Regulatory Mechanisms of Secretory Phospholipases A2

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Description (applicant's description): Our long-term goal is to identify the structural basis for the activation of secretory phospholipases A2 (PLA2) upon binding to the aggregated substrate (interfacial activation). PLA2s hydrolyze phospholipids to free fatty acids and lysolipids and thus initiate the biosynthesis of eicosanoids and platelet activating factor, potent mediators of inflammation, allergy, apoptosis and tumorigenesis. The project is focused on secretory PLA2s, including human pancreatic and synovial PLA2s, because these enzymes are of great biological and clinical importance and their regulatory mechanisms are poorly understood. Our specific aims are: 1. Identify the roles of membrane electrostatics, membrane binding strength, orientation of membrane-bound PLA2 and membrane-induced structural changes in PLA2 activation. To test our hypothesis that membrane surface properties and membrane-induced structural changes in PLA2 act synergistically in activation of PLA2, a relationship will be established between the membrane surface potential, membrane binding of PLA2, structural changes in the enzyme and PLA2 activity. The reciprocal effects of PLA2 on membrane surface properties and the roles of the fatty acid and lysolipid in PLA2 activation will be studied. The 3D orientation of membrane-bound PLA2 before and after activation will be determined based on the infrared dichroism of unlabeled and segment-13C-labeled PLA2 to test the hypothesis that reorientation of PLA2 at the membrane surface may contribute to PLA2 activation. 2. Characterize conformational changes in secretory PLA2s of different groups during membrane binding and activation. To resolve conformational changes in PLA2s that are induced by surface adsorption, we will study the secondary and dynamic structural changes in PLA2s upon binding to phospholipid membranes or micelles using FTIR, NMR, time-resolved fluorescence spectroscopy and circular dichroism. By using FTIR and NMR, we will test our hypothesis that membrane binding of PLA2 induces a conversion of standard alphaI helices to less stable alphaII helices. We will check by FTIR spectroscopy whether alphaI-alphaII transition can be reverted by high pressures or low temperatures. 3. Identify the specific regions of PLA2 that undergo structural changes upon membrane binding of PLA2 and contribute to its activation. A multidisciplinary approach, using segmental isotopic labeling of PLA2s, FTIR, NMR, fluorescence spectroscopy and amide hydrogen exchange, will be applied to accomplish this aim. Global and site-specific changes in the secondary and dynamic structure of PLA2 during interfacial activation will be determined by 13C-isotope-edited FTIR and heteronuclear NMR. The fast dynamics of the main chain of PLA2 during its interfacial activation will be measured using 15N nuclear relaxation techniques. Collectively, our results will help develop new strategies for regulation of PLA2s, enzymes of profound biomedical importance.