One important function of intracellular protein degradation is to selectively destroy misfolded or damaged proteins, as accumulate in various human diseases and during aging. Our primary goal is to understand further the functioning of the ubiquitin proteasome pathway and the involvement of molecular chaperons in this process. Most protein breakdown in mammalian cells is catalyzed by the 26S proteasome, whose 19S regulatory particle contains six ATPases which function as molecular chaperones. To understand how these ATPases unfold and translocate substrates into the core 20S proteasome, we are also studying the simpler, homologous ATPase complex, PAN, which activates proteolysis by 20S proteasomes in archaea. We recently discovered that upon ATP-binding, a conserved C-terminal sequence (HbYX motif) in these ATPases docks into pockets in the 20S's outer ring and like a "key-in-a-lock" triggers opening of its gated channel for substrate entry. A major goal will be to elucidate further the molecular events for gate-opening and its structural basis. In the eukaryotic 26S, the six ATPases and seven 20S ?-subunits differ, and gate opening is restricted to two ATPases. We hope to learn which C-termini dock into which 20S pockets and whether other proteasome regulators (BLM10, PI131) and p97/cdc48 that contain the HbYX motif activate 20S proteasomes similarly. We have dissociated proteasomal degradation into a series of ATP-requiring steps, and hope to elucidate further this multistep reaction sequence. We shall explore whether the eukaryotic 26S utilizes additional reaction steps in degrading ubiquitin-conjugated proteins. We've found that the six ATPase subunits interact through negative cooperativity and identified in PAN two substrate-binding domains whose function is nucleotide-dependent. We hope to clarify the functional significance in protein degradation of these cooperative interactions and the associated conformational changes. We recently found that increased temperatures and oxygen radicals in yeast cause the degradation specifically of newly synthesized proteins, without affecting breakdown of most cell proteins. We hope to clarify further the roles of different ubiquitination enzymes and molecular chaperones in their degradation and to identify the newly synthesized proteins most sensitive to such damage. In harsh conditions that damage cell proteins (e.g. heat-shock, oxidative stress, near-freezing temperatures), yeast produce large amounts of the "chemical chaperone", trehalose, which enhances cell viability. We recently found that trehalose is also required for efficient protein degradation, even in normal cells. The mechanisms of this unexpected new protective function of trehalose will be investigated.

Public Health Relevance

A prominent feature of many inherited and neurodegenerative diseases and of the aging process is the accumulation of misfolded proteins, as may result from mutations or free-radical damage. The overall goal of our studies is to clarify the biochemical mechanisms by which such abnormal proteins are selectively degraded by the ubiqutin-proteasome pathway. These studies are focusing on the molecular mechanisms of the proteasome-regulatory ATPases that bind protein substrates and translocate them into the 20S proteasome for degradation and also on the functions of molecular chaperones in this degradative process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051923-16
Application #
8197829
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Wehrle, Janna P
Project Start
1995-08-01
Project End
2012-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
16
Fiscal Year
2012
Total Cost
$545,966
Indirect Cost
$223,862
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Rock, Kenneth L; Farfán-Arribas, Diego J; Colbert, Jeff D et al. (2014) Re-examining class-I presentation and the DRiP hypothesis. Trends Immunol 35:144-52
Gavrish, Ekaterina; Sit, Clarissa S; Cao, Shugeng et al. (2014) Lassomycin, a ribosomally synthesized cyclic peptide, kills mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2. Chem Biol 21:509-18
Besche, Henrike C; Sha, Zhe; Kukushkin, Nikolay V et al. (2014) Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates. EMBO J 33:1159-76
Sha, Zhe; Goldberg, Alfred L (2014) Proteasome-mediated processing of Nrf1 is essential for coordinate induction of all proteasome subunits and p97. Curr Biol 24:1573-83
Qian, Min-Xian; Pang, Ye; Liu, Cui Hua et al. (2013) Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis. Cell 153:1012-24
Nathan, James A; Kim, Hyoung Tae; Ting, Lily et al. (2013) Why do cellular proteins linked to K63-polyubiquitin chains not associate with proteasomes? EMBO J 32:552-65
Peth, Andreas; Nathan, James A; Goldberg, Alfred L (2013) The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome. J Biol Chem 288:29215-22
Peth, Andreas; Kukushkin, Nikolay; Bosse, Marc et al. (2013) Ubiquitinated proteins activate the proteasomal ATPases by binding to Usp14 or Uch37 homologs. J Biol Chem 288:7781-90
Nathan, James A; Spinnenhirn, Valentina; Schmidtke, Gunter et al. (2013) Immuno- and constitutive proteasomes do not differ in their abilities to degrade ubiquitinated proteins. Cell 152:1184-94
Raju, Ravikiran M; Unnikrishnan, Meera; Rubin, Daniel H F et al. (2012) Mycobacterium tuberculosis ClpP1 and ClpP2 function together in protein degradation and are required for viability in vitro and during infection. PLoS Pathog 8:e1002511

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