Stablization of Fragile Human Transfer RNAs

脆弱人类转移 RNA 的稳定

• 批准号: 9769070
• 负责人: PAUL R SCHIMMEL
• 金额: $ 38.7万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-23 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9769070
• 关键词: 3-Dimensional; Alanine-tRNA Ligase; Amino Acids; Amino Acyl-tRNA Synthetases; Aminoacylation; Appearance; Bacteria; Base Pairing; Binding; Binding Proteins; Cells; Defect; Detection; Disease; Elbow; Environment; Enzymes; Evolution; Future; Genes; Genome; Goals; Homeostasis; Human; Laboratories; Link; Metabolism; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial Proteins; Molecular Chaperones; Mutation; Myopathy; Neurodegenerative Disorders; Nuclear; Oxidative Phosphorylation; Pathology; Predisposition; Production; Protein Biosynthesis; Proteins; Publishing; RNA Splicing; Recombinant Proteins; Recombinants; Respiration; Stress; Structure; Testing; Therapeutic; Therapeutic Intervention; Transfer RNA; Translations; Variant; Work; base; design; disease-causing mutation; experimental study; insight; mitochondrial genome; mutant; novel; nuclease

Project Summary This proposal is aimed at understanding enough to eventually have a therapeutic intervention for a large group of human mitochondrial diseases associated with transfer RNAs. These diseases arise from mutations in mitochondrial tRNAs that cause instability and susceptibility to nuclease degradation. We focus on stabilizing these mutant tRNAs by identifying and utilizing a pan-specific mitochondrial tRNA binding protein that stabilize the fragile tRNA structure. We have found one such natural protein from the recently discovered ensemble of human tRNA synthetase splice variants. Because defects in mitochondrial (mt) protein synthesis have an immediate impact on cellular metabolism, it is perhaps not surprising that more than 50% of all identified disease-causing mutations in mtDNA are located within mtDNA genes for tRNAs. And yet, these genes constitute only 10% of the mitochondrial genome. A significant number of mt disease-linked mutations, which cause myopathies, neurodegenerative diseases, and multisystemic disorders, are located in the mt tRNAs. Our laboratory has published extensively on aaRSs from bacteria to humans. However, we did not previously have the insight to suggest a path to stabilize disease-causing mutant tRNAs. aaRSs have progressively acquired new domains in evolution. These new domains are dispensable for the aminoacylation function and are mobilized for specific, novel functions outside of translation. We discovered over 250 splice variants (SVs) of human aaRSs. The majority ablate the catalytic activity but retain the novel motifs. Importantly, most are stable as recombinant proteins. Amongst the SVs, we focus on the few that have the appearance of being chaperones for tRNAs, that is, proteins that bind to and stabilize tRNAs, but with a structure- but not sequence-specific recognition of the outside corner (elbow) of the L-shaped tRNA structure. The goal is to select one or more easy- to-purify, stable, recombinant corner- binding splice variants. These will be tested for their ability, when added in trans, to bind defective mutant mitochondrial tRNAs. Another criterion is that the chosen domain can be applied exogenously to cells and enter the mitochondria. In preliminary work we identified at least one SV that fulfills the criteria we established. If successful, this proposal will suggest a new path for therapeutic intervention of the most prevalent human mitochondrial diseases. !

项目摘要 这项提议的目的是了解足够多的知识，最终进行治疗干预。 用于与转移RNA相关的一大组人类线粒体疾病。这些疾病 源于线粒体tRNA的突变，导致不稳定和对核酸酶降解的敏感性。 我们专注于通过鉴定和利用泛特异的线粒体tRNA来稳定这些突变的tRNA。 稳定脆弱tRNA结构的结合蛋白。我们已经发现了一种这样的天然蛋白质，来自 最近发现了人类tRNA合成酶剪接变异体。 因为线粒体(MT)蛋白质合成缺陷会对细胞产生直接影响 新陈代谢，也许并不令人惊讶的是，在所有已发现的致病突变中，超过50% 在线粒体DNA中，位于tRNA的mtDNA基因内。然而，这些基因只占基因总数的10% 线粒体基因组。大量与多发性肌萎缩侧索硬化症相关的突变，导致肌病， 神经退行性疾病和多系统疾病位于mt tRNA中。我们的 实验室已经发表了大量关于从细菌到人类的AARS的文章。然而，我们没有 以前有洞察力提出了一条稳定致病突变tRNA的途径。 AARS在进化中逐渐获得了新的领域。这些新域名是 对于氨基酰化功能来说是必不可少的，并被动员起来用于特定的、新的功能之外 翻译。我们发现了250多个人类AARs的剪接变异体(Svs)。大多数都会消融 具有催化活性，但保留了新的基序。重要的是，大多数作为重组蛋白是稳定的。 在SVS中，我们关注的是少数几个看起来像是tRNA的伴侣，即 是与tRNA结合并稳定的蛋白质，但具有结构-但不是序列特异性识别 位于L形tRNA结构的外角(肘部)。目标是选择一个或多个容易的- 提纯稳定的重组角结合剪接变异体。这些人将接受能力测试，当 加入反式，以结合有缺陷的突变线粒体tRNA。另一个标准是被选中的 结构域可以外源作用于细胞并进入线粒体。在前期工作中，我们 确定了至少一个符合我们建立的标准的SV。如果成功，这项提议将 为人类最常见的线粒体疾病的治疗干预提供了新的途径。 好了！