Regulation of Peripheral Protein-Membrane Interactions by Lipid Second Messengers

描述(申请人提供)：在细胞和细胞外水平，心血管疾病和其他疾病的特征是脂类结构(如脂蛋白和细胞膜)的富含磷脂的表面发生动态重塑。这种重塑包括在表面产生脂类代谢物，称为脂类第二信使，例如二酰甘油。对一种产生这些代谢物的脂肪酶的研究表明，它们的存在是脂肪酶从周围的水介质转移到其发挥作用的表面的关键要求。此外，脂肪酶转位需要这些脂质第二信使的水平超过临界值。低于此值，甘油二酯与表面磷脂形成络合物，转位受到抑制。我们假设，这种调节机制不仅对脂肪酶的功能至关重要，而且对细胞内稳态和疾病过程至关重要的许多其他结构无关的蛋白质也是必不可少的。该项目的目标是通过确定二酰甘油调节特定蛋白质表面寻找的独立折叠单位(即结构域)向高度受控的模型脂质表面的移位的程度和机制来检验这一假说。目的1研究确定二酰甘油和其他脂类物种在表面预先存在的分布如何调节结构域转移到表面的能力。目的2研究确定结构域与表面二酰甘油的侧向相互作用如何调节其在表面停留的时间。目的3项研究确定了存在于表面的多结构域蛋白的一个结构域如何与二酰甘油相互作用，从而产生一个“自组装受体”，从而影响该蛋白的另一个寻求表面结构域的移位。这些蛋白结构域是载脂蛋白E、MUNC 13-1和胰腺脂肪酶的一部分，并作为一系列其他生物重要蛋白的模型，这些蛋白具有共同的结构域基序。能够进行这些研究是为该项目开发的新方法和独特的新工具。该项目的完成将使人们更好地了解甘油二酯调节基本生物过程的机制，如胆固醇清除(载脂蛋白E)和神经传递(MUNC 13-1)。此外，研究结果有望为调节严重依赖于脂质表面组织的其他过程提供策略。

DESCRIPTION (provided by applicant): At both cellular and extracellular levels, cardiovascular and other diseases are characterized by the dynamic remodeling of the phospholipid-rich surfaces of lipid-based structures, like lipoproteins and cell membranes. This remodeling involves the generation in the surface of lipid metabolites, termed lipid second messengers, exemplified by diacylglycerols. Studies of one lipase that generates these metabolites have shown that their presence is a key requirement for the lipase to translocate from the surrounding aqueous medium to the surface where it functions. Moreover, lipase translocation requires a level of these lipid second messengers that exceeds a critical value. Below this value diacylglycerols are complexed with surface phospholipids and translocation is inhibited. We hypothesize that this regulatory mechanism is essential to the functions of not only lipases but many other, structurally-unrelated proteins that are crucial to cellular homeostasis and disease processes. The goal of this project is to test that hypothesis by determining the extent to which and mechanism by which diacylglycerol regulates the translocation of surface-seeking, independent folding units of selected proteins, i.e. domains, to and from highly controlled model lipid surfaces. Aim 1 studies determine how preexisting distribution of diacylglycerol and other lipid species in the surface regulates the ability of a domain to translocate to the surface. Aim 2 studies determine how lateral interaction of the domain and diacylglycerol within the surface regulates the time it spends at the surface. Aim 3 studies determine how the presence of one domain of a multi-domain protein residing in a surface can interact with diacylglycerol to produce a "self-assembled receptor" for effecting the translocation of another surface-seeking domain of that protein. The protein domains are parts of apolipoprotein E, munc 13-1 and pancreatic lipase and serve as models for a range of other biologically important proteins that share their domain motifs. Enabling these studies to be carried out is new methodology and unique new instrumentation developed for this project. The completion of this project will provide better understanding of the mechanisms by which diacylglycerols regulate essential biological processes, like cholesterol clearance (apolipoprotein E) and nerve transmission (munc 13-1). Moreover, results are expected to suggest strategies for regulating other processes which depend critically on the lipid surface organization.