Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

中文翻译：

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C9orf72 聚 (PR) 的相分离和毒性取决于精氨酸的交替分布

富含 Arg (R) 的二肽重复蛋白（DPR；poly(PR)：Pro-Arg 和 poly(GR)：Gly-Arg）由 C9ORF72 基因中的六核苷酸扩展编码，可诱导肌萎缩侧索硬化症 (ALS) 中的神经变性。尽管富含 R 的 DPR 会经历液-液相分离 (LLPS)，从而影响多种生物过程，但 DPR 的 LLPS 背后的机制仍然难以捉摸。在这里，使用计算机、体外和纤维素方法，我们确定带电精氨酸残基的分布调节与阴离子肽和核酸的复杂凝聚。蛋白质组学分析表明，poly(PR) 中的交替 Arg 分布有助于通过 LLPS 捕获带有酸性基序的蛋白质。核仁核磷蛋白 (NPM1) 的转录、翻译和扩散会受到具有交替电荷分布的聚 (PR) 的损害，但不会受到具有连续电荷分布的聚 (PR) 变体的损害。我们认为，富含 R 的 DPR 的致病性是通过序列控制的多价蛋白质-蛋白质相互作用捕获在相分离液滴中，从而扰乱蛋白质来介导的。