Structural Plasticity of ApoAI & Lipid-binding Activity

描述(申请人提供)：载脂蛋白A-I(ApoAI)是一种243个残基的可交换载脂蛋白，在清除人体血液中的“坏胆固醇”方面起着关键作用。根据脂化程度的不同，ApoAI可能采用四种不同的构象之一，包括：(1)。无脂ApoAI构象(特异性磷脂结合)。(2)。载脂蛋白AI对前β-高密度脂蛋白(特异性胆固醇结合)的影响。(3)。盘状高密度脂蛋白上的载脂蛋白AI(特异性LCAT激活)。(4)。ApoAI在球形高密度脂蛋白上(特异性SR-BI，高密度脂蛋白受体，结合)。这种构象可塑性确实使载脂蛋白AI能够执行多种功能，调节/指导高密度脂蛋白的形成、成熟、运输和代谢。目前，我们还不清楚ApoAI结构域是如何实现这种结构可塑性的。本建议集中在确定无脂和前-β-高密度脂蛋白结合的载脂蛋白A1的核磁共振结构/动力学。根据初步数据和其他实验室发表的结果，提出了以下假设：(1)无脂ApoAI主要采用螺旋束结构。(2)。磷脂结合导致ApoAI的构象发生戏剧性的变化，使胆固醇结合的疏水部位暴露出来。(3)。载脂蛋白AI/DPC模拟载脂蛋白AI/前β-高密度脂蛋白的结构和功能，可能对动脉粥样硬化有治疗意义。为了验证这些假设，我们提出了求解ApoAI在三种不同状态下的核磁共振结构：(1)。脱脂状态，(2)。ApoAI/DPC状态，(3)。载脂蛋白AI/前β-高密度脂蛋白状态。在胆固醇结合活性方面，还将进行载脂蛋白AI/DPC和载脂蛋白AI/前β-高密度脂蛋白的功能表征。此外，还将利用核磁共振技术研究载脂蛋白AI在这三种状态下的结构动力学。这些研究一起可能使我们能够解决结构转换机制，该机制将apoA1的构象从一种转换为另一种。预计无脂和结合前β-高密度脂蛋白的载脂蛋白AI的结构将有助于理解载脂蛋白AI是如何招募脂质来启动高密度脂蛋白的形成，以及载脂蛋白AI如何促进前β-高密度脂蛋白为高密度脂蛋白的成熟招募更多的中性脂质。由于低水平的血浆高密度脂蛋白和功能受损的高密度脂蛋白是代谢紊乱/疾病的共同主线，包括动脉粥样硬化、糖尿病、肥胖、中风和阿尔茨海默病，这项提议的结果应该对新药干预治疗这些代谢紊乱/疾病具有重要意义。

DESCRIPTION (provided by applicant): Apolipoprotein A-I (apoAI) is a 243-residue exchangeable apolipoprotein that plays a key role in clearing the "bad cholesterol" from the human blood stream. Depending on the extent of lipidation, apoAI may adopt one of four distinct conformations, including: (1). Lipid-free apoAI conformation (Specific phospholipid-binding). (2). ApoAI on pre-beta-HDL (Specific cholesterol binding). (3). ApoAI on discoidal HDL (Specific LCAT activation). (4). ApoAI on spherical HDL (Specific SR-BI, the HDL-receptor, binding). The conformational plasticity indeed enables apoAI to perform multiple functions which regulate/direct HDL formation, maturation, transport and metabolism. Currently we do not have a clear understanding of how the apoAI structural domains allow for this structural plasticity. This proposal concentrates on determining the NMR structures/dynamics of the lipid-free and pre-beta-HDL-bound apoAl. Based on preliminary data and published results by other laboratories, the following hypotheses are proposed: (1). Lipid-free apoAI mainly adopts a helix-bundle structure. (2). Phospholipid-binding induces a dramatic conformational change of apoAI that exposes the hydrophobic sites for cholesterol binding. (3). ApoAI/DPC mimics the structure and functions of apoAI/pre-beta-HDL, thus may have therapeutical implications to atherosclerosis. In order to verify these hypotheses, we propose to solve the NMR structures of apoAI in three different states: (1). Lipid-free state, (2). ApoAI/DPC state, (3). ApoAI/pre-beta-HDL state. Functional characterizations of apoAI/DPC and apoAI/pre-beta-HDL will also be performed, in terms of cholesterol-binding activity. In addition, NMR techniques will be utilized to study structural dynamic of apoAI in these three states. These studies together may allow us to address the structural switching mechanism, which converts apoAl's conformation from one to another. It is anticipated that the structures of lipid-free and pre-beta-HDL bound apoAI will help in the understanding of how apoAI recruits lipid to initiate the HDL formation, and how apoAI promotes pre-beta-HDL to recruit more neutral lipids for the maturation of HDL. Since a low level of plasma HDL and a compromised HDL function are the common thread of metabolic disorders/diseases including: atherosclerosis, diabetes, obesity, stroke, and Alzheimer's disease, the results obtained from this proposal should have significant implications for the intervention of new medicine to treat these metabolic disorders/diseases.