northern blot analysis was performed using the COP-1 cDNA probe. Blots containing poly A-selected mRNA from various adult tissues (Clontech, Palo Alto, Calif.) Were hybridized with a ³² P-labeled COP-1 cDNA probe. The probe is a 570 bp long cDN containing the 5'untranslated region of the COP, the entire ORF, and the 3'untranslated region.
A was shown. The COP-1 probe (derived from the EST clone corresponding to AA070591 obtained from IMAGE Consortium (Washington Medical College, St. Louis, Mo.)) was excised from the plasmid by restriction digestion with EcoRI and XhoI, gel-purified, and Radiolabeled with a kit of ³² P] dCTP and Ambion (Austin, Tex.). After hybridization, heat-denatured probe was 68 ° C with QuickHyb hybridization solution (Stratagene, La Jolla, CA).
Annealed for 1 hour at 2 ° C, then blotted with 2xSSC, solution containing 0.1% (w / v) SDS (15 min at 25 ° C, 2 times each), followed by 0.1 × SSC, 0.1% (w / v) Washed with a solution containing SDS (twice for 10 minutes at 40 ° C). Bands were visualized by autoradiography.

Hybridizing bands of approximately 0.6 kbp, 1.5 kbp and 2.6 kbp were identified and
The bp band was shown to be the most abundant of these transcripts and probably corresponds to the total splice COP-1 mRNA. Larger 1.5kbp and lesser 2.
The 6 kbp transcript may represent an unspliced precursor. Alternatively, the 2.6 kbp mRNA may represent pro-caspase-1 mRNA resulting from probe cross-hybridization. The 0.6 kbp COP-1 mRNA was most abundant in the spleen, followed by the liver, placenta, and peripheral blood leukocytes (PBL). However, most tissues (heart, muscle, colon, kidney, small intestine and lung) were shown to contain at least some detectable 0.6 kbp COP-1 mRNA.

To confirm Northern blot analysis, mRNA expression in COP-1 human adult tissues was also investigated using RT-PCR and COP specific primers. CDNA samples from multiple human adult tissues (Clontech, Palo Alto, Calif.) Contain one set of COPs
Specific primer (forward primer (SEQ ID NO: 147) and reverse primer (SEQ ID NO: 148)). The resulting PCR product was size-fractionated by electrophoresis in a 1.5% agarose gel and then stained with ethidium bromide for UV photography. In some cases, the bands were excised from the gel, purified and sequenced to verify that the RT-PCR assay amplified the correct product.

RT-PCR analysis showed that COP-1 mRNA was expressed in all tissues analyzed (brain, heart, muscle, colon, spleen, kidney, liver, small intestine, placenta, lung and PBL) except thymus. It was shown that it was done. Parallel RT-PCR analysis of β-actin mRNA served as a control. In general, R
The relative levels of COP-1 mRNA detected by T-PCR were consistent with Northern blot data.

14.2 COP-1 Interactions The pro-domain of pro-caspase-1 is required for dimerization and activation of this zymogen. Since the COP-1 prodomain shares a high degree of amino acid sequence identity with the caspase-1 prodomain, COP-1 has been shown to co-immunoprecipitate in the pro-caspase-1
Tested for the probability of interacting with. COP-1 itself, RIP2, Bcl-10, cIAP1, cIAP
Interactions with multiple other CARD-containing proteins, including 2 and pro-caspase-9, were also tested.

In these experiments, the primers Was used to amplify the entire open reading frame (ORF) of COP-1 by PCR. The COP-1 PCR product was digested with EcoRI / XhoI and ligated into the mammalian expression vectors pcDNA3-Myc, pcDNA-HA and pcDNA3-Flag at the EcoRI / XhoI cloning site. Plasmids encoding wild-type pro-caspase-1, RIP2 and pro-IL-1β are described by Tome et al., Curr. Biol. 8: 885-888 (1998); Nett-Fiordalisi et al. J. Leukoc. Biol. 58: 717-724 (1995);
And Wang et al., J. Biol. Chem. 271: 20580-20587 (1996).

The pro-caspase-1 Cys 285 Ala mutant is a commercially available kit (Stratagene,
La Jolla, California) and primers Was prepared from a wild-type caspase-1 plasmid by site-directed mutagenesis. A truncation mutant of pro-caspase-1, which has a stop codon introduced downstream of CARD, is used as a primer. Was created using.

