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.
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