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

细胞的功能依赖于包括蛋白质和脂类在内的一系列生物分子的协调沟通和行动。脂质通过其相互作用的受体表现出不同的生物学效应。含有脂质结合域的蛋白质，一旦与其脂质配体结合，就会发生构象变化，从而导致与新伙伴的相互作用或移位到另一个亚细胞室。这种多组分的络合物是瞬变的，或者在特定条件下是动态的，很难研究。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，而且是在一次一个的基础上进行的。因此，在这项提案中，我们计划开发一个高通量平台，利用交联剂、脂类下拉和蛋白质组学技术的组合，实时捕捉空间和时间上的全球脂质-蛋白质相互作用网络的图像。然后将使用定量蛋白质组学方法检查已识别的相互作用对蛋白质功能和细胞活动的影响。这项研究中将重点关注的一类脂质是氧固醇类。氧化甾醇是胆固醇的氧化衍生物，其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络合物的组装和拆卸。这些结果可能有助于阐明生物过程，理解氧化甾醇在健康状态下的重要性，以及它们的失衡可能是疾病的原因。

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.