The Cullin-RING ubiquitin E3 ligase (CRLs) super-family is responsible for much of the signal-dependent protein turnover in eukaryotes. CRL specificity derives from the identity of the substrate adaptor, which often interacts with the substrate in a modification-dependent manner. With more than 200 distinct CRL substrate adaptor proteins for the 7 human cullins, the CRL system controls many facets of biology that impinge on disease and aging. However, our understanding of CRL-substrate relationships is largely limited to a few well- studied adaptors, with many CRL adaptors remaining unstudied. We have taken a multi-pronged approach that uses Global Protein Stability (GPS) profiling, quantitative diGLY capture proteomics, and substrate capture by interaction proteomics using specific CRL substrate adaptors to identify more than 600 high priority candidate CRL substrates. Nevertheless, thus far only a portion of the human proteome has been sampled for substrates and that vast majority of candidate CRL substrates have yet to be paired with the appropriate CRL substrate adaptor protein (such as an F-box protein). To address these major limitations, we propose the following aims: 1) AIM 1 will provide a more comprehensive database of candidate CRL substrates in human cells through further development and screening of a v3.0 GPS system, thereby accessing components of the proteome that are not accessible through the current v2.0 system. Improvements will include new C-terminally tagged GPS libraries, bar-coding, as well as Next-gen sequencing of sorted libraries, thereby avoiding limitations of microarray hybridization methods currently employed. 2) AIM 2 will utilize focused high throughput screening and quantitative diGLY proteomics to specifically link high priority candidate substrates to specific cullins and will use both GPS and RNAi screening, as well as interaction proteomics, to link CRL1 substrates with specific F-box protein adaptors, followed by extensive validation. This analysis will provide the first large-scale analysis of the CRL1-F-box system in mammals. 3) AIM 3 will produce a web-based database for dissemination and analysis of CRL-substrate relationships by the community. In addition, selected high priority candidate substrates and the corresponding F-box adaptor proteins will be analyzed through biochemical and cell biological methods to place these proteins into regulatory and physiological pathways with an initial focus on a BACH1, a signal-dependent transcriptional repressor of NRF2-dependent genes and a novel substrate of SCFFBXL17 discovered via our proteomics platform. Together, this work will substantially improve our understanding of the CRL-substrate landscape and will set the stage for in-depth studies that further define signals that dynamically control the proteome via CRLs.
Regulated protein stability is involved in virtually every cellular process and underlies cell cycle control, cell cycle checkpoints, and fundamental processes including cancer and aging. In this proposal, we will employ multiple methods we have recently developed to provide a global understanding of the substrates of one of the largest sub-families of E3 ubiquitin ligases, the cullin-RING ubiquitin ligases (CRLs). We will apply these methods across a wide cross-section of the human proteome and the CRL network will develop a website which facilitates dissemination of CRL-substrate relationships, and will perform detailed mechanistic analyses on a small group of selected high priority substrates involved in cell cycle control, checkpoints, and cancer.
|Gu, Xin; Orozco, Jose M; Saxton, Robert A et al. (2017) SAMTOR is an S-adenosylmethionine sensor for the mTORC1 pathway. Science 358:813-818|
|Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban et al. (2017) Quantitative Phospho-proteomic Analysis of TNF?/NF?B Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death. Mol Cell Proteomics 16:1200-1216|
|Scott, Daniel C; Hammill, Jared T; Min, Jaeki et al. (2017) Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase. Nat Chem Biol 13:850-857|
|Wang, Bin; Jie, Zuliang; Joo, Donghyun et al. (2017) TRAF2 and OTUD7B govern a ubiquitin-dependent switch that regulates mTORC2 signalling. Nature 545:365-369|
|Liu, Lijun; Michowski, Wojciech; Inuzuka, Hiroyuki et al. (2017) G1 cyclins link proliferation, pluripotency and differentiation of embryonic stem cells. Nat Cell Biol 19:177-188|
|Borkent, Marti; Bennett, Brian D; Lackford, Brad et al. (2016) A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2. Stem Cell Reports 6:704-716|
|Harper, J Wade; Bennett, Eric J (2016) Proteome complexity and the forces that drive proteome imbalance. Nature 537:328-38|
|Brown, Nicholas G; VanderLinden, Ryan; Watson, Edmond R et al. (2016) Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C. Cell 165:1440-1453|
|Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L et al. (2016) Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks. Proc Natl Acad Sci U S A 113:E3667-75|
|Scott, Daniel C; Rhee, David Y; Duda, David M et al. (2016) Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation. Cell 166:1198-1214.e24|
Showing the most recent 10 out of 128 publications