Elucidation Of Cellular Damage During Exposure To Oxidat

NHLBI生物化学实验室酶科的研究旨在阐明在暴露于氧化应激期间产生细胞损伤的基本机制以及这种损伤对衰老和疾病的贡献。为此，我们目前的研究涉及以下探索领域的研究：(a)老化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，蛋白质对金属催化氧化非常敏感，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一个广泛使用的标志，并导致证明氧化蛋白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，我们现在已经开发出鉴定和分析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，老年大鼠肝脏中蛋白质的赖氨酸、精氨酸和脯氨酸残基的氧化至少占蛋白质羰基的50%。(b)蛋白质蛋氨酸残基的抗氧化作用。蛋白质表面暴露的蛋氨酸残基极易被几乎所有活性氧（ROS）氧化。但是，与其他种类的蛋白质氧化（除了半胱氨酸残基的氧化）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来修复，该酶催化硫氧还蛋白依赖的蛋氨酸亚砜还原回蛋氨酸。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转化为非活性产物，我们提出蛋白质蛋氨酸残基的循环氧化/还原可能是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主要形式的突变小鼠。与野生型亲本菌株相比，突变体表现出对氧化应激（暴露于100%氧气）的敏感性增强，在正常和高氧条件下寿命都较短，6个月后出现不典型（踮脚）行走行为，在氧化应激条件下积累了更高水平的氧化蛋白（羰基衍生物），并且在氧化应激条件下表现出异常的硫氧还蛋白还原酶表达模式。因此，蛋氨酸亚砜还原酶可能在衰老和神经系统疾病中起重要作用。

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.