Effect of Apolipoprotein Structural Adaptability

描述（由申请人提供）：描述我们研究的长期目标是了解脂蛋白和细胞表面受体之间的分子相互作用如何影响脂质代谢和疾病易感性。载脂蛋白（apo）E和低密度脂蛋白受体（LDLR）家族成员的代谢作用的知识将与现有的结构信息相结合，在设计的实验策略，解剖生产性结合相互作用的决定因素。一种新的策略，被称为表达蛋白连接，将被用来引入一个特定的，预定的，区域的载脂蛋白E，这是必需的LDLR结合稳定的同位素。要进行的研究包括三个具体目标。1)在设计用于区分脂质结合apoE的替代结构模型的实验中，将通过多维异源NMR光谱分析与脂质复合的分段同位素标记的apoE 3-N-末端结构域。2)将表征apoE和LDLR之间的接触位点。假设分析与功能性LDLR微型受体复合的节段性同位素标记的apoE 3-NT 7 DMPC将产生其结合相互作用的分子细节。3)研究apoE与LDL受体相关蛋白6（LRP 6）的相互作用。将评价位于LRP 6跨膜序列附近的LDL-A重复序列参与apoE配体结合的假设。此外，将测试与人类受试者的冠状动脉疾病相关的LRP 6（R611 C）突变导致pH依赖性apoE配体释放缺陷的假设。这些实验的结果将扩展知识的LDLR家族，载脂蛋白E介导的脂蛋白代谢和调节血脂稳态。基于LDLR通路中的畸变与心血管疾病的发作呈正相关，apoE表现出对神经退行性疾病的亚型特异性易感性，我们预计从这些研究中获得的新知识将为调节健康和疾病中关键代谢过程的分子机制提供见解。 公共卫生相关性：载脂蛋白（apo）E是全身脂质稳态的重要调节剂。apoE的生物学功能通过与细胞表面受体家族的相互作用而显现。拟议的研究将表征载脂蛋白E的受体活性结构以及其与低密度脂蛋白受体家族的两个成员的结合相互作用。研究结果将有助于进一步了解载脂蛋白E如何调节脂质代谢及其与疾病易感性的关系。

DESCRIPTION (provided by applicant): Description The long-term goal of our research is to understand how molecular interactions between lipoproteins and cell surface receptors affect lipid metabolism and disease susceptibility. Knowledge of the metabolic roles of apolipoprotein (apo) E and low-density lipoprotein receptor (LDLR) family members will be combined with available structural information in the design of an experimental strategy to dissect determinants of a productive binding interaction. A novel strategy, termed expressed protein ligation, will be employed to introduce stable isotopes into a specific, predetermined, region of apoE that is essential for LDLR binding. The research to be pursued includes three specific aims. 1) Segmental isotope labeled apoE3-N-temrinal domain, in complex with lipid, will be analyzed by multidimensional heteronuclear NMR spectroscopy in experiments designed to distinguish between alternate structural models of the lipid-bound apoE. 2) The contact sites between apoE and the LDLR will be characterized. It is hypothesized that analysis of segmental isotope labeled apoE3-NT7DMPC in complex with a functional LDLR mini-receptor will yield molecular details of their binding interaction. 3) The interaction of apoE with LDL receptor related protein 6 (LRP6) will be studied. The hypothesis that LDL-A repeats located near the transmembrane spanning sequence of LRP6 are involved in apoE ligand binding will be evaluated. In addition, the postulate that a mutation in LRP6 (R611C), associated with coronary artery disease in human subjects, causes a defect in pH dependent apoE ligand release will be tested. The results of these experiments will extend knowledge of the LDLR family, apoE-mediated lipoprotein metabolism and regulation of plasma lipid homeostasis. Based on the fact that aberrations in the LDLR pathway are positively correlated to onset of cardiovascular disease and apoE manifests isoform-specific susceptibility to neurodegenerative diseases, we anticipate that new knowledge gained from these studies will provide insight into molecular mechanisms that regulate key metabolic processes in health and disease. PUBLIC HEALTH RELEVANCE: Apolipoprotein (apo) E is an important modulator of whole body lipid homeostasis. Biological functions of apoE are manifest through interactions with a family of cell surface receptors. Proposed research will characterize the receptor-active structure of apoE as well as its binding interaction with two members of the low-density lipoprotein receptor family. Results obtained will improve understanding of how apoE regulates lipid metabolism and its association with disease susceptibility.