Department of Biochemistry, Purdue University, West Lafayette, Indiana, USA.
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
Protein Sci. 2021 Nov;30(11):2338-2345. doi: 10.1002/pro.4176. Epub 2021 Sep 13.
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 -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 -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 is better reflected by the degradation of CODD than that of HIF1α, especially under conditions where CRL2-independent degradation of HIF1α is active.
通过催化关键调节蛋白的泛素化，Cullin-RING 泛素连接酶（CRLs）发挥着重要的生物学作用，其活性受到多种机制的控制，包括 NEDD8 与 Cullin 的连接（Neddylation）。在 Neddylation 后，CRL1 等基于 CUL1 的 CRL 会发生构象变化，从而加速底物的泛素化。鉴于 CRLs 及其底物的亚家族之间存在结构多样性，Neddylation 对单个 CRL 活性的调节程度仍有待评估。在这里，我们通过体外重建 CRL2 的泛素化反应，表明 Neddylation 促进了全长 HIF1α 和 HIF1α 降解结构域肽的 CRL2 依赖性降解，导致底物泛素化的速率增加了 10 倍以上。一致地，pevonedistat（也称为 MLN4924），一种 Neddylation 的抑制剂，在稳定表达 VHL 的 RCC4 细胞中抑制环磷酰胺追踪实验中 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，进一步发现这是由于在 HEK293 细胞中存在的 CRL2 非依赖性降解，而在 RCC4 细胞中不存在。在将 HIF1α 截断至其羧基末端氧依赖性降解（CODD）结构域后，我们表明 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 中起着重要作用，而 CRL2 的细胞活性通过 CODD 的降解比 HIF1α 的降解更好地反映出来，特别是在 CRL2 非依赖性降解 HIF1α 活跃的情况下。
