A high-resolution structural approach to understanding HDL biogenesis

描述（由申请人提供）：尽管高密度脂蛋白（HDL）血浆水平升高与心血管疾病（CVD）保护密切相关，但新分泌的无脂质载脂蛋白如何转变为脂化HDL颗粒的分子细节仍未知。这种理解上的差距是一个关键的障碍，因为制药行业正在努力确定提高血浆HDL水平的疗法。我们的长期目标是了解HDL是如何产生的，以及这个过程是否可以被操纵以获得治疗益处。根据这一点，我们的目标是在这里得到的两个原型HDL载脂蛋白：载脂蛋白（apo）A-I和apoA-IV的脂质结合结构转变的分子理解。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的机制主题，将适用于更广泛的载脂蛋白家族。我们的中心假设是，载脂蛋白的自我关联是一个重要的功能，决定了一个给定的蛋白质的能力，以产生高密度脂蛋白。这是基于载脂蛋白普遍自缔合的事实，以及我们的初步数据显示了它们如何实现这一点的明确和高度新颖的机制。我们的基本原理是，了解脂质结合过程中载脂蛋白的结构适应将为体内高密度脂蛋白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）确定无脂质的apoA-I和apoA-IV的结构和自缔合机制; 2）确定apoA-I和apoA-IV在其脂质结合状态下的结构; 3）确定自缔合的重要性并测试apoA-I和apoA-IV脂蛋白颗粒组装的分子折叠途径。在目标1和2中，我们将通过X射线晶体学，辅以基于溶液的光谱和交联实验，在HDL颗粒组装过程的开始和结束时获得急需的这些载脂蛋白的高分辨率结构。这一知识将用于目标3，以改变apoA-I和apoA-IV的自缔合特性，并通过自发脂质重组或通过基于细胞的HDL颗粒形成测定来测试对生成HDL颗粒的能力的影响。我们还将引入二硫键限制来测试两种蛋白质所进行的脂质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的自缔合和其他载脂蛋白。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，它提供了一个明确的基础，载脂蛋白在HDL颗粒中采用的安排。最后，这项工作是重要的，因为它将阐明HDL形成过程中发生的分子事件以及载脂蛋白从预形成的HDL颗粒中解离的机制，这两个关键过程都是HDL代谢的关键过程。这种理解将有助于指导旨在提高血浆HDL以预防CVD的治疗策略的发展，CVD是美国的头号杀手。

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.