Biochemical Mechanisms of Enzyme Action and Cellular Regulation

在这个项目中，我们重点关注了以下项目： (I)RNA氧化。越来越多的证据表明，RNA氧化与许多与年龄相关的神经退行性疾病有关，包括最近的研究结果表明，在ALS的运动神经元退化过程中，mRNA的氧化发生在早期。我们之前的研究表明，尽管氧化后的信使核糖核酸在结合多聚体方面表现出与未氧化信使核糖核酸相似的亲和力，但氧化后的信使核糖核酸会导致翻译保真度的降低。我们最近的研究表明，在体外，细胞色素c(Cytc)/H_2O_2或Fe(II)/抗坏血酸/H_2O_2体系催化的RNA氧化产生了不同的共价修饰的RNA衍生物。我们发现，在细胞色素c(Cytc)介导的氧化中，RNA中的鸟苷是被氧化的主要核糖核苷，而Fe(II)/抗坏血酸体系氧化所有的核糖核苷，没有明显的选择性。GC/MS和LC/MS分析表明，鸟嘌呤碱基不仅被氧化，而且还被净化成碱性糖基。碱性位点上的乙醛部分与蛋白质中的氨基形成席夫碱，并产生交联物，如氧化的RNA与细胞色素c之间的交联物。有趣的是，氧化的RNA与细胞色素c之间的交联物的形成促进了含心磷脂的脂质体释放细胞色素c，这可能代表细胞色素c从线粒体释放到胞浆中。因此，RNA的氧化修饰，包括交联，不仅导致RNA的正常功能受损，而且还获得了一种保护性信号，促进了细胞在氧化应激下的凋亡。 (Ii)调节过氧化还蛋白的谷胱甘肽基化作用。蛋白质谷胱甘肽可逆化是一种氧化还原敏感的调节机制，在细胞调节、细胞信号转导和抗氧化防御中发挥着重要作用。这一机制涉及调节过氧化氢酶的功能，过氧化氢酶是一种普遍表达的硫醇特异性过氧化物酶家族。我们早些时候报道过，过氧化还蛋白I可以在它的三个半胱氨酸残基C52、C83和C173上发生谷胱甘肽基化，而去谷胱甘肽基化是由硫氧还蛋白催化的。过氧化还蛋白在其催化活性的半胱氨酸上的谷胱甘肽基化不仅提供了支持其过氧化物酶活性的还原当量，而且还保护了过氧化还蛋白免受不可逆的过度氧化。此外，当过氧化还蛋白I以十聚体和/或更高分子质量的复合体存在时，它还可以作为分子伴侣发挥作用。我们发现谷胱甘肽的作用调节了过氧化还蛋白的四级结构。过氧化氧还蛋白I在C83位的谷胱甘肽基化可将十聚体的过氧化氧还蛋白转化为其二聚体，同时失去其伴侣活性。研究发现，二聚体/寡聚体结构特异性的过氧化还蛋白I结合蛋白，其中磷酸酶和张力蛋白同源蛋白(PTEN)和哺乳动物Ste20样激酶-1(MST1)分别调节细胞周期和细胞凋亡，提示谷胱甘肽与这些信号通路之间可能存在联系。 (Iii)对大肠杆菌硒磷酸合成酶催化机理的结构洞察。硒磷酸合成酶(SPS)催化合成硒磷酸，为硒半胱氨酸和硒-tRNA中2-硒尿苷残基的生物合成提供硒供体。硒半胱氨酸在翻译过程中被结合到蛋白质中，形成调节各种细胞过程的硒蛋白。SPS以三磷酸腺苷和硒化物为底物，以镁和钾为辅因子，催化形成硒磷酸盐。在该反应中，三磷酸腺苷的伽马磷酸盐转移到硒化物上生成亚硒酸盐，而二磷酸腺苷则被水解生成正磷酸盐和AMP。目前关于这个酶系统的知识来自于对E.ColiSPS的研究。为了深入了解该酶的结构和催化机理，对大肠杆菌SPS的C17S突变体(EcSPSC17S)的晶体结构进行了研究。EcSPSC17S结晶为同源二聚体。分析超速离心实验证实了该酶在溶液中的二聚体结构。其富含甘氨酸的N-末端区域(残基1-47)以开放的构象存在，并且在两个结构中大部分是有序的，每个单体的活性部位都有一个镁结合，涉及四个保守的天冬氨酸残基，D51，D68，D91和D227。这些保守的天冬氨酸残基与活性部位的保守N87一起突变为丙氨酸，完全消除了AMP的产生，突显了它们在酶的催化作用中的重要作用。根据本文报道的ECSPS的结构和生化分析，结合从Aquifex aeolicus和人类获得的SPS同源物的研究数据，提出了ECSPS催化合成磷酸硒的催化机理。

In this program, we focused on the following projects: (i) RNA oxidation. Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, including the recent finding showing that mRNA oxidation occurs early in motor neuron deterioration in ALS. We previously showed that oxidized mRNA causes a reduction of translation fidelity despite the fact that the oxidized mRNA exhibits a similar affinity as non-oxidized mRNA for its capacity to bind polysomes. Our recent study revealed that in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H2O2 or by the Fe(II)/ascorbate/H2O2 system yielded different covalently modified RNA derivatives. We found that guanosine in RNA was the predominant ribonucleoside oxidized in cytochrome c (cyt c)-mediated oxidation, while Fe(II)/ascorbate system oxidized all ribonucleoside with no obvious preference. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but it also depurinated to form an abasic sugar moiety. The aldehyde moieties on the abasic site formed Schiff base with the amino groups in the proteins and generated cross-linking products, such as that between oxidized RNA and cyt c. Interestingly, the formation of the cross-linking product between oxidized RNA and cyt c facilitates the release of cyt c from cardiolipin-containing liposomes, which may represent the release of cyt c from the mitochondria to the cytosol. Thus, the oxidative modification of RNA, including cross-linking, leads not only to impair RNA normal functions, but