The ubiquitin system is a major pathway for selective protein degradation (Finley D. et al. (1991) Annu. Rev. Cell Biol. 7: 25-69). Degradation by this system is instrumental in a variety of cellular functions such as DNA repair, cell cycle progression, signal transduction, transcription, and antigen presentation. The ubiquitin pathway also eliminates proteins that are misfolded, misplaced, or that are in other ways abnormal. This pathway requires the covalent attachment of ubiquitin (E1), a highly conserved 76 amino acid protein, to defined lysine residues of substrate proteins.
Substrate recognition by this pathway involves a specialized recognition and targeting apparatus, the ubiquitin-conjugating system. Ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3), either independently or in conjunction, catalyze isopeptide formation between the carboxyl terminus of ubiquitin and amino groups of internal lysine residues of target proteins (Scheffner M. et al. (1995) Nature 373: 81-83). Ubiquitin-protein conjugates are then recognized and degraded by a specific protease complex, the 26S proteasome. Both E2 and E3 exist as protein families, and their pattern of expression is thought to determine substrate specificity (Nuber U. et al. (1996) J. Biol. Chem. 271: 2795-2800). For example, E6 oncoprotein of the cancer-associated human papillomavirus types 16 and 18, inactivates the tumor suppressor protein p53 via the ubiquitin protein degradation pathway. An E3 protein, E6-AP, and an E2 protein, either UbcH5 or UbcH7, complex with E6 and specifically conjugate ubiquitin to p53 (Scheffner M. et al. (1993) Cell 75: 495-505; Nuber et al., supra). Other E2 proteins are not sufficient for p53 ubiquitination, thus UbcH5 and UbcH7 appear to be involved in the specific targeting of p53 for degradation.
The yeast ubiquitin-conjugating enzyme Ubc3 (also known as CDC34) plays a crucial role in the progression of the cell cycle from the G1 to S phase and the correct positioning of ubiquitin on a surface of Ubc3 is a requirement for this cell cycle transition (Prendergast J. A. et al. (1995) J. Biol. Chem. 270: 9347-9352). Mutation studies have suggested that amino acids S-73, S-97, and S-139 of Ubc3 may be critical for substrate specificity, while C-95 is the site of catalytic activity (Liu Y. et al. (1995) Mol. Cell Biol. 15: 5635-5644). An alteration in C-95 and another highly conserved amino acid, L-99, resulted in a dominant negative mutation (Banerjee A. et al. (1995) J. Biol. Chem. 270: 26209-26215). Overexpression of this mutation of Ubc3 was found to block cell growth in otherwise wild type strains.
A decrease in muscle mass, known as muscle wasting or cachexia, has been shown to be associated with the ubiquitin-dependent proteolytic system. Rats bearing the Yoshida AH-130 ascites hepatoma for 7 days showed a significant decrease in muscle mass in relation to non-tumor bearing controls (Llovera M. et al. (1995) Int. J. Cancer 61: 138-141). The muscle wasting was found to be associated with an increased proteolytic rate related to the ubiquitin-dependent proteolytic system. Muscle wasting is common among human cancer patients. In addition to cancer, ubiquitin-dependent muscle wasting is also influenced by nutritional manipulation (such as fasting and dietary protein deficiency), muscle activity and disuse, AIDS, and the pathological conditions, sepsis, trauma, and acidosis (Attaix D. et al. (1994) Reprod. Nutr. Dev. 34: 583-597). In a rat model for long lasting sepsis, researchers found that E2 mRNA levels increase during the acute and chronic disease phases and parallel a rise in muscle protein breakdown (Voisin L. et al. (1996) J. Clin. Invest. 97: 1610-1617).
The presence of ubiquitin and ubiquitin conjugates has been detected in patients affected by neurodegenerative diseases such as Alzheimer's disease. Whereas the intracellular amyloid beta-protein precursor (APP) did not show appreciable ubiquitin-mediated degradation, three extracellular APP forms were degraded by this proteolytic pathway, suggesting a potential regulatory role for the ubiquitin-dependent system in the in vivo APP metabolic pathway (Gregori L. et al. (1994) Biochem. Biophys. Res. Commun. 203: 1731-1738). Paired helical filaments (PHF) are fibrillar structures that accumulate in degenerating neurons in the brains of Alzheimer's disease patients. One component of PHF, the PHF-smear, consists of the tau protein fragment bound to ubiquitin (Morishima M. et al. (1994) Dementia 5: 282-288).
Evidence from experiments on mouse and rabbit reticulocytes indicate that ubiquitin conjugation is a key rate-limiting step in antigen presentation (Grant E.P. et al. (1995) J. Immunol. 155: 3750-3758). The rates of degradation of beta-galactosidase constructs correlated with the rates of class I antigen presentation in vivo. This shows that ubiquitin degradation pathway may have a critical role in generating major histocompatibility complex (MHC) class I-presented peptides.
Depletion of specific cellular proteins may have many medical and agricultural benefits. Redirecting the ubiquitin-dependent proteolytic pathway may facilitate specific proteolytic removal. Gosink M. M. et al. (1995, Proc. Natl. Acad. Sci. 92: 9117-9121) report examples in which E2s target recognition was redefined by engineering E2s to contain appropriate protein-binding peptides fused to their C termini. Thus, it may be possible to design E2s capable of directing the selective removal of many intracellular proteins, such as those implicated in the pathogenesis of Alzheimer's disease.
The ability to selectively modulate E2 activity may provide the means for treating diseases associated with protein degradation, such as Alzheimer's disease, muscle wasting syndrome or a means by which undesired proteins may be targeted for degradation, such as in the case of viral infections and cancer. A new E2 having different specificity could target a unique set of proteins for degradation.
The discovery of polynucleotides encoding ubiquitin-conjugating enzymes, and the enzymes themselves, provides the means to investigate protein degradation pathways. Discovery of molecules related to ubiquitin-conjugating enzyme satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in monitoring or modulating protein degradation in patients with Alzheimer's disease, muscle wasting syndrome, viral infections, or cancer.
USPTO Backgrounds
USPTO Backgrounds