Elucidation Of Cellular Damage During Exposure To Oxidat

阐明暴露于氧化剂期间的细胞损伤

• 批准号: 6541599
• 负责人: EARL R STADTMAN
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6541599
• 关键词: 3T3 cells; antioxidants; apoptosis; biological signal transduction; carbonyl compound; cellular pathology; chemical aggregate; cysteine endopeptidases; cytotoxicity; enzyme activity; free radical oxygen; laboratory mouse; laboratory rat; metal metabolism; methionine; nitric oxide; oxidation; oxidative stress; peroxides; prions; protein metabolism; protein structure function

Research in the Section on Enzymes in the Laboratory of Biochemistry, NHLBI, is directed toward elucidation of basic mechanisms involved in the production of cellular damage during exposure to oxidative stress and the contributions of such damage to aging and disease. To this end, our current research involves studies in the following areas of exploration: (a) Metal-catalyzed oxidation of proteins in aging and disease. Previous studies in this laboratory led to the discovery that proteins are highly susceptible to metal-catalyzed oxidation and that this oxidation leads to conversion of the side chains of some amino acid residues to carbonyl derivatives. Based on this finding, the carbonyl content of protein has become a widely used marker of oxidative stress-mediated cellular damage and has led to the demonstration that the accumulation of oxidized protein is associated with aging and a number of age-related diseases. Taking advantage of sophisticated mass spectroscopic and high pressure liquid chromatographic technologies, we have now developed procedures for the identification and assay of those protein carbonyl derivatives known to be formed by metal-catalyzed reactions. Results of these studies demonstrate that oxidation of lysine, arginine, and proline residues of proteins account for at least 50% of the protein carbonyl groups in liver from old rats. (b) Antioxidant role of methionine residues of proteins. Surface-exposed methionine residues of proteins are highly susceptible to oxidation by almost every kind of reactive oxygen species (ROS). But, unlike other kinds of protein oxidation (except the oxidation of cysteine residues), the oxidation of methionine residues of proteins can be repaired by the action of methionine sulfoxide reductase that catalyzes the thioredoxin-dependent reduction of methionine sulfoxide back to methionine. Because the overall oxidation-reduction of methionine residues of proteins leads to conversion of various forms of ROS to unreactive products, we proposed that the cyclic oxidation/reduction of methionine residues of proteins may constitute an important antioxidant mechanism of cellular defense. To test this hypothesis, a mutant strain of mice was developed that lacked the dominant form of methionine sulfoxide reductase. Compared to the wild-type parental strain, the mutant exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen), has a shorter life span under both normal and hyperoxic conditions, develops an atypical (tip-toe) walking behavior after six months of age, accumulated higher levels of oxidized protein (carbonyl derivatives) under oxidative stress, and exhibits abnormal patterns of