Molecules of the card-related protein family and uses thereof

Details Molecules of the card-related protein family and uses thereof Molecules of the card-related protein family and uses thereof

2001-05-25

2003-09-02

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S007100, C435S004000, C536S023100, C530S300000, C530S350000

Reexamination Certificate

active

06613521

ABSTRACT:

BACKGROUND OF THE INVENTION
In multicellular organisms, homeostasis is maintained by balancing the rate of cell proliferation against the rate of cell death. Cell proliferation is influenced by numerous growth factors and the expression of proto-oncogenes, which typically encourage progression through the cell cycle. In contrast, numerous events, including the expression of tumor suppressor genes, can lead to an arrest of cellular proliferation.
In differentiated cells, a particular type of cell death called apoptosis occurs when an internal suicide program is activated. This program can be initiated by a variety of external signals as well as signals that are generated within the cell in response to, for example, genetic damage. For many years, the magnitude of apoptotic cell death was not appreciated because the dying cells are quickly eliminated by phagocytes, without an inflammatory response.
The mechanisms that mediate apoptosis have been intensively studied. These mechanisms involve the activation of endogenous proteases, loss of mitochondrial function, and structural changes such as disruption of the cytoskeleton, cell shrinkage, membrane blebbing, and nuclear condensation due to degradation of DNA. The various signals that trigger apoptosis are thought to bring about these events by converging on a common cell death pathway that is regulated by the expression of genes that are highly conserved from worms, such as
C. elegans
, to humans. In fact, invertebrate model systems have been invaluable tools in identifying and characterizing the genes that control apoptosis. Through the study of invertebrates and more evolved animals, numerous genes that are associated with cell death have been identified, but the way in which their products interact to execute the apoptotic program is poorly understood.
Caspases, a class of proteins central to the apoptotic program, are cysteine protease having specificity for aspartate at the substrate cleavage site. These proteases are primarily responsible for the degradation of cellular proteins that lead to the morphological changes seen in cells undergoing apoptosis. For example, one of the caspases identified in humans was previously known as the interleukin-1&bgr; (IL-1&bgr;) converting enzyme (ICE), a cysteine protease responsible for the processing of pro-IL-1&bgr; to the active cytokine. Overexpression of ICE in Rat-1 fibroblasts induces apoptosis (Miura et al., Cell 75:653 [1993]).
Many caspases and proteins that interact with caspases possess domains of about 60 amino acids called a caspase recruitment domain (CARD). Hofmann et al. (TIBS 22:155 [1997]) and others have postulated that certain apoptotic proteins bind to each other via their CARDs and that different subtypes of CARDs may confer binding specificity, regulating the activity of various caspases, for example.
The functional significance of CARDs have been demonstrated in recent publications. Duan et al. (Nature 385:86 [1997]) showed that deleting the CARD at the N-terminus of RAIDD, a newly identified protein involved in apoptosis, abolished the ability of RAIDD to bind to caspases. In addition, Li et al. (Cell 91:479 [1997]) showed that the N-terminal 97 amino acids of apoptotic protease activating factor-1 (Apaf-1) was sufficient to confer caspase-9-binding ability. Inohara et al. (J. Biol. Chem. 273:12296-12300, 1998) showed that Apaf-1 can bind several other caspases such as caspase-4 and caspase-8. Apaf-1 can interact with caspases via CARD-CARD interaction (Li et al., supra, Hu et al., PNAS, 95:4386-4391, 1998).
