Interactions of C-Reactive Protein with Highly Curved Lipid Membranes

描述(申请人提供)：通过利用纳米颗粒模拟低密度脂蛋白的脂质结构，C反应蛋白(CRP)如何区分受损的低密度脂蛋白和完整的低密度脂蛋白的机制将被阐明。这项研究的中心假设是，低密度脂蛋白脂膜的高曲率导致脂头基团暴露，然后被CRP识别。与这些暴露的头部基团的结合导致CRP结构的变化，揭示了促进补体激活的C1q结合部位和限制补体炎症途径的因子H的结合部位。这项研究的一个主要和新颖的工具是一系列脂质包裹的纳米颗粒，这些纳米颗粒经过调整，以匹配低密度脂蛋白变体的大小和组成，例如小而致密的低密度脂蛋白和氧化低密度脂蛋白，它们都与心血管疾病的风险增加相关。在脂质体上模拟的纳米颗粒的用途是能够独立地控制脂类成分和膜曲率。第一个特定的目标是表征C反应蛋白与具有不同程度弯曲程度的高脂膜结合后二级和三级结构的变化。具体地说，这个目标将利用荧光光谱和圆二色谱来测试CRP是否以相似的方式响应所有的膜，或者每个曲率是否导致蛋白质的不同构象。在第二个具体目标中，两项技术将探索当遇到受损的膜时，CRP的四级结构如何变化。C反应蛋白由5个相同的亚基组成，排列在一个五聚体中。当蛋白质结合到氧化的低密度脂蛋白等受损的膜上时，蛋白质的五聚体四级结构被破坏，但解离的机制尚不清楚。使用小分子探针，结合质谱学，将有可能评估蛋白质的四级结构在结合到具有不同曲率和不同脂质成分的膜上时如何变化。荧光能量转移实验将类似地探测四元结构的变化，并导致一种用于对CRP进行显微镜检查的工具。第三个具体目标是将C1q和因子H结合位点的暴露与特定膜结构的相互作用联系起来。这将揭示是脂质氧化还是脂膜形状的变化对确定CRP是否触发促炎或抗炎反应更为关键。这个项目创新性地使用纳米颗粒来解决一个非常具有挑战性的问题，即膜的性质如何影响蛋白质结合，而不同于脂类的性质。这将给患有心脏病的患者带来相当大的长期影响。每个目标都将提供有关CRP的新信息，这些信息可用于设计减轻慢性炎症和心脏病进展的药物。具体地说，被识别和表征的新的CRP构象状态可以被视为药物设计的目标，以最大限度地减少与心脏病风险较高相关的慢性炎症。

DESCRIPTION (provided by applicant): By utilizing nanoparticles that mimic the lipid structure of LDL, the mechanism of how C-reactive protein (CRP) distinguishes damaged LDL from intact LDL will be elucidated. The central hypothesis of this research is that high curvature in the lipid coating of LDL results in exposure of the lipid head groups that are then recognized by CRP. Binding to these exposed head groups results in a change to the CRP structure, revealing a C1q binding site that promotes complement activation and a binding site for factor H that limits the inflammatory pathway of complement. A primary and novel tool for this study is a series of lipid-coated nanoparticles that are tuned to match the size and composition of LDL variants, such as small dense LDL and oxidized LDL that are both correlated with increased risk for cardiovascular disease. The utility of a nanoparticle mimic over a liposome is the ability to independently control lipid composition and membrane curvature. The first specific aim is to characterize changes to the secondary and tertiary structure of CRP after it binds to highly lipid membranes with varying degrees of curvature. Specifically, this aim will utilize fluorescence spectroscopy and circular dichroism to test whether CRP responds to all membranes in a similar manner or whether each curvature results in a different conformation of the protein. In the second specific aim, two techniques will explore how the quaternary structure of CRP changes when it encounters a damaged membrane. CRP is composed of 5 identical subunits arranged in a pentamer. The pentameric quaternary structure of the protein is disrupted when the protein binds to damaged membranes such as oxidized LDL, but the mechanism of the dissociation is unclear. Using a small molecule probe, combined with mass spectrometry, it will be possible to assess how the quaternary structure of the protein changes when bound to membranes that have different curvatures and different lipid compositions. A fluorescence energy transfer experiment will similarly probe changes to the quaternary structure and lead to a tool for microscopy of CRP. The third specific aim is to correlate the exposure of C1q and factor H binding sites to interactions with specific membrane structures. This will reveal whether lipid oxidation or changes in lipid membrane shape are more critical to determining whether CRP triggers a pro- or anti- inflammatory response. This project makes an innovative use of nanoparticles to address the very challenging question of how membrane properties, distinct from lipid properties, influence protein binding. This will result in a substantial long-term impac for patients suffering from heart disease. Each aim will provide new information about CRP that can be utilized in the design of drugs that mitigate the effect of chronic inflammation and the progression of heart disease. Specifically, new conformational states of CRP that are identified and characterized can be considered as targets for the design of drugs that minimize the chronic inflammation associated with higher risk for heart disease.