High levels of high density lipoprotein-cholesterol (HDL-C) are associated with lowered risk for cardiovascular disease (CVD) in epidemiological studies. However, recent genetic and drug studies have shown that HDL-C itself is probably not causal in reducing CVD risk. Instead, a consensus is building that HDL functions may protect against CVD, and that treating for the biomarker of HDL-C may not always coincide with increased HDL function. One of the functions of HDL that may play a role in its protective effect is its role in the reverse cholesterol transport (RCT) pathway, in which cholesterol is removed from peripheral tissues and transferred to the liver for excretion. HDL and its major protein constituent, apolipoprotein-AI (apoA1), are critical components of this process. In the first step of the RCT pathway, lipid-poor apoA1 acts as an acceptor for cell cholesterol and phospholipids via the cell membrane protein ABCA1, generating nascent HDL through a mechanism which is not understood at the molecular level. ABCA1 has several well characterized activities, the outward translocation of phosphatidylserine, and the cell surface binding of its ligand apoA1. In the last cycle of this grant we characterized two novel activities of ABCA1, the outward translocation of phosphatidylinositol (4,5) bis-phosphate (PIP2) that is responsible for apoA1 binding to ABCA1 expressing cells, and the recruitment of the lysosomal vacuolar ATPase (V-ATPase) to the plasma membrane leading to local acidification of apoA1 that promotes its unfolding and HDL biogenesis. Building on these discoveries we propose to develop a cell-free ABCA1 reconstituted system, allowing a better understanding of the mechanism by which ABCA1 assembles nascent HDL.
In Aim 2, we explore the mechanism by which ABCA1 recruits V- ATPase to the plasma membrane. Successful completion of the proposed studies will increase our knowledge of the mechanism of de novo HDL production, which may yield insights into new strategies to increase HDL biogenesis and HDL function, and aid in the prevention of CVD.
Although recent genetic and drug studies have shown that HDL-cholesterol may not itself be protective against coronary heart disease (CHD), a consensus is building that HDL function may be protective, particularly the ability of apoAI, the major HDL protein, to accept cholesterol from cells in the arterial wall. Nascent HDL is formed when apoAI is lipidated in a reaction mediated by the plasma membrane protein ABCA1; however, the mechanism for ABCA1 lipidation of apoAI is not understood. The proposed studies will test novel hypotheses for the mechanism of ABCA1's lipidation of apoAI, which may lead to a better understanding of HDL biogenesis and provide the basis for future therapeutic strategies to prevent CHD.
| Lorkowski, Shuhui Wang; Brubaker, Gregory; Gulshan, Kailash et al. (2018) V-ATPase (Vacuolar ATPase) Activity Required for ABCA1 (ATP-Binding Cassette Protein A1)-Mediated Cholesterol Efflux. Arterioscler Thromb Vasc Biol 38:2615-2625 |
| Halawani, Dalia; Gogonea, Valentin; DiDonato, Joseph A et al. (2018) Structural control of caspase-generated glutamyl-tRNA synthetase by appended noncatalytic WHEP domains. J Biol Chem 293:8843-8860 |
| Gulshan, Kailash; Brubaker, Gregory; Conger, Heather et al. (2016) PI(4,5)P2 Is Translocated by ABCA1 to the Cell Surface Where It Mediates Apolipoprotein A1 Binding and Nascent HDL Assembly. Circ Res 119:827-38 |
