Apolipoprotein conformation in amyloid and cardiovascular diseases

项目总结/摘要 这项资助的总体目标是从分子的细节上了解载脂蛋白（APO）的作用 和脂蛋白在健康和疾病中的作用。目前的焦点是人apoA-I，血浆中的主要蛋白质 高密度脂蛋白（HDL，a.k.a.好胆固醇），从细胞中清除胆固醇， 心血管疾病ApoA-I可以以不稳定的贫脂/游离状态从HDL中释放，其是ApoA-I的前体。 淀粉样蛋白，并可引起两种形式的系统性人类淀粉样变性。在获得性形式中，apoA-I沉积在 动脉纤维化，这加剧了动脉粥样硬化。在遗传形式中，突变型apoA-I的片段存款 重要器官（肾脏、肝脏、神经等）并损坏它们。这种危及生命的疾病无法治愈 目前唯一的治疗方法就是器官移植为了确定治疗目标，我们必须了解 决定了淀粉样蛋白前体的产生、其清除及其 错误折叠，从游离apoA-I中的天然螺旋束结构到淀粉样蛋白中的不溶性交叉折叠。 揭示蛋白质错误折叠和蛋白质稳态的复杂过程以阻断系统性淀粉样变性已经成为 很有挑战性但并非不可能为此，我们整合了高分辨率和低分辨率的结构和 光谱方法（圆二色性、荧光、氢氘交换等）与生化 和计算工具。对几种致病突变体的分析使我们能够提出第一个分子 apoA-I错误折叠的机制。我们推测，淀粉样蛋白“热点”的扰动包装与 天然折叠的结构完整性使蛋白质淀粉样变性。这个想法可以推广到其他亲- 它有助于解释为什么许多球状蛋白质的结构不稳定既不必要也不充分 导致淀粉样疾病这些和其他新的想法将在下一轮赠款中得到严格的检验。 目的1阐明遗传性淀粉样变性中apoA-I错误折叠的分子机制。我们将确定 致病突变如何扰乱敏感片段中的天然蛋白质构象， 聚集与蛋白水解。基于细胞的研究将揭示为什么突变携带者的风险较低， 动脉粥样硬化，尽管血浆HDL水平低。目标2将确定 遗传性和获得性淀粉样变性的分子基础，并将建立结构-毒性关系， apoA-I的聚集形式。目的3将明确脂质在apoA-I错误折叠中的作用。这一目标将考验我们的 假设降脂方法对apoA-I淀粉样变性具有治疗潜力。 这项研究开辟了一个新的前沿，超越了蛋白质稳定性，以确定蛋白质错误折叠的关键驱动因素 in vivo.这些结果将揭示apoA-I的淀粉样蛋白生成和心脏保护特性之间的联系， 找到apoA-I淀粉样变性的治疗方法，对其他突出的apo错误折叠产生更清晰的见解， 在人类淀粉样变性中，并且对由其他球状蛋白引起的错误折叠疾病具有广泛的影响。

PROJECT SUMMARY / ABSTRACT The overall goal of this grant has been to understand in molecular details the action of apolipoproteins (apos) and lipoproteins in health and disease. The current focus is on human apoA-I, the major protein of plasma high-density lipoproteins (HDL, a.k.a. good cholesterol) that remove cholesterol from cells and protect against cardiovascular disease. ApoA-I can be released from HDL in a labile lipid-poor/free state that is the precursor of amyloid, and can cause two forms of systemic human amyloidosis. In the acquired form, apoA-I deposits in the arteries as fibrils, which augments atherosclerosis. In the hereditary form, fragments of mutant apoA-I deposit in vital organs (kidney, liver, nerves, etc.) and damage them. There is no cure for this life-threatening disease and the only current treatment is organ transplant. To pinpoint therapeutic targets, we must understand what determines the dynamic equilibrium between the generation of the amyloid precursor, its clearance, and its misfolding, from the native helix-bundle structure in free apoA-I to the insoluble cross--sheet in amyloid. Unraveling the complex process of protein misfolding and proteostasis to block systemic amyloidosis has been very challenging but not impossible. To this end we have integrated high- and low-resolution structural and spectroscopic methods (circular dichroism, fluorescence, hydrogen-deuterium exchange, etc.) with biochemical and computational tools. Analysis of several disease-causing mutants enabled us to propose the first molecular mechanism of apoA-I misfolding. We postulated that perturbed packing in amyloid `hot spots' combined with the structural integrity of the native fold make the protein amyloidogenic. This idea can be extended to other pro- teins; it helps explain why structural destabilization of many globular proteins is neither necessary nor sufficient to cause amyloid disease. These and other new ideas will be rigorously tested in the next cycle of this grant. Aim 1 will elucidate the molecular mechanism of apoA-I misfolding in hereditary amyloidosis. We will determine how the disease-causing mutations perturb the native protein conformation in sensitive segments to promote -aggregation vs. proteolysis. Cell-based studies will unveil why mutation carriers are at a low risk of atherosclerosis despite low levels of plasma HDL. Aim 2 will identify the similarities and differences in the molecular basis for hereditary and acquired amyloidoses, and will establish the structure-toxicity relationship in aggregated forms of apoA-I. Aim 3 will define the role of lipids in apoA-I misfolding. This aim will test our hypothesis that lipid-lowering approaches hold therapeutic potential for apoA-I amyloidoses. This research opens a new frontier that goes beyond protein stability to identify key drivers of protein misfolding in vivo. The results will unveil the link between amyloidogenic and cardioprotective properties of apoA-I, help find therapies for apoA-I amyloidoses, yield sharper insights into the misfolding of other apos that are prominent in human amyloidoses, and have broad implications for misfolding diseases caused by other globular proteins.