Effect of Apolipoprotein Structural Adaptability

描述(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代谢和疾病易感性。了解载脂蛋白(Apo)E和低密度脂蛋白受体(LDLR)家族成员的代谢作用，将与现有的结构信息相结合，设计一种实验策略，分析有效结合相互作用的决定因素。一种名为表达蛋白连接的新策略将被用于将稳定的同位素引入到载脂蛋白E的特定的、预定的区域，该区域是LDLR结合所必需的。将进行的研究包括三个具体目标。1)片段同位素标记的apoE3-N-端粒酶结构域，在与脂质结合的复合体中，将在实验中用多维异核核磁共振进行分析，以区分脂结合的apoE的不同结构模型。2)将描述apoE和LDLR之间的接触位置。假设对片段同位素标记的apoE3-NT7DMPC与功能LDLR微型受体的复合物的分析将获得它们结合作用的分子细节。3)研究载脂蛋白E与低密度脂蛋白受体相关蛋白6(LRP6)的相互作用。将评估位于LRP6跨膜跨越序列附近的LDL-A重复参与apoE配体结合的假设。此外，将测试与人类冠状动脉疾病相关的LRP6(R611C)突变导致pH依赖的apoE配体释放缺陷的假设。这些实验结果将扩大对低密度脂蛋白受体家族、载脂蛋白E介导的脂蛋白代谢和调节血浆脂平衡的知识。基于LDLR通路的异常与心血管疾病的发病呈正相关，以及apoE表现出对神经退行性疾病的同型特异性易感性，我们预计从这些研究中获得的新知识将为深入了解调节健康和疾病中关键代谢过程的分子机制提供帮助。 公共卫生相关性：载脂蛋白(Apo)E是一种重要的全身脂肪平衡调节剂。载脂蛋白E的生物学功能是通过与细胞表面受体家族的相互作用而表现出来的。拟议的研究将表征载脂蛋白E的受体活性结构以及它与低密度脂蛋白受体家族的两个成员的结合作用。所获得的结果将加深对载脂蛋白E如何调节脂代谢及其与疾病易感性的关系的理解。

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.