Biochemistry: phospholipase A2, signal transduction in macrophages, lipid maps, prostaglandin regulation, mass spec of lipids and proteins.
Harvard Medical School
Awards and Academic Honors
Editor-in-Chief, Journal of Lipid Research
Amer. Soc. Biochemistry and Molecular Biology Avanti Award
Our laboratory is leading the national NIGMS LIPID MAPS Glue Grant Consortium which is developing LC/MS based lipidomics analysis to map the lipidome by detecting and quantizing the numerous molecular species of lipids in the murine macrophage. We are determining the lipid changes upon cell stimulation by various agonists such as TLR receptor agonists. Our laboratory is focused on identifying the known and discovering new fatty acids and eicosanoid metabolites (prostaglandins and leukotrienes) in these cells and using LC/MS to follow their production upon cell stimulation and determining lipid metabolite fluxes at the cellular level.
Historically, our laboratory has focused on discovering the regulation of lipid second messengers and signal transduction processes and especially the role of various phospholipases in their generation. Special attention is paid to the cytosolic, secreted, and membrane-bound phospholipase A2s (PLA2) responsible for the control of eicosanoid biosynthesis in macrophage cells. These molecules are produced from arachidonic acid released by PLA2 upon cell stimulation. Our goal is to characterize and elucidate the regulatory mechanisms of various phospholipase A2s both in vitro and in the intact cell and the LC/MS lipidomics efforts are currently being utilized to study their regulation.
PLA2s are currently receiving a great deal of attention because they include the smallest (molecular weight 13,000) and perhaps the simplest enzymes of complex lipid metabolism known and are ideally suited for in vitro mechanistic studies. PLA2s have several kinds of phospholipid binding sites including activator sites, interfacial sites, and catalytic sites. It is important to define the precise role of the amino acid residues involved in their interactions with phospholipids. Our laboratory is now utilizing mass spectrometric deuterium exchange analysis (HD/MS) to map the surface interactions of various pure recombinant human PLA2s with phospholipids.
Our laboratory also designs and synthesizes chemical inhibitors of phospholipase A2. Many different inhibitor classes have been developed and are being studied using in vitro, ex vivo and in vivo animal models. In summary, our laboratory utilizes organic synthetic approaches, enzyme kinetics, molecular biology,, cell and tissue culture, and mass spectrometric techniques as well as traditional biochemical approaches in attacking phospholipase and membrane problems.
Primary Research Area
- Arachidonic Derived Dihomo-prostaglandin Production Observed in Endotoxin Stimulate Macrophage-like Cells. With Harkewicz,R., Fahy,E., and Andreyev,A., J.Biol.Chem. 282 (2007).
- Differential Inhibition of Group IVA and Group VIA Phospholipases A2 by 2-Oxoamides. With Stephens,D.L., Barbayianni,E., Constantinou-Kokotou,V., Peristeraki,A., Cooper,J., Harkewicz,R., and Deems,R.A., and Kokotos,G., J.Med.Chem, 49, 2821-2828 (2006).
- Kdo2-Lipid A Escherichia coli, a Defined Endotoxin That Activates Macrophages Via TLR-4. With Raetz,C.R.H., Garrett,T.A., Reynolds,C.M., Shaw,W., Moore,J.D., Smith,D.C., Ribeiro,A.A., Murphy,R.C., Ulevitch,R.J., Fearns,C., Reichart,D., Glass,C.K., Benner,C., Subramaniam,S., Harkewicz,R., Bowers-Gentry,R.C., Buczynski,M.W., Cooper,J., and Deems,R., Journal of Lipid Research, 47, 1097-1111 (2006).
- Lipopolysaccharide Induced Cyclooxygenase-2 Expression in Human U937 Macrophages is Phosphatidic Acid Phosphohydrolase-1 Dependent. With Grkovich,A. and Johnson,C.A., J.Biol.Chem., 281, 32,978-32,987 (2006).
- The Phospholipase A2 Superfamily And Its Group Numbering System. WithSchaloske,R.H., Biochim.Biophys.Acta, 1761, 1246-1259 (2006).