Interactions of C-Reactive Protein with Highly Curved Lipid Membranes

描述（由申请人提供）：通过利用模拟LDL脂质结构的纳米颗粒，将阐明C-反应蛋白（CRP）如何区分受损LDL与完整LDL的机制。本研究的中心假设是LDL脂质涂层的高曲率导致脂质头部基团暴露，然后被CRP识别。与这些暴露的头部基团结合导致CRP结构的变化，揭示了促进补体激活的C1 q结合位点和限制补体炎症途径的H因子结合位点。这项研究的一个主要和新颖的工具是一系列脂质包覆的纳米颗粒，这些纳米颗粒经过调整以匹配LDL变体的大小和组成，例如小密度LDL和氧化LDL，这两者都与心血管疾病风险增加相关。与脂质体相比，纳米颗粒模拟物的效用是独立控制脂质组成和膜曲率的能力。第一个具体目标是表征CRP与具有不同曲率程度的高脂膜结合后其二级和三级结构的变化。具体而言，该目标将利用荧光光谱和圆二色性来测试CRP是否以类似的方式对所有膜做出响应，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，两种技术将探索CRP的四级结构在遇到受损膜时如何变化。CRP由5个相同的亚基组成，排列成五聚体。当蛋白质与受损的膜如氧化的LDL结合时，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，结合质谱法，将有可能评估蛋白质的四级结构在与具有不同曲率和不同脂质组成的膜结合时如何变化。荧光能量转移实验将类似地探测四级结构的变化，并导致CRP的显微镜检查工具。第三个具体目标是将C1 q和H因子结合位点的暴露与特定膜结构的相互作用相关联。这将揭示脂质氧化或脂质膜形状的变化是否对确定CRP触发促炎或抗炎反应更关键。该项目创新性地使用纳米颗粒来解决非常具有挑战性的问题，即与脂质性质不同的膜性质如何影响蛋白质结合。这将对患有心脏病的患者造成长期的严重影响。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。具体而言，被鉴定和表征的CRP的新构象状态可以被认为是用于设计药物的靶标，所述药物最小化与心脏病的较高风险相关的慢性炎症。

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.