Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
J Biol Chem. 2020 Mar 6;295(10):3148-3158. doi: 10.1074/jbc.RA119.010239. Epub 2020 Feb 3.
() is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Although the precise mechanisms of mutant (SOD1)-induced motoneuron toxicity are still unclear, defects in SOD1 proteostasis are known to have a critical role in ALS pathogenesis. We previously reported that the SOD1 adopts a conformation that exposes a Derlin-1-binding region (DBR) and that DBR-exposed SOD1 interacts with Derlin-1, leading to motoneuron death. We also found that an environmental change, zinc depletion, induces a conformational change in WT SOD1 (SOD1) to the DBR-exposed conformation, suggesting the presence of an equilibrium state between the DBR-masked and DBR-exposed states even with SOD1 Here, we conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1 conformational change, and we used a genome-wide siRNA screen to search for regulators of SOD1 proteostasis. This screen yielded 30 candidate genes that maintained an absence of the DBR-exposed SOD1 conformation. Among these genes was one encoding DDB1- and CUL4-associated factor 4 (DCAF4), a substrate receptor of the E3 ubiquitin-protein ligase complex. Of note, we found that DCAF4 mediates the ubiquitination of an ALS-associated protein and autophagy receptor, optineurin (OPTN), and facilitates autophagic degradation of DBR-exposed SOD1. In summary, our screen identifies DCAF4 as being required for proper proteostasis of DBR-exposed SOD1, which may have potential relevance for the development of therapies for managing ALS.
() 是一种与毁灭性神经退行性疾病肌萎缩性侧索硬化症（ALS）相关的基因。尽管突变型（SOD1）诱导运动神经元毒性的确切机制尚不清楚，但 SOD1 蛋白稳态的缺陷已知在 ALS 发病机制中起关键作用。我们之前报道过，SOD1 采用一种暴露 Derlin-1 结合区域（DBR）的构象，并且暴露 DBR 的 SOD1 与 Derlin-1 相互作用，导致运动神经元死亡。我们还发现环境变化，锌耗竭，诱导 WT SOD1（SOD1）构象发生变化，暴露 DBR，表明即使在 SOD1 中，DBR 掩蔽和 DBR 暴露状态之间也存在平衡状态。在这里，我们进行了基于时间分辨 FRET 的高通量筛选，以进一步研究 SOD1 构象变化，并使用全基因组 siRNA 筛选来搜索 SOD1 蛋白稳态的调节剂。该筛选产生了 30 个候选基因，它们保持不存在暴露 DBR 的 SOD1 构象。这些基因中有一个编码 DDB1 和 CUL4 相关因子 4（DCAF4），它是 E3 泛素蛋白连接酶复合物的底物受体。值得注意的是，我们发现 DCAF4 介导 ALS 相关蛋白和自噬受体 OPTN 的泛素化，并促进暴露 DBR 的 SOD1 的自噬降解。总之，我们的筛选确定 DCAF4 是暴露 DBR 的 SOD1 正确蛋白稳态所必需的，这可能与开发治疗 ALS 的疗法有关。
