Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
1994-03-18
2003-12-09
Navarro, Mark (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C514S001000, C514S002600
Reexamination Certificate
active
06660268
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for reducing the cellular content and activity of NF-&kgr;B by use of inhibitors of proteasome function or ubiquitin conjugation.
2. Description of Related Art
The transcription factor NF-&kgr;B and other members of the rel family of protein complexes play a central role in the regulation of a remarkably diverse set of genes involved in the immune and inflammatory responses (Grilli et al.,
International Review of Cytology
143:1-62 (1993)). For example, NF-&kgr;B is required for the expression of a number of immune response genes, including the Ig-&kgr; light chain immunoglobulin gene, the IL-2 receptor &agr; chain gene, the T cell receptor &bgr; chain gene, and class I and II major histocompatibility genes. In addition, NF-&kgr;B has been shown to be required for a number of genes involved in the inflammatory response, such as the TNF-&agr; gene and the cell adhesion genes, E-selectin, I-cam, and V-cam. NF-&kgr;B is also required for the expression of a large number of cytokine genes such as IL-2, IL-6, G-CSF, and IFN-&bgr;. Finally, NF-&kgr;B is essential for the expression of the human immunodeficiency virus (HIV).
In the cytosol, there is a soluble proteolytic pathway that requires ATP and involves covalent conjugation of the cellular proteins with the small polypeptide ubiquitin (“Ub”) (Hershko et al.,
A. Rev. Biochem
. 61:761-807 (1992); Rechsteiner et al.,
A. Rev. Cell. Biol
. 3:1-30 (1987)). Thereafter, the conjugated proteins are hydrolyzed by a 26S proteolytic complex containing a 20S degradative particle called the proteasome (Goldberg,
Eur. J. Biochem
. 203:9-23 (1992); Goldberg et al.,
Nature
357:375-379 (1992)). This multicomponent system is known to catalyze the selective degradation of highly abnormal proteins and short-lived regulatory proteins. However, the system also appears to be responsible for the breakdown of most proteins in maturing reticulocytes (Boches et al.,
Science
215:978-980 (1982); Spenser et al.,
J. Biol. Chem
. 257:14122-14127 (1985)), in growing fibroblasts (Ciechanover et al.,
Cell
37:57-66 (1984); Gronostajski et al.,
J. Biol. Chem
. 260:3344-3349 (1985)), and in atrophying skeletal muscle.
The first step in degradation of many proteins involves their conjugation to Ub by an ATP-requiring process. The ubiquitinated proteins are then degraded by an ATP-dependent proteolytic complex, referred to above, known as the 26S proteasome complex.
The precise nature of the 26S proteasome complex is unclear, although it has been shown that the 1000-1500 kDa (26S) complex can be formed in extracts of energy-depleted reticulocytes by an ATP-dependent association of three components, referred to as CF-1, CF-2, and CF-3 (Ganoth, D. et al.,
J. Biol. Chem
. 263:12412-12419 (1988)). A large (~700 kDa) multimeric protease found in the cytoplasm and nucleus of eukaryotic cells, referred to as the proteasome, is a component (CF-3) (Driscoll et al.,
J. Biol. Chem
. 265:4789-4792 (1992); Eytan et al.,
Proc. Natl. Acad. Sci. USA
86:7751-7755 (1989); Orlowski,
Biochemistry
29:10289-10297 (1990) and Rivett,
Arch. Biochem. Biophys
. 268:1-8 (1989)).
The proteasome is believed to make up the catalytic core of the large 26S multisubunit cytoplasmic particle necessary for the ubiquitin-dependent pathway of intracellular proteolysis (Driscoll et al.,
J. Biol. Chem
. 265:4789-4692 (1990); Eytan et al.,
Proc. Natl. Acad. Sci. U.S.A
. 86:7751-7755 (1989); Hough et al.,
Biochemistry
262:8303-8313 (1987); McGuire et al.,
Biochim. Biophys. Acta
967:195-203 (1988); Rechsteiner et al.,
A. Rev. Cell. Biol
. 3:1-30 (1987); Waxman et al.,
J. Biol. Chem
. 262:2451-2457 (1987)). By itself, the proteasome is unable to degrade ubiquitinated proteins, but provides most of the proteolytic activity of the 26S proteasome complex.
There is another ATP-dependent protease that is involved in degradation of ubiquitinated proteins, forms a complex with the proteasome, and appears to be part of the 26S proteasome complex, which rapidly degrades proteins conjugated to ubiquitin. This protease, referred to as multipain, has been identified in muscle and plays an essential role in the ATP-ubiquitin-dependent pathway.
