Development of a clinically-viable measure of HDL functionality
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.
The experimental approachIn 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.