The mammalian multi-tRNA synthetase complex

哺乳动物多tRNA合成酶复合物

基本信息

批准号：
10331178
负责人：
PAUL L FOX
金额：
$ 49.72万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-01 至 2026-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10331178
关键词：
3-Dimensional Alleles Amino Acids Amino Acyl-tRNA Synthetases Architecture Atrophic Binding Biochemical Genetics Cell physiology Cells Central Nervous System Diseases Cerebrum Child Cognitive Complex Computer software Coupled Cytoplasmic Protein DNA Sequence Alteration Data Data Analyses Defect Disease Etiology Event Exhibits Fluorescence Foxes Functional disorder Genes Genetic Translation Glutamine-tRNA ligase Goals Grant Health Human Lead Length Mammalian Cell Mass Spectrum Analysis Messenger RNA Methods Microcephaly Modeling Molecular Motor Multiprotein Complexes Mutate Mutation Myelin Neurodegenerative Disorders Nomenclature Pathologic Pathology Peptides Photobleaching Positioning Attribute Protein Biosynthesis Proteins Reporting Ribosomes Role Scaffolding Protein Sodium Chloride Specificity Stimulus Structural Models Structure Sulfoxide Testing Translations crosslink density design experimental study fluorescence imaging improved insight instrumentation interest knock-down leukodystrophy molecular modeling neuropathology programs protein complex single molecule spatial relationship stoichiometry

项目摘要

Project Summary/Abstract Mammalian cells contain a cytoplasmic multi-tRNA synthetase complex (MSC) consisting of 8 aminoacyl-tRNA synthetases (AARSs) and 3 non-synthetase proteins. AARSs in the MSC function as “gene decoders” during mRNA translation, but also exhibit non-canonical functions outside the MSC. However, the assembly, structure, and function of the MSC are poorly understood. Importantly, mutations in genes encoding 7/11 constituents cause central nervous system (CNS) disorders – five cause hypomyelinating leukodystrophy (HLD), and two others cause progressive microcephaly. We will utilize state-of-the-art molecular approaches to improve our understanding of the MSC, and its potential role in neuropathology. Our proposed Multiple-PI program takes advantage of the expertise of two highly collaborative PI's – Paul Fox (Contact PI), a molecular biologist with long-term interest in tRNA synthetases and the MSC, and Valentin Gogonea (Multiple PI), a physical chemist with expertise in analysis and molecular modeling of multi-protein complexes. We will determine the quaternary structure of the MSC by cross-linking mass spectrometry (XL-MS), a state-of-the-art method that facilitates analysis of otherwise intractable complexes. To date we have found 19 inter-protein cross-links between all 11 MSC constituents, and 118 intra-protein cross-links. We have generated an initial model of the MSC that will be refined here by XL-MS experiments with expanded amino acid specificity, and by SiMPull (single-molecule pulldown) coupled with single-molecule fluorescence to determine stoichiometry. In addition, we will investigate the mechanism of assembly of the MSC. Constitutive, multi-protein complexes are thought to be assembled by domain-specific interactions between fully-formed, mature constituents (“post-translational assembly”). However, assembly of some complexes utilizes a “co-translational assembly” mechanism in which a mature constituent interacts with the nascent peptide of a partner constituent as it emerges from the ribosome. In preliminary data we show at least 10 pairs of MSC constituents interact co-translationally. We will apply these mechanistic approaches to elucidate the role of two MSC constituents in CNS diseases – genetic defects in QARS1 and EPRS1 that cause microcephaly and HLD, respectively. Our preliminary studies indicate that constituent mutation or suppression can lead to extra-MSC accumulation. Our preliminary studies have led us to propose the following hypothesis: The mammalian MSC is a compact structure assembled in part by an orderly sequence of co-translational interactions, however, mis-assembly or mutation can induce extra-MSC accumulation of constituents, with potentially deleterious downstream consequences. We will test this hypothesis by (1) determining MSC quaternary structure and component stoichiometry, and (2) determining the role of co-translational interactions in MSC formation and integrity. We anticipate that elucidation of the structure and assembly of the MSC will provide insights into mechanisms by which molecular defects in MSC constituents can cause severe pathological disturbances, in particular, debilitating disorders of the CNS.

项目概要/摘要哺乳动物细胞含有由 8 个氨酰基-tRNA 组成的细胞质多 tRNA 合成酶复合物 (MSC) MSC 中的合成酶 (AARS) 和 3 个非合成酶蛋白在过程中充当“基因解码器”。 mRNA 翻译，但也表现出 MSC 之外的非规范功能。重要的是，人们对 MSC 的功能和编码知之甚少。导致中枢神经系统 (CNS) 疾病 – 五种导致低髓鞘性脑白质营养不良 (HLD)，两种其他会导致进行性小头畸形，我们将利用最先进的分子方法来改善我们的症状。了解 MSC 及其在神经病理学中的潜在作用。充分利用两位高度协作的 PI 的专业知识——Paul Fox（联系人 PI），一位分子生物学家，对 tRNA 合成酶和 MSC 的长期兴趣，以及物理化学家 Valentin Gogonea（多 PI）凭借多蛋白质复合物分析和分子建模方面的专业知识，我们将确定四元。通过交联质谱 (XL-MS) 分析 MSC 的结构，这是一种最先进的方法，有助于迄今为止，我们已经发现所有 11 个蛋白质之间有 19 个蛋白质间交联。 MSC 成分和 118 个蛋白质内交联我们已经生成了 MSC 的初始模型。通过具有扩展氨基酸特异性的 XL-MS 实验和 SiMPull（单分子 Pulldown）与单分子荧光相结合以确定化学计量。此外，我们将进行研究。 MSC 的组成性多蛋白复合物的组装机制被认为是由完全形成的成熟成分之间的特定领域相互作用（“翻译后组装”）。然而，一些复合物的组装利用了“共翻译组装”机制，其中成熟的当它从核糖体中出现时，成分与伙伴成分的新生肽相互作用。初步数据显示，至少有 10 对 MSC 成分通过共翻译相互作用，我们将应用这些数据。阐明两种 MSC 成分在中枢神经系统疾病中的作用的机制方法——遗传缺陷我们的初步研究表明，QARS1 和 EPRS1 分别导致小头畸形和 HLD。我们的初步研究表明，成分突变或抑制可能导致额外的 MSC 积累。提出以下假设：哺乳动物间充质干细胞是一种紧凑的结构，部分由共翻译相互作用的有序序列，然而，错误组装或突变可以诱导额外的 MSC 成分的积累，可能会产生有害的下游后果。我们将对此进行测试。通过 (1) 确定 MSC 四级结构和成分化学计量，以及 (2) 确定我们期望阐明共翻译相互作用在 MSC 形成和完整性中的作用。 MSC 的结构和组装将有助于深入了解 MSC 中分子缺陷的机制成分可引起严重的病理紊乱，特别是中枢神经系统衰弱性疾病。