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.

中文翻译：

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neddylation 对 CRL2VHL 底物泛素化和降解影响的定量分析

通过催化关键调控蛋白的泛素化，cullin-RING 泛素连接酶 (CRL) 发挥着重要的生物学作用，其活性受多种机制控制，包括 neddylation、NEDD8 与 cullin 的结合。在 neddylation 后，CRL，例如基于 CUL1 的 CRL1，会发生构象变化，从而加速底物泛素化。鉴于 CRL 及其底物亚家族的结构多样性，neddylation 在多大程度上调节单个 CRL 的活性仍有待评估。在这里，通过在体外重构 CRL2 泛素化反应，我们表明 neddylation 促进 CRL2 ^VHL全长 HIF1α 和 HIF1α 的 degron 肽的 -依赖性降解，导致底物泛素化率增加 10 倍以上。一直以来，pevonedistat（也称为 MLN4924）是一种neddylation 抑制剂，在放线菌酮追踪试验中抑制稳定表达 VHL 的 RCC4 细胞中 HIF1α 的降解。然而，在HEK293细胞中没有观察到pevonedistat对HIF1α降解的这种抑制作用，这进一步被发现是由于CRL2 ^VHL- 在 HEK293 而非 RCC4 细胞中活跃的独立降解。在将 HIF1α 截断到其羧基末端氧依赖性降解 (CODD) 域后，我们发现 Pevonedistat 抑制了 CODD 的降解，并将其在 HEK293 细胞中的半衰期延长了六倍。我们的结果表明，neddylation 在激活 CRL2 中起重要作用，并且 CRL2 ^VHL的细胞活性通过 CODD的降解比 HIF1α 的降解更好地反映，尤其是在 CRL2 独立的 HIF1α 降解活跃的条件下。