Biochemical Mechanisms of Enzyme Action and Cellular Regulation

酶作用和细胞调节的生化机制

• 批准号: 8746536
• 负责人: P. BOON Chock
• 金额: $ 29.23万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8746536
• 关键词: Active Sites; Affinity; Aging; Alanine; Aldehydes; Anabolism; Antioxidants; Apoptosis; Aspartate; Binding; Binding Proteins; Biochemical; Biological Process; C83; Cardiolipins; Cell Cycle; Cell physiology; Complex; Cysteine; Cytosol; Data Reporting; Deterioration; Disease; Enzymes; Escherichia coli; Etiology; Exhibits; Family; Free Radicals; Glycine; Guanine; Guanosine; Human; Hydrogen Peroxide; In Vitro; Knowledge; Link; Liposomes; Magnesium; Mediating; Messenger RNA; Mitochondria; Modification; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Weight; Motor Neurons; Mutation; N-terminal; Neurodegenerative Disorders; Orthologous Gene; Orthophosphate; Oxidation-Reduction; Oxidative Stress; PTEN gene; Peroxidases; Phosphotransferases; Physiological; Play; Polyribosomes; Production; Proteins; RNA; Reaction; Reactive Oxygen Species; Regulation; Reporting; Research; Ribonucleosides; Role; Schiff Bases; Selenium; Selenocysteine; Signal Pathway; Signal Transduction; Site; Solutions; Structure; Sulfhydryl Compounds; System; Translations; age related; amino group; analytical ultracentrifugation; ascorbate; base; cell growth regulation; cofactor; crosslink; cytochrome c; dimer; enzyme mechanism; inorganic phosphate; insight; liquid chromatography mass spectrometry; monomer; mutant; oxidation; peroxiredoxin; peroxiredoxin I; preference; programs; research study; response; seleno-tRNA; selenophosphate; selenophosphate synthetase; selenoprotein; sugar

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.

在这个项目中，我们专注于以下项目： (i)RNA氧化。越来越多的证据表明，RNA氧化与许多年龄相关的神经退行性疾病相关，包括最近的发现表明，mRNA氧化发生在ALS运动神经元退化的早期。我们以前表明，氧化的mRNA导致翻译保真度的降低，尽管事实上，氧化的mRNA表现出类似的亲和力，作为非氧化的mRNA的能力，以结合多核糖体。我们最近的研究表明，在体外细胞色素c（cyt c）/H2 O2或Fe（II）/抗坏血酸/H2 O2系统催化的RNA氧化产生不同的共价修饰的RNA衍生物。我们发现，在细胞色素c（cyt c）介导的氧化中，RNA中的鸟苷是主要的核糖核苷氧化，而Fe（II）/抗坏血酸体系氧化所有的核糖核苷没有明显的偏好。GC/MS和LC/MS分析表明，鸟嘌呤碱基不仅被氧化，但它也脱嘌呤，形成脱碱基糖部分。脱碱基位点上的醛部分与蛋白质中的氨基形成席夫碱，并产生交联产物，例如氧化的RNA和cyt c之间的交联产物。有趣的是，氧化的RNA和细胞色素c之间的交联产物的形成促进细胞色素c从含心磷脂的脂质体中释放，这可能代表细胞色素c从线粒体释放到胞质溶胶中。因此，RNA的氧化修饰，包括交联，不仅导致RNA正常功能受损，而且获得保护性信号以促进细胞凋亡以响应氧化应激。 (ii)蛋白谷胱甘肽化在过氧化物酶调节中的作用。可逆的蛋白质谷胱甘肽化是一种氧化还原敏感的调节机制，在细胞调节、细胞信号传导和抗氧化防御中起着重要作用。这种机制涉及调节过氧化物酶的功能，过氧化物酶是一种广泛表达的巯基特异性过氧化物酶家族。我们之前报道过，peroxiredoxin I可以在其三个半胱氨酸残基C52，C83和C173处被谷胱甘肽化，并且去谷胱甘肽化由sulfiredoxin催化。过氧化物氧还蛋白在其催化活性半胱氨酸处的谷胱甘肽化不仅提供还原当量以支持其过氧化物酶活性，而且保护过氧化物氧还蛋白免于不可逆的过度氧化。此外，当过氧化物氧还蛋白I作为十聚体和/或更高分子量的复合物存在时，它也起到分子伴侣的作用。我们发现谷胱甘肽化调节过氧化物氧化还原酶的四级结构。过氧化物氧还蛋白I在C83处的谷胱甘肽化将十聚体过氧化物氧还蛋白转化为其二聚体，同时丧失其伴侣活性。二聚体/寡聚体结构特异性过氧化物氧还蛋白I结合蛋白，其中，磷酸酶和张力蛋白同源物（PTEN）和哺乳动物Ste 20样激酶-1（MST 1），分别调节细胞周期和细胞凋亡，这一发现表明谷胱甘肽化和这些信号通路之间可能存在联系。 (iii)对E.大肠杆菌硒磷酸合成酶。硒磷酸合成酶（SPS）催化硒磷酸的合成，硒磷酸是硒tRNA中硒代半胱氨酸和2-硒尿苷残基生物合成的硒供体。硒代半胱氨酸在翻译过程中掺入蛋白质中形成硒蛋白，其调节多种细胞过程。SPS催化硒磷酸盐的形成，使用ATP和硒化物作为底物，镁和钾作为辅因子。 在该反应中，ATP的γ磷酸被转移到硒化物以形成硒磷酸，而ADP被水解以形成正磷酸和AMP。目前对这种酶系统的了解来自于使用E. coli SPS。为了深入了解该酶的结构和催化机制，对大肠杆菌SPS的C17 S突变体的晶体结构进行了分析。coli（EcSPSC 17 S）中的表达。EcSPSC 17 S结晶为同二聚体。该酶在溶液中的二聚体结构通过分析超离心实验证实。其富含甘氨酸的N-末端区域（残基1- 47）被发现处于开放构象，并且在两种结构中大部分是有序的，其中镁结合在每个单体的活性位点，涉及四个保守的天冬氨酸残基，D51，D 68，D91和D227。 这些保守的天冬氨酸残基，沿着保守的N87，在活性位点突变为丙氨酸，完全消除了AMP的产生，突出了它们在酶的催化作用中的重要作用。基于EcSPS的结构和生化分析，连同从Aquifex aeolicus和人类的SPS直系同源物获得的研究报告的数据，EcSPS催化硒磷酸合成的催化机制提出。