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.
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.
|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|
|Yu, Clinton; Yang, Yingying; Wang, Xiaorong et al. (2016) Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation. Mol Cell Proteomics 15:2279-92|
|Liu, Jinqiang; Yu, Clinton; Hu, Xichan et al. (2015) Dissecting Fission Yeast Shelterin Interactions via MICro-MS Links Disruption of Shelterin Bridge to Tumorigenesis. Cell Rep 12:2169-80|
|Yu, Clinton; Mao, Haibin; Novitsky, Eric J et al. (2015) Gln40 deamidation blocks structural reconfiguration and activation of SCF ubiquitin ligase complex by Nedd8. Nat Commun 6:10053|
|Kandur, Wynne V; Kao, Athit; Vellucci, Danielle et al. (2015) Design of CID-cleavable protein cross-linkers: identical mass modifications for simpler sequence analysis. Org Biomol Chem 13:9793-807|
|Kaake, Robyn M; Wang, Xiaorong; Burke, Anthony et al. (2014) A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells. Mol Cell Proteomics 13:3533-43|
|Yu, Clinton; Kandur, Wynne; Kao, Athit et al. (2014) Developing new isotope-coded mass spectrometry-cleavable cross-linkers for elucidating protein structures. Anal Chem 86:2099-106|
|Kaake, Robyn M; Kao, Athit; Yu, Clinton et al. (2014) Characterizing the dynamics of proteasome complexes by proteomics approaches. Antioxid Redox Signal 21:2444-56|