Lecithin:cholesterol acyltransferase (LCAT) esterifies cholesterol in high density lipoprotein (HDL), which promotes HDL maturation in reverse cholesterol transport, the process by which cholesterol is moved from arterial plaques to the liver. Mutations in LCAT cause two genetic diseases, both of which are characterized by low HDL-cholesterol (HDL-C) levels. Furthermore, clinical studies have shown that low HDL-C is associated with high risk for coronary heart disease (CHD). Consequently, administration of recombinant LCAT and LCAT activating compounds are being investigated as treatments to increase HDL in both CHD and LCAT genetic disease. However, improvement of these therapeutics is limited due to a lack of structural information on LCAT. The objective of this proposal is to determine the molecular basis for LCAT activation and the structural elements required for cholesterol recognition through the use of X-ray crystallography and cryo-electron microscopy in parallel with functional studies.
The first aim will focus on functional characterization of a new crystal form of LCAT wherein a retractable lid that seems to block the active site. Interestingly, the lid interacts with residues on the catalytic core of LCAT known to be important for activation by apolipoprotein A-I (ApoA-I), the major protein component of HDL particles, suggesting that ApoA-I binding may displace the lid to stimulate LCAT activity. In the second aim, I will investigate the molecular basis for LCAT acyl acceptor selectivity and for activation by a small molecule activator through combined crystallographic and biochemical studies. LCAT crystal structures will be determined in complex with acceptor analogs and/or small molecule activators, and residues in the active site that differ between LCAT and a closely related enzyme will be mutated to test whether substrate selectivity can be exchanged. Overall, these studies are expected to pave the way for the design of more potent small molecule activators as well as more catalytically efficient LCAT for use in enzyme replacement therapy for the treatment of CHD and/or genetic disease.
The enzyme lecithin:cholesterol acyltransferase (LCAT) is responsible for maturation of high density lipoprotein (HDL) in the process of reverse cholesterol transport. In this study, we aim to determine atomic resolution models of LCAT so we can better understand how it binds to cholesterol, is activated by HDLs, and how a recently discovered small molecule is able to make LCAT more active. These studies will ultimately help design better therapeutics with the clinical goal of increasing HDL levels in people with heart disease or disease-causing genetic mutations in LCAT.
|Hinkovska-Galcheva, Vania; Kelly, Robert; Manthei, Kelly A et al. (2018) Determinants of pH profile and acyl chain selectivity in lysosomal phospholipase A2. J Lipid Res 59:1205-1218|
|Freeman, Lita A; Demosky Jr, Stephen J; Konaklieva, Monika et al. (2017) Lecithin:Cholesterol Acyltransferase Activation by Sulfhydryl-Reactive Small Molecules: Role of Cysteine-31. J Pharmacol Exp Ther 362:306-318|
|Manthei, Kelly A; Ahn, Joomi; Glukhova, Alisa et al. (2017) A retractable lid in lecithin:cholesterol acyltransferase provides a structural mechanism for activation by apolipoprotein A-I. J Biol Chem 292:20313-20327|