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)蛋白质蛋氨酸残基的循环氧化和还原的抗氧化作用。蛋白质中蛋氨酸残基的活性氧氧化导致蛋氨酸亚砜的R-和S-立体异构体的外消旋混合物的形成。在以前的研究中，我们发现细菌、酵母和哺乳动物中都含有一种叫做蛋氨酸亚砜还原酶(MSRA)的酶，该酶能催化麦硫氨酸的S异构体还原为蛋氨酸，而缺乏MSRA的小鼠突变株比野生株对氧化应激诱导的蛋白图像更敏感，最长寿命也更短。在持续的研究中，我们发现了第二种形式的蛋氨酸亚砜还原酶(MSRB)，它专用于还原MetO的R-异构体。我们进一步发现，与MSRA不同的是，这种R-特异性还原酶在催化部位含有一个硒半胱氨酸残基。通过定点突变的方法，我们在大肠杆菌中表达了小鼠硒-MSRB，结果表明，半胱氨酸取代该酶中的硒半胱氨酸会导致酶的催化活性显著下降(&gt；90%)，而硒半胱氨酸被丙氨酸或丝氨酸取代会导致活性完全丧失。在进一步的研究中，我们将尝试确定同时缺失和过度表达这两种形式的还原酶对小鼠寿命和对氧化应激敏感性的影响。 (B)甲硫氨酸亚砜转录和/或翻译的调控。为了确定参与MSRA蛋白表达调控的蛋白质，对部分纯化的野生型和零突变酵母核蛋白与蛋氨酸亚砜还原酶基因(MSRA)启动子区域结合的能力进行了检测。与野生型相比，零突变体的延伸因子(EF)-γ、核G蛋白和延伸因子-3与MSRA启动子DNA具有更强的结合活性。延伸因子-1伽马被证明与MSRA启动子3‘末端的39个碱基对片段结合。野生型酵母菌株在MSRA基因及其启动子区域导入后，其MSRA的表达水平是缺失因子1-γ突变菌株的3倍，证实了其对MSRA合成的调控作用。 (C)Caspase-12在细胞信号转导和锰诱导的细胞凋亡中的作用。我们早些时候报道，高浓度的锰通过非线粒体介导的机制诱导3T3细胞凋亡，其中caspase12起关键作用。为了阐明caspase-12基因调控的基本机制，我们现在分离了大鼠caspase-12表达中包含基本启动子和对血清饥饿反应的调控元件的DNA片段。此外，caspase-12的转录起始点已经确定。进一步的研究旨在鉴定血清中调节该酶表达的生长因子、细胞因子或转录因子。此外，caspase12基因已经在Pichi pasterns酵母中过度表达，在那里它被排泄到生长介质中，并从那里进行纯化。我们希望能够使该蛋白结晶，并检测其与已知与细胞凋亡有关的潜在调控蛋白相互作用的能力。 (D)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，我们开展了旨在阐明相关机制的研究。正如去年的报告指出的那样，初步结果表明，氯胺衍生物是中间体。然而，进一步的研究表明，其他尚未阐明的机制也参与其中。 (E)衰老和疾病中金属催化的蛋白质氧化。本实验室以往的研究表明，金属催化的蛋白质氧化是蛋白质氧化为羰基衍生物的主要机制，而赖氨酸和精氨酸残基是这些金属催化反应的主要靶标。我们现在已经启动了一个项目来产生针对氧化形式的精氨酸和赖氨酸残基的抗体，这些抗体可以用来检查金属催化的氧化对衰老和各种疾病的贡献。

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 the cyclic oxidation and reduction of protein methionine residues. Reactive oxygen-mediated oxidation of methionine residues of proteins leads to the formation of a racemic mixture of the R- and S- stereo isomers of methionine sulfoxide (MetO). In previous studies, we showed that bacteria, yeast, and mammals contain an enzyme, methionine sulfoxide reductase (MsrA), that can catalyze reduction of the S-isomer of MetO back to methionine, and that mutant strains of mice lacking MsrA are more sensitive to oxidative stress-induced protein image and have a shorter maximum life span than the wild type strain. In continuing studies, we identified a second form of methionine sulfoxide reductase (MsrB) that is specific for the reduction of the R-isomer of MetO. We found further that in contrast to MsrA this R-specific reductase contains a selenocysteine residue at the catalytic site. By means of site-directed mutagenesis we expressed the mouse seleno-MsrB in Escherichia coli, demonstrated that substitution of cysteine for selenocysteine in this enzyme leads to a substantial (>90%) decrease in catalytic activity, and that replacement of the selenocysteine with either alanine or serine leads to complete loss of activity. In further studies, we will attempt to determine the effects of simultaneous deletions and overexpressions of both forms of reductase in mice on their longevity and sensitivity to oxidative stress. (b) Regulation of methionine sulfoxide transcription and /or translation. To identify proteins that are involved in the regulation of MsrA protein expression, partially purified nuclear proteins from both wild type and null mutant strains of yeast were tested for their ability to bind to the promoter region of the methionine sulfoxide reductase gene (msrA). The elongation factor (EF)-gamma, nuclear G-protein, and elongation factor-3, were among those that exhibited greater binding activity to msrA promoter DNA from the null mutant as compared to that from the wild-type. The elongation factor-1gamma was shown to bind to the 39 base pair segment at the 3'-prime end of the msrA promoter. Its role in the regulation of MsrA synthesis was confirmed by the demonstration that after their transfection with the msrA gene together with its promoter region, the level of MsrA in a wild type yeast strain was three-fold greater than that in a mutant strain lacking factor 1-gamma. (c) Role of Caspase-12 in cell signaling and manganese-induced apoptosis. We reported earlier that high concentrations of manganese induces apoptosis in 3T3 cells by a non-mitochondrial-mediated mechanism in which caspase 12 plays a key role. To elucidate basic mechanisms involved in the regulation of caspase-12 gene regulation, we have now isolated the DNA fragments containing the basal promoter and the regulatory element responsive to serum starvation in rat caspase-12 expression. In addition, the transcriptional start site of caspase-12 has been identified. Further studies are directed toward identification of the growth factors, cytokines, or transcription factors in serum that regulate expression of this enzyme. Furthermore, the caspase 12 gene has been over expressed in the Pichi pasterns yeast where it is excreted into the growth medium, from which it is being purified. We hope to be able to crystallize the protein and examine its ability to interact with potential regulatory proteins known to be implicated in apoptosis. (d) Inflammation-induced oxidation of methionine residues of proteins. The biosynthesis of hypochlorous acid by neutrophils and macrophage represents a major mechanism for antibacterial action in mammals. Because methionine residues of proteins are particularly sensitive to oxidation by hypochlorous acid, we have carried out studies aimed at elucidation of the mechanism involved. As noted in last years report, preliminary results demonstrated that chloramine derivatives are intermediates. However, further studies have shown that other mechanisms not yet elucidated are also involved. (e) Metal-catalyzed oxidation of proteins in aging and disease. Previous studies in this laboratory led to the demonstration that metal-catalyzed oxidation of proteins is the major mechanism involved in the oxidation of proteins to carbonyl derivatives, and that lysine and arginine residues are major targets for these metal-catalyzed reactions. We have now initiated a program to generate antibodies against oxidized forms of arginine and lysine residues that can be used to examine the contribution of metal-catalyzed oxidation to aging and various diseases.