Structural Plasticity of ApoAI & Lipid-binding Activity

描述（由申请人提供）：载脂蛋白A-I（apoAI）是一种243个残基的可交换载脂蛋白，在清除人体血流中的“坏胆固醇”中起关键作用。根据脂质化的程度，apoAI可以采用四种不同的构象之一，包括：（1）。无脂质apoAI构象（特异性磷脂结合）。（二）、ApoAI对前β-HDL（特异性胆固醇结合）的影响。（三）、ApoAI对盘状HDL的影响（特异性LCAT活化）。（四）、ApoAI对球形HDL（特异性SR-BI，HDL受体，结合）。这种构象可塑性确实使apoAI能够执行多种功能，调节/指导HDL的形成、成熟、运输和代谢。目前，我们还没有一个清晰的了解apoAI结构域如何允许这种结构可塑性。该建议集中于确定无脂质和前β-HDL结合的apoAl的NMR结构/动力学。 基于初步数据和其他实验室发表的结果，提出以下假设：（1）。无脂质apoAI主要采用螺旋束结构。 （二）、磷脂结合诱导apoAI的巨大构象变化，暴露胆固醇结合的疏水位点。 （三）、ApoAI/DPC模拟apoAI/pre-beta-HDL的结构和功能，因此可能对动脉粥样硬化具有治疗意义。 为了验证这些假设，我们提出了解决apoAI在三种不同状态下的NMR结构：（1）。无脂质状态，（2）。ApoAI/DPC状态，（3）. ApoAI/前β-HDL状态。还将在胆固醇结合活性方面进行apoAI/DPC和apoAI/前β-HDL的功能表征。此外，核磁共振技术将用于研究apoAI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的结构转换机制，转换apoAl的构象从一个到另一个。预计无脂质和前β-HDL结合的apoAI的结构将有助于理解apoAI如何募集脂质以启动HDL形成，以及apoAI如何促进前β-HDL募集更多中性脂质以使HDL成熟。由于低水平的血浆HDL和受损的HDL功能是包括动脉粥样硬化、糖尿病、肥胖症、中风和阿尔茨海默病的代谢紊乱/疾病的共同线索，因此从该提议获得的结果对于治疗这些代谢紊乱/疾病的新药物的干预应该具有重要意义。

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.