Plasmids submitted to AddGene.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.

大型蛋白质包涵体的存在是神经退行性疾病的标志性特征，但其形成的确切分子机制仍不甚明晰。以往利用易聚集蛋白质开展的筛选研究，通常以下游毒性作为检测指标，而非直接检测聚集物的形成。本研究将全基因组CRISPR敲除筛选与脉冲形状分析（一种基于荧光激活细胞分选（FACS）的包涵体检测方法）相结合，旨在鉴定人类细胞中调控TDP-43聚集的直接修饰因子。筛选结果不仅揭示了经典的蛋白质稳态基因，还发掘出功能尚未明确的SRRD蛋白，将其鉴定为蛋白质包涵体形成的核心调控因子。APEX生物素标记实验显示，SRRD与参与二硫键形成与断裂的蛋白质以及中间丝蛋白邻近，提示其可能参与调控中间丝网络的空间动态变化。实验证实，SRRD缺失会导致蛋白毒性应激下中间丝纤维异常，以及聚集体形成受损，包括波形蛋白笼状结构的钝化。有趣的是，SRRD还可定位于聚集体与未折叠蛋白，并能在酵母中缓解蛋白毒性，且其N端低复杂度结构域足以介导这一效应。综上，本研究表明SRRD在细胞骨架组织与细胞蛋白质稳态中发挥了此前未被认知的广泛作用。