also to gain a protective signal to facilitate cellular apoptosis in response to oxidative stress. (ii) Protein glutathionylation in the regulation of peroxiredoxins. Reversible protein glutathionylation, a redox-sensitive regulatory mechanism, plays an important role in cellular regulation, cell signaling, and antioxidant defense. This mechanism is involved in regulating the functions of peroxiredoxins, a family of ubiquitously expressed thiol-specific peroxidase enzymes. We reported earlier that peroxiredoxin I can be glutathionylated at three of its cysteine residues, C52, C83, and C173, and the deglutathionylation is catalyzed by sulfiredoxin. Glutathionylation of peroxiredoxins at their catalytically active cysteines not only provide the reducing equivalents to support their peroxidase activity but also protect peroxiredoxins from irreversible hyperoxidation. In addition, peroxiredoxin I also functions as a molecular chaperone when it exists as a decamer and/or higher molecular weight complexes. We showed that glutathionylation regulates the quaternary structure of peroxiredoxins. Glutathionylation of peroxiredoxin I at C83 converts the decameric peroxiredoxin to its dimers with the loss of its chaperone activity. The findings that dimer/oligomer structure-specific peroxiredoxin I binding proteins, among them, phosphatase and tensin homolog (PTEN) and mammalian Ste20-like kinase-1 (MST1), regulate cell cycle and apoptosis, respectively, suggest a possible link between glutathionylation and those signaling pathways. (iii) Structural insights into the catalytic mechanism of E. coli selenophosphate synthetase. Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine is incorporated into proteins during translation to form selenoproteins which regulate a variety of cellular processes. SPS catalyzes the formation of selenophosphate using ATP and selenide as substrates and a magnesium and a potasium as cofactors. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Current knowledge of this enzyme system is derived from studies using the E. coli SPS. To gain the structural insights and the catalytic mechanism of this enzyme, the crystal structure of the C17S mutant of SPS from E. coli (EcSPSC17S) was investigated. EcSPSC17S crystallizes as a homodimer. The dimeric structure in solution of this enzyme was confirmed by analytical ultracentrifugation experiments. Its glycine-rich N-terminal region (residues 1- 47) was found in an opened conformation and was mostly ordered in both structures, with a magnesium bound at the active site of each monomer involving four conserved aspartate residues, D51, D68, D91 and D227. Mutation of these conserved aspartate residues, along with the conserved N87, at the active site, to alanine completely abolished AMP production, highlighting their essential role in the catalytic action of the enzyme. Based on the structural and biochemical analysis of EcSPS reported here, together with the data reported from studies obtained with SPS orthologs from Aquifex aeolicus and humans, a catalytic mechanism was proposed for the selenophosphate synthesis catalyzed by EcSPS.