A high-resolution structural approach to understanding HDL biogenesis

描述(由申请人提供)：尽管血浆高密度脂蛋白(高密度脂蛋白)水平升高与预防心血管疾病(CVD)密切相关，但新分泌的无脂载脂蛋白如何转变为有脂肪的高密度脂蛋白颗粒的分子细节仍不清楚。这种认识上的差距是一个严重的障碍，因为制药业正在进行一项重大努力，以确定提高血浆高密度脂蛋白水平的疗法。我们的长期目标是了解高密度脂蛋白是如何产生的，以及这一过程是否可以被操纵以获得治疗益处。在此基础上，我们的目标是从分子水平上理解两种典型的高密度脂蛋白载脂蛋白的脂结合结构转变：载脂蛋白A-I和载脂蛋白A-IV。由于这些蛋白质以不同的方式与脂质相互作用，我们的目标是定义一系列适用于更广泛的载脂蛋白家族的机制主题。我们的中心假设是，载脂蛋白的自结合是决定特定蛋白质产生高密度脂蛋白能力的关键特征。这是基于载脂蛋白普遍自结合的事实，以及我们的初步数据显示，他们如何实现这一点的明确和高度新颖的机制。我们的理论基础是，了解载脂蛋白在脂结合过程中的结构适应将为体内高密度脂蛋白的形成机制提供新的见解。为了验证我们的假设，我们将追求三个具体目标：1)确定无脂apoA-I和apoA-IV的结构和自结合机制；2)确定apoA-I和apoA-IV在脂结合状态下的结构；3)确定自结合的重要性并测试apoA-I和apoA-IV脂蛋白颗粒组装的分子折叠途径。在目标1和目标2中，我们将通过X射线结晶学在高密度脂蛋白颗粒组装过程的开始和结束时获得这些载脂蛋白急需的高分辨率结构，并辅之以基于溶液的光谱和交联实验。这些知识将在目标3中用于改变apoA-I和apoA-IV的自结合属性，并测试通过自发的脂质重组或通过基于细胞的高密度脂蛋白颗粒形成试验对生成高密度脂蛋白颗粒能力的影响。我们还将引入二硫键限制条件来测试这两种蛋白质进行的脂结合结构转变的详细方案。这项工作是创新的，因为我们发现了一种新的“螺旋交换”机制，可以解释apoA-IV的自我结合，也可能解释其他载脂蛋白。这提供了一个新的概念框架，在此基础上可以更好地了解载脂蛋白的功能，并为载脂蛋白在高密度脂蛋白颗粒中的排列提供了明确的基础。最后，这项工作意义重大，因为它将阐明在高密度脂蛋白形成期间发生的分子事件，以及从预先形成的高密度脂蛋白颗粒中解离载脂蛋白的机制，这两个关键过程都控制着高密度脂蛋白的新陈代谢。这一认识将有助于指导旨在提高血浆高密度脂蛋白的治疗策略的开发，以预防美国头号杀手心血管疾病。 公共卫生相关性：拟议的研究与公共健康相关，因为对高密度脂蛋白(高密度脂蛋白)生物发生的了解的增加将有助于指导旨在提高血浆高密度脂蛋白以预防心血管疾病的治疗策略的开发。心血管疾病是美国的头号杀手。因此，拟议的研究与NIH使命的一部分相关，该使命与开发基础知识以了解人类疾病的原因、预防和最终治愈有关。

DESCRIPTION (provided by applicant): Despite the strong relationship of elevated plasma levels of high density lipoproteins (HDL) with protection from cardiovascular disease (CVD), the molecular details of how newly secreted lipid-free apolipoproteins transition to lipidated HDL particles remains unknown. This gap in understanding is a critical hindrance as the pharmaceutical industry is in the midst of a major effort to identify therapies that raise plasma HDL levels. Our long term goal is to understand how HDL is generated and whether this process can be manipulated for therapeutic benefit. Pursuant to this, our objective here is to derive a molecular understanding of the lipid binding structural transitions of two prototypical HDL apolipoproteins: apolipoprotein (apo)A-I and apoA-IV. Since these proteins interact with lipid in distinct ways, we aim to define a range of mechanistic themes that will be applicable to the broader apolipoprotein family. Our central hypothesis is that apolipoprotein self-association is a critical feature that dictates a given protein's ability to generate HDL. This is based on the fact that apolipoproteins universally self-associate as well as our preliminary data showing a clear and highly novel mechanism for how they accomplish this. Our rationale is that understanding apolipoprotein structural adaptations during lipid binding will provide new insights into the mechanism of HDL formation in vivo. To test our hypothesis, we will pursue three specific aims: 1) Determine the structure and self-association mechanism of lipid-free apoA-I and apoA-IV; 2) Determine the structure of apoA-I and apoA-IV in their lipid-bound states; and 3) Determine the importance of self-association and test molecular folding pathways for apoA-I and apoA- IV lipoprotein particle assembly. In Aims 1 and 2 we will derive badly needed high-resolution structures of these apolipoproteins at the beginning and end of the HDL particle assembly process by X-ray crystallography, complemented by solution-based spectroscopic and cross-linking experiments. This knowledge will be used in Aim 3 to alter apoA-I and apoA-IV self-association properties and test the effects on ability to generate HDL particles either by spontaneous lipid reorganization or via cell-based HDL particle formation assays. We will also introduce disulfide constraints to test detailed schemes for the lipid-binding structural transitions undertaken by both proteins. This work is innovative because we have discovered a novel "helix swapping" mechanism that explains apoA-IV self-association and likely other apolipoproteins as well. This provides a new conceptual framework upon which a better understanding of apolipoprotein function can be built and it offers a clear basis for the arrangements that apolipoproteins adopt in HDL particles. Finally, the work is significant because it will illuminate the molecular events that occur during HDL formation as well as mechanisms for dissociation of apolipoproteins from pre-formed HDL particles, both critical processes governing HDL metabolism. This understanding will help guide the development of therapeutic strategies designed to raise plasma HDL for protection against CVD, the number 1 killer in the U.S. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because an increased understanding of the biogenesis of high density lipoproteins (HDL) will help guide the development of therapeutic strategies designed to raise plasma HDL for protection against cardiovascular disease, the # 1 killer in the U.S. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing fundamental knowledge for understanding the causes, prevention and eventually a cure for human diseases.