We previously identified an ensemble of proteasome components which had been largely unrecognized for years because they are released from proteasomes by the high salt used in conventional purifications. We now propose to focus more narrowlyon two such factors: Hul5, a ubiquitin-protein ligase, and Ubp6, a deubiquitinating enzyme. We have found that Ubp6 has a second, highly unexpected function: noncatalytic inhibition of protein degradation. Moreover,when the proteasome is inhibited by UbpG, the other deubiquitinating enzyme of the proteasome, Rpn11, is suppressed. Since Ubp6 trims ubiquitin chains progressively, while Rpn11 removes chains en bloc, the presence of Ubp6 imposes a switch in the modeof substrate deubiquitination. Interestingly, Ubp6 was also found to be regulated through a novel ubiquitin stress response. We found that Hul5 serves to extend the chains of proteasome-bound ubiquitin conjugates, acting in particular on degradative substrates such as cyclin B. Accordingly, hu!5 mutants show stabilization of many substrates. Hul5 binding to proteasomes is enhanced by UbpG, and functional opposition between them is suggested by suppression of ubp6 by the hu!5 null mutation. Moreover,products of Hul5 conjugation can be rapidly disassembled by proteasome-bound Ubp6. We propose that, by regulating the lengths of proteasome-bound ubiquitin chains, the balance of Hul5 and Ubp6 activity regulates substrate commitment to degradation. The degradation delay caused by Ubp6 might serve to ensure that the deubiquitinating activity of Ubp6 and the ubiquitin-ligating activity of Hul5 have adequate time to refashion substrate-bound ubiquitin chains. We will more fully characterize noncatalytic proteasome inhibition by Ubp6 with the benefit of a quantitative degradation assay, and determine the mechanism of inhibition. We recentlyfound that Hul5 also has a prominent noncatalytic influence on proteasomefunction, and this will be further characterized. We will also test the following hypotheses: that UbpG's activity of deubiquitination also mediates proteasome inhibition; and that there is a functional opposition between Hul5 and Ubp6, resulting in ubiquitin chains being in a dynamic state when bound to the proteasome. The proteasome is a key cellular regulator and is important for human health, as proteasome inhibitors are in clinical use in the treatment of a growing list of cancers. Thus, by providing a deeper understanding of this enzyme, our studies may facilitate the development of other useful drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM065592-05A1S1
Application #
7494185
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Ikeda, Richard A
Project Start
2003-03-01
Project End
2011-06-30
Budget Start
2007-09-01
Budget End
2008-06-30
Support Year
5
Fiscal Year
2007
Total Cost
$133,791
Indirect Cost
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Finley, Daniel; Chen, Xiang; Walters, Kylie J (2016) Gates, Channels, and Switches: Elements of the Proteasome Machine. Trends Biochem Sci 41:77-93
Schmidt, Marion; Finley, Daniel (2014) Regulation of proteasome activity in health and disease. Biochim Biophys Acta 1843:13-25
Tar, Krisztina; Dange, Thomas; Yang, Ciyu et al. (2014) Proteasomes associated with the Blm10 activator protein antagonize mitochondrial fission through degradation of the fission protein Dnm1. J Biol Chem 289:12145-56
Chen, Ping-Chung; Bhattacharyya, Bula J; Hanna, John et al. (2011) Ubiquitin homeostasis is critical for synaptic development and function. J Neurosci 31:17505-13
Lee, Min Jae; Lee, Byung-Hoon; Hanna, John et al. (2011) Trimming of ubiquitin chains by proteasome-associated deubiquitinating enzymes. Mol Cell Proteomics 10:R110.003871
Park, Soyeon; Tian, Geng; Roelofs, Jeroen et al. (2010) Assembly manual for the proteasome regulatory particle: the first draft. Biochem Soc Trans 38:6-13
Isasa, Marta; Katz, Elijah J; Kim, Woong et al. (2010) Monoubiquitination of RPN10 regulates substrate recruitment to the proteasome. Mol Cell 38:733-45
Lee, Byung-Hoon; Lee, Min Jae; Park, Soyeon et al. (2010) Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467:179-84
Finley, Daniel (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78:477-513
Kleijnen, Maurits F; Kirkpatrick, Donald S; Gygi, Steven P (2007) The ubiquitin-proteasome system regulates membrane fusion of yeast vacuoles. EMBO J 26:275-87

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