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聚集体是诸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，均可检测到RNA结合蛋白TDP-43的胞质聚集体。但针对这类疾病中的多数而言，究竟是蛋白聚集体本身，还是其他形式的该蛋白对细胞具有毒性，仍是一个悬而未决的核心问题。

本研究针对TDP-43的这一问题展开探究，通过对该蛋白开展系统性诱变，并量化其对细胞毒性的影响。在针对内在无序朊病毒样结构域（intrinsically disordered prion-like domain, PRD）构建的包含50000余个突变的数据集当中，疏水性与聚集潜能的变化可高度预测细胞毒性的改变。但令人意外的是，疏水性增强与胞质聚集反而会降低细胞毒性。

突变在PRD的中央区域展现出最强的调控效应：其中可提升细胞毒性的变异，会促进细胞形成更具动态性的类液凝聚体。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，并可在活体状态下对蛋白聚集与细胞毒性产生可检测的影响。

本研究结果证实，深度诱变（deep mutagenesis）是探究内在无序蛋白序列-功能关系及其体内结构构象的高效研究手段。同时本研究还揭示，TDP-43的聚集并非具有毒性，反而可对细胞起到保护作用，其核心机制大概率是通过将蛋白从有毒的类液相中隔离出来。

【实验总体设计】：本研究系统性测定了TDP-43蛋白290-373位氨基酸区域内50000个突变的细胞毒性效应。其中，Input 5-8为TDP-43氨基酸290-331突变体文库的生物学重复样本；Input 1-4为TDP-43氨基酸332-373突变体文库的生物学重复样本。每一份Input样本均提取自完成转化但未表达TDP-43的培养细胞。编号一致的Output样本为技术重复样本，均源自同一份Input样本，且均提取自在表达TDP-43约6代后培养的细胞的基因组DNA。