Department of Materials Science and Engineering, School of Materials and Chemical Technology, Tokyo Institute of Technology, Tokyo, Japan.
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan.
J Cell Biol. 2021 Nov 1;220(11). doi: 10.1083/jcb.202103160. Epub 2021 Sep 9.
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.
富含精氨酸的二肽重复蛋白（DPRs；聚（PR）：脯氨酸-精氨酸和聚（GR）：甘氨酸-精氨酸），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）的神经变性。尽管富含 R 的 DPR 会发生液-液相分离（LLPS），从而影响多种生物学过程，但 DPRs 的 LLPS 机制仍不清楚。在这里，我们使用计算、体外和细胞内方法，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，聚（PR）中交替的 Arg 分布通过 LLPS 促进了带有酸性基序的蛋白质的捕获。核仁核磷蛋白（NPM1）的转录、翻译和扩散被交替电荷分布的聚（PR）所抑制，但不会被连续电荷分布的聚（PR）变体所抑制。我们提出，富含 R 的 DPRs 的致病性是通过通过序列控制的多价蛋白质-蛋白质相互作用，通过相分离液滴中的捕获，干扰蛋白质来介导的。
