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 这项建议的目的是了解足够的最终有一个治疗干预 与转移RNA相关的一大组人类线粒体疾病。这些疾病 由线粒体tRNA的突变引起，其引起不稳定性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性线粒体tRNA来稳定这些突变体tRNA 稳定脆弱的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， 最近发现的人类tRNA合成酶剪接变体的集合。 由于线粒体（mt）蛋白质合成的缺陷对细胞的 代谢，这也许并不奇怪，超过50%的所有确定的致病突变 在线粒体DNA中位于tRNA的线粒体DNA基因内。然而，这些基因只占10%， 线粒体基因组大量与mt疾病相关的突变导致肌病， 神经退行性疾病和多系统性疾病位于mttRNA中。我们 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 先前有洞察力，提出了一种稳定致病突变tRNA的途径。 aaRS在进化中逐渐获得了新的领域。这些新领域是 氨酰化功能的酶，并被动员用于特定的，新的功能以外的 翻译.我们发现了超过250种人类aaRS的剪接变体（SV）。大多数人都切除了 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SV中，我们关注的是少数几个看起来像是tRNA分子伴侣的SV， 即结合并稳定tRNA的蛋白质，但具有结构特异性识别，而不是序列特异性识别 L形tRNA结构的外角（肘部）。我们的目标是选择一个或多个简单的- 以纯化稳定的重组角结合剪接变体。他们的能力将受到考验， 以反式添加，以结合有缺陷的突变线粒体tRNA。另一个标准是， 结构域可以外源地应用于细胞并进入线粒体。在前期工作中， 确定至少一个SV符合我们制定的标准。如果成功的话，这一提议将 提出了一种新的途径，治疗干预最普遍的人类线粒体疾病。 !