expression of thioredoxin reductase under conditions of oxidative stress. Thus, it appears that methionine sulfoxide reductase may play an important role in aging and neurological disorders. (c) Oxidation of the prion protein. Studies on the oxidation of Syrian hamster SHa(29-231) prion protein were initiated because this protein binds copper with high affinity and could therefore be highly susceptible to metal-catalyzed oxidation. Indeed, exposure of the prion protein to the ascorbate/oxygen/copper mixed function oxidation system led to rapid oxidation of the protein and to its aggregation, similar to that observed during conversion of the prion protein to its pathogenic counterpart. Because the prion protein contains numerous surface-exposed methionine residues, structural changes associated with the oxidation of these residues is also under investigation. It was established that exposure of the protein to hydrogen peroxide in the absence of copper leads to rapid oxidation of methionine residues 109 and 112, which are known to be essential for the properties of the toxic peptide, the fibrillogenic prion fragment PrP 106-126. Several other residues, including Met 129, were also oxidized. In contrast to the metal-catalyzed oxidation, this oxidation did not result in aggregation. (d) Regulation of methionine sulfoxide reductase transcription. Results of studies described in the above section and results of earlier studies in this laboratory with yeast and bacteria demonstrate that methionine sulfoxide reductase serves an important biological function as an antioxidant under conditions of oxidative stress. To identify which proteins are involved in the regulation of methionine sulfoxide reductase gene (msrA) transcription, nuclear proteins were isolated from both wild-type and null mutant strains of yeast and their ability to bind msrA promoter DNA was determined by electrophoretic mobility shift assays. By using this technique, several proteins that are candidates for a role in msrA transcription have been detected. Some of these have been cloned and antibodies are being prepared to confirm their binding specificities. Further studies are needed to establish their roles, if any, in msrA transcription. (e) Oxidation of methionine residues by hypochlorous acid. The biosynthesis of hyporchlorous acid by neutrophils and macrophages represents a major mechanism for antibacterial action in mammals. Hypochlorous acid is also able to oxidize methionine residues of proteins. Results of preliminary studies indicate that oxidation of free methionine by hypochlorous acid proceeds by an oxygen-independent mechanism in which chloramine derivatives are intermediates. However, if the alpha-amino group of methionine is acylated, as occurs in proteins, then the oxidation proceeds by a mechanism that does not involve a chloramine intermediate. Further studies are designed to determine whether the oxidation of methionine residues in proteins involves direct transfer of oxygen from hypochlorous acid to form methionine sulfoxide or if it involves interactions with water or molecular oxygen. (f) Role of reactive oxygen species in apoptosis. The activation of one or more proteases (caspases) is fundamental to