Interactions of C-Reactive Protein Isoforms With Oxidized Lipid Membranes Abstract: C-reactive protein (CRP) rises in serum in response to inflammation and is a promising marker for identifying patients at risk for cardiovascular disease (CVD). Elevated CRP levels have a strong correlation to myocardial infarction, ischemic stroke, and death. Fundamental questions about the function of CRP remain unanswered, limiting our understanding of why CRP levels predict CVD. It is known that CRP binds to oxidized phosopholipids on cell membranes at sites of inflammation. We are designing model systems that mimic the size, shape and ligand presentation of a CRP substrate, oxidized low density lipoprotein. These models will improve our understanding of proteins like CRP and their interactions with oxidized membranes at sites of inflammation. CRP has two forms: native, pentameric CRP (nCRP) and modified, monomeric CRP (mCRP). Each has distinct physiological roles with nCRP appearing to be more pro-inflammatory. However there are no commercially available tests that distinguish between these isoforms in immunohistochemistry, microscopy or cytometry. Understanding CRP as a predictor of CVD will require bioanalytical techniques for measuring each isoform and a better understanding of how these isoforms interact with membranes that contain oxidized lipids. The first specific aim of this research is to synthesize lipid-coated metal nanoparticle probes that mimic the size, curvature, and oxidation state of oxLDL. These nanoparticles will be used in conjunction with fluorescent aptamers in anisotropy-based sandwich assays. Natural lipoprotein particles change size and shape as their molecular composition changes. A benefit of this nanoparticle based approach is that the nanoparticles have a rigid core and their lipid composition can be changed without altering the size and shape of the metal core. These probes will be used to separately assess both the chemical and structural requirements for binding of CRP to oxidized membranes. The second specific aim is to design sensors capable of measuring the mCRP:nCRP ratio and to study how exposure to oxidized lipids alters this ratio. Aptamer based fluorescent probes that interact and undergo Fluorescence Resonance Energy Transfer will be used to measure the isoform ratio. In the third specific aim we will image the interaction of CRP with cell membranes using aptamer probes and test the nanoparticle sensors using serum samples. This project will provide innovative new tools that will improve understanding of how CRP predicts risk of CVD and will improve upon available diagnostics for this important protein.
Metal nanoparticle-lipid conjugates will be prepared as bioanalytical probes that mimic the properties of oxidized membranes. Fluorescent assays that distinguish the different CRP isoforms will be developed and tested on apoptotic cell surfaces. This research will provide innovative new tools for understanding how CRP predicts risk of cardiovascular disease and will lead to improved diagnostics for the protein.
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