Human embryo kidney 293T cells were cultured at 37 ° C. in a Dulbecco's modified Eagle medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) under a 5% CO ₂ atmosphere. Cells in log phase are treated with Superfect Transfection Reagent (Qiage
n, Valencia, Calif.) was used to transfect 60 mm diameter dishes containing the expression plasmid (5 μg total DNA). Cells were harvested after 2 days and were treated with 1 mM DTT, 12
Ice-cold NP- supplemented with .5 mM β-glycerophosphate, 1 μM Na ₃ VO ₄ , 1 mM PMSF, and 1x Protease Inhibitor Mix (Roche, Indianapolis, IN)
It was dissolved in 40 lysis buffer (10 mM HEPES [pH 7.4], 142.5 mM KCl, 0.2% NP-40, 5 mM EGTA). Clarify the cell lysate by centrifugation at 16,000 xg for 5 minutes and
Immunization with Myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-Flag antibody (Sigma, St. Louis, MO) in combination with 15 μl of protein A-Sepharose or protein G-Sepharose (Zymed, South San Francisco, CA) Subjected to settling.

The immunocomplex was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS
-PAGE) and fractionated on a nitrocellulose membrane. The resulting blot is treated with anti-H
A antibody (1: 1000v / v; Roche, Indianapolis, Indiana), anti-Myc antibody (1: 100v / v; Santa Cruz Biotechnology, Santa Cruz, CA)
And anti-Flag antibody (1: 1000 v / v; Sigma, St. Louis, Mo.) were incubated with each species antibody, followed by horseradish peroxidase-conjugated secondary antibody, and enhanced chemiluminescence (ECL) method (Amersham-Pharmacia, Piscataway). , NJ
) Detected. Alternatively, after normalization for total protein content, lysates were analyzed directly by immunoblotting.

The co-immunoprecipitation results showed HA-COP-1 co-immunoprecipitation with Myc-COP, indicating that this protein is capable of self-association. Furthermore, HA-COP-1 was co-immunoprecipitated not only by Myc-tagged pro-caspase-1 (C285A mutant) but also by a fragment of pro-caspase-1 containing only its CARD-bearing prodomain. Therefore, COP-1 binds to pro-caspase-1 through its CARD domain. In these co-immunoprecipitation experiments, the active site cysteine of pro-caspase-1 was mutated to avoid the induction of apoptosis that could occur when overexpressing this protease. Furthermore, Myc-COP-1 co-immunoprecipitated with Flag-RIP2. On the other hand, CO
P-1 does not co-immunoprecipitate with CARD-containing proteins Bcl-10, cIAP1, cIAP2 or pro-caspase-9, demonstrating the specificity of these results.

RIP2 binds to and activates caspase-1 through its CARD interaction, resulting in oligomerization of pro-caspase-1 and its activation through an “induced proximity” mechanism. It is shown. Thus, data showing that COP-1 binds both pro-caspase-1 and RIP2 suggested that COP-1 acts as a modifier of RIP2-induced pro-caspase-1 oligomerization.

To test this hypothesis, Myc-tagged pro-caspase-1 (C285A mutant) or HA-tagged pro-caspase with or without Flag-tagged RIP2 and COP.
Experiments were performed to transiently transfect 293T cells with an expression plasmid encoding -1 (C285A mutant), and then Myc-pro-caspase-1 and HA-pro-caspase-1 association by co-immunoprecipitation assay. I monitored.

As determined by this co-immunoprecipitation assay, pro-caspase-1 self-associated, which was increased by co-expression of RIP2. However, when COP-1 was also co-expressed, this RIP-2 mediated effect on pro-caspase-1 self-association was denied. These findings suggest a possible mechanism of competition, in which COP-1 competes with RIP2 for binding to pro-caspase-1. Therefore, to test this hypothesis, Flag-RIP2 and Myc-tagged pro-caspase-1 (C285A mutant) were HA-tagged
Transfection experiments were performed expressing in 293T cells in the presence of increasing amounts of P-1. Next, the effect of COP-1 on the association of RIP2 with pro-caspase-1 was evaluated by co-immunoprecipitation assay. In the immunoprecipitation assay, immunoprecipitation was performed using anti-Flag antibody to recover Flag-RIP2 protein, and the resulting immune complex was analyzed by SDS-PAGE / immunoblotting with anti-Myc antibody and associated. Myc-pro-caspase-1 was detected.

