The mammalian multi-tRNA synthetase complex

哺乳动物多tRNA合成酶复合物

• 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
• 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

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.

项目摘要/摘要 哺乳动物细胞含有由8个氨基酰基-TRNA组成的细胞质多-TRNA合成酶复合酶（MSC） 合成酶（AARSS）和3种非核酶蛋白。 MSC中的AARS在“基因解码器”中的功能 mRNA翻译，但也暴露了MSC之外的非典型功能。但是，组件，结构， 理学硕士的功能知之甚少。重要的是，编码7/11的基因中的突变构成 引起中枢神经系统（CNS）疾病 - 五个导致白细胞营养不良（HLD）的降低责任和两个 其他人会导致渐进性。我们将利用最先进的分子方法来改善我们的 了解MSC及其在神经病理学中的潜在作用。我们提出的多重PI计划需要 两个高度协作PI的专业知识的优势 - Paul Fox（联系PI），他是分子生物学家 对TRNA合成酶和MSC的长期兴趣，以及Valentin Gogonea（多个PI），一名物理化学家 具有多蛋白质复合物的分析和分子建模方面的专业知识。我们将确定第四纪 通过交联质谱法（XL-MS）的结构，这是一种促进的最先进方法 分析原本棘手的复合物。迄今为止，我们发现了所有11个之间的19个蛋白质间交联 MSC构成118个蛋白质交联。我们已经生成了MSC的初始模型 可以通过具有扩展的氨基酸特异性的XL-MS实验和Simpull（单分子 下拉）与单分子荧光结合以确定化学计量。此外，我们将调查 MSC组装机制。宪法，多蛋白络合物被认为是由 完整形成的成熟一致性之间的域特异性相互作用（“翻译后组装”）。 但是，一些配合物的组装利用了“共译组装”机制，其中成熟 成分与伴侣成分的新生意大利辣香肠相互作用，因为它是从核糖体出来的。在 初步数据我们显示至少10对MSC构成共同翻译。我们将应用这些 阐明两种MSC在中枢神经系统疾病中的作用的机理方法 - 遗传缺陷 QARS1和EPRS1分别引起小头畸形和HLD。我们的初步研究表明 组成突变或抑制会导致超过MSC的积累。我们的初步研究使我们 提出以下假设：哺乳动物MSC是一种紧凑的结构，部分由 然而，有序的共同相互作用序列，错误组装或突变会诱导超级序列 成分的积累，可能会带来有害的下游后果。我们将测试这个 通过（1）确定MSC第四纪结构和分量化学计量的假设，以及（2）确定 共同相互作用在MSC形成和完整性中的作用。我们预计会阐明 MSC的结构和组装将提供有关MSC中分子缺陷的机制的见解 成分可能引起严重的病理疾病，特别是使中枢神经系统的疾病使人衰弱。