Cytochrome c mechanism of ROS signaling in apoptosis

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DESCRIPTION (provided by applicant): Apoptosis plays an essential role in embryonic development, tissue homeostasis and various pathological conditions including inflammation and ischemia. Our long term goal is to elucidate mechanisms of reactive oxygen species (ROS) signaling at the early phase of mitochondria-dependent apoptosis. The specific hypothesis is that cytochrome c (cyt c), when attached to inner membrane of mitochondria, can catalytically oxidize cardiolipin upon reaction with hydrogen peroxide. Accumulation of cardiolipin hydroperoxides induces detachments of cyt c from membrane and its subsequent release from mitochondria that inevitably initiates apoptosis. This hypothesis is based on the observations that 1) cyt c in mitochondria exist predominantly in membrane-bound form; 2) membrane-bound cyt c possess significant peroxides-like activity; 3) accelerated rate of ROS production is associated with apoptosis; 4) oxidation of cardiolipin is associated with cyt c release. This joint research project is focused on the catalytic mechanism of ROS-dependent cardiolipin oxidation and on the role of peroxidized cardiolipin in cyt c release. The specific aims are to: 1) identify binding characteristics of cyt c associated with its pro-oxidant activity and determine conditions that amplify or inhibit this activity; 2) establish the catalytic mechanism of cyt c pro-oxidant activity and determine its preferential substrates; 3) determine signaling role of the cyt c pro-oxidant activity in mitochondria during apoptosis. Using biophysical and biochemical approaches including HPLC, ESI mass spectroscopy, EPR spectroscopy and array of quantitative fluorescence and spectroscopic assays we plan to estimate peroxidase activity of membrane-bound cyt c in mitochondria, to characterize products of cyt c-dependent phospholipid oxidation, and, finally, to establish a link between these oxidative reactions and cyt c release from mitochondria. Thus, these experiments will improve our understanding of the signaling role of ROS and products of phospholipid oxidation in apoptosis. This research will be done primarily in Moscow (Russian Federation) at the Research Institute of Physico-Chemical Medicine in collaboration with Dr. Grigory Borisenko as an extension of NIH grant # R01 HL070755 entitled "Pulmonary Phosphatidylserine Oxidation in Apoptosis".