Many processes in cells are mediated by large protein assemblies or multi-protein complexes that work together in a highly regulated manner to maintain normal homeostasis. Aberrations in multi-subunit protein complexes can lead to various disease states. In order to understand how """"""""protein machines"""""""" function in various cellular processes and to aid future drug development, mapping interaction networks of protein complexes has become one of the major endeavors in modern proteomics research. In order to capture protein interactions of all types in living cells, we have developed the QTAX method to allow effective capture, purification, and quantitative identification of stable, weak and/or transient protein interactions of protein complexes. This strategy allows generating an authentic snapshot of protein interaction networks as they exist in living cells. The ubiquitin-proteasome system (UPS) represents the major pathway for regulated degradation of intracellular proteins in eukaryotes, which helps control numerous essential physiological processes. Aberration of the UPS is known to lead to a variety of human diseases including cancer. The first class of anticancer drugs acts through general inhibition of the UPS and is effective but cannot be used for long-term treatment. This is because general proteasome inhibition will block the entire degradation process and affect many processes nonspecifically. In order to develop more effective and less toxic therapeutics, it is necessary to determine proteasome structural and functional heterogeneity and obtain a better understanding of the molecular mechanisms of UPS regulation and substrate specificity. Toward these goals, this proposal aims to investigate two previously uncharacterized subgroups of human 26S proteasome complexes in mammalian cells and to advance QTAX-based protein interaction study to new levels by defining structural topologies of in vivo protein complexes. The development of these novel strategies will be an exciting technological advancement in proteomics research.
The specific aims i nclude: 1) To unravel structural and functional differences of DNA-bound and non-DNA bound human proteasome complexes using the QTAX strategy;2) To develop the next generation of the QTAX strategy for mass spectrometric characterization of in vivo protein interaction topologies of protein complexes;3) To determine quantitative differences in ubiquitin receptor- associated proteasome complexes by a split-tag strategy and quantitative mass spectrometry.

Public Health Relevance

The ubiquitin-proteasome system affects many important cellular processes and has been implicated in a variety of human diseases including cancer. Comprehensive analysis of the human 26S proteasome to elucidate its structure, function and regulation will allow direct identification of potential molecular targets for improved cancer therapeutics targeting proteasome inhibition.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074830-07
Application #
8309143
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
2005-08-01
Project End
2015-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
7
Fiscal Year
2012
Total Cost
$299,596
Indirect Cost
$99,813
Name
University of California Irvine
Department
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
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Kim, Jin-Kwang; Liu, Jinqiang; Hu, Xichan et al. (2017) Structural Basis for Shelterin Bridge Assembly. Mol Cell 68:698-714.e5
Scott, Harry; Kim, Jin-Kwang; Yu, Clinton et al. (2017) Spatial Organization and Molecular Interactions of the Schizosaccharomyces pombe Ccq1-Tpz1-Poz1 Shelterin Complex. J Mol Biol 429:2863-2872
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Zhang, Liye; Sement, Francois M; Suematsu, Takuma et al. (2017) PPR polyadenylation factor defines mitochondrial mRNA identity and stability in trypanosomes. EMBO J 36:2435-2454
Lv, Yiqing; Meng, Bo; Dong, Hao et al. (2016) Upregulation of GSK3? Contributes to Brain Disorders in Elderly REG?-knockout Mice. Neuropsychopharmacology 41:1340-9
Gutierrez, Craig B; Yu, Clinton; Novitsky, Eric J et al. (2016) Developing an Acidic Residue Reactive and Sulfoxide-Containing MS-Cleavable Homobifunctional Cross-Linker for Probing Protein-Protein Interactions. Anal Chem 88:8315-22
Fang, Lei; Chen, Danqi; Yu, Clinton et al. (2016) Mechanisms Underlying Acrolein-Mediated Inhibition of Chromatin Assembly. Mol Cell Biol 36:2995-3008
Suematsu, Takuma; Zhang, Liye; Aphasizheva, Inna et al. (2016) Antisense Transcripts Delimit Exonucleolytic Activity of the Mitochondrial 3' Processome to Generate Guide RNAs. Mol Cell 61:364-378

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