The research is devoted to host defense mechanisms against infections and inflammation. It is focused on the molecular mechanism regulating phagocyte NADPH oxidase and cytosolic phosphlipase A2alfa (cPLA2) activities, the relationship between both enzymes and their role in health and disease in human as well as in mouse models of different inflammatory disorder. Both NADPH oxidase and cPLA2 are crucial in physiological conditions but their elevated activities participate in accelerating inflammatory diseases. NADPH oxidase is found in high levels in phagocytes, like neutrophils and monocytes, where the elicited production of superoxides play an important role in protection against microbial infections. cPLA2 is the main enzyme liberating arachidonic acid, the precursor for eicosanoids and is involved in gene regulation. In addition, a series of our previous studies, provided evidence indicating an essential requirement for cPLA2 in activation of the NADPH oxidase in neutrophils. Activated phagocytes in pathological conditions, in particular neutrophils, release superoxides that cause cell damage at the site of inflammation. cPLA2 is also an important player in inflammation as it participates in both the regulation of NADPH oxidase and the production of eicosanoids that play a role in recruiting activated neutrophils to the site of inflammation.
• The role of NADPH oxidase and cytosolic phospholipase A2 in vitro and in vivo in inflammatory diseases, in neurodegeneration and in cancer.
• Developing new therapeutic venues against infections and inflammation in animal models of inflammation such as collagen induced arthritis in mice.
• The role of NADPH oxidase, cytosolic phospholipase A2 and neutrophils in inflammation and metabolic syndrome in obesity.
Funding: Israel Academy of Sciences.
|Abstracts of Current Research:
- A novel binding site via cytosolic phospholipase A2alfa to p47phox domain in the assembled NADPH oxidase.J. Bio. Chem. 2008 : We have previously demonstrated a physical interaction between cytosolic phospholipase A2 (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, FRET analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using sub-fragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify peptides within these two domains that inhibit the interaction between cPLA2 and the NADPH oxidase. Hydrophobic peptides corresponding to each domain inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, while the single mutant I67A was highly effective. This study identified Ile67 of the cPLA2-C2 domain as a critical, centrally positioned residue in a hydrophobic interaction with p47phox-PX domain.
- Elgazar-carmon, V., Rudich, A., Hadad, N. and Levy, R. Neutrophils transiently
infiltrate intra-abdominal fat early in the course of high fat feeding J Lipid Res. 49: 1894-903 (2008)
- Shmelzer, Z., Karter, M., Eisenstien M., Leto L.T., Hadad, N., Ben-Menahem, D., Gitler D., Wolach B., Rotem M. and Levy R. Cytosolic phospholipase A2a is targeted to the p47phox-PX domain of the assembled NADPH Oxidase via a novel binding site in its C2 domain J. Biol. Chem. 283: 31898-908 (2008)
- Raichel, L., Berger, Y., Kachko, L., Hadad, N., Karter, M., Solodkin, I., Williams, IR., Feldmann, M. and Levy, R. Reduction of cPLA2a overexpression– an efficient anti-inflammatory therapy for collagen induced arthritis. Eur. J. Immunol. 38 : 1-12 (2008)
- Rachel Levy. The role of cytosolic phospholipase A2-alfa in regulation of phagocytic functions.
Biochim Biophys Acta BA Molecular and Cell Biology of Lipids 1761: 1323-1334 (2006)
- Hazan-Eitan Z., Weinstein Y., Hadad N., Konforty A. and Levy R. . Induction of FcgRIIA expression in myeloid PLB cells during differentiation depends on cytosolic phospholipase A2 activity and is regulated via activation of CREB by PGE2. Blood 108: 1758-66 (2006)
- Shmelzer, Z., Haddad, N., Admon, E., Pessach, I., Leto, T.L., Eitan-Hazan, Z., Hershfinkel M. and Levy, R. . Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes. J. Cell Biol. 162: 693-701 (2003)