One of the major goals in current proteomics research is to elucidate protein functions by globally mapping protein-protein interactions. A key step toward gaining a full understanding of the function of a macromolecular protein complex is to characterize protein complex composition and map their interaction networks. Mass spectrometry-based interaction proteomics has become the method of choice for analyzing functional protein complexes. To capture all protein-protein interactions occurring in intact cells, we will develop a novel integrated mass spectrometry-based proteomics approach to decipher the dynamics of protein interaction networks. The 26S proteasome is a macromolecular machine responsible for ubiquitin/ATP dependent protein degradation in both cytosol and nucleus. Ubiquitin-proteasome-mediated protein degradation is essential in regulating many biological processes including cell division, transcription, cell signaling and development. Disruption of normal ubiquitin-proteasome degradation pathways has been implicated in a wide range of human disease. Despite intensive research, many key questions remain unanswered, especially the mechanisms of how ubiquitinated substrates are recognized by and translocated to 26S proteasome. To address these questions, we hypotheses that multiple ubiquitin receptors or alternative pathways are present and responsible for regulating substrate recognition by and transport to the 26S proteasome for degradation. To test the hypothesis, we intend to apply a novel integrated mass spectrometry-based proteomics approach to decipher proteome-wide proteasome interacting networks and determine the molecular linkage between the proteasome and its substrates.
The specific aims are: 1) to develop and optimize a novel integrated proteomics approach to capture and identify the dynamic proteasome interacting networks using in vivo cross-linking, affinity purification and mass spectrometry analysis; 2) to identify specific proteasome interacting proteins related to different phases of cell cycle and in a checkpoint-induced cell cycle arrest; 3) to identify the protein interaction interface between proteasome subunits and their interacting partners by mapping cross-linked peptide sequences and generate the spatial organization of the proteasome complexes and interaction linkage with their interacting partners to fully elucidate their functions.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
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Edmonds, Charles G
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University of California Irvine
Anatomy/Cell Biology
Schools of Arts and Sciences
United States
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Gutierrez, Craig B; Block, Sarah A; Yu, Clinton et al. (2018) Development of a Novel Sulfoxide-Containing MS-Cleavable Homobifunctional Cysteine-Reactive Cross-Linker for Studying Protein-Protein Interactions. Anal Chem 90:7600-7607
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Wang, Xiaorong; Cimermancic, Peter; Yu, Clinton et al. (2017) Molecular Details Underlying Dynamic Structures and Regulation of the Human 26S Proteasome. Mol Cell Proteomics 16:840-854
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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