SCF and the cullin-RING ubiquitin ligases (CRLs) comprise the largest family of ubiquitin ligases in humans. These ~250 enzymes have a major impact on human biology through their ability to specify the half-lives of key regulatory proteins that control processes from basic metabolism to circadian rhythms. In keeping with their broad impact on biology, CRLs also play a major role in human health and cancer chemotherapy. Substrate specificity of CRLs is determined by an interchangeable substrate receptor subunit (SRS). Several CRL SRSs, including Vhl and Fbxw7, are prominent tumor-suppressors, whereas others (Skp2) are proto- oncogenic and yet another, Crbn, is targeted by the drugs that are most commonly used to treat multiple myeloma. Moreover, Nedd8 conjugation, which regulates CRL assembly and activity, is the target of a cancer drug in clinical development. Given their extraordinary impact on human biology and health, CRLs have been studied intensively. However, there is a great deal that we still do not understand about these enzymes. For example, we do not fully understand how exchange of SRSs occurs and we do not know how the exchange process is regulated to ensure that cells possess an appropriate repertoire of CRL complexes. Even very basic questions remain unaddressed, such as: what is the fraction of SRSs that assemble to form CRLs? Do all SRSs assemble with equal efficiency?; do all cells express the same repertoire of CRL complexes?; and so forth. This application proposes development of new assays and methodologies that will enable us to address these fundamental questions. The first two Aims investigate the steady-state structure and dynamics of the cellular network of SCF complexes through development and application of a set of `Selected Reaction Monitoring' (SRM) mass spectrometry assays to identify and quantify all known SCF subunits and regulators.
The third Aim focuses on development and application of new assays to monitor the dynamic association of the SRS exchange factor Cand1 with SCF. FRET and single molecule approaches will explore how Cand1 promotes SRS exchange, and how this process is regulated. Understanding the regulation of CRL assembly will reveal how the network of CRLs is controlled in normal and diseased cells.

Public Health Relevance

The enzymes that are the focus of this grant - cullin-RING ubiquitin ligases (CRLs) - have a deep relationship with human disease and therapy. Mutations in the CRL FBXW7 are one of the most common causes of the adult lymphocytic leukemia, whereas another CRL, CRBN, mediates the anti-cancer activity of the multiple myeloma drugs Revlimid and Pomalyst. We seek to study how CRLs are assembled and controlled, which may help in the development of new therapies directed towards these enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM065997-13
Application #
8964613
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Gerratana, Barbara
Project Start
2002-07-01
Project End
2019-04-30
Budget Start
2015-07-01
Budget End
2016-04-30
Support Year
13
Fiscal Year
2015
Total Cost
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Nguyen, Thang Van; Li, Jing; Lu, Chin-Chun Jean et al. (2017) p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates. Proc Natl Acad Sci U S A 114:3565-3571
Reitsma, Justin M; Liu, Xing; Reichermeier, Kurt M et al. (2017) Composition and Regulation of the Cellular Repertoire of SCF Ubiquitin Ligases. Cell 171:1326-1339.e14
Reiterer, Veronika; Figueras-Puig, Cristina; Le Guerroue, Francois et al. (2017) The pseudophosphatase STYX targets the F-box of FBXW7 and inhibits SCFFBXW7 function. EMBO J 36:260-273
Nguyen, T Van; Lee, J Eugene; Sweredoski, Michael J et al. (2016) Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon. Mol Cell 61:809-20
Kleiger, Gary; Deshaies, Raymond (2016) Tag Team Ubiquitin Ligases. Cell 166:1080-1081
Pulvino, Mary; Chen, Luojing; Oleksyn, David et al. (2015) Inhibition of COP9-signalosome (CSN) deneddylating activity and tumor growth of diffuse large B-cell lymphomas by doxycycline. Oncotarget 6:14796-813
Deshaies, Raymond J (2015) Protein degradation: Prime time for PROTACs. Nat Chem Biol 11:634-5
Honarpour, Narimon; Rose, Christopher M; Brumbaugh, Justin et al. (2014) F-box protein FBXL16 binds PP2A-B55? and regulates differentiation of embryonic stem cells along the FLK1+ lineage. Mol Cell Proteomics 13:780-91
Pierce, Nathan W; Lee, J Eugene; Liu, Xing et al. (2013) Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell 153:206-15
Emberley, Ethan D; Mosadeghi, Ruzbeh; Deshaies, Raymond J (2012) Deconjugation of Nedd8 from Cul1 is directly regulated by Skp1-F-box and substrate, and the COP9 signalosome inhibits deneddylated SCF by a noncatalytic mechanism. J Biol Chem 287:29679-89

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