Capturing the organisation of the Mycoplasma pneumoniae proteome with in-cell crosslinking mass spectrometry

利用细胞内交联质谱法捕获肺炎支原体蛋白质组的组织

• 批准号: 426290502
• 负责人: Professor Dr. Juri Rappsilber
• 金额: --
• 依托单位: Fachgebiet Bioanalytik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/426290502?language=en
• 关键词: Capturing; organisation; Mycoplasma; pneumoniae; proteome

Cells have complex internal organization which cannot be fully captured via ex vivo experiments. For example, phase transitions and “membrane-less organelles” have been shown by fluorescence microscopy and cryo-electron tomography. A recent paper involving us identified “protein communities” as a potential level of cellular organisation above that of “core” protein complexes that is often lost during purification (Kastritis, O’Reilly et al. 2017) and is ideally studied in situ. Very little is known about this higher-order organisation of the proteome. Currently our most comprehensive understanding for how the proteome is organised has come from large-scale proteomics studies that identified protein complexes by affinity-purification/mass spectrometry or co-fractionation. Importantly these techniques do not produce topological information for the complexes discovered and so complexes have needed to be purified for further structural analysis, potentially causing associated factors to be lost. Additionally, insoluble or membrane associated complexes are usually missed. These are issues that in-cell crosslinking mass spectrometry (CLMS) can address. In this proposal, we will analyze the topology of the proteome of the simple model pathogenic organism Mycoplasma pneumoniae and discover how far biological processes are organised independent of organelles. In particular, we will investigate the expressosome as a previously identified ‘higher-order’ complex in M. pneumoniae for which little structural information is known. Immunoprecipitation experiments have shown that there is direct coupling of transcription and translation in M. pneumoniae. However no in situ structural investigation has been conducted so any associated factors necessary for this interaction are unknown.In-cell CLMS combined with integrative structural modelling techniques will build an organisational model of the M. pneumoniae proteome in situ. The M. pneumoniae proteome is particularly useful here as a model organism for two reasons: firstly, it is simple enough to allow for comprehensive analysis by CLMS; secondly, many previous system-wide studies have been performed and so are a valuable resource for validation. Our preliminary work has already shown that we can detect a large number of previously unknown interactions and can further structurally validate selected interactions through the powerful combination with in-cell cryo-electron tomography in collaboration with the group of Julia Mahamid at EMBL.The systems-level structural insights gathered in this study, while interesting in general for cell biologists, will also indicate potential targets for novel therapies against this pathogen.

细胞具有复杂的内部组织，不能通过体外实验完全捕获。例如，荧光显微镜和低温电子断层扫描显示了相变和“无膜细胞器”。最近一篇涉及我们的论文确定了“蛋白质群落”是一种潜在的细胞组织水平，高于“核心”蛋白质复合体的水平，这种复合体在纯化过程中经常丢失(Kasttis，O‘Reilly等人)。2017)，是理想的现场研究对象。人们对蛋白质组的这种更高层次的组织知之甚少。目前，我们对蛋白质组如何组织的最全面的了解来自大规模的蛋白质组学研究，这些研究通过亲和纯化/质谱学或共分馏鉴定蛋白质复合体。重要的是，这些技术不会为所发现的络合物产生拓扑信息，因此需要对络合物进行纯化以进行进一步的结构分析，这可能会导致相关因素的丢失。此外，不溶性或与膜相关的络合物通常被遗漏。这些都是细胞内交联质谱(CLMS)可以解决的问题。在这项提案中，我们将分析简单的模式致病生物肺炎支原体的蛋白质组的拓扑结构，并发现生物过程在多大程度上是独立于细胞器组织的。特别是，我们将研究作为以前在肺炎支原体中发现的‘更高阶’复合体的扁平体，它的结构信息知之甚少。免疫沉淀实验表明肺炎支原体中存在转录和翻译的直接偶联。然而，还没有进行原位结构研究，因此这种相互作用所需的任何相关因素都是未知的。细胞内CLMS与综合结构建模技术相结合将建立原位肺炎支原体蛋白质组的组织模型。肺炎支原体蛋白质组在这里作为模式生物特别有用，原因有两个：第一，它足够简单，可以用CLMS进行全面分析；第二，以前已经进行了许多系统范围的研究，因此是一个宝贵的验证资源。我们的初步工作已经表明，我们可以检测到大量先前未知的相互作用，并可以通过与细胞内冷冻电子断层扫描的强大组合，进一步在结构上验证选定的相互作用。这项研究收集的系统水平的结构见解，虽然一般对细胞生物学家感兴趣，但也将指明针对这种病原体的新疗法的潜在靶点。