Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-06-02
2001-12-04
Caputa, Anthony C. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S001001, C536S001110, C536S018700, C536S022100, C536S023100, C536S023500, C530S350000
Reexamination Certificate
active
06326481
ABSTRACT:
BACKGROUND OF THE INVENTION
The human body contains various tissues that continually undergo a process of self-renewal, whereby older cells in the tissue die and are replaced by new cells. In order to maintain a constant number of cells within a particular tissue, it is important that the number of newly produced cells equals the number of cells that die. This homeostasis is maintained by committing differentiated cells to a deliberate and genetically controlled cellular process known as programmed cell death or apoptosis. Apoptotic cells undergo characteristic morphological changes including cell shrinkage, loss of mitochondrial function, and both nuclear condensation and fragmentation. These cellular alterations provide structures suitable for recognition and clearence by proximal phagocytosing cells. Importantly, apoptosis occurs without inducing an inflammatory response and without damage to surrounding cells.
Apoptosis can be induced by a number of unrelated stimuli. However, recent evidence suggests that regardless of the initiating stimulus, apoptosis is signalled through a common pathway. Numerous genes associated with this pathway have been identified, but the way in which their products interact to execute the apoptotic program is still poorly understood.
Defects in the apoptotic pathway can contribute to the onset or progression of various pathological conditions. In humans, the failure of cells to undergo appropriate apoptosis can lead to cancer, autoimmune diseases and viral infection. Conversely, accelerated rates of apoptosis can lead to e.g., neurodegenerative disorders and osteoporosis. Thus, controlling inappropriate cell death or cell survival is important for the treatment of a variety of human diseases.
Amongst the few proteins known to inhibit cell death are certain members of the Bcl-2 family of proteins (Reed,
Nature
387:773-776). Recently, another family of anti-apoptotic proteins, IAP (inhibitor of apoptosis), was identified (Clem and Duckett,
Trends in Cell Biology
7:337-339).
The first IAP gene was identified in baculovirus and since then cellular homologues of IAP have been identified in Drosophila, chickens and humans (Hay et al.,
Cell
, 83, 1253-1262, 1995; Duckett et al.,
EMBO J
., 15:2685-2689, 1996; Liston et al.,
Nature
379:349-353, 1996). The IAP proteins are highly conserved through evolution and characteristically contain two types of sequence motifs/domains (Reed, supra). The C-terminus of an IAP typically contains a RING finger motif. This motif is a type of zinc finger motif, and is thought to be involved in protein-protein interactions. However the exact function of the RING finger motif remains elusive (Saurin et al., Trends Biochem Sci 21:208-214, 1996).
The other common sequence motif common to many of the IAPs is a BIR (baculovirus IAP repeat). BIRs, which are situated at the N-terminus of the IAP, normally comprise 2-3 imperfect repeats of approximately 65 amino acid residues each and contain a number of absolutely conserved residues, including CysX
2
Cys and CysX
6
His motifs (where X is any amino acid). Recent evidence suggests that the BIRS mediate anti-death activity through their involvement in protein-protein interactions (Ambrosini et al., Nature Med 3:917-921, 1997). To date, all IAPs have been found to contain at least one BIR motif. Furthermore, all but two members of the IAP protein family, NAIP (neuronal apoptosis inhibitory protein) and survivin (human IAP homologue), have been found to contain a RING finger motif.
The mechanism of action of an IAP protein is complex. While viral IAP homologues block apoptotic cell death, this is not always the case for the cellular homologues. However, several cellular homologues do possess the ability to block apoptosis. For example, two human IAPs, c-IAP1 and c-IAP2, have been identified as components of the TNF (tumor necrosis factor) receptor signalling complex. These c-IAPs interact with the TRAF-N domain of TRAF1 (TNF receptor associated factor 2) and TRAF2 via the BIRs (Rothe et al.,
Cell
, 83:1243-1252, 1995). While the exact function of this interaction is still unknown, recent reports show that c-IAP2 is involved in protecting cells from TNF-induced cell death by activating NF-&kgr;B (Chu et al.,
PNAS
, 94:10057-10062, 1997). Additionally, c-IAP1 and c-IAP2 inhibit cell death by interfering with specific members of the caspase family of cell death proteases, thereby promoting cell survival (Roy et al.,
EMBO J
, 16:6914-6925, 1997).
SUMMARY OF THE INVENTION
The present invention is based, at least in part, on the discovery of a gene encoding an apoptotic inhibitor protein 6 (AIP-6), an intracellular protein that is predicted to be a member of the IAP superfamily. The AIP-6 cDNA described below (SEQ ID NO:1) has a 1116 nucleotide open reading frame (nucleotides 104-1219 of SEQ ID NO:1; SEQ ID NO:3) which encodes a 372 amino acid protein (SEQ ID NO:2). AIP-6 protein possesses a RING finger domain (amino acids 324-358; SEQ ID NO:4). The AIP-6 protein is unique among other IAP proteins in that it does not contain a BIR motif.
The AIP-6 molecules of the present invention are predicted to play a role in apoptosis. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding AIP-6 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of AIP-6-encoding nucleic acids.
The invention features a nucleic acid molecule which is at least 55%, (65%, 75%, 85%, 95%, or 98%) identical to the nucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence of the cDNA insert of the plasmid deposited with ATCC as Accession Number (the “cDNA of ATCC 209860”), or a complement thereof.
The invention features a nucleic acid molecule which includes a fragment of at least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1300, 1600, 1900 and 2026) nucleotides of the nucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence of the cDNA ATCC 209860, or a complement thereof.
The invention also features a nucleic acid molecule which includes a nucleotide sequence encoding a protein having an amino acid sequence that is at least 45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA of ATCC 209860.
In a preferred embodiment, a AIP-6 nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:1, or SEQ ID NO:3, or the nucleotide sequence of the cDNA of ATCC 209860.
Also within the invention is a nucleic acid molecule which encodes a fragment of a polypeptide having the amino acid sequence of SEQ ID NO:2, the fragment including at least 15 (25, 30, 50, 100, 150, 300, or 372) contiguous amino acids of SEQ ID NO:2 or the polypeptide encoded by the cDNA of ATCC Accession Number 209860.
The invention includes a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or an amino acid sequence encoded by the cDNA of ATCC Accession Number 209860, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or SEQ ID NO:3 under stringent conditions.
Also within the invention is an isolated AIP-6 protein having an amino acid sequence that is at least about 85%, 95%, or 98% identical to the RING finger domain of SEQ ID NO:2 (e.g., about amino acid residues 324 to 358 of SEQ ID NO:2; SEQ ID NO:4).
Also within the invention are: an isolated AIP-6 protein which is encoded by a nucleic acid molecule having a nucleotide sequence that is at least about 65%, preferably 75%, 85%, or 95% identical to SEQ ID NO:3 or the cDNA of ATCC 209860; an isolated AIP-6 protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 65% preferably 75%, 85%, or 95% identical the RING finger domain encoding portion of SEQ ID NO:1 (e.g., about nucleotides 1077 to 1179 of SEQ ID NO:1); and an isolate
Caputa Anthony C.
Fish & Richardson P.C.
Harris Alana M.
Millennium Pharmaceuticals. Inc.
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