| Summary: | Epidemiological studies consistently demonstrate an inverse relationship between HDL levels and cardiovascular disease (CVD), independent of LDL and triglyceride levels. Due to the crucial role ABCA1 plays in HDL biogenesis, increasing ABCA1 expression is considered an attractive strategy to increase plasma HDL levels. In this thesis we attempt to identify novel post-transcriptional and post-translational mechanisms that regulate ABCA1 expression and/or function. Prior to translation, ABCA1 protein expression is regulated by non-coding RNA molecules known as microRNAs which bind and inhibit translation of mature mRNA transcripts in the cytoplasm. In this study we used bioinformatic prediction programs to identify potential microRNA regulators of ABCA1. Using reporter constructs, protein expression analysis by immunoblotting, and cholesterol efflux assays, we validated microRNA-145 as a novel repressor of ABCA1 translation. The inhibition of endogenous microRNA-145 in HepG2 cells increases both ABCA1 protein levels and cholesterol efflux activity. The inhibition of this microRNA in the liver is a potential strategy to increase HDL levels. Following translation, numerous post-translational modifications and protein-protein interactions are required for the ABCA1 protein to function properly. In this study we identified palmitoylation as a novel post-translational modifier of ABCA1. The majority of ABCA1-mediated cholesterol efflux and HDL biogenesis occurs at the cell surface. We show that palmitoylation is a crucial lipid addition for proper ABCA1 plasma membrane localization. We also identify a number of enzymes that mediate the incorporation of radio-labeled palmitate onto ABCA1, and demonstrate that the overexpression of the palmitoyl transferase enzyme DHHC8 increases ABCA1 palmitoylation and cholesterol efflux activity. The increase of ABCA1 palmitoylation in the liver is a novel strategy to increase HDL levels. In this thesis, we have contributed to the understanding of ABCA1 biology by the identification of two novel regulators of ABCA1 expression and/or function. === Medicine, Faculty of === Medical Genetics, Department of === Graduate
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