The results from these experiments showed that COP-1 inhibits the association of pro-caspase-1 with RIP2 in a dose-dependent manner. Immunoblot analysis of lysates with these same cells demonstrated that COP-1 did not affect the total level of pro-caspase-1 or RIP2, but rather its association. Therefore, these results confirm that COP-1 can interfere with the binding of pro-caspase-1 to RIP2.

14.3 Inhibition of Caspase-1-Mediated Activation of Pro-IL-1β by COP-1 Active Caspase-1 cleaves pro-IL-1β, resulting in bioactive IL- secreted from cells. 1β was generated. COP-1 suppresses caspase-1-induced pro-IL-1β treatment and thus reduces IL-1β secretion.

To test this hypothesis, various amounts (total DNA = 2.0 μg) of mouse pro-IL-1β
, Human caspase-1, RIP2 or COP-1, lipofectamine plus reagent (G
IBCO BRL, Grand Island, NY), COS-7, 293T or 2
93HEK cells were co-transfected in 12-well (22 mm diameter) plates. One day after transfection, the supernatant is collected and stored at -80 ° C or according to the manufacturer's protocol (R & D systems, Minneapolis, Minnesota), the secretion of mature mouse IL-1β into the medium by an ELISA assay. Used immediately for quantification.

By co-expression of pro-caspase-1 and pro-IL-1β in COS-7 cells, 80 pg / ml ~
Secretion of mature IL-1β in the range of 250 pg / ml resulted, which was proportional to the amount of pro-caspase-1 plasmid used (FIG. 17). This secretion of IL-1β is RIP2
Improved by co-expression of the plasmid. In contrast, COP-1 was replaced with pro-caspase-1
, Pro-IL-1β and RIP2, resulted in a dose-dependent decrease in mature IL-1β secretion, which was proportional to the amount of COP-1-encoding plasmid used (FIG. 6). ). Similar results were obtained with 293T or 293HEK. These results indicate that COP-1 can suppress caspase-1-mediated secretion of IL-1β.

15.0 Identification of COP-2 A human CARD-containing protein called COP-2, which is an abbreviation for CARD-only protein 2, was identified, and the gene and cDNA were cloned. The predicted protein of COP-2 has high similarity to the CARD domain of human caspase-1. In COP-2, caspase-
Two primers based on 15 genomic sequences, one in the middle of the CARD domain And a primer in the catalytic domain Designed. RT-PTR was performed and one band was observed, but the size of the band was smaller than expected for caspase-15. The PCR product was sequenced and found to have two exons deleted and the catalytic domain directly linked to the CARD domain. However, frameshifting resulted in a stop codon immediately following the CARD domain, resulting in a truncated protein and no translation of the catalytic domain.

A primer having a genomic sequence at the most N-terminal of the CARD domain containing ATG for cloning the N-terminal region Designed. RT-PCR was performed and the PCR product was sequenced and found to be identical to genomic DNA. PCR of combined constructs containing both N-terminal fragment and CARD domain sequences
Created by

The identified COP-2 cDNA sequence contains 321 nucleotides (SEQ ID NO: 89) and the deduced amino acid sequence (SEQ ID NO: 90) has a high level of identity with caspase-1. Was there. The alignment of COP-2 (SEQ ID NO: 90) and caspase-1 (SEQ ID NO: 87) is shown in Figure 5, and the consensus sequence (SEQ ID NO: 91) is shown above the sequence compared sequences. The black shaded amino acids are identical. The shaded dots represent matches within 3 distal units. COP-2 is encoded by the caspase-15 gene (Figure 3), but COP-2 is a CARD-only protein lacking the caspase catalytic domain.

Since the COP-2 cDNA with a stop codon downstream encodes a polypeptide, it has no associated catalytic protease domain and results in a shorter protein containing the CARD domain. Therefore, COP-2 is a CARD in pro-enzymes such as pro-caspase-1.
It is expected to function as a transdominant inhibitor, which tends to prevent caspase activation by binding to the domain (pro-domain).

COP-2 polypeptides are expected to function as regulators of caspase-1 activation by enhancing or repressing caspase-1 activation. COP-2
Binding activity can be measured, for example, by COP-2 in order to determine binding interactions with caspase-1.
And make epitope-tagged fusions with Caspase 1 or test by co-immunoprecipitation. Antibodies specific for COP-2 will also be generated.

