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Development of a clinically-viable measure of HDL functionality

The question
Can “exchangeability” of apoA-I be used clinically as a measure of HDL functionality and as a predictor of a patient’s risk of developing CVD?

The hypothesis
Cholesterol-laden macrophages primarily release cholesterol to lipid-free or lipid-poor apoA-I via the ATP binding cassette A1 (ABCA1). This pathway is believed to be one of the mechanisms whereby apoA-I reduces atherosclerosis progression. However, lipid-free/lipid-poor apoA-I is barely detectable in plasma, where the majority of apoA-I is HDL-associated. The mechanism by which lipid-free/lipid-poor apoA-I is formed in the sub-endothelial space of arteries is poorly understood.

Our hypothesis is that reduced apoA-I “exchangeability” in dysfunctional HDL leads to lower local availability of lipid-free apoA-I and lower cellular lipid release through the ABCA-1 pathway.
Our prediction is that low apoA-I “exchangeability” can be used as a measure of reduced HDL ability to promote cellular lipid release.

The experimental approach

In our lab, we generate lipidation-state sensitive apoA-Is using advanced protein chemistry techniques such as expressed protein ligation and click chemistry in order to label apoA-I with specific fluorophores in selected positions. Making use of the fluorescence resonance energy transfer (FRET) properties of the fluorophores, we can then determine whether the apoA-I is lipid-free or HDL-associated. The fluorescent and structural characterizations of the protein variants are performed by fluorescence and circular dichroism spectroscopies, and mass-spectrometry. The fluorescent apoA-I variants are used to quantify apoA-I “exchangeability” in HDL from plasma samples. In parallel, the functionality of HDL is evaluated using cell culture techniques to measure ABCA1-mediated cellular lipid release and scavenger receptor class B1 (SR-B1)-mediated cellular cholesteryl esters selective uptake.



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