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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=667952
• 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 F2α 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(H_2O_2)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，细胞已经开发出复杂的抗氧化系统来清除过氧化氢。我的实验室研究一种称为膜联蛋白的细胞内钙和磷脂结合蛋白家族。已经在人类中发现了12种膜联蛋白。在植物中，膜联蛋白可以抵御光诱导的氧化胁迫，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具有活性半胱氨酸残基(Cys-8)，该残基很容易被H_2O_2氧化并被硫氧还蛋白体系还原。我们提出ANXA2是一种新型的细胞内过氧化氢清除剂，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干扰来耗尽ANXA2的细胞，并确定ANXA2的耗尽是否也会导致过氧化氢对DNA、细胞蛋白质和膜脂的氧化增加。对照和ANXA2耗竭的细胞系(Time，MCF-7，LLC，HT1080，A549)将通过与过氧化氢孵育而受到氧化应激，pHyPer载体表达系统将被用于在氧化应激过程中特异性地定量细胞内过氧化氢的水平。8-异前列腺素F2α的脂质过氧化作用采用酶免疫分析法，8-羟基-2‘-脱氧鸟苷(8-OHdG)的EL ISA法测定。我们还将比较野生型和ANXA2基因敲除小鼠肺和肝组织中的整体蛋白质、脂肪和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。我们将使用抗ANXA2抗体免疫沉淀对照细胞和氧化应激细胞内的ANXA2，并通过质谱仪和表面等离子共振鉴定ANXA2结合伙伴。第三个目标是研究ANXA2的过氧化物酶活性。因此，我们将在体外用重组ANXA2和标准过氧化物酶测定ANXA2与H_2O_2相互作用的动力学和机制。这一方法将确定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对过氧化氢的消耗是否以足够快的速度进行到具有生理意义。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细胞如何保护细胞免受膜联蛋白氧化损伤的了解，为该领域提供原创性和创新性的贡献。