Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-04-22
2001-01-30
McKelvey, Terry (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
Reexamination Certificate
active
06180772
ABSTRACT:
BACKGROUND OF THE INVENTION
Nuclear factor &kgr;B (NF-&kgr;B) is a eukaryotic transcription factor that exerts pleiotropic effects on diverse cellular genes involved in the immediate early steps of immune activation and inflammation. Additionally, NF-&kgr;B has been implicated in the transcriptional activation of several viruses, including HIV-1 (for a review, see Siebenlist et al.
Annu. Rev. Cell. Biol.
10:405-455 (1994)).
Nuclear expression and consequent biological action of the eukaryotic NF-&kgr;B transcription factor complex is tightly regulated through its cytoplasmic retention by an ankyrin-rich inhibitory protein termed I&kgr;B&agr;. I&kgr;B&agr; specifically binds to and masks the nuclear localization signal of the Rel A subunit of NF-&kgr;B, thereby effectively sequestering this transcription factor complex in the cytoplasm. Specific cellular activation signals lead to the rapid proteolytic degradation of I&kgr;B&agr; and the concomitant nuclear translocation of NF-&kgr;B. Such signals include, for example, mitogens such as phorbol esters, cytokines such as tumor necrosis factor alpha (TNF-&agr;) and interleukin-1 (IL-1), and the Tax protein from the type I human T cell leukemia virus (HTLV-1). Activation of NF-&kgr;B by these and other inducers appears to involve the transient phosphorylation and subsequent proteolytic degradation of I&kgr;B&agr; which permits nuclear translocation of the liberated NF-&kgr;B complex. Nuclear expression of the NF-&kgr;B complex leads to transcriptional activation of a broad array of cellular genes involved in immune stimulation, inflammation, and cell growth.
Mutant I&kgr;B&agr; molecules have been constructed to investigate the regions in I&kgr;B&agr; essential for signaling and degradation (Brown et al.
Science
267:1485-1487 (1995); Brockman et al.
Mol. Cell. Biol.
15:2809-2818 (1995)).
SUMMARY OF THE INVENTION
A class of dominant negative I&kgr;B&agr; mutants was constructed that retains full inhibitory function on NF-&kgr;B yet fails to undergo stimulus induced degradation. These mutants can be used to inhibit NF-&kgr;B activation in specific target cells, thereby reducing or eliminating undesirable consequences of NF-&kgr;B activation.
One aspect of the invention is a method of inhibiting NF-&kgr;B activation in a cell comprising introducing into the cell nucleic acid encoding a dominant negative mutant I&kgr;B&agr; polypeptide, wherein the I&kgr;B&agr; mutant polypeptide is expressed in the cell.
A further aspect of the invention is a composition comprising an I&kgr;B&agr; polypeptide, wherein residues 1 through 36 and 278-317 are deleted.
REFERENCES:
Hatada et al, The EMBO Journal, vol. 12(7): pp. 2781-2788, 1993.
Ernst et al, Molecular and Cellular Biology, vol. 15(2): pp. 872-882, Jan. 23, 1995.
Brockman, et al., “Coupling of a Signal Response Domain in I&kgr;B-&agr; to Multiple Pathways for NF-&kgr;B Activation”,Mol. Cell. Biol.15: 2809-2818 (1995).
Brown, et al., “Control of I&kgr;B-&agr; Proteolysis by Site-Specific, Signal-Induced Phosphorylation”,Science267: 1485-1487 (1995).
Orkin, et al., “Report and Recommendation of the Panel to Assess the NIH Investment in Research on Gene Therapy”, unnumbered pages, (1995).
Rodriquez, M.S., et al., “Inducible Degradation of I&kgr;B&agr; In Vitro and In Vivo Requires the Acidic C-Terminal Domain of the Protein”,Mol. Cell. Biol.15(5): 2413-2419 (1995).
Siebenlist, et al., “Structure, Regulation and Function of Nf-&kgr;B”,Annu. Rev. Cell. Biol.10: 405-455 (1994).
Traenckner, E.B., et al., “Phosphorylation of human I&kgr;B-&agr; on serines 32 and 36 controls I&kgr;B-&agr; proteolysis and NF-&kgr;B activation in response to diverse stimuli,”EMBO J.14(12): 2876-2883 (1995).
Whiteside, S.T., et al., “N-and C-Terminal Sequences Control Degradation of MAD3/I&kgr;B&agr; in Response to Inducers of NF-&kgr;B Activity,”Mol. Cell. Biol.15(10): 5339-5345 (1995).
Ganchi Parham
Greene Warner C.
Sun Shao-Cong
J. David Gladstone Institutes
McKelvey Terry
Townsend and Townsend / and Crew LLP
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