Elucidation Of Cellular Damage During Exposure To Oxidat

在NHLBI生物化学实验室的酶部分的研究，旨在阐明在氧化应激下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几个方面的探索： (A)蛋白质的循环氧化和蛋氨酸残基还原的抗氧化作用。活性氧介导的蛋白质蛋氨酸残基氧化形成 蛋氨酸亚砜的R-和S-立体异构体的外消旋混合物。大多数生物体系含有两种形式的蛋氨酸亚砜还原酶：一种是在催化部位含有半胱氨酸部分，专用于将亚砜的S异构体还原为蛋氨酸；另一种是在催化部位含有硒半胱氨酸，专用于R-异构体的还原。为了研究硒对蛋氨酸相互转化抗氧化活性的影响，用低硒饲料喂养小鼠。硒缺乏导致组织中氧化蛋白水平的大幅增加，以蛋氨酸亚砜含量和蛋白质羰基含量来衡量。关于硒缺乏在蛋白质氧化中的作用以及蛋氨酸亚砜还原酶在小鼠体内过度表达的影响的研究正在进行中。 (B)炎症引起的蛋白质蛋氨酸残基的氧化。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。由于蛋白质的蛋氨酸残基对次氯酸的氧化特别敏感，我们开展了相关研究，以阐明相关机制。鉴于次氯酸的反应可能会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液体系中进行的，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，次氯酸很容易与碳酸氢盐缓冲液的成分反应，形成一种中间体，该中间体也能够氧化蛋白质的蛋氨酸残基。进一步的研究将致力于确定这种中间体的结构，并鉴定蛋白质的蛋氨酸残基在次氯-碳酸氢盐依赖的氧化过程中形成的产物。 (C)细胞凋亡在衰老中的作用。当动物成熟时，各种组织的大小是固定的。因此，这些组织中细胞的氧化损伤将导致组织功能的丧失，除非破坏的细胞被移除，然后被良好的细胞取代。值得注意的是，在低浓度下，活性氧能够激活细胞信号通路，导致通过凋亡去除受损的细胞，以及参与激活细胞复制的通路。这些信号过程为维持哺乳动物组织的完整性提供了一种机制，这一可能性得到了初步研究结果的支持，这些结果表明，在培养的白血病NB4细胞中，细胞凋亡受到抑制，随后细胞暴露在氧化应激下，导致氧化损伤蛋白在这些细胞中积累。 (D)核糖核酸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疾病有关。为了研究核糖核酸氧化对其翻译效率的影响，将编码荧光素酶基因的核糖核酸用过氧化氢氧化，并检测其在网织红细胞裂解液中产生荧光素酶的能力。初步实验结果表明，由氧化核糖核酸翻译的荧光素酶蛋白的活性明显低于正常的荧光素酶制剂。将进行进一步的研究，以确定氧化核糖核酸产生的荧光素酶的修饰形式是否反映了氨基酸的缺失。 (E)caspase-12转录调控。我们早些时候发现，高浓度的锰通过caspase-12介导的机制诱导NIH3T3细胞凋亡。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5‘侧翼区和5’非翻译区的两个片段，并将它们克隆到荧光素酶基因上游的pGL3无启动子载体中。我们还分离了位于caspase-12基因下游的caspase-12 3‘非编码区，并将其插入到受SV40启动子控制的荧光素酶基因下游的pGL3中。在有或无10%血清培养的NIH3T3细胞中，观察了5‘非编码区和3’非编码区构建的影响。对5‘端非编码区结构的研究结果表明，血清中含有转录荧光素酶mRNA和下调caspase-12基因所需的因子。相反，对3‘非编码区结构的研究表明，血清中含有一种荧光素酶基因下调所需的因子。总之，这些发现代表了caspase 12基因5‘和3’非编码区的第一个特征，应该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.