Role of Annexins in Redox Regulation

膜联蛋白在氧化还原调节中的作用

• 批准号: RGPIN-2018-05316
• 负责人: Waisman, David
• 金额: $ 2.33万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=713327
• 关键词: Role; Annexins; Redox; Regulation

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.

过氧化氢 (H2O2) 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，细胞开发出了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。已在人类中鉴定出十二种膜联蛋白。在植物中，膜联蛋白可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) 来定义哪些膜联蛋白参与氧化还原调节。我们观察到 ANXA2 具有反应性半胱氨酸残基 (Cys-8)，很容易被 H2O2 氧化并被硫氧还蛋白系统还原。我们提出 ANXA2 是一种新型细胞内 H2O2 清除剂，可保护细胞的磷脂、蛋白质和/或核酸成分免受氧化损伤。第一个目标是利用 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 耗尽的细胞系（TIME、MCF-7、LLC、HT1080、A549）将通过与 H2O2 一起孵育来承受氧化应激，并且 pHyPer 载体表达系统将用于特异性定量氧化应激过程中 H2O2 的细胞水平。通过 SDS-PAGE 和蛋白质侧链羰基的蛋白质印迹分析来定量蛋白质氧化，通过 8-异前列腺素 F2 的酶免疫测定来定量脂质过氧化，通过 8-羟基-2'-脱氧鸟苷 (8-OHdG) 的 ELISA 测量来定量 DNA 氧化。我们还将比较野生型和 ANXA2 敲除小鼠的肺和肝组织中的整体蛋白质、脂质和 DNA 氧化。第二个目标是确定 ANXA2 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 ANXA2 抗体从对照细胞和氧化应激细胞中免疫沉淀细胞内 ANXA2，并通过质谱和表面等离振子共振鉴定 ANXA2 结合伴侣。第三个目标是表征 ANXA2 的过氧化物酶活性。因此，我们将使用重组 ANXA2 和标准过氧化物酶测定来测量 ANXA2 与 H2O2 体外相互作用的动力学和机制。该方法将确定 ANXA2 过氧化物酶活性是否受到还原剂、硫氧还蛋白或谷胱甘肽的刺激，以及 ANXA2 对 H2O2 的消耗是否以足够快的速度进行以具有生理意义。在未来的研究中，我们将研究其他潜在的氧化还原调节膜联蛋白，例如膜联蛋白 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细胞如何免受膜联蛋白氧化损伤的了解，为该领域提供原创和创新的贡献。