The effect of COP-2 on caspase-1 proteolytic activity is also tested. Methods for measuring caspase activity are well known (eg Thornberry, Na
ture 356: 768-774 (1992); Thornberry and Molinox (
Molineaux), Protein Science 4: 3-12 (1995); Rano et al., Chem. Biol.
.4: 149-155 (1997); Fletcher et al., J. Interferon Cytokine
Res. 15: 243-248 (1995)) and above.

All journal articles, references, and patent citations above, with or without parentheses, whether previously shown or not, are incorporated herein by reference in their entirety.

Although the present invention has been described with reference to the embodiments set forth above, it should be understood that various modifications can be made without departing from the spirit of the invention.

[Brief description of drawings]

FIG. 1A is on the chromosome 2 (accession number: AL121653) and Homo sapiens chromosome 2 working draft sequence (accession number: NT_005194.1) inferred from BAC 164M19, which is derived from the SPG4 candidate region in 2p21 to 2p22. CLAN (CARD 4
/ 5X) shows the gene composition. B indicates mRNA splicing producing CLAN A, B, C and D. C is the splice-type inference domain structure of CARD4 / 5X (CLAN A, B,
C and D) are shown.

FIG. 2 shows an alignment of protein sequences of isoforms of CLAN (
Designated CLAN A, B, C and D; SEQ ID NOs: 97, 99, 103 and 101, respectively)
. Black boxes indicate identical amino acids and white boxes indicate conserved amino acids.

FIG. 3 Amino acid sequences of CARD-A, CARD-B and NB-ARC domains of CARD3X (
SEQ ID NOs: 170, 172 and 174) are shown respectively.

FIG. 4 shows an alignment of COP-1 (SEQ ID NO: 86) and caspase-1 (SEQ ID NO: 87). The black shaded amino acids are identical.

FIG. 5 shows an alignment of COP-2 (SEQ ID NO: 90) and Caspase-1 (SEQ ID NO: 87), with a consensus sequence (SEQ ID NO: 91) above the aligned sequences.
Indicates. The black shaded amino acids are identical.

FIG. 6 shows the secretion of IL-1β by COS7 cells transfected with the indicated amounts of an expression vector encoding the indicated proteins.

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 48/00 A61P 1/04 4C084 A61P 1/00 7/02 4C085 1/04 9/04 4C086 7/02 9/10 4H045 9/04 103 9/10 13/08 103 17/00 13/08 17/02 17/00 19/02 17/02 19/08 19/02 25/00 19/08 25/16 25 / 00 25/28 25/16 29/00 25/28 31/04 29/00 31/18 31/04 35/00 31/18 37/06 35/00 37/08 37/06 43/00 105 37/08 C07K 14/47 43/00 105 16/18 C07K 14/47 19/00 16/18 C12N 1/15 19/00 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 21 / 08 5/10 C12Q 1/02 C12P 21/02 1/68 A 21/08 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33/53 D G01N 33/15 M 33/50 33 / 566 33/53 C12N 15/00 ZNAA 5/00 A 33/566 A61 37/02 (31) Priority claim number 60 / 275,980 (32) Priority date March 14, 2001 (March 14, 2001) (33) Country of priority claim United States (US) (31) Priority Claim number 09 / 864,921 (32) Priority date May 23, 2001 (23 May 2001) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ , BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, C , CO, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU , SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Gosic Adam San Diego, CA, USA Buckwheat Street 9184 (72) Inventor Sterik Christian United States San Diego Apartments California California Charmant Drive 7535 (72) Inventor Damiano Jason et . United States La Jolla Bonaire Street, California 329 (72) Inventor Lee Soo Hung South Korea Seoul Seo Choo Jam Wong Dong Han Shin Apartment 321-701 (72) Inventor Oliviera Bascoey. M. San Diego Apartment, California 317 Noble Drive 3929 (72) Inventor Hideki Hayashi 4-2-202 Mihara, Nagasaki City, Nagasaki Prefecture (72) Inventor Kurdish Stov Paulowski Sweden Malmo Kramerswagen 10: 1061 F Term (reference) 2G045 AA25 AA40 BB03 BB20 CB01 CB21 DA12 DA13 DA14 DA36 DA37 DA77 FB02 FB03 4B024 AA01 AA11 BA41 BA80 CA04 DA01 DA02 DA05 DA11 EA04 GA01 GA11 HA08 HA11 4B063 QA01 QA18 QQ02 QQ08 QQ21 QQ42 QQ53 QQ79 QR15 QR32 QR42 QR48 QR50 QR55 QR72 QR77 QS32 QS33 QS34 QS36 QX02 4B064 AG01 AG26 AG27 CA02 CA05 CA10 CA11 CA19 CA20 CC24 DA01 DA13 4B065 AA01X AA57X AA87X AA90Y AB01 AB02 BA02 BA08 CA24 CA25 CA44 CA46 4C084 ZAZA ZA ZA 522 MAA MA MA MA 22 MA16 MA22 MA16 MA22 MA16 MA22 MA16 MA22 MA16 MA22 ZA812 ZA892 ZA962 ZB022 ZB082 ZB112 ZB132 ZB262 ZB352 4C085 AA13 AA14 AA16 BB11 BB15 BB16 BB41 BB43 BB44 DD62 DD88 4C086 AA01 AA02 AA03 EA16 MA01 MA04 MA17 MA22 MA23 MA24 MA66 NA14 ZA15 ZA16 ZA22 ZA37 ZA40 ZA66 ZA68 ZA81 ZA89 ZA96 ZB08 ZB11 ZB13 ZB21 ZB26 ZB33 ZB35 4H045 AA10 AA11 AA20 AA30 BA09 BA41 CA40 DA75 DA76 FA74EA20

