Characterization of the Post-Translational Regulation of the ABCA1 and ABCG1 Lipid Transporters by E3 Ligases

This thesis present a series of experimental approaches that aim to characterize the roles of three E3 ubiquitin ligase enzymes in the post-translational regulation of two ABC lipid transporters; ABCA1 and ABCG1. These E3 ligases, namely HECTD1 ( Homologous to E6Ap C-terminus Domain 1), HUWE1 (HECT, UBA, and WWE domain containing 1), and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), were identified during a previous work in our laboratory using peptide mass spectrometry analysis as associate proteins with ABCG1. The experiments presented in the following chapters investigate the effect of these ligases on the ABC transporters' protein expression and cholesterol activity. In addition, the possible mechanism underlying the mode of interaction between these ligases and the ABC transporters is also investigated in attempt to provide a more specific approach to future targeting strategies. Chapter1 provides an overview of atherosclerosis and the essential roles of ABCA1 and ABCG1 lipid transporters in maintaining cellular lipid homeostasis and mediating the macrophage reverse cholesterol transport (RCT) pathway. This chapter also presents a general overview regarding the developed anti-atherogenic therapeutic strategies, including those that attempted to upregulate these ABC transporters, although they were precluded due to drawbacks, indicating the need for a novel specific therapeutic strategy. In addition, it discusses the different regulatory pathways of ABCA1 and ABCG1, in particular the ubiquitin proteasomal pathway, and the role of E3 ligases that determine the specificity of the overall pathway. Chapter 2 describes the main experimental procedures that have been used throughout this thesis, including optimization of siRNA silencing as well as transient overexpression of the ligases in cultured cells, measurement of the ABC transporters' protein expression, immunoprecipitation approaches that detect the interaction between each ligase and ABC transporter, and cholesterol efflux assays that measure the activity of the transporter in exporting cholesterol from cultured cells to extracellular acceptors. Chapter 3 presents experiments investigating the role of the first E3 ligase, HECTD1, in the regulation of ABCA1 and ABCG1. These experiments propose that this newly discovered E3 ligase specifically regulates ABCA1 expression and activity, in a manner that is independent of any change in ABCG1. Silencing of HECTD1 substantially increased ABCA1 expression and cholesterol efflux from THP-1 macrophages to apoA-I, without affecting the ABCG1-mediated cholesterol export to HDL from these cells. The role of the other E3 ligases, HUWE1 and NEDD4-1, are addressed via experiments presented in chapter 4. These two E3 ligases seemed to have an additive effect on ABCG1 protein expression and activity, but not ABCA1. Simultaneous silencing of both ligases significantly increased ABCG1 protein levels and substantially more than the individual silencing. These effects on ABCG1 protein levels were translated to upregulation of the transporter activity. Silencing of both ligases significantly increased the ABCG1- mediated cholesterol export to HDL from cells overexpressing the transporter as well as from THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant decrease in ABCG1 protein levels, confirming their roles in the regulation of the transporter degradation. Chapter 5 illustrates a research strategy used to understand how NEDD4-1 can interact with ABCG1 and also to identify potential candidate adaptor proteins that may perhaps mediate this interaction. This strategy involved looking for specific proline-rich consensus sequences, which are essential for NEDD4-1 interaction with its substrate, in the original mass spectrometry peptide hits. Out of twenty three potential candidates that were investigated, four candidates fulfilled the requirements to act as an adaptor proteins. These findings highlight the probability that one of the identified candidate proteins might recruitNEDD4-1 to ABCG1 and thus facilitate ABCG1 ubiquitination. In conclusion, the work presented in this thesis explores the roles of three E3 ubiquitin ligases in the post-translational regulation of ABC lipid transporters. Identifying these ligases as specific molecular determinates for ABCA1 and ABCG1 ubiquitination and subsequent degradation would provide a new insight into the post-translational regulation of these lipid transporters, as well as into the discovery of a selective therapeutic approach to promote their upregulation