The E3 Ubiquitin Ligase UBE3C Enhances Proteasome Processivity by Ubiquitinating Partially Proteolyzed Substrates
The E3 Ubiquitin Ligase UBE3C Enhances Proteasome Processivity by Ubiquitinating Partially Proteolyzed Substrates
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DOI：
10.1074/jbc.m113.499350
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发表时间：
2013-11-29
影响因子：
4.8
通讯作者：
Wandless, Thomas J.
中科院分区：
文献类型：
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作者：
Chu, Bernard W.;Kovary, Kyle M.;Wandless, Thomas J.
背景：RNAi 筛选确定 UBE3C 是模型未折叠蛋白降解的关键参与者。结果：UBE3C 敲低导致相对稳定的底物不完全降解。结论：UBE3C 增强蛋白酶体的持续合成能力，防止潜在有害蛋白质片段的积累。意义：这增进了我们对蛋白酶体持续合成能力及其缺陷后果的理解。为了维持蛋白质稳态，细胞必须平衡蛋白质合成与蛋白质降解。错误折叠或部分降解的蛋白质的积累可能导致病理性蛋白质聚集体的形成。在这里，我们报告使用不稳定结构域（其折叠状态可以使用高亲和力配体可逆调节的蛋白质）作为模型底物，以使用正向遗传筛选来询问哺乳动物细胞中的细胞蛋白质质量控制机制。 UBE3C（一种 E3 泛素连接酶，一种由与 GFP 融合的不稳定结构域组成的报告蛋白）敲低后，蛋白酶体降解速度更慢且不完全。当 UBE3C 存在但由于其活性位点已突变、无法与蛋白酶体结合或底物缺乏赖氨酸残基而无法泛素化底物时，也会观察到部分蛋白水解。 UBE3C 敲除也会导致底物多泛素化减少。最后，敲低使细胞对 Hsp90 抑制剂 17-AAG 更敏感，这表明 UBE3C 可以防止因蛋白酶体底物不完全降解而产生的蛋白质片段的有害积累。
Background: An RNAi screen identified UBE3C as a key player in degradation of a model unfolded protein. Results: UBE3C knockdown results in incomplete degradation of relatively stable substrates. Conclusion: UBE3C enhances proteasome processivity to prevent the accumulation of potentially harmful protein fragments. Significance: This advances our understanding of proteasome processivity and the consequences of defects therein.To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.