How are signalling complexes assembled and regulated on cellular membranes

许多细胞过程，包括生长、分化和趋化性，都是由大的多组分蛋白复合物在脂质膜上的选择性募集控制的。特定蛋白的正确募集到单个膜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伙伴，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固有的困难，研究蛋白质如何在膜上被招募、组装和调节的分子细节仍然是一个巨大的挑战。**研究项目目标*我们的目标是利用新型蛋白质组学技术的前沿组合，特别是氢氘交换质谱，以及仔细的生物物理和生化表征，确定膜招募，组装和信号酶调节的分子细节。我们将专门针对Ras超家族的小G蛋白，因为这些蛋白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器到膜表面。该计划旨在确定它们如何招募和激活下游效应物的机制，特别是磷酸肌苷3-激酶家族蛋白，以及Ras超家族成员如何通过鸟嘌呤核苷酸交换因子（gef）的作用被激活的机制。这项工作的关键将是这些蛋白质的生物化学重构成蛋白质膜纳米盘，这将为我们进行详细的生物化学和结构研究提供一个稳定的平台。与此方法相结合，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的Ras超家族结合蛋白。该系统的主要技术优势在于，我们可以创建具有各种不同脂质组成的纳米圆盘，因此能够识别需要特定蛋白质和膜组分存在的新型Ras超家族结合伙伴。**本研究具有广泛的影响，因为本提案中基于蛋白质组学的新方法的开发和应用将能够应用于各种蛋白质/膜系统。小G蛋白的Ras超家族构成了最广泛使用的信号通路之一，然而关于它们如何在膜表面被调节仍然存在许多问题。许多调节或受Ras超家族小gtpase调节的酶在从无活性可溶性形式转变为活性膜结合形式时发生了很大的构象变化。该计划使我们能够探索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。确定小G蛋白信号的新结合伙伴也将在推进这些信号通路控制的基本细胞信号通路的知识方面发挥关键作用。

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.