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 CRL2VHL‐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 CRL2VHL‐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 CRL2VHL is better reflected by the degradation of CODD than that of HIF1α, especially under conditions where CRL2‐independent degradation of HIF1α is active.

Potassium Effects on NCC Are Attenuated during Inhibition of Cullin E3–Ubiquitin Ligases

“…Activity of cullins depends on their neddylation status, with cycling between neddylated and deneddylated forms changing the stability of the ligase, with the degree of neddylation having a direct correlation with their activity and ubiquitin-mediated degradation of their target substrates [ 31 , 32 , 33 , 34 , 35 , 36 ]. This neddylation status can be observed in the reducing conditions of Western blotting, where cullin proteins are detected as a doublet ( Figure 1 A), the lower molecular weight band representing the non-neddylated (inactive) form and the higher molecular weight band representing the neddylated (active) form.…”

Investigation of the ubiquitin proteasome system in pulmonary arterial hypertension

“…Hypoxia-inducing factor (HIF) is one of the key regulators of oxygen homeostasis, and it is a heterodimer complex composed of oxygen-sensitive subunits (HIF-1α, 2α, 3α) and oxygen-insensitive subunits (HIF-β). In oxygen-containing cells, HIF-α triggered the recognition of the polymeric E3 ubiquitin ligase complex CRL2 VHL, and subsequently targeted hydroxylated HIF-α for proteasomal degradation 61 . Hydroxylated HIF-α is weakened under hypoxia, leading to the stabilization of HIF-α, and this activated HIF initiates an adaptive response to hypoxia by adjusting a large number of genes involved in vascular tone, angiogenesis, cellular metabolism and autophagy responses 62 .…”

Roles of Cullin-RING Ubiquitin Ligases in Cardiovascular Diseases

“…Similar to ubiquitination, the process of cullin neddylation requires consecutive enzymatic reactions involving NEDD8 E1 activating enzyme (NAE), NEDD8 E2 conjugating enzyme, and NEDD8 E3 ligase [18]. Neddylation of cullins efficiently promotes the activity of CRLs, leading to increased ubiquitination of substrates [178][179][180][181]. The mechanism by which neddylation activates CRLs has been revealed from both biochemical and structural perspectives.…”