Lysosomal phospholipase A2 (LPLA2) plays a major role in lipid degradation and is believed to underlie drug-induced phospholipidosis, which commonly occurs in patients taking cationic lipophilic drugs such as the antiarrhythmic amiodarone. Aberrant LPLA2 activity may also be involved in development of autoimmune disease and atherosclerosis. LPLA2 is 50% identical in sequence to lecithin-cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport from arterial plaque macrophages via high density lipoproteins (HDL). Genetic mutations in LCAT are responsible for Familial LCAT Deficiency (FLD), a devastating disease characterized by low serum cholesterol ester levels and renal failure. There are no reported atomic models for either LPLA2 or LCAT, which do not have significant homology to other proteins of known structure. Thus, the molecular bases for their substrate selectivity, regulation, and disease phenotypes remain poorly understood. In this proposal, we address this critical gap in knowledge via functional analysis of our new 1.8 ? crystal structure of LPLA2, determination of the atomic structure of LCAT, imaging LCAT bound to HDL particles by electron microscopy, mapping somatic mutations known to cause genetic disease, and investigating the structural basis for differences in acyl acceptor selectivity. In support of our aims, we provide multiple high resolution structure of LPLA2 in various ligand states, negative stained images of LCAT-HDL complexes, and a low resolution crystal structure of fully glycosylated LCAT. The expected outcome of these studies is a better mechanistic understanding of a structurally uncharacterized family of eukaryotic enzymes that play key roles in lipid metabolism. Our structural and functional studies will help explain the molecular basis for genetic disease and ultimately assist in the design of improved biotherapeutics and small molecule LCAT activators to treat lipid-related disorders such as atherosclerosis and LCAT deficiency.

Public Health Relevance

Lysosomal phospholipase A2 (LPLA2) is a recently discovered enzyme that catabolizes glycerophospholipids and breaks down lung surfactants. Inhibition of LPLA2 correlates with phospholipidosis during long-term administration of the antiarrhythmic drug amiodarone. LPLA2 is closely related to lecithin-cholesterol acyltransferase (LCAT), a plasma protein that plays a critical role in reverse cholesterol transport via HDLs to the liver. Mutations in LCAT are responsible for a devastating disease known as familial LCAT deficiency. Both enzymes are believed to be important for amelioration of atherosclerosis. This proposal seeks to determine the molecular basis for substrate selectivity, catalysis, small molecule activation, and disease in this structurally uncharacterized family of lipid metabolizing enzymes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL122416-03
Application #
9174909
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Liu, Lijuan
Project Start
2014-11-04
Project End
2017-08-13
Budget Start
2016-11-01
Budget End
2017-08-13
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pharmacology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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
Shayman, James A; Tesmer, John J G (2018) Lysosomal phospholipase A2. Biochim Biophys Acta Mol Cell Biol Lipids :
Waldschmidt, Helen V; Bouley, Renee; Kirchhoff, Paul D et al. (2018) Utilizing a structure-based docking approach to develop potent G protein-coupled receptor kinase (GRK) 2 and 5 inhibitors. Bioorg Med Chem Lett 28:1507-1515
Beyett, Tyler S; Gan, Xinmin; Reilly, Shannon M et al. (2018) Design, synthesis, and biological activity of substituted 2-amino-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid derivatives as inhibitors of the inflammatory kinases TBK1 and IKK? for the treatment of obesity. Bioorg Med Chem 26:5443-5461
Beyett, Tyler S; Gan, Xinmin; Reilly, Shannon M et al. (2018) Carboxylic Acid Derivatives of Amlexanox Display Enhanced Potency toward TBK1 and IKK? and Reveal Mechanisms for Selective Inhibition. Mol Pharmacol 94:1210-1219
Sakr, Moustafa; Li, Xiao-Yan; Sabeh, Farideh et al. (2018) Tracking the Cartoon mouse phenotype: Hemopexin domain-dependent regulation of MT1-MMP pericellular collagenolytic activity. J Biol Chem 293:8113-8127
Bouley, Renee; Waldschmidt, Helen V; Cato, M Claire et al. (2017) Structural Determinants Influencing the Potency and Selectivity of Indazole-Paroxetine Hybrid G Protein-Coupled Receptor Kinase 2 Inhibitors. Mol Pharmacol 92:707-717
Yao, Xin-Qiu; Cato, M Claire; Labudde, Emily et al. (2017) Navigating the conformational landscape of G protein-coupled receptor kinases during allosteric activation. J Biol Chem 292:16032-16043
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
Abe, Akira; Hiraoka, Miki; Ohguro, Hiroshi et al. (2017) Preferential hydrolysis of truncated oxidized glycerophospholipids by lysosomal phospholipase A2. J Lipid Res 58:339-349

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