Lipid-protein interactions: Characterisation of the oxysterol mediated protein complexes by chemical proteomics

细胞功能依赖于包括蛋白质和脂质在内的生物分子集合的协调通信和作用。脂质通过其相互作用受体表现出多种生物学效应。含有脂质结合结构域的蛋白质在与其脂质配体结合后可以经历构象变化，这导致与新的伴侣相互作用或易位到另一个亚细胞区室。这样的多组分复合物在特定条件下是瞬时的或动态的，并且难以研究。研究脂质-蛋白质相互作用的常规生物化学方法通常需要大量材料，并且在“一次一个”的基础上进行。因此，在这项提案中，我们计划开发一个高通量的平台，在真实的时间捕获的空间和时间的“全球”脂质-蛋白质相互作用网络的图片，使用交联，脂质下拉，和蛋白质组学技术的组合。然后将使用定量蛋白质组学方法检查所鉴定的相互作用对蛋白质功能和细胞活性的影响。在本研究中重点关注的脂质类别是氧固醇。氧固醇是胆固醇的氧化衍生物，它们的失衡与动脉粥样硬化和神经退行性疾病有关。我们将使用开发的新方法来识别氧固醇受体，并揭示它们参与蛋白质的组装和拆卸。这些结果将有助于阐明生物学过程，了解氧固醇在健康状态中的重要性以及它们的不平衡如何可能是疾病的原因。

Cellular function relies on the coordinated communication and action of a collection of biomolecules including proteins and lipids. Lipids exhibit diverse biological effect via their interacting receptors. Proteins that contain a lipid binding domain can, upon binding of their lipid ligand, undergo a conformational change, which leads to interaction with new partners or translocation to another subcellular compartment. Such multicomponent complexes are transient, or dynamic under specific conditions, and difficult to investigate. Conventional biochemical methods to study lipid-protein interactions often require large amounts of material and are carried out on a 'one-at-a-time' basis. Thus, in this proposal we plan to develop a high throughput platform to capture in real time a picture of the spatial and temporal 'global' lipid-protein interaction network using a combination of cross-linking, lipid pull-down, and proteomics technologies. The identified interactions will then be examined for their effect on protein function and cellular activities using a quantitative proteomics approach. The class of lipids will be focused on in this study are oxysterols. Oxysterols are oxidized derivatives of cholesterol and their imbalance is implicated in atherosclerosis and neurodegenerative diseases. We will use the developed novel approach to identify oxysterol receptors and reveal their involvement in protein complexe assembly and disassembly. The results will potentially aid in the elucidation of biological process, the understanding of the importance of oxysterols in the healthy state and how their imbalance may be the cause of disease.