Cullin-RING ligases (CRLs) represent the largest family of E3 ubiquitin ligases in eukaryotes and they are involved in regulating a wide array of biological processes including development, cell cycle progression, signal transduction, and DNA damage. CRLs are key players in the ubiquitin-proteasome system (UPS) and control protein fate by marking specific substrates for ubiquitination and subsequent proteasomal degradation. Unlike other RING E3 ligases, cullins do not bind substrates directly;instead, CRL specificity relies mainly on substrate receptors that dynamically interact with core CRL complexes through protein-protein interactions. In mammals, there are multiple cullin family members and specific adaptors, as well as a large number of substrate receptors, which can assemble in a modular manner to form over 400 CRLs in vivo. CRLs are estimated to target nearly 20% of proteasomal degradation substrates including many oncogenes and tumor suppressor genes. Given its critical importance in the UPS and cell biology, dis-regulation of CRLs'function can lead to many different diseases including cancer. Therefore, detailed understanding of the CRL biology would not only further our understanding of how they are regulated during cancer development but also provide novel molecular targets for future cancer therapeutics targeting at selective protein degradation. This would be a much more attractive strategy for developing more effective and less toxic cancer treatment in comparison to current drugs targeting the UPS through general proteasome inhibition. Protein-protein interactions are essential for establishing CRLs'diverse assemblies and activities. We hypothesize that mapping the CRL interaction networks in vivo not only would allow us to obtain a complete description of CRL system in living cells but also has important implications for the identification of pharmacological agents that affect particular CRL pathways. To obtain the CRL landscape in living cells, we propose to employ the QTAX strategy to define in vivo protein interactome and dynamics of CRLs, aiming to identify new regulators of the CRL complexes. Here are our specific aims: 1) To define the in vivo interactome of CRL complexes as they occur in living cells;2) To unravel the in vivo interaction dynamics of CRL4 complexes during DNA damage response to elucidate their function and regulation.

Public Health Relevance

Mis-regulation of the Cullin-Ring Ligases can lead to many human diseases including cancer. The CRLs are multi-subunit complexes that are involved in regulating turmorigenesis through modulating protein degradation of oncogenes and tumor suppressor genes. Detailed characterization of the CRL interactomes in vivo and their dynamics in response to DNA damage will allow the identification of potential molecular targets for improved cancer therapeutics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZGM1-CBB-0 (MI))
Program Officer
Gindhart, Joseph G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of California Irvine
Organized Research Units
United States
Zip Code
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
Sement, Fran├žois M; Suematsu, Takuma; Zhang, Liye et al. (2018) Transcription initiation defines kinetoplast RNA boundaries. Proc Natl Acad Sci U S A 115:E10323-E10332
Yu, Clinton; Huang, Lan (2018) Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology. Anal Chem 90:144-165
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
Wang, Xiaorong; Chemmama, Ilan E; Yu, Clinton et al. (2017) The proteasome-interacting Ecm29 protein disassembles the 26S proteasome in response to oxidative stress. J Biol Chem 292:16310-16320
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
Yu, Clinton; Huszagh, Alexander; Viner, Rosa et al. (2016) Developing a Multiplexed Quantitative Cross-Linking Mass Spectrometry Platform for Comparative Structural Analysis of Protein Complexes. Anal Chem 88:10301-10308
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

Showing the most recent 10 out of 17 publications