How are lipid signalling complexes assembled on membranes

大多数细胞对信号的反应都有一个共同的调节成分，源于蛋白质复合体在膜上的协调募集。这个过程的协调对于细胞生死的几乎每一个方面都是必不可少的。用标准的生物物理方法研究蛋白质如何与脂膜相互作用是极具挑战性的，也是在分子水平上理解信号的主要障碍。在NSERC支持的过去六年(2014-2019年)，我在定义如何监管脂质信号系统方面取得了巨大进展。我提议的计划将在这一成功的成功记录的基础上继续并扩大，以检查脂质信号蛋白的调节。这项研究计划的长期愿景是了解如何招募、组装和调节脂膜上的大分子组件的分子细节。该研究计划的具体目标如下：目标1.确定膜上鸟嘌呤核苷酸交换因子(GEF)调节的分子机制2.确定磷脂酶C(PLC)家族如何在膜上调节方法本计划中研究的生物系统都将使用一种独特的协同最先进的结构生物学方法，包括HDX-MS，电子显微镜(EM)和XRC。这种方法在加拿大是独一无二的，为学员提供了一个特殊的培训机会。许多正在研究的复合体非常大(&gt；500 kDa)，并且由多种不同的蛋白质组成。这些方法的串联应用对于研究这些复杂的系统是必不可少的，重要的是这些工具将以协同的方式应用，因此不同的方法可以提供新的战略，以优化在获得高分辨率结构信息方面的成功可能性。具体地说，这种方法允许研究这些大的蛋白质复合体是如何组装的高分辨率结构信息，以及使用HDX-MS来定义膜表面发生的构象变化的动态研究。这种方法将允许对调节脂质信号的分子机制有重要的洞察力。影响和意义这项研究计划描述了我们在分子水平上了解脂质信号的长期目标，以及研究极端复杂的脂质信号系统的一套明确和可实现的中短期目标。这项研究将对理解膜上脂质信号复合体的调控机制起到至关重要的作用，这涉及到细胞生物学的几乎所有方面。这一建议对加拿大的直接影响是培训高素质的高级技术人员，包括分子生物学、HDX-MS、EM和XRC，这些都是生物技术部门的高需求。

Most cellular responses to signals have a common component of regulation arising from the coordinated recruitment of protein complexes to membranes. The coordination of this process is essential to almost every aspect of a cell's life and death. Studying how proteins interact with lipid membranes is extremely challenging by standard biophysical approaches, and has been a major hurdle in understanding signalling at the molecular level. In the last six years of NSERC support (2014-2019), I have made tremendous progress in defining how lipid signalling systems are regulated. The program I am proposing will continue and expand on this strong track record of success to examine the regulation of lipid signalling proteins. The long-term vision of this research program is to understand the molecular details of how large macromolecular assemblies are recruited, assembled, and regulated on lipid membranes. The specific objectives of the research program are as follows: Objectives  1. Define the molecular mechanism of regulation of guanine nucleotide exchange factors (GEFs) on membranes 2. Define how the Phospholipase C (PLC) family of enzymes is regulated on membranes Approach The biological systems under study in this program will be all be approached using a unique synergy of state of the art structural biology approaches, including HDX-MS, Electron Microscopy (EM), and XRC. This approach is unique within Canada, and provides trainees with an exceptional training opportunity. Many of the complexes under study are extremely large (>500 kDa), and are composed of multiple different proteins. The tandem application of these approaches is essential to the study of these complicated systems, and importantly these tools will be applied in a synergistic way, so that the different approaches can inform novel strategies to optimise the likelihood of success in obtaining high resolution structural information. Specifically, this approach allows for the study of high resolution structural information on how these large protein complexes are assembled, together with dynamic studies using HDX-MS to define conformational changes that occur on membrane surfaces. This approach will allow for critical insight into the molecular mechanisms regulating lipid signalling. Impact and significance This research program describes a long term vision of our goals to understand lipid signalling at the molecular level, as well as a set of clear and achievable medium and short term objectives to study lipid signalling systems of extreme complexity. This research will play a vital role in understanding the mechanisms by which lipid signalling complexes are regulated on membranes, which touches on almost all aspects of cell biology. The immediate impact of this proposal on Canada is the training of highly qualified personnel in advanced techniques including molecular biology, HDX-MS, EM, and XRC, which are all in high demand in the biotechnology sector.