Claims (30)

[Claims] 1. A CARD-containing polypeptide, or CARD or NB-ARC derived therefrom.
, ANGIO-R, LRR, or an isolated nucleic acid molecule encoding a SAM domain, wherein (a) SEQ ID NO: 12, 168, 188, 170, 172, 174, 176, 97, 99, 101, 103, 178
, 180, 182, 184, 86 and 90, which encodes a polypeptide, and (b) hybridizes to (a) DNA under moderately stringent conditions.
An isolated nucleic acid molecule which is NA and is selected from DNA encoding a bioactive polypeptide. 2. The nucleotide sequence of the nucleic acid molecule is SEQ ID NO: 11, 167, 187, 16
2. The nucleic acid molecule according to claim 1, comprising any of 9, 171, 173, 175, 96, 98, 100, 102, 177, 179, 181, 183, 85 and 89. 3. The nucleic acid molecule according to claim 1, which is a cDNA. 4. A vector containing the nucleic acid molecule according to claim 1. 5. A recombinant cell containing the nucleic acid molecule of claim 1. 6. An isolated oligonucleotide comprising at least 15 contiguous nucleotides of the nucleic acid molecule of claim 2. 7. The oligonucleotide of claim 6, which is labeled with a detectable marker. 8. Comprising at least one of the oligonucleotides of claim 6,
A kit for detecting the presence of a CARD-encoding nucleic acid molecule. 9. SEQ ID NO: 12, 168, 188, 170, 172, 174, 176, 97, 99, 10
An isolated CARD-containing polypeptide, or a CARD derived therefrom, comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of any of 1, 103, 178, 180, 182, 184, 86 and 90. NB-ARC, ANGIO-R, LRR or SAM domain. 10. SEQ ID NOs: 11, 167, 187, 169, 171, 173, 175, 96, 98,
10. A CARD-containing polypeptide according to claim 9 encoded by the nucleotide sequence according to any of 100, 102, 177, 179, 181, 183, 85 and 89. 11. A peptide comprising at least 10 contiguous amino acids of the polypeptide of claim 9. 12. A method of producing a CARD-containing polypeptide, which is in vitro or intracellular under conditions suitable for the expression of said polypeptide.
Including the step of expressing, wherein the cells are bacterial cells, yeast cells, plant cells, animal cells,
A method selected from the group consisting of mammalian cells and insect cells. 13. An isolated anti-CARD antibody having specific reactivity with the CARD-containing polypeptide of claim 9. 14. The antibody according to claim 13, which is a monoclonal antibody. 15. A cell line producing the monoclonal antibody according to claim 14. 16. The antibody according to claim 13, which is a polyclonal antibody. 17. A method of identifying a nucleic acid molecule encoding a CARD-containing polypeptide, which comprises contacting a sample containing nucleic acid with an oligonucleotide according to claim 6 carried out under highly stringent hybridization conditions. And the step of identifying a nucleic acid molecule that hybridizes to it. 18. A method of detecting the presence of a CARD-containing polypeptide in a sample, comprising contacting a test sample with the antibody of claim 13, detecting the presence of an antibody: CARD complex, and Detecting the presence of a human CARD-containing polypeptide in said test sample by. 19. A method of identifying a CARD associated polypeptide (CAP) comprising the steps of: (a) contacting the CARD-containing polypeptide of claim 9 with a candidate CAP; (b) the CARD-containing polypeptide. Detecting the association of the peptide with the CAP. 20. A method of identifying an effective agent that alters the association of a CARD containing polypeptide with a CARD associated polypeptide (CAP), comprising the steps of: (a) claim 9. Contacting the CARD-containing polypeptide with the CAP under conditions capable of associating the CARD-containing polypeptide with the CAP with an agent believed to be capable of altering the association of the CARD-containing polypeptide with the CAP. And (b) detecting altered association of the CARD-containing polypeptide with the CAP, wherein the altered association identifies an effective agent. 21. A method of identifying an agent effective in altering the association of a CARD-containing polypeptide with a CARD associated polypeptide (CAP), the method comprising the steps of: (a) Contacting the described CARD-containing polypeptide and the CAP under conditions capable of associating the CARD-containing polypeptide and the CAP with an agent that is believed to be capable of altering the association of the CARD-containing polypeptide and the CAP. And (b) detecting an altered association of the CARD-containing polypeptide with the CAP, wherein the altered association identifies an effective agent, the CAP comprising:
