Elucidation Of Cellular Damage During Exposure To Oxidat

在生物化学实验室，NHLBI的酶部分的研究，是针对在暴露于氧化应激过程中细胞损伤的产生所涉及的基本机制的阐明，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)蛋白质甲硫氨酸残基循环氧化还原的抗氧化作用。活性氧介导的蛋白质甲硫氨酸残基的氧化导致 甲硫氨酸亚砜的R和S立体异构体的外消旋混合物。大多数生物系统含有两种形式的甲硫氨酸亚砜还原酶：一种在催化位点含有半胱氨酸部分并且特异性地将亚砜的S-异构体还原回甲硫氨酸，另一种在催化位点含有硒代半胱氨酸并且特异性地还原R-异构体。为了研究硒对蛋氨酸互变的抗氧化活性的影响，小鼠在硒缺乏的饮食中生长。硒缺乏导致氧化蛋白的组织水平大幅增加，蛋氨酸亚砜含量和蛋白质羰基含量。硒缺乏在蛋白质氧化中的作用和小鼠蛋氨酸亚砜还原酶过表达的影响正在努力澄清中。 (b)炎症诱导的蛋白质蛋氨酸残基氧化。嗜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。由于蛋白质的蛋氨酸残基对次氯酸的氧化特别敏感，我们进行了研究，以阐明所涉及的机制。鉴于次氯酸的反应可能受到体外实验中使用的缓冲液种类的影响，以及迄今为止大多数研究都是在非生理缓冲系统中进行的，我们使用生理碳酸氢盐缓冲液进行了研究。这些研究的结果表明，次氯酸容易与碳酸氢盐缓冲液的组分反应，形成中间体，该中间体也能够氧化蛋白质的甲硫氨酸残基。进一步的研究将致力于确定该中间体的结构，并鉴定蛋白质的蛋氨酸残基的次氯酸-碳酸氢盐依赖性氧化中形成的产物。 (c)细胞凋亡在衰老中的作用。当动物达到成熟时，各种组织的大小是固定的。因此，对这些组织中细胞的氧化损伤将导致组织功能丧失，除非将受损细胞去除，然后用好细胞代替。值得注意的是，在低浓度下，活性氧物质能够激活细胞信号传导途径，导致通过细胞凋亡去除受损细胞，以及激活细胞复制的途径。这些信号传导过程提供了维持哺乳动物组织完整性的机制的可能性得到了初步研究结果的支持，初步研究结果显示，在培养的白血病NB 4细胞中抑制细胞凋亡，随后将细胞暴露于氧化应激，导致氧化损伤蛋白在这些细胞中积累。 (d)核糖核酸（RNA）氧化对翻译效率和准确性的影响。核糖核酸的氧化修饰与几种神经系统疾病有关。为了研究核糖核酸氧化对其翻译效力的影响，将编码荧光素酶基因的核糖核酸通过过氧化氢进行氧化，并检查其在网织红细胞裂解物中孵育时产生荧光素酶的能力。初步实验的结果表明，从氧化核糖核酸翻译的荧光素酶蛋白的活性显著低于正常荧光素酶制剂的活性。将进行进一步研究以确定由氧化核糖核酸产生的荧光素酶的修饰形式是否反映氨基酸的错误掺入。 (e)caspase-12转录的调节。我们先前发现，高浓度的锰诱导NIH 3 T3细胞凋亡的caspase-12介导的机制。为了阐明caspase-12基因转录调控的基本机制，我们分离并测序了小鼠caspase-12启动子的5'侧翼区和5'非翻译区（5' UTR）的两个片段，并将它们克隆到pGL 3荧光素酶基因上游的无启动子载体中。我们还分离了caspase-12基因下游的caspase-12 3 'UTR，并将其插入到SV 40启动子控制下的荧光素酶基因下游的pGL 3中。在NIH 3 T3细胞中监测5' UTR和3' UTR构建体的作用，所述NIH 3 T3细胞在存在和不存在10%血清的情况下生长。5 'UTR构建体的研究结果表明，血清含有荧光素酶mRNA翻译和caspase-12基因下调所需的因子。相反，用3 'UTR构建体的研究表明，血清含有下调荧光素酶基因所需的因子。总之，这些发现代表了caspase 12基因的caspase 5'和3' UTR区域的第一个特征，并应有助于更好地理解转录和翻译控制。

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) Antioxidant role of cyclic oxidation and reduction of methionine residues of proteins. Reactive oxygen-mediated oxidation of methionine residues of proteins leads to formation of a racemic mixture of the R- and S-stereo isomers of methionine sulfoxide. Most biological systems contain two forms of methionine sulfoxide reductase: one that contains a cysteine moiety at the catalytic site and is specific for the reduction of the S- isomer of the sulfoxide back to methionine, and an another that contains selenocysteine at the catalytic site and is specific for reduction of the R-isomer. To investigate the effects of selenium on the antioxidant activity of methionine interconversion, mice were grown on a selenium deficient diet. Selenium deficiency led to large increases in the tissue levels of oxidized protein, as measured by methionine sulfoxide content and the protein carbonyl content. Efforts to clarify the role of selenium deficiency in protein oxidation and the effect of over-expression of methionine sulfoxide reductase in mice are in progress. (b) Inflammation-induced oxidation of methionine residues of proteins. The biosynthesis of hypochlorous acid by neutrophils and macrophages represents a major mechanism for antibacterial action in mammals. Because methionine residues of proteins are particularly sensitive to oxidation by hypochlorous acid, we carried out studies to elucidate the mechanisms involved. In view of the fact that the reactions of hypochlorous acid might be influenced by the kind of buffer used in in vitro experiments and the fact that most studies to date have been carried out in non-physiological buffer systems, we carried out studies using the physiological bicarbonate