The long term goal of this research is to comprehensively understand the mechanisms and regulation of intracellular protein degradation. Protein degradation regulates nearly every aspect of normal cellular function. Dysfunction of protein degradation underlies many diseases including cancer, muscle wasting, and neuropathologies. Most protein degradation in eukaryotic cells is catalyzed by the 26S proteasome, the ATP dependent protease of the ubiquitin system. The immediate goal of this project is to determine the mechanisms by which ATP binding and hydrolysis mediate proteasome function.
Specific Aim 1 will characterize biochemical features of ATP binding and hydrolysis by the 26S proteasome and define the relative qualitative and quantitative contributions of six different AAA ATPase subunits of the 26S proteasome to these processes. These experiments will provide essential new information about these subunits and support subsequent mechanistic studies on the roles of ATP and the AAA subunits in proteasome function.
Specific Aim 2 will determine molecular mechanisms by which ATP binding to AAA subunits mediates both the assembly of the 26S proteasome from protease (20S proteasome) and regulatory (PA700/19S) sub complexes, and the activation of protease activity upon assembly, using biochemically defined in vitro systems. These experiments will test the hypothesis that proteasome assembly and activation require separate ATP binding events and define roles of individual AAA subunits in these processes. These experiments will distinguish between alternative models in which the six AAA subunits have either distinct/dedicated roles or multiple/redundant roles.
Specific Aim 3 will define the role of ATP hydrolysis in 26S proteasome catalyzed protein degradation. These experiments will compare relative rates of degradation of variants of a model substrate that differ systematically in structural stability and proteasome targeting elements, such as structurally defined polyubiquitin chains. By comparing the qualitative and quantitative requirements for ATP hydrolysis and proteolysis, these experiments will deconvolute the multiple roles of ATP consumption in sub processes of proteolysis such as substrate binding, unfolding, translocation, and deubiquitylation. Completion of these aims will provide detailed information about mechanisms of proteolysis by the 26S proteasome and elucidate the fundamental role of ATP in mediating proteasome function. The long term goal of this research is to comprehensively understand the mechanisms and regulation of intracellular protein degradation. Protein degradation regulates nearly every aspect of normal cellular function. Dysfunction of protein degradation underlies many diseases including cancer, muscle wasting, and neuropathologies. Most protein degradation in eukaryotic cells is catalyzed by the 26S proteasome, the ATP dependent protease of the ubiquitin system. The immediate goal of this project is to determine the mechanisms by which ATP binding and hydrolysis mediate proteasome function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK046181-17
Application #
8271396
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Haft, Carol R
Project Start
1994-07-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
17
Fiscal Year
2012
Total Cost
$315,446
Indirect Cost
$114,525
Name
University of Texas Sw Medical Center Dallas
Department
Physiology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Li, Xiaohua; Thompson, David; Kumar, Brajesh et al. (2014) Molecular and cellular roles of PI31 (PSMF1) protein in regulation of proteasome function. J Biol Chem 289:17392-405
Demartino, George N (2012) Reconstitution of PA700, the 19S regulatory particle, from purified precursor complexes. Methods Mol Biol 832:443-52
Kim, Young-Chan; DeMartino, George N (2011) C termini of proteasomal ATPases play nonequivalent roles in cellular assembly of mammalian 26 S proteasome. J Biol Chem 286:26652-66
Agarwal, Anil K; Xing, Chao; DeMartino, George N et al. (2010) PSMB8 encoding the ýý5i proteasome subunit is mutated in joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy syndrome. Am J Hum Genet 87:866-72
Kumar, Brajesh; Kim, Young-Chan; DeMartino, George N (2010) The C terminus of Rpt3, an ATPase subunit of PA700 (19 S) regulatory complex, is essential for 26 S proteasome assembly but not for activation. J Biol Chem 285:39523-35
Lewis, Karen A; Yaeger, Arynn; DeMartino, George N et al. (2010) Accelerated formation of alpha-synuclein oligomers by concerted action of the 20S proteasome and familial Parkinson mutations. J Bioenerg Biomembr 42:85-95
Li, Xiaohua; Demartino, George N (2009) Variably modulated gating of the 26S proteasome by ATP and polyubiquitin. Biochem J 421:397-404
Ikeda, Yukio; Demartino, George N; Brown, Michael S et al. (2009) Regulated endoplasmic reticulum-associated degradation of a polytopic protein: p97 recruits proteasomes to Insig-1 before extraction from membranes. J Biol Chem 284:34889-900
Thompson, David; Hakala, Kevin; DeMartino, George N (2009) Subcomplexes of PA700, the 19 S regulator of the 26 S proteasome, reveal relative roles of AAA subunits in 26 S proteasome assembly and activation and ATPase activity. J Biol Chem 284:24891-903
Gillette, Thomas G; Kumar, Brajesh; Thompson, David et al. (2008) Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome. J Biol Chem 283:31813-22

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