The mutational landscape of a Prion-like domain. The mutational landscape of a Prion-like domain

Specific insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, cytoplasmic aggregates of the RNA-binding protein TDP-43 are observed in 97% of cases of Amyotrophic Lateral Sclerosis (ALS). However, whether the protein aggregates themselves or other forms of the proteins are toxic to cells is still a very open question for many of these diseases. Here we address this question for TDP-43 by systematically mutating the protein and quantifying the effects on cellular toxicity. In a dataset of >50,000 mutations in the intrinsically disordered prion-like domain (PRD), changes in hydrophobicity and aggregation potential are highly predictive of changes in toxicity. Surprisingly, however, increased hydrophobicity and cytoplasmic aggregation reduce toxicity. Mutations have their strongest effects in a central region of the PRD, with variants that increase toxicity promoting the formation of more dynamic liquid-like condensates. Moreover, the genetic interactions in double mutants indicate that specific secondary structures form in this region and have detectable effects on aggregation and toxicity in vivo. Our results demonstrate that deep mutagenesis is a powerful approach for probing the sequence-function relationships of intrinsically disordered proteins, as well as their in vivo structural conformations. Moreover, they reveal that aggregation of TDP-43 is not toxic but actually protects cells, most likely by titrating the protein away from a toxic liquid-like phase. Overall design: Systematic measurment of the toxic effects of 50.000 mutations in the region 290-373 of TDP-43 Input 5-8 are biological replicates for library TDP aa 290-331; Input 1-4 are biological replicates for library TDP aa 332-373. Each input corresponds to DNA extracted from cells growing after transformation and without expression of TDP-43. Outputs named with the same number are technical replicates and they originate from the same input. Each output corresponds to DNA extracted from cells growing after having expressed TDP-43 for roughly 6 generations.

特异性不可溶性蛋白质聚集体是众多神经退行性疾病的标志性病理特征。例如，在97%的肌萎缩侧索硬化症（Amyotrophic Lateral Sclerosis, ALS）病例中，均可观察到RNA结合蛋白TDP-43的细胞质聚集体。然而，对于这类疾病中的多数而言，究竟是蛋白质聚集体本身，还是蛋白质的其他构象形式对细胞具有毒性，目前仍是一个尚未定论的开放性问题。本研究针对TDP-43的相关问题，通过对该蛋白进行系统性诱变并定量分析其对细胞毒性的影响展开研究。在包含超过50000个突变的固有无序朊蛋白样结构域（intrinsically disordered prion-like domain, PRD）数据集内，疏水性与聚集潜能的变化可高度预测毒性水平的改变。但令人意外的是，疏水性增强以及细胞质聚集反而会降低细胞毒性。诱变效应在PRD的中央区域最为显著，其中可增强毒性的突变体能够促进形成更具动态性的液态样凝聚体。此外，双突变体的遗传互作分析表明，该区域可形成特定的二级结构，并在体内对聚集过程与毒性产生可检测的影响。本研究结果证实，深度诱变是解析固有无序蛋白质序列-功能关系及其体内结构构象的有效手段。此外，研究结果还揭示，TDP-43的聚集并非具有毒性，反而可对细胞起到保护作用，其机制极有可能是通过将蛋白从毒性液态相中分离移除。实验整体设计：针对TDP-43的290-373区域内的50000个突变，系统性测定其毒性效应。输入样本5-8为TDP氨基酸290-331文库的生物学重复样本；输入样本1-4为TDP氨基酸332-373文库的生物学重复样本。每一份输入样本均提取自完成转化且未表达TDP-43的培养细胞基因组DNA。编号相同的输出样本为技术重复样本，其均来源于同一份输入样本。每一份输出样本均提取自表达TDP-43约6个世代的培养细胞基因组DNA。