The mammalian multi-tRNA synthetase complex

项目总结/摘要 哺乳动物细胞含有由8个氨酰-tRNA组成的细胞质多tRNA合成酶复合物（MSC）， 合成酶（AARS）和3种非合成酶蛋白。MSC中的AARS在细胞增殖过程中起“基因解码器”的作用。 mRNA的翻译，但也表现出非典型的MSC外的功能。然而，组件、结构、 对MSC的功能和功能了解甚少。重要的是，编码7/11组分的基因突变 引起中枢神经系统（CNS）疾病-五个引起低髓鞘化脑白质营养不良（HLD）， 另一些引起进行性小头畸形。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 两个高度合作的PI的专业知识的优势-保罗福克斯（接触PI），分子生物学家， 长期对tRNA合成酶和MSC感兴趣，物理化学家Valentin Gogonea（多个PI） 在多蛋白质复合物的分析和分子建模方面具有专业知识。我们将确定第四纪 通过交联质谱法（XL-MS）分析MSC的结构，这是一种最先进的方法， 分析其他棘手的复杂问题。到目前为止，我们已经发现了19个蛋白质间的交联之间的所有11个 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， 在此通过具有扩展的氨基酸特异性的XL-MS实验和通过SiMPull（单分子 下拉）与单分子荧光偶联以确定化学计量。此外，我们将调查 MSC的组装机制。组成性的多蛋白质复合物被认为是由 完全形成的成熟成分之间的结构域特异性相互作用（“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，其中成熟的复合物被翻译成蛋白质。 当伴侣成分的新生肽从核糖体中出现时，它与伴侣成分的新生肽相互作用。在 初步数据显示至少有10对MSC成分协同相互作用。我们将应用这些 阐明两种MSC成分在CNS疾病中的作用的机制方法- 分别导致小头畸形和HLD的QARS 1和EPRS 1。我们的初步研究表明， 成分突变或抑制可导致额外MSC积累。我们的初步研究 提出了以下假设：哺乳动物MSC是一种紧凑的结构，部分由一个 有序序列的共翻译相互作用，然而，错误组装或突变可以诱导额外的MSC 成分的积累，具有潜在的有害下游后果。我们将测试这个 通过（1）确定MSC四级结构和组分化学计量，和（2）确定MSC的分子量， 共翻译相互作用在MSC形成和完整性中的作用。我们预计， MSC的结构和组装将提供对MSC中的分子缺陷 这些成分可引起严重的病理学紊乱，特别是中枢神经系统的衰弱性疾病。

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.