Role of Annexins in Redox Regulation

过氧化氢（H2O2）是DNA损伤、蛋白质氧化和脂质过氧化的主要因素。为了防止细胞损伤，细胞已经发展出复杂的抗氧化系统来清除H2O2。我的实验室研究细胞内Ca2+和磷脂结合蛋白家族，称为膜联蛋白。已经在人体中发现了12种膜联蛋白。在植物中，膜联蛋白可以防止光诱导的氧化应激，并具有过氧化物酶活性，但人类膜联蛋白在氧化还原调节中的作用尚不清楚。我们建议通过初步研究膜联蛋白A2 （ANXA2）来确定哪些膜联蛋白参与氧化还原调节。我们已经观察到ANXA2具有活性半胱氨酸残基（Cys-8），它很容易被H2O2氧化并被硫氧还蛋白系统还原。我们提出ANXA2是一种新的细胞内H2O2清除剂，用于保护细胞的磷脂、蛋白质和/或核酸成分免受氧化损伤。第一个目标是使用RNA干扰来消耗细胞中的ANXA2，并确定ANXA2的消耗是否也会导致H2O2对DNA、细胞蛋白和膜脂的氧化增加。对照和anxa2缺失细胞系（TIME, MCF-7， LLC, HT1080, A549）将通过H2O2孵育受到氧化应激，pHyPer载体表达系统将用于特异性定量氧化应激期间细胞中H2O2的水平。蛋白质氧化将通过SDS-PAGE和蛋白质侧链羰基的western blot分析，脂质过氧化通过8-异前列腺素F2的酶免疫分析，DNA氧化通过ELISA检测8-羟基-2'-脱氧鸟苷（8-OHdG）。我们还将比较野生型和ANXA2基因敲除小鼠肺和肝脏组织中的整体蛋白质、脂质和DNA氧化。第二个目的是确定ANXA2是否可以直接与氧化细胞蛋白相互作用并降低氧化细胞蛋白。我们将使用抗ANXA2抗体从对照和氧化应激细胞中免疫沉淀细胞内的ANXA2，并通过质谱和表面等离子体共振鉴定ANXA2的结合伙伴。第三个目的是表征ANXA2的过氧化物酶活性。因此，我们将通过重组ANXA2和标准过氧化物酶实验来测量ANXA2与H2O2在体外相互作用的动力学和机制。该方法将确定还原剂、硫氧还蛋白或谷胱甘肽是否刺激了ANXA2过氧化物酶活性，以及ANXA2对H2O2的消耗是否足够快，从而具有生理意义。在未来的研究中，我们将研究其他潜在的氧化还原调节的膜联蛋白，如膜联蛋白V和膜联蛋白VI。总的来说，我们的研究计划将通过增强我们对膜联蛋白如何保护细胞免受氧化损伤的认识，为该领域提供原创和创新的贡献。

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.