The complex formed between multipain and proteasome in vitro appears very similar or identical to the 1500 kDa Ub-conjugate, degrading enzyme, or 26S proteolytic complex, isolated from reticulocytes and muscle. The complexes contain the characteristic 20-30 kDa proteasome subunits, plus a number of larger subunits, including the six large polypeptides found in multipain. The complex formed contains at least 10-12 polypeptides of 40-150 kDa.
A 40 kDa polypeptide regulator of the proteasome, which inhibits the proteasome's proteolytic activities has been purified from reticulocytes and shown to be an ATP-binding protein whose release appears to activate proteolysis. The isolated regulator exists as a 250 kDa multimer and is quite labile (at 42° C.). It can be stabilized by the addition of ATP or a nonhydrolyzable ATP analog, although the purified regulator does not require ATP to inhibit proteasome function and lacks ATPase activity. The regulator has been shown to correspond to an essential component of the 1500 kDa proteolytic complex. The regulator appears identical to CF-2 by many criteria. These findings suggest that the regulator plays a role in the ATP-dependent mechanism of the 26S proteasome complex.
There is also a system in the cytosol that generates antigenic particles from endogenously synthesized cellular and viral proteins (Moore et al.,
Cell
54:777-785 (1988); Morrison et al.,
J. Exp. Med
. 163:903-921 (1986); Powis et al.,
Nature
354:529-531 (1991); Spies et al.,
Nature
351:323-324 (1991); Townsend et al.,
Cell
42:457-467 (1985); Townsend et al.,
Nature
324:575-577 (1986); Monaco et al.,
Proc. Natl. Acad. Sci. U.S.A
. 79:3001-3005 (1982); Monaco,
Immun. Today
13:173-179 (1992); Yewdell et al.,
Adv. Immun
. 52:1-123 (1992); Townsend et al.,
J. Exp. Med
. 168:1211-1224 (1988)). Indirect evidence suggests a role for proteolytic particles closely resembling and perhaps identical to the proteasome (Goldberg et al.,
Nature
357:375-379 (1992); Monaco,
Immun. Today
13:173-179 (1992); Parham,
Nature
348:674-675 (1990); Yang et al.,
Proc. Natl. Acad. Sci. U.S.A
. 89:4928-4932 (1992); (Brown et al.,
Nature
353:355-357 (1991)). It has been shown that the proteasome is responsible for cytoplasmic processing of MHC class I antigen molecules.
The 20S proteasome is composed of about 15 distinct 20-30 kDa subunits. It contains at least three different peptidases that cleave specifically on the carboxyl side of the hydrophobic, basic, and acidic amino acids (Goldberg et al.,
Nature
357:375-379 (1992); Goldberg,
Eur. J. Biochem
. 203:9-23 (1992); Orlowski,
Biochemistry
29:10289-10297 (1990); Rivett et al.,
Archs. Biochem. Biophys
. 218:1 (1989); Rivett et al.,
J. Biol. Chem
. 264:12,215-12,219 (1989); Tanaka et al.,
New Biol
. 4:1-11 (1992)). These peptidases are referred to as the chymotrypsin-like peptidase, the trypsin-like peptidase, and the peptidylglutamyl peptidase. Which subunits are responsible for these activities is unknown, although the cDNA's encoding several subunits have been cloned (Tanaka et al.,
New Biol
. 4:1-11 (1992)).
Recent studies have found that the 20S proteasomes resemble in size and subunit composition the MHC-linked LMP particles (Driscoll et al.,
Cell
68:823 (1992); Goldberg et al.,
Nature
357:375-379 (1992); Matthews et al.,
Proc. Natl. Acad. Sci. U.S.A
. 86:2586 (1989); Monaco et al.,
Human Immunology
15:416 (1986); Parham,
Nature
348:674-675 (1990); Martinez et al.,
Nature
353:664 (1991); Oritz-Navarette et al.,
Nature
353:662 (1991); Glynne et al.,
Nature
353:357 (1991); Kelly et al.,
Nature
353:667 (1991); Monaco et al.,
Proc. Natl. Acad. Sci. U.S.A
. 79:3001 (1982); Brown et al.,
Nature
353:355 (1991); Goldberg,
Eur. J. Biochem
. 203:9-23 (1992); Tanaka et al.,
New B
Goldberg Alfred L.
Maniatis Thomas P.
Palombella Vito J.
Rando Oliver
Hale and Dorr LLP
Navarro Mark
The President and Fellows of Harvard College
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