Role of Annexins in Redox Regulation

过氧化氢（H2 O2）是DNA损伤、蛋白质氧化和脂质过氧化的主要贡献者。为了防止细胞损伤，细胞已经开发出复杂的抗氧化系统来吸收H2 O2。我的实验室研究一个家族的细胞内钙和磷脂结合蛋白称为膜联蛋白。在人类中已发现12种膜联蛋白。在植物中，膜联蛋白保护免受光诱导的氧化应激，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。我们建议定义哪些膜联蛋白参与氧化还原调节，通过初步研究膜联蛋白A2（ANXA 2）。我们已经观察到，ANXA 2具有一个反应性的半胱氨酸残基（Cys-8），它很容易被H2 O2氧化，并被硫氧还蛋白系统还原。我们提出ANXA 2是一种新型的细胞内H2 O2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ANXA 2的细胞，并确定ANXA 2的消耗是否也会导致DNA、细胞蛋白和膜脂质被H2 O2氧化的增加。通过与H2 O2孵育，对对照和ANXA 2耗尽的细胞系（TIME、MCF-7、LLC、HT 1080、A549）进行氧化应激，并使用pHyPer载体表达系统特异性定量氧化应激期间H2 O2的细胞水平。通过蛋白质侧链羰基的SDS-PAGE和蛋白质印迹分析定量蛋白质氧化，通过8-异前列腺素F2的酶免疫测定定量脂质过氧化，通过8-羟基-2 '-脱氧鸟苷（8-OHdG）的ELISA测量定量DNA氧化。我们还将比较野生型和ANXA 2敲除小鼠肺和肝组织中的整体蛋白质，脂质和DNA氧化。第二个目的是确定ANXA 2是否可以直接与氧化的细胞蛋白相互作用并还原氧化的细胞蛋白。我们将使用抗ANXA 2抗体从对照和氧化应激细胞中免疫沉淀细胞内ANXA 2，并通过质谱和表面等离子体共振鉴定ANXA 2结合伴侣。第三个目的是表征ANXA 2的过氧化物酶活性。因此，我们将使用重组ANXA 2和标准过氧化物酶测定来测量ANXA 2与H2 O2在体外相互作用的动力学和机制。该方法将确定ANXA 2过氧化物酶活性是否被还原剂、硫氧还蛋白或谷胱甘肽刺激，以及ANXA 2对H2 O2的消耗是否以足够快的速率进行以具有生理学意义。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知识，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.