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:10531618
- 负责人:
- 金额:$ 48.19万
- 依托单位:
- 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:
- 关键词:3-DimensionalAllelesAmino AcidsAmino Acyl-tRNA SynthetasesArchitectureAtrophicBindingBiochemicalCell physiologyCellsCentral Nervous System DiseasesCerebrumChildComplexComputer softwareCoupledCritical ThinkingCrosslinkerCytoplasmCytoplasmic ProteinDNA Sequence AlterationDataData AnalysesDefectDiseaseEtiologyEventExhibitsFluorescenceFunctional disorderGenesGeneticGlutamine-tRNA ligaseGoalsGrantHealthHumanImpaired cognitionLengthLigaseMammalian CellMass Spectrum AnalysisMessenger RNAMethodsMicrocephalyModelingMolecularMultiprotein ComplexesMutateMutationMyelinNeurodegenerative DisordersNomenclaturePathologicPathologyPeptidesPhotobleachingPositioning AttributeProtein BiosynthesisProteinsReportingRibosomesRoleScaffolding ProteinSeizuresSodium ChlorideSpecificityStimulusStructural ModelsStructureSulfoxideTestingTranslationscrosslinkdensitydesignexperimental studyfluorescence imagingimprovedinsightinstrumentationinterestknock-downleukodystrophymRNA Translationmolecular modelingmotor impairmentneuropathologyprogramsprotein complexprotein crosslinksingle moleculespatial relationshipstoichiometrytool
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,装配、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-
QARS 1和EPRS 1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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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PAUL L FOX其他文献
PAUL L FOX的其他文献
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{{ truncateString('PAUL L FOX', 18)}}的其他基金
The Untranslated 3'End of SARS-CoV-2 RNA as a Determinant of Obesity-Accelerated Infectivity
SARS-CoV-2 RNA 的非翻译 3 末端是肥胖加速感染的决定因素
- 批准号:
10318871 - 财政年份:2021
- 资助金额:
$ 48.19万 - 项目类别:
The Untranslated 3'End of SARS-CoV-2 RNA as a Determinant of Obesity-Accelerated Infectivity
SARS-CoV-2 RNA 的非翻译 3 末端是肥胖加速感染的决定因素
- 批准号:
10689137 - 财政年份:2021
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Assay Development for Discovery of a Small Molecule Inhibitor of a Novel Metabolic Pathway that Drives Obesity
发现导致肥胖的新型代谢途径的小分子抑制剂的检测方法开发
- 批准号:
10320035 - 财政年份:2020
- 资助金额:
$ 48.19万 - 项目类别:
Assay Development for Discovery of a Small Molecule Inhibitor of a Novel Metabolic Pathway that Drives Obesity
发现导致肥胖的新型代谢途径的小分子抑制剂的检测方法开发
- 批准号:
10115720 - 财政年份:2020
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Multisite phosphorylated S6K1 directs a regulatory module determining adipocyte lipid metabolism
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10349543 - 财政年份:2020
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