How are signalling complexes assembled and regulated on cellular membranes

信号复合物如何在细胞膜上组装和调​​节

• 批准号: RGPIN-2014-05218
• 负责人: Burke, John
• 金额: $ 3.79万
• 依托单位: University of Victoria
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=646548
• 关键词: How; signalling; complexes; assembled; regulated

Many cellular processes including growth, differentiation, and chemotaxis are controlled by the selective recruitment of large multi component protein complexes to lipid membranes. The correct recruitment of specific proteins to individual membrane compartments plays a major role in controlling the identity and function of different cellular organelles. The localisation of membrane binding proteins can be controlled by a number of factors including specific membrane and protein binding partners, as well as by more general features of the membrane including surface charge and curvature. Studying the molecular details of how proteins are enlisted, assembled, and regulated at membranes has remained a tremendous challenge due to the inherent difficulties of stabilising and reconstituting these complex protein membrane systems. **Research program objectives*Our goal is to identify the molecular details of membrane recruitment, assembly, and regulation of signalling enzymes using a cutting edge combination of novel proteomics based technology, specifically hydrogen deuterium exchange mass spectrometry, together with careful biophysical and biochemical characterisation. We will specifically target the Ras superfamily of small G proteins, as these proteins are membrane bound molecular switches that are able to selectively recruit a large number of protein effectors to membrane surfaces. This program aims to identify the mechanism of how they recruit and activate downstream effectors, specifically the phosphoinositide 3-kinase family of proteins, along with the mechanism of how the Ras superfamily members themselves are activated through the action of guanine nucleotide exchange factors (GEFs). Critical to this work will be the biochemical reconstitution of these proteins into protein membrane nanodiscs that will provide a stable platform for us to carry out detailed biochemical and structural studies. *In tandem with this approach we also plan to use the protein membrane nanodiscs as a platform to identify novel Ras superfamily binding proteins from cellular lysates using mass spectrometry. The major technical advantage of this system is that we can create nanodiscs with a variety of different lipid compositions, and hence are able to identify novel Ras superfamily binding partners that require the presence of both specific protein and membrane components. **This research is of broad impact as the development and application of novel proteomics based methodology in this proposal will be able to be applied to a wide variety of protein/membrane systems. The Ras supefamily of small G proteins constitute one of the most broadly utilised signalling pathways, yet many questions still remain in how they are regulated on membrane surfaces. Many enzymes that regulate or are regulated by Ras superfamily small GTPases undergo large conformational changes upon shifting from an inactive soluble form to an active membrane bound form. This program allows us to probe the molecular basis of this process, and will have wide ranging impact on the mechanism of how signalling complexes are assembled and regulated on membranes. Determining novel binding partners of small G protein signalling will also play a key role in advancing knowledge of fundamental cellular signalling pathways controlled by these signalling pathways.

许多细胞过程，包括生长，分化和趋化性是由大的多组分蛋白质复合物的选择性招募到脂质膜控制。特定蛋白质的正确募集到各个膜隔室在控制不同细胞器的身份和功能方面起着重要作用。膜结合蛋白的定位可以由许多因素控制，包括特定的膜和蛋白质结合伴侣，以及膜的更一般的特征，包括表面电荷和曲率。由于稳定和重建这些复杂的蛋白质膜系统的固有困难，研究蛋白质如何在膜上被征募、组装和调节的分子细节仍然是一个巨大的挑战。** 研究计划目标 * 我们的目标是确定膜招聘的分子细节，装配，并使用基于新的蛋白质组学技术，特别是氢氘交换质谱法的前沿组合调节信号酶，连同仔细的生物物理和生化表征。我们将专门针对Ras超家族的小G蛋白，因为这些蛋白质是膜结合的分子开关，能够选择性地招募大量的蛋白质效应物到膜表面。该计划旨在确定它们如何招募和激活下游效应物的机制，特别是蛋白质的磷酸肌醇3-激酶家族，沿着Ras超家族成员本身如何通过鸟嘌呤核苷酸交换因子（GEF）的作用被激活的机制。这项工作的关键是将这些蛋白质生化重组成蛋白质膜纳米盘，这将为我们进行详细的生化和结构研究提供一个稳定的平台。* 与这种方法相结合，我们还计划使用蛋白质膜纳米盘作为平台，使用质谱法从细胞裂解物中鉴定新型Ras超家族结合蛋白。该系统的主要技术优势是，我们可以创建具有各种不同脂质成分的纳米盘，因此能够识别需要特定蛋白质和膜组分存在的新型Ras超家族结合伴侣。** 本研究具有广泛的影响，因为本提案中基于新蛋白质组学的方法的开发和应用将能够应用于各种蛋白质/膜系统。Ras超家族的小G蛋白构成了最广泛使用的信号通路之一，但许多问题仍然存在于它们如何在膜表面上进行调节。调节Ras超家族小GTP酶或受Ras超家族小GTP酶调节的许多酶在从无活性可溶形式转变为活性膜结合形式时经历大的构象变化。该计划使我们能够探测这一过程的分子基础，并将对信号复合物如何在膜上组装和调节的机制产生广泛的影响。确定新的结合伙伴的小G蛋白信号也将发挥关键作用，在推进知识的基本细胞信号通路控制这些信号通路。