buffer. Results of these studies indicate that hypochlorous acid reacts readily with components of the bicarbonate buffer to form an intermediate which is also able to oxidize methionine residues of proteins. Further studies will be directed toward determination of the structure of this intermediate and to identify the products formed in the hypochlorous-bicarbonate dependent oxidation of methionine residues of proteins. (c) Role of apoptosis in aging. When animals reach maturity, the size of various tissues is fixed. Therefore, oxidative damage to cells in these tissues will lead to loss of tissue function unless the damaged cells are removed and then replaced by good cells. Significantly, at low concentrations, reactive oxygen species are able to activate cell signaling pathways leading to the removal of damaged cells by apoptosis and also pathways involved in the activation of cell replication. The possibility that these signaling processes provide a mechanism for maintaining the integrity of mammalian tissues is supported by results of preliminary studies showing the inhibition of apoptosis in cultured leukemia NB4 cells, followed by exposure of the cells to oxidative stress, leads to accumulation of oxidatively damaged protein in these cells. (d) Effect of ribonucleic acid (RNA) oxidation on translational efficiency and accuracy. Oxidative modification of ribonucleic acid is associated with several neurological disorders. To study the effect of ribonucleic acid oxidation on its translational efficacy, ribonucleic acid encoding the luciferase gene was subjected to oxidation by hydrogen peroxide and its ability to produce luciferase when incubated in reticulocyte lysate was examined. Results of preliminary experiments indicate that activity of the luciferase protein translated from oxidized ribonucleic acid is considerably lower than that of normal luciferase preparations. Further studies will be made to determine if the modified form of luciferase generated by the oxidized ribonucleic acid reflects miss-incorporation of amino acids. (e) Regulation of caspase-12 transcription. We showed earlier that high concentrations of manganese induces apoptosis in NIH 3T3 cells by a caspase-12-mediated mechanism. To elucidate basic mechanisms involved in the regulation of caspase-12 gene transcription, we isolated and sequenced two fragments of the 5' flanking region and the 5' untranslated region (5' UTR) of the mouse caspase-12 promoter and cloned them into the pGL3 promoter-less vector upstream of the luciferase gene. We also isolated the caspase-12 3'UTR which is downstream of the caspase-12 gene and inserted into pGL3 downstream from the luciferase gene which is under control of the SV40 promoter. The effects of the 5' UTR and 3' UTR constructs were monitored in NIH 3T3 cells which were grown in the presence and absence of 10% serum. Results of the studies with the 5'UTR construct suggest that serum contains factors that are required for translation of the luciferase mRNA and for down regulation of the caspase-12 gene. In contrast, studies with the 3'UTR construct showed that serum contains a factor that is required for the down regulation of the luciferase gene. Together, these findings represent the first characterization of the caspase 5' and 3' UTR region of the caspase 12 gene and should facilitate a better understanding of transcriptional and translational control.