Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-11-24
2001-10-23
Huff, Sheela (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S387900, C514S012200, C424S138100, C424S139100
Reexamination Certificate
active
06307021
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to nucleic acid and amino acid sequences of a novel human apoptosis regulator protein and to the use of these sequences in the diagnosis, prevention, and treatment of diseases associated with decreased or increased apoptosis.
BACKGROUND OF THE INVENTION
Normal development, growth, and homeostasis in multicellular organisms require a careful balance between the production and destruction of cells in tissues throughout the body. Cell division is a carefully coordinated process with numerous checkpoints and control mechanisms. These mechanisms are designed to regulate DNA replication and to prevent inappropriate or excessive proliferation. In contrast, apoptosis is the genetically controlled process by which unneeded or damaged cells can be eliminated without causing the tissue destruction and inflammatory responses that are often associated with acute injury and necrosis.
The term “apoptosis” was first used by Kerr, J. F. et al. (1972; Br. J. Cancer 26:239-257) to describe the morphological changes that characterize cells undergoing programmed cell death. Apoptotic cells have a shrunken appearance with an altered membrane lipid content and highly condensed nuclei. Apoptotic cells are rapidly phagocytosed by neighboring cells or macrophages without leaking their potentially damaging contents into the surrounding tissue or triggering an inflammatory response.
The processes and mechanisms regulating apoptosis are highly conserved throughout the phylogenetic tree, and much of our current knowledge about apoptosis is derived from studies of the nematode,
Caenorhabditis elegans
and the fruit fly,
Drosophila melanogaster
(cf., Steller, H. (1995) Science 267:1445-1449, and references therein). Dysregulation of apoptosis has recently been recognized as a significant factor in the pathogenesis of human disease. For example, inappropriate cell survival can cause or contribute to many diseases such as cancer, autoimmune diseases, and inflammatory diseases. In contrast, increased apoptosis can cause immunodeficiency diseases such as AIDS, neurodegenerative disorders, and myelodysplastic syndromes (Thompson, C. B. (1995) Science 267:1456-1462).
A variety of ligands and their cellular receptors, enzymes, tumor suppressors, viral gene products, pharmacological agents, and inorganic ions have important positive or negative roles in regulating and implementing the apoptotic destruction of a cell. Although some specific components of the apoptotic pathway have been identified and characterized, many interactions between the proteins involved are undefined, leaving major aspects of the pathway unknown (Steller, H., supra; Thompson, C. B., supra).
The adenovirus E1B 19K gene product and the cellular oncogene Bcl-2 protein have been shown to prevent apoptotic cell death. The E1B 19K protein suppresses apoptosis in cells exposed to agents such as adenovirus, tumor necrosis factor &agr;, ultraviolet radiation, and overexpression of p53. The Bcl-2 protein can substitute for EIB 19K in adenovirus infected cells and provides similar protection against apoptosis due to a variety of stimuli. The mechanism by which this protection occurs is not known, but various reports (Boyd, J. M. (1994) Cell 79:341-351, Farrow, S. N. et al. (1995) Nature 374:731-739, and Sentman, C. L. (1991) Cell 67:879-888) suggest that EIB 19K and Bcl-2 may mediate cell survival by interactions with a certain subset of cellular proteins.
Three human proteins that interact with E1B 19K and Bcl-2 have been isolated using the two-hybrid screen in yeast. This screening system contains three components: a chimeric vector expressing a fusion protein consisting of the yeast GAL4 DNA-binding domain and the E1B 19K protein, a human cDNA expression library tagged with the GAL4 activation domain, and a GAL1 UAS-reporter construct. Upon cotransformation, the binding of proteins from the cDNA library with the E1B 19K protein reconstitutes GAL4 function. GAL4 then binds to the GAL1 UAS and results in transcription of the reporter gene. Using this system, Boyd (supra) isolated Nip1, Nip2, and Nip3 that specifically interact with the E1B 19K protein.
Upon further analysis, these three proteins were shown to associate with sequences in Bcl-2 that are homologous to motifs in E1B 19K. In vitro binding and immunoprecipitation assays demonstrated that the Nip proteins bind to domains in Bcl-2 and E1B 19K that are required for suppression of apoptosis. Immunotocalization studies show that the Nip proteins colocalize with Bcl-2 or E1B 19K at the nuclear envelope of cells. Furthermore, E1B 19K mutants that are defective for suppression of apoptosis are also defective for interaction with the Nip proteins. These results suggest a correlation between interaction of the Nip proteins with the E1B 19K protein and suppression of apoptosis (Boyd, J. M. supra).
The discovery of polynucleotides encoding human apoptosis regulator protein, and the molecules themselves, provides a means to investigate the regulation of programmed cell to death and apoptosis. Discovery of molecules related to human Nip proteins satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the detection, prevention, and treatment of cancer, autoimmune diseases, lymphoproliferative disorders, atherosclerosis, AIDS, immunodeficiency diseases, ischemic injuries, neurodegenerative diseases, osteoporosis, myelodysplastic syndromes, toxin-induced diseases, and viral infections.
SUMMARY OF THE INVENTION
The present invention features a novel human apoptosis regulator hereinafter designated APRG and characterized as having similarity to human Nip3 (GI 558845).
Accordingly, the invention features a substantially purified APRG having the amino acid sequence shown in SEQ ID NO:1.
One aspect of the invention features isolated and substantially purified polynucleotides that encode APRG. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2.
The invention also relates to a polynucleotide sequence comprising the complement of SEQ ID NO:2 or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2.
The invention additionally features nucleic acid sequences encoding polypeptides, digonucleotides, peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and expression vectors and host cells comprising polynucleotides that encode APRG. The present invention also features antibodies which bind specifically to APRG, and pharmaceutical compositions comprising substantially purified APRG. The invention also features the use of agonists and antagonists of APRG.
REFERENCES:
patent: 0 733 706 A2 (1996-09-01), None
Kerr, J.F. et al., “Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.”Br. J. Cancer, 26 (4) :239-257, (1972).
Steller, H., “Mechanisms and Genes of Cellular Suicide”,Sciencem, 267:1445-1450, (1995).
Thompson, C.B., “Apoptosis in the Pathogenesis and Treatment of Disease”,Science, 267:1456-1461, (1995).
Boyd, J.M. et al., “Adenovirus E1B 19 kDa and Bc1-2 Proteins Interact with a Common Set of Cellular Proteins”,Cell, 79:341-351, (1994).
Farrow, S.N. et al., “Cloning of a bcl-2 homologue by interaction with adenovirus E1B 19K.”,Nature, 374:731-739, (1995).
Sentman, C.L. et al., “bc1-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes.”,Cell, 67:879-888, (1991).
Stanger, B.Z. et al., “RIP: a novel protein containing a death domain that interacts with Fas/APO-1 (CD95) in yeast and causes cell death.”,Cell, 81 (4):513-523, (1995).
Bandman Olga
Goli Surya K.
Hillman Jennifer L.
Huff Sheela
Incyte Genomics Inc.
Incyte Genomics, Inc.
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