Grant: Discovery Grants Program - Individual

Hydrogen peroxide (H2O2) is a major contributor to DNA damage, protein oxidation and lipid peroxidation. To prevent cellular damage, cells have developed complex antioxidant systems to scavenge H2O2. My laboratory studies a family of intracellular Ca2+-and phospholipid-binding proteins called the annexins. Twelve annexins have been identified in humans. In plants, the annexins protect against light-induced oxidative stress and possess peroxidase activity, however the role of human annexins in redox regulation is unclear. We propose to define which annexins participate in redox regulation, by initially studying annexin A2 (ANXA2). We have observed that ANXA2 possesses a reactive cysteine residue (Cys-8) that is readily oxidized by H2O2 and reduced by the thioredoxin system. We are proposing that ANXA2 is a novel intracellular H2O2 scavenger that serves to protect phospholipid, protein, and/or nucleic acid components of the cell from oxidative damage. The first objective is to use RNA interference to deplete cells of ANXA2 and determine if depletion of ANXA2 also results in the increased oxidation of DNA, cellular proteins and membrane lipids by H2O2. Control and ANXA2-depleted cell lines (TIME, MCF-7, LLC, HT1080, A549) will be subjected to oxidative stress by incubation with H2O2 and the pHyPer vector expression system will be used to specifically quantitate cellular levels of H2O2 during oxidative stress. Protein oxidation will be quantitated by SDS-PAGE and western blot analysis of protein side-chain carbonyl groups, lipid peroxidation by enzyme immunoassay of 8-iso-prostaglandin F2 and DNA oxidation by ELISA measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG). We will also compare global protein, lipid and DNA oxidation in the lung and liver tissues from wild-type and ANXA2 knockout mice. The second objective is to determine if ANXA2 can directly interact with and reduce oxidized cellular proteins. We will use anti-ANXA2 antibodies to immunoprecipitate intracellular ANXA2 from control and oxidatively stressed cells and identify ANXA2 binding partners by mass spectrometry and surface plasmon resonance. The third objective is to characterize the peroxidase activity of ANXA2. Accordingly, we will measure the kinetics and mechanism of interaction of ANXA2 with H2O2 in vitro using recombinant ANXA2 and standard peroxidase assays. This approach will establish if ANXA2 peroxidase activity is stimulated by the reductants, thioredoxin or glutathione and if the consumption of H2O2 by ANXA2 proceeds at a sufficiently rapid rate to be of physiological significance. In future studies, we will study other potential redox-regulated annexin proteins, such as annexin V and annexin VI. Collectively, our research program will provide original and innovative contributions to the field by enhancing our knowledge of how cells are protected against oxidative damage by the annexin proteins.