Nuclear factor-(kappa)B (NF-&kgr;B) is a transcription factor expressed in many cell types and which activates homologous or heterologous genes that have kB sites in their promoters. Quiescent NF-&kgr;B resides in the cytoplasm as a heterodimer between proteins referred to as p50 and p65 and is complexed with the regulatory protein IkB. NF-&kgr;B binding to IkB causes NF-&kgr;B to remain in the cytoplasm. At least two dozen stimuli that activate NF-&kgr;B are known (New England Journal of Medicine 336:1066 [1997]) and they include cytokines, protein kinase C activators, oxidants, viruses, and immune system stimuli. NF-&kgr;B activating stimuli activate specific IkB kinases that phosphorylate IkB leading to its degradation. Once liberated from IkB, NF&kgr;B translocates to the nucleus and activates genes with kB sites in their promoters. How all of these NF-&kgr;B activating stimuli act is unknown at the present time and it is presumed that novel NF-&kgr;B pathway components are involved. NF-&kgr;B and the NF-&kgr;B pathway has been implicated in mediating chronic inflammation in inflammatory diseases such as asthma, ulcerative colitis, rheumatoid arthritis (New England Journal of Medicine 336:1066 [1997]) and inhibiting NF-&kgr;B or NF-&kgr;B pathways may be an effective way of treating these diseases. NF-&kgr;B and the NF-&kgr;B pathway has also been implicated in atherosclerosis (American Journal of Cardiology 76:18C [1995]), especially in mediating fatty streak formation, and inhibiting NF-&kgr;B or NF-&kgr;B pathways may be an effective therapy for atherosclerosis.
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of genes encoding CARD-3 and CARD-4. The CARD-4 gene can express a long transcript that encodes CARD-4L, a short transcript that encodes partial CARD-4S, or two CARD-4 splice variants. A murine full length cDNA sequence for the murine ortholog of CARD-4L is also presented. CARD-3 and CARD-4 are intracellular proteins that are predicted to be involved in regulating caspase activation. CARD-4 is found to activate the NF-KB pathway and to enhance caspase 9-mediate cell death. In addition, proteins that bind to CARD-4 are presented including CARD-3, hNUDC, caspase 9, BCLX, and CARD-4.
The CARD-3 cDNA described below (SEQ ID NO:1) has a 1620 open reading frame (nucleotides 214 to 1833 of SEQ ID NO:1; SEQ ID NO:3) which encodes a 540 amino acid protein (SEQ ID NO:2). CARD-3 contains a kinase domain which extends from amino acid 1 to amino acid 300 of SEQ ID NO:2; SEQ ID NO:4, followed by a linker domain at amino acid 301 to amino acid 431 of SEQ ID NO:2; SEQ ID NO:5 and a CARD at amino acid 432 to amino acid 540 of SEQ ID NO:2; SEQ ID NO:6.
At least four forms of CARD-4 exist in the cell, a long form, CARD-4L, a short form, CARD-4S, and two splice variants, CARD-4Y and CARD-4Z. The cDNA of CARD-4L described below (SEQ ID NO:7) has a 2859 nucleotide open reading frame (nucleotides 35 245-3103 of SEQ ID NO:7; SEQ ID NO:9) which encodes a 953 amino acid protein (SEQ ID NO:8). CARD-4L protein possesses a CARD domain (amino acids 15-114; SEQ ID NO:10). The nucleotide sequence of the full length cDNA corresponding to the murine ortholog of human CARD-4L is presented (SEQ ID NO:42) as is the predicted amino acid sequence of murine CARD-4L (SEQ ID NO:43). A comparison between the predicted amino acid sequences of human CARD-4L and murine CARD-4L is also depicted in
FIGS. 17A-17B
.
Human CARD-4L is also predicted to have a nucleotide binding domain which extends from about amino acid 198 to about amino acid 397 of SEQ ID NO:8; SEQ ID NO:11, a Walker Box “A”, which extends from about amino acid 202 to about amino acid 209 of SEQ ID NO:8; SEQ ID NO:12, a Walker Box “B”, which extends from about amino acid 280 to about amino acid 284, of SEQ ID NO:8; SEQ ID NO:13, a kinase 3a subdomain, which extends from about amino acid 327 to about amino acid 338 of SEQ ID NO:8; SEQ ID NO:14, and ten Leucine-rich repeats which extend from about amino acid 674 to about amino acid 950 of SEQ ID NO:8. The first Leucine-rich repeat extends from about amino acid 674 to about amino acid 701 of SEQ ID NO:8; SEQ ID NO:15. The second Leucine-rich repeat extends from about amino acid 702 to about amino acid 727 of SEQ ID NO:8; SEQ ID NO:16. The third Leucine-rich repeat extends from about amino acid 728 to about amino acid 754 of SEQ ID NO:8; SEQ ID NO:17. The fourth Leuci

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