The mammalian multi-tRNA synthetase complex

项目摘要/摘要 哺乳动物细胞含有一个细胞质多tRNA合成酶复合体(MSC)，该复合体由8个氨基酰-tRNA组成 合成酶(AARs)和3种非合成酶蛋白。间充质干细胞中的AARs在 MRNA的翻译，但也表现出非典范功能外的MSC。然而，组装、结构、 对MSC的功能了解甚少。重要的是，编码7/11成分的基因突变 引起中枢神经系统(CNS)障碍--五种引起髓鞘减少性白质营养不良(HLD)，两种 另一些则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 了解间充质干细胞及其在神经病理学中的潜在作用。我们建议的多PI计划需要 利用两位高度合作的派的专业知识-保罗·福克斯(联系派)，一位具有 对tRNA合成酶和MSC的长期兴趣，以及物理化学家Valentin Gogonea(多PI) 在多蛋白质复合体的分析和分子建模方面具有专业知识。我们将确定第四纪 通过交联质谱(XL-MS)对MSC的结构进行分析，这是一种最先进的方法，有助于 对原本难以处理的复合体进行分析。到目前为止，我们已经在所有11个蛋白质之间发现了19个蛋白质间的交叉连接 MSC组分和118个蛋白质内交联物。我们已经生成了MSC的初始模型，它将 通过扩展氨基酸专一性的XL-MS实验和SiMPull(单分子)进行提纯 下拉)与单分子荧光联用以确定化学计量比。此外，我们还将调查 MSC的组装机制。构成的多蛋白质复合体被认为是由 完全形成的成熟成分之间特定领域的相互作用(“翻译后组装”)。 然而，一些复合体的组装利用了一种“共翻译组装”机制，在这种机制中，成熟的 当配对成分从核糖体中出现时，它与配对成分的新生多肽相互作用。在……里面 初步数据显示，至少有10对MSC成分相互作用。我们将应用这些 阐明两种间充质干细胞成分在中枢神经系统疾病中的作用的机制--遗传缺陷 QARS1和EPRS1分别引起小头畸形和肝豆状核变性。我们的初步研究表明 成分突变或抑制可导致MSC外积聚。我们的初步研究使我们 提出以下假设：哺乳动物的MSC是一个紧凑的结构，部分由一个 然而，有序的共翻译相互作用序列，错误组装或突变可以诱导额外的MSC 成分的积累，可能对下游造成有害后果。我们将对此进行测试 通过(1)确定MSC的四元结构和组分化学计量比，以及(2)确定 共翻译相互作用在MSC形成和完整性中的作用。我们期待对这一事件的澄清 MSC的结构和组装将提供对MSC中分子缺陷的机制的见解 成分可引起严重的病理障碍，特别是中枢神经系统的衰弱障碍。

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.