the elimination of damaged (non-functional) cells in animal tissues by a process referred to as apoptosis. We demonstrated previously that activation of caspase-3 like activity of HeLa cells is induced by hydrogen peroxide and that this induction is inhibited by a general caspase inhibitor and also a caspase-3 specific inhibitor. Results of current investigations indicate that the caspase-3 activation does not involve either the caspase-9 (mitochondrial-dependent) or the caspase-8 (death receptor-dependent) mechanism, but may involve an actin-dependent focal adhesion process. Further studies are designed to confirm this possibility. (g) Manganese-induced apoptosis. We reported earlier that at high concentrations manganese (Mn) induces apoptosis by a non-mitochondrial-mediated mechanism. In continuing studies, we have demonstrated that Mn-induced activation of caspase-3 like activity in 3T3 cells is suppressed by calpain inhibitors I, II, and by the p38 inhibitor, SB 202190. However, after activation has occurred, these inhibitors have no effect on caspase-3 like activity. Further studies are directed toward explanation of the linkages between p38, calpain, and caspase-12 in Mn-induced apoptosis.

NHLBI 生物化学实验室酶科的研究旨在阐明氧化应激过程中细胞损伤产生的基本机制，以及这种损伤对衰老和疾病的影响。为此，我们目前的研究涉及以下探索领域的研究：（a）衰老和疾病中金属催化的蛋白质氧化。该实验室之前的研究发现蛋白质对金属催化的氧化高度敏感，并且这种氧化导致一些氨基酸残基的侧链转化为羰基衍生物。基于这一发现，蛋白质的羰基含量已成为氧化应激介导的细胞损伤的广泛使用的标志物，并证明氧化蛋白质的积累与衰老和许多与年龄相关的疾病有关。利用先进的质谱和高压液相色谱技术，我们现在开发了用于鉴定和测定已知由金属催化反应形成的蛋白质羰基衍生物的程序。这些研究结果表明，蛋白质赖氨酸、精氨酸和脯氨酸残基的氧化至少占老年大鼠肝脏中蛋白质羰基的50%。 (b)蛋白质的蛋氨酸残基的抗氧化作用。蛋白质表面暴露的蛋氨酸残基非常容易被几乎每种活性氧 (ROS) 氧化。但是，与其他类型的蛋白质氧化（半胱氨酸残基的氧化除外）不同，蛋白质的蛋氨酸残基的氧化可以通过蛋氨酸亚砜还原酶的作用进行修复，该还原酶催化甲硫氨酸亚砜依赖硫氧还蛋白还原成蛋氨酸。由于蛋白质蛋氨酸残基的整体氧化还原导致各种形式的ROS转化为非反应产物，因此我们提出蛋白质蛋氨酸残基的循环氧化/还原可能构成细胞防御的重要抗氧化机制。为了验证这一假设，开发了一种缺乏蛋氨酸亚砜还原酶的主要形式的突变小鼠品系。与野生型亲本品系相比，突变体对氧化应激（暴露于100%氧气）表现出增强的敏感性，在正常和高氧条件下寿命较短，六个月后出现非典型（踮起脚尖）行走行为，在氧化应激下积累更高水平的氧化蛋白（羰基衍生物），并表现出硫氧还蛋白还原酶表达异常模式 在氧化应激条件下。因此，蛋氨酸亚砜还原酶似乎在衰老和神经系统疾病中发挥重要作用。 (c) 朊病毒蛋白的氧化。人们开始对叙利亚仓鼠 SHa(29-231) 朊病毒蛋白的氧化进行研究，因为该蛋白以高亲和力结合铜，因此对金属催化的氧化非常敏感。事实上，朊病毒蛋白暴露于抗坏血酸/氧/铜混合功能氧化系统导致蛋白质快速氧化及其聚集，类似于朊病毒蛋白转化为其致病对应物期间观察到的情况。由于朊病毒蛋白含有大量表面暴露的蛋氨酸残基，因此与这些残基氧化相关的结构变化也在研究中。已经确定，在没有铜的情况下，将蛋白质暴露于过氧化氢会导致蛋氨酸残基 109 和 112 快速氧化，已知这对于有毒肽（纤维化朊病毒片段 PrP 106-126）的特性至关重要。包括 Met 129 在内的其他几种残留物也被氧化。与金属催化的氧化相反，这种氧化不会导致聚集。 (d)甲硫氨酸亚砜还原酶转录的调节。上一节所述的研究结果以及本实验室早期对酵母和细菌的研究结果表明，蛋氨酸亚砜还原酶在氧化应激条件下作为抗氧化剂发挥着重要的生物功能。为了确定哪些蛋白质参与甲硫氨酸亚砜还原酶基因 (msrA) 转录的调节，从野生型和无效突变酵母菌株中分离出核蛋白，并通过电泳迁移率变动测定测定它们结合 msrA 启动子 DNA 的能力。通过使用这种技术，已经检测到了几种在 msrA 转录中发挥作用的候选蛋白质。其中一些已经被克隆，并且正在制备抗体以确认它们的结合特异性。需要进一步的研究来确定它们在 msrA 转录中的作用（如果有的话）。 (e)次氯酸对甲硫氨酸残基的氧化。中性粒细胞和巨噬细胞生物合成次氯酸是哺乳动物抗菌作用的主要机制。次氯酸还能够氧化蛋白质的蛋氨酸残基。初步研究结果表明，次氯酸对游离蛋氨酸的氧化是通过不依赖氧的机制进行的，其中氯胺衍生物是中间体。然而，如果甲硫氨酸的α-氨基被酰化（如蛋白质中发生的那样），则氧化通过不涉及氯胺中间体的机制进行。进一步的研究旨在确定蛋白质中蛋氨酸残基的氧化是否涉及氧从次氯酸直接转移形成蛋氨酸亚砜，或者是否涉及与水或分子氧的相互作用。 (f)活性氧在细胞凋亡中的作用。一种或多种蛋白酶（半胱天冬酶）的激活是通过称为细胞凋亡的过程消除动物组织中受损（无功能）细胞的基础。我们之前证明，HeLa 细胞的 caspase-3 样活性的激活是由过氧化氢诱导的，并且这种诱导被一般 caspase 抑制剂和 caspase-3 特异性抑制剂抑制。目前的研究结果表明，caspase-3的激活不涉及caspase-9（线粒体依赖性）或caspase-8（死亡受体依赖性）机制，但可能涉及肌动蛋白依赖性粘着斑过程。进一步的研究旨在证实这种可能性。 (g) 锰诱导的细胞凋亡。我们之前报道过，高浓度的锰 (Mn) 通过非线粒体介导的机制诱导细胞凋亡。在持续的研究中，我们证明了 3T3 细胞中锰诱导的 caspase-3 样活性激活可被钙蛋白酶抑制剂 I、II 和 p38 抑制剂 SB 202190 抑制。然而，激活发生后，这些抑制剂对 caspase-3 样活性没有影响。进一步的研究旨在解释 p38、钙蛋白酶和 caspase-12 在 Mn 诱导的细胞凋亡中的联系。