Ionic Charge and pH Regulation of Apoptotic Signaling

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This proposal is in response to PAR-98-087, "Small grant program for K08 recipients," and is linked to NIH K08 DK02537. Endothelial cell apoptosis has an essential role in the pathogenesis of hemolytic uremic syndrome and other causes of acute renal failure. The studies in this proposal will advance our understanding of the biochemistry of endothelial cell apoptosis by examining the molecular mechanism by which ionic charge and alkaline conditions inhibit cytochrome c- mediated activation of caspase-3. We have developed an in vitro assay of cytochrome c-dependent activation of cytosolic caspase-3 allowing discrete steps in the process to be identified and characterized. In this assay cytochrome c added to cytosol initiates the formation of apoptosomes (oligomers of cytochrome c, apoptosis activating factor-1, and dATP/ATP) resulting in activation of caspase-9, which subsequently activates caspase-3. We showed that both alkaline pH and cations, at normal intracellular concentrations, inhibited activation of caspase-3. Excess cytochrome c suppressed the inhibition by high pH, but did not affect the inhibition by cations, suggesting that pH and cationic strength act at two different steps in caspase-3 activation. This proposal will test two overall hypotheses derived from these observations. First, that alkaline pH affects the formation of apoptosomes, whereas ionic charge prevents caspase-9 activation. Second, that cellular accumulation of a non-cationic osmotic solute is required for the decrease in cationic strength necessary for caspase-3 activation. The Specific Aims are 1) to develop an in vitro assay for caspase-9 activation, and determine the effect of alkalinity and high cationic strength on activation; 2) to study the formation and constituents of apoptosomes, using a size separation column, under high ionic charge and alkaline conditions; and 3) to examine the role of non- cationic solute accumulation in apoptosis by testing if potential osmoles, such as amino acids and mono- and di-saccharides, interfere with caspase-3 activation in our in vitro assay, and by comparing cytosol from nonapoptotic and apoptotic cells using liquid chromatography for differences in osmotic solute content. This grant will also enhance the research productivity, fiscal independence and career development of the applicant furthering his advancement towards becoming an independent investigator.