Effect of neurodegenerative disease mutations and post-translational modifications on hnRNPA2 structure, function, and interactions in phase-separated ribonucleoprotein granules

神经退行性疾病突变和翻译后修饰对相分离核糖核蛋白颗粒中 hnRNPA2 结构、功能和相互作用的影响

• 批准号: 9794017
• 负责人: Veronica Hanley Ryan
• 金额: $ 4.3万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-16 至 2020-08-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9794017
• 关键词: Affect; Alleles; Amyotrophic Lateral Sclerosis; Architecture; Arginine; Binding; Binding Proteins; Biological Assay; Caenorhabditis elegans; Cells; Cytoplasmic Granules; Cytoskeleton; Data; Degenerative Disorder; Dendrites; Disease; Disease model; Drug Design; Drug Targeting; Elements; Enzymes; Exons; Future; Genes; Genetic; Heterogeneous-Nuclear Ribonucleoproteins; Human; Immediate-Early Genes; In Vitro; Inclusion Bodies; Induced Mutation; Knowledge; Lead; Liquid substance; Mediating; Messenger RNA; Methylation; Methyltransferase; Microscopy; Microtubule-Associated Proteins; Microtubules; Modeling; Molecular; Muscle; Mutate; Mutation; Myelin Basic Proteins; Myopathy; NMR Spectroscopy; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Neurons; Nuclear Magnetic Resonance; Organelles; Organism; Orthologous Gene; Osteitis Deformans; Pathologic; Patients; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Post-Translational Protein Processing; Protein Methyltransferases; Protein Tyrosine Phosphatase; Proteins; RNA-Binding Proteins; Ribonucleoproteins; Role; Scaffolding Protein; Site; Stress; Structure; Tissues; Transcript; Translations; Tyrosine; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; bone; disease phenotype; experimental study; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genetic analysis; in vivo; insight; mutant; novel; overexpression; prevent; protein aggregation; protein protein interaction; src-Family Kinases; stress granule; structural biology; therapeutic target; tumor

PROJECT SUMMARY/ABSTRACT A number of RNA binding proteins with disordered low complexity domains have recently been shown to undergo liquid-liquid phase separation which underlies membraneless organelle architecture both in cells and in vitro. Several of these proteins are mutated and form inclusions in the neurodegenerative diseases amyotrophic lateral sclerosis and multisystem proteinopathy. One such protein, heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2), is a major component neuronal mRNA transport granules that carry transcripts, including immediate early gene activity-regulated cytoskeleton associated protein Arc, to sites of local translation in dendrites. Using in vitro structural studies including nuclear magnetic resonance spectroscopy and a novel disease model in C. elegans, I will determine the molecular contacts and protein-protein interactions underlying granule formation, as well as the effect of post-translational modifications and disease mutations on physiological interactions, pathological aggregation, and neurodegeneration. Aim 1 will determine the effects of the disease mutation in vitro and develop a disease model of neurodegeneration by replacing the third exon of the C. elegans gene with the corresponding human sequence. I hypothesize that the disease mutants will cause aggregation in vitro and neurodegeneration in vivo. Preliminary results have shown that hnRNPA2 undergoes liquid-liquid phase separation mediated by its low complexity domain and that disease mutants aggregate within phase-separated droplets. Aim 2 will determine the effects of post-translational modifications, including asymmetric arginine dimethylation and tyrosine phosphorylation on phase separation and aggregation as well as the genetic interaction in C. elegans of hnRNPA2 with the methyltransferase, kinase, and phosphatase responsible for the post-translational modifications. I hypothesize that tyrosine phosphorylation will decrease phase separation and aggregation of the disease mutant and that loss of each enzyme will change the degree of neurodegeneration in vivo. Aim 3 will examine the interaction between hnRNPA2 and ch-TOG (CKAP5), a microtubule binding protein that was previously shown to bind hnRNPA2 in myelin basic protein mRNA transport granules. I hypothesize that a) TOG acts as a multivalent scaffolding protein that binds hnRNPA2 at tyrosine residues, b) that tyrosine phosphorylation will disrupt this interaction, and c) that loss of TOG will increase neurodegeneration. Results from this proposal will provide critical understanding of the molecular interactions underlying granule formation, how those interactions are disrupted by disease mutations, and will lead to possible drug targets to prevent protein aggregation and associated neurodegeneration.

项目总结/摘要 最近已经证明，许多具有无序低复杂性结构域的RNA结合蛋白， 经历液-液相分离，这是细胞中无膜细胞器结构的基础， 体外这些蛋白质中的几种发生突变并在神经退行性疾病中形成包涵体 肌萎缩侧索硬化症和多系统蛋白质病。一种这样的蛋白质， 核糖核蛋白A2（hnRNPA 2）是神经元mRNA转运颗粒的主要成分，其携带 转录本，包括立即早期基因活性调节的细胞骨架相关蛋白Arc， 树突中的局部平移。使用体外结构研究，包括核磁共振 光谱和一种新的疾病模型在C.我将确定分子接触， 蛋白质-蛋白质相互作用的颗粒形成，以及翻译后的影响， 修饰和疾病突变对生理相互作用，病理聚集， 神经变性目的1将确定体外疾病突变的影响，并发展一种疾病 通过替换C.与相应的人类基因 顺序我假设疾病突变体会在体外引起聚集，在体内引起神经变性。 vivo.初步结果表明，hnRNPA 2经历由其介导的液-液相分离。 低复杂性结构域和疾病突变体在相分离的液滴内聚集。目标2将 确定翻译后修饰的影响，包括不对称精氨酸二甲基化， 酪氨酸磷酸化对相分离和聚集的影响以及C. elegans hnRNPA 2与甲基转移酶，激酶和磷酸酶负责翻译后 修改.我假设酪氨酸磷酸化将减少相分离和聚集， 疾病突变体和每种酶的缺失将改变体内神经变性的程度。目标3 将研究hnRNPA 2和ch-TOG（CKAP 5）之间的相互作用，ch-TOG是一种微管结合蛋白， 先前显示结合髓鞘碱性蛋白mRNA转运颗粒中的hnRNPA 2。我假设a） TOG作为多价支架蛋白，在酪氨酸残基处结合hnRNPA 2，B）酪氨酸残基结合hnRNPA 2。 磷酸化将破坏这种相互作用，和c）TOG的损失将增加神经变性。结果 从这个建议将提供关键的理解的分子相互作用的颗粒形成， 这些相互作用如何被疾病突变破坏，并将导致可能的药物靶点，以防止 蛋白质聚集和相关的神经变性。