Effect of Apolipoprotein Structural Adaptability

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，设计一个实验策略来剖析一个有效的结合相互作用的决定因素。将采用一种称为表达蛋白连接的新策略，将稳定的同位素引入对LDLR结合至关重要的apoE的特定预定区域。要进行的研究包括三个具体目标。1)在与脂质结合的apoE的不同结构模型的实验中，将使用多维异核磁共振波谱分析标记apoe3 - n -末端结构域的片段同位素。2)确定apoE与LDLR的接触位点。假设分析标记为apoE3-NT7DMPC的片段同位素与功能性LDLR微型受体的复合物将获得它们结合相互作用的分子细节。3)研究apoE与LDL受体相关蛋白6 （LRP6）的相互作用。我们将评估位于LRP6跨膜序列附近的LDL-A重复序列参与载脂蛋白e配体结合的假设。此外，与人类受试者冠状动脉疾病相关的LRP6 （R611C）突变导致pH依赖性载脂蛋白e配体释放缺陷的假设将进行测试。这些实验的结果将扩展LDLR家族，载脂蛋白e介导的脂蛋白代谢和血浆脂质稳态调节的知识。基于LDLR通路的畸变与心血管疾病的发病正相关以及apoE对神经退行性疾病表现出异构体特异性易感性这一事实，我们预计从这些研究中获得的新知识将为了解调节健康和疾病关键代谢过程的分子机制提供见解。

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.