Quantitative Characterization of Composition and Regulation of Cullin-RING Ubiquitin Ligases

Author: Reichermeier, Kurt Michael

Year: 2020

Degree: Dissertation (Ph.D.)

Advisors: Deshaies, Raymond Joseph; Zinn, Kai George

Committee Members: Chan, David C.; Deshaies, Raymond Joseph; Sternberg, Paul W.; Shan, Shu-ou; Zinn, Kai George

Option: Biology

DOI: 10.7907/0WX7-2Q56

Abstract

Induced proteolysis of pathogenic proteins via degrader molecules, such as Proteolysis Targeting Chimeras (PROTACs), is emerging as a promising therapeutic strategy. In particular, induced proximity of Cullin-RING ubiquitin Ligases (CRLs) with various neo-substrates has proven successful in mediating proteasomal degradation of previously undruggable proteins. Hijacking enzymes to carry out biochemical reactions on neo-substrates stands in stark contrast to conventional pharmacological approaches and exposes degrader molecules to unusually complex pharmacodynamics. While the first PROTACs entered the clinic in 2019, much about the organization and regulation of the frequently co-opted CRLs remains elusive. In particular, the COP9 Signalosome (CSN) is essential to regulate CRL activity and assembly through cleaving Nedd8 from cullin scaffolds, yet it remains unknown how CSN becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation, detailing the kinetic picture of the deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network. Furthermore, we establish Protein Interaction Kinetics and Estimation of Stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1 acts as an exchange factor to remodel the CRL4 ligase pool. Integrating quantitative data and model simulations of CRL-mediated substrate turnover, we show that high substrate receptor levels can enhance the potency of degraders.

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