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，这是由蛋白质复合物向膜的协调募集引起的。这一过程的协调对于细胞生与死的几乎每一个方面都是至关重要的。研究蛋白质如何与脂质膜相互作用是非常具有挑战性的标准生物物理方法，并已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）中，我在定义脂质信号系统如何调节方面取得了巨大进展。我所提议的项目将继续并扩大这一成功的良好记录，以研究脂质信号蛋白的调节。这项研究计划的长期愿景是了解大分子组装体如何在脂质膜上招募，组装和调节的分子细节。研究计划的具体目标如下：目标1。明确鸟嘌呤核苷酸交换因子在细胞膜上调控的分子机制2.定义磷脂酶C（PLC）家族的酶是如何在膜上调节的方法在这个程序中研究的生物系统将全部使用最先进的结构生物学方法，包括HDX-MS，电子显微镜（EM）和XRC的独特协同作用。这种方法在加拿大是独一无二的，为受训人员提供了一个特殊的培训机会。研究中的许多复合物都非常大（>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这些复杂系统的研究至关重要，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告知新的策略，以优化成功获得高分辨率结构信息的可能性。具体而言，这种方法允许研究这些大蛋白质复合物如何组装的高分辨率结构信息，以及使用HDX-MS的动态研究，以确定发生在膜表面上的构象变化。这种方法将允许关键的洞察调节脂质信号的分子机制。该研究计划描述了我们的目标的长期愿景，以了解脂质信号在分子水平上，以及一组明确的和可实现的中期和短期目标，研究脂质信号系统的极端复杂性。这项研究将在理解脂质信号复合物在膜上调节的机制方面发挥至关重要的作用，这几乎涉及细胞生物学的所有方面。该提案对加拿大的直接影响是培训先进技术的高素质人才，包括分子生物学，HDX-MS，EM和XRC，这些都是生物技术领域的高需求。

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.