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Published November 2021 | Published + Supplemental Material
Journal Article Open

Quantitative analyses for effects of neddylation on CRL2^(VHL) substrate ubiquitination and degradation

Abstract

Through catalyzing the ubiquitination of key regulatory proteins, cullin-RING ubiquitin ligases (CRLs) play essential biological roles and their activities are controlled by multiple mechanisms including neddylation, the conjugation of NEDD8 to cullins. Upon neddylation, a CRL, such as the CUL1-based CRL1, undergoes conformational changes that accelerate substrate ubiquitination. Given the structural diversity across subfamilies of CRLs and their substrates, to what extent neddylation modulates the activity of individual CRLs remains to be evaluated. Here, through reconstituting the CRL2 ubiquitination reaction in vitro, we showed that neddylation promotes CRL2^(VHL)-dependent degradation of both full-length HIF1α and the degron peptide of HIF1α, resulting in more than 10-fold increase in the rate of substrate ubiquitination. Consistently, pevonedistat (also known as MLN4924), an inhibitor of neddylation, inhibits the degradation of HIF1α in RCC4 cells stably expressing VHL in cycloheximide chase assays. However, such inhibitory effect of pevonedistat on HIF1α degradation was not observed in HEK293 cells, which was further found to be due to CRL2^(VHL)-independent degradation that was active in HEK293 but not RCC4 cells. After truncating HIF1α to its Carboxy-terminal Oxygen-Dependent Degradation (CODD) domain, we showed that pevonedistat inhibited the degradation of CODD and increased its half-life by six-fold in HEK293 cells. Our results demonstrate that neddylation plays a significant role in activating CRL2, and the cellular activity of CRL2^(VHL) is better reflected by the degradation of CODD than that of HIF1α, especially under conditions where CRL2-independent degradation of HIF1α is active.

Additional Information

© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Issue Online: 18 October 2021; Version of Record online: 13 September 2021; Accepted manuscript online: 30 August 2021; Manuscript accepted: 23 August 2021; Manuscript revised: 16 August 2021; Manuscript received: 14 June 2021. We thank Dr. Jörg Klug and Dr. Andreas Meinhardt (Justus-Liebig Universität, Giessen, Germany) for their mentorship to Kurt M. Reichermeier. We thank Dr. Raymond Deshaies and Dr. Rati Verma (Amgen Inc., formerly California Institute of Technology) for critical reading of the manuscript. This study was supported by National Institutes of Health grant R35 GM138016 (to Xing Liu) and American Heart Association Career Development Award (to Xing Liu). Author Contributions: Kankan Wang: Data curation (equal); formal analysis (lead); investigation (lead); methodology (lead); validation (lead); writing – original draft (lead); writing – review and editing (supporting). Kurt M. Reichermeier: Conceptualization (supporting); data curation (supporting); investigation (supporting); methodology (supporting); writing – original draft (supporting). Xing Liu: Conceptualization (lead); data curation (equal); formal analysis (supporting); funding acquisition (lead); methodology (supporting); project administration (lead); supervision (lead); writing – review and editing (lead). The authors declare no conflict of interest.

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Additional details

Created:
August 22, 2023
Modified:
October 23, 2023