The step of being the described CARD-containing polypeptide. 22. A method of altering the level of a biochemical process regulated by a CARD containing polypeptide comprising the steps of: (a) introducing the nucleic acid molecule of claim 1 into a cell. And (b) expressing the nucleic acid molecule in the cell, thereby altering the level of a biochemical process in which expression of the nucleic acid is regulated by a CARD-containing polypeptide. 23. Biochemical processes regulated by CARD-containing polypeptides consist of apoptosis, NF-kB induction, cytokine processing, cJun N-terminal kinase induction, caspase-mediated proteolysis, transcription, inflammation, and cell adhesion. 23. The method of claim 22, selected from the group. 24. A live regulated by a CARD-containing polypeptide comprising the step of introducing into the cell an antisense nucleotide sequence that specifically hybridizes to the nucleic acid molecule encoding the CARD-containing polypeptide of claim 11. A method of altering the level of a chemical process whereby hybridization reduces or inhibits expression of said CARD-containing polypeptide in said cell. 25. A method comprising contacting a sample with an agent that effectively changes the association between the CARD-containing polypeptide of claim 9 and a CARD-associated polypeptide.
A method of altering the level of a biochemical process regulated by an RD-containing polypeptide, whereby the level of a biochemical process regulated by a CARD-containing polypeptide is altered. 26. A method of diagnosing or predicting the clinical prognosis of a condition characterized by an increase or decrease in the level of CARD-containing polypeptide in a subject, the method comprising the steps of: (a) the subject Obtaining a test sample from (b) the test sample under an agent capable of binding to the CARD-containing polypeptide according to claim 9, and under the condition that the agent can specifically bind to the CARD-containing polypeptide. Contacting; and (c) comparing the amount of specific binding in the test sample with the amount of specific binding in a reference sample in the test sample compared to the reference sample. Increasing or decreasing the amount of said specific binding diagnoses or predicts the clinical prognosis of the condition. 27. A composition comprising a CARD-containing polypeptide, a functional fragment derived therefrom, and a compound selected from the group consisting of anti-CARD antibodies; and a pharmaceutically acceptable carrier. 28. A method of treating a condition characterized by abnormal cell proliferation, abnormal cell death, or inflammation, comprising administering to an individual an effective amount of the composition of claim 27. 29. A chimeric polypeptide comprising a domain selected from the group consisting of SEQ ID NOs: 168, 170, 172, 174, 176, 178, 180, 182 and 184. 30. A method of identifying an agent effective in modulating the activity of the NB-ARC domain of a CARD-containing polypeptide, comprising: (a) the NB- according to any of SEQ ID NOs: 174 or 180. Contacting a pre-peptide containing the ARC domain with an agent known or believed to modulate the activity of the NB-ARC domain; and (b) measuring the activity of the NB-ARC domain. And thereby increasing or decreasing the activity comprises identifying the agent as an agent that modulates the activity of the NB-ARC domain of the CARD-containing polypeptide, wherein NB-of the CARD-containing polypeptide. A method wherein the activity of the ARC domain is selected from homo-oligomerization, hetero-oligomerization, nucleotide hydrolysis and nucleotide binding.