Biochemical Mechanisms of Enzyme Action and Cellular Regulation

酶作用和细胞调节的生化机制

• 批准号: 9353081
• 负责人: P. BOON Chock
• 金额: $ 37.25万
• 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/9353081
• 关键词: Accounting; Actins; Acute; Aging; Aldehydes; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Apoptosis; Attention; Bacteria; Binding; Biochemical; Bioinformatics; Biological Process; C-terminal; Cardiolipins; Catalysis; Cell Adhesion; Cells; Complex; Cytosol; DNA Sequence; Data; Degradation Pathway; Density Gradient Centrifugation; Deterioration; Dimerization; Disease; Dissociation; Early Diagnosis; Enzymes; Epidemic; Etiology; Exhibits; Fever; Frameshift Mutation; Free Radicals; Gene Expression; Genes; Genomic DNA; Gram-Negative Bacteria; Guanine; Guanosine; Histone-Lysine N-Methyltransferase; Hydrogen Peroxide; In Vitro; Kinetics; Knowledge; Liposomes; Lysine; Mass Fragmentography; Mediating; Membrane; Membrane Proteins; Messenger RNA; Methods; Methylation; Methyltransferase; Microarray Analysis; Mitochondria; Modification; Molecular; Mono-S; Motor Neurons; Nature; Neurodegenerative Disorders; Oligonucleotides; Oxidative Stress; Pathogenicity; Pathway interactions; Physiological; Polyribosomes; Proteins; RNA; Reactive Oxygen Species; Reporting; Research; Resolution; Ribonucleosides; Ribosomes; Rickettsia; Rickettsia Infections; Rickettsia prowazekii; Rickettsia typhi; Role; S-Adenosylhomocysteine; S-Adenosylmethionine; Schiff Bases; Scrub Typhus; Signal Transduction; Site; Site-Directed Mutagenesis; Specificity; Spottings; Structure; Substrate Interaction; Sucrose; System; Translating; Translations; Typhus; Vaccines; Virulence; Virulent; age related; amino group; ascorbate; base; cell growth regulation; cofactor; coping; crosslink; cytochrome c; enzyme mechanism; gene product; insight; liquid chromatography mass spectrometry; mRNA Decay; monomer; overexpression; oxidation; oxidative damage; preference; programs; response; sugar; trimethyllysine

In this program, we focused on the following projects: (i) RNA oxidation. Oxidative damage to RNA has received relatively little attention despite the fact that growing evidence indicates that mRNA oxidation is correlated with a number of age-related neurodegenerative diseases, including Alzheimer's disease and the finding reveals that mRNA oxidation occurs early in motor neuron deterioration in Amyotrophic lateral sclerosis. We have shown previously that oxidized mRNA causes a reduction of translation fidelity. In additional study we 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. Guanosine in RNA was the predominant ribonucleoside oxidized in cytochrome c-mediated oxidation, while Fe(II)/ascorbate system oxidized all ribonucleoside with no obvious preference. GC/MS and LC/MS analyses showed 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 led to the formation of cross-linking products, e.g. between oxidized RNA and cyt c. The formation of this cross-linking product facilitated 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, led not only to impair RNA normal functions, but it might also gain a protective signal to facilitate cellular apoptosis in response to oxidative stress. To investigate the molecular basis of this observation led us to carry out microarray analysis of oxidized mRNA species in Neuro2a cells. Our results suggest the involvement of a translation-dependent mRNA oxidation regulatory mechanism, consistent with the notion of mRNA surveilliance mechanisms, e. g. No-go mediated mRNA decay pathway, may participate to cope with damaged mRNA. To investigate whether the level of oxidized mRNA is correlated with ribosome, we subjected the cell lysates of Neuro2 cells to sucrose density gradient centrifugation to separate mRNA into three fractions, polysome, oligo- to mono-some, and free fractions. The RNA was isolated in each fraction, and analyzed their oxidation levels. The results revealed that under normal culture conditions, the mRNA in free fraction exhibited the highest levels, while mRNA in oligosome/monosome fraction yielded the lowest level (i.e. in -actin, polysome: 0.22 %, oligosome/monosome: 0.01 %, free: 0.93 %). It is believed that substantial amount of oxidized mRNA was likely eliminated by degradation pathway. When the cell lysates after treated with 0.2 mM H2O2 for 30 min were analyzed, the results showed the oxidized mRNA levels were elevated in the three fractions to a different levels (i.e. in -actin, polysome: 0.96 %, oligosome/monosome: 0.59 %, free: 1.33 %). The oxidation levels were induced in the oligosome/monosome fraction, with a 3-fold increase in the total amount of mRNA in the fraction, likely derived from the polysome mRNA due to partial dissociation of polysome. Note the oxidation levels in polysome fraction was also increased substantially (4 folds), even though the total mRNA in the fraction was reduced (50 % reduction). It has been suggested that a part of the polysome associated oxidized mRNA was accumulated in the polysome instead of dissociating under acute oxidative stress. However, it remained to be shown whether the oxidized mRNA in the polysome are actively translated or associated with multiple ribosomes stalled. Together these observations showed distinctly the oxidized mRNA are associated to ribosome, implying that oxidized mRNA is metabolized or degraded with translation machinery. (ii) Rickettsiae are obligatory intracellular infectious Gram-negative bacteria that responsible for major rickettsiosis, which include epidemic typhus, spotted fever, and scrub typhus, without the availability of vaccine or early detection method. The outer membrane of Rickettsia is largely composed of an outer membrane protein called OmpB that accounts for up to 15 % of its total cellular proteins. OmpB is known to involve in cell adhesion, attachment, and invasion. We and others showed that methylation of lysyl residues in rickettsial OmpB correlated with bacterial virulence. Methylation profiles analysis using LC-MS/MS methods revealed a high correlation in methylation sites between those detected in native proteins purified from bacteria and those attained from in vitro methylation, except the methylation level was found significantly higher in the native proteins relative to those methylated using purified methtyltransferases with overexpressed OmpB fragments as substrates. In addition, the natively purified OmpB from the avirulent strain Madrid E of Rickettsia prowazekii does not contain any trimethyllysine. An observation consistent with the report that the gene encoding the trimethyltransferase, RP027-028, in Madrid E has been interrupted by a frameshift mutation to generate the inactive trimethyltransferase, the RP027 and RP028 fragments. However, OmpB from the highly virulent Rickettsia prowazekii strains Breinl, and RP22 each contains clusters of trimethyllysines located at a relatively close proximity. Thus, the state and type of OmpB methylation is correlated with rickettsial pathogenicity. To this end, knowledge on the enzyme(s) that catalyzes OmpB methylation and on the nature of the methylated OmpB could provide new insight on OmpB-methylation and its role on virulent effect. Through bioinformatics analysis of genomic DNA sequences of Rickettsia, Dr. Yangs lab has revealed five potential sequences of putative protein lysine methylatransferases. Syntthesis and expression of these genes, follow with purification and characterization of the gene products revealed the presence two distinct types of protein lysine methyltransferases. They are the PKMT1 and PKMT2, which catalyzes predominantly monomethylation and trimethylation, respectively. Among known protein lysine methyltransferases, rickettsial PKMT1 and PKMT2 are unique in that their substrates appears to be limited to OmpB and both methyltransferases are capable of methylating multiple lysyl residues with broad sequence specificity. To better understand the mechanism by which PKMT1 and PKMT2 differentially catalyze OmpB methylation, we carry out crystal structural analysis for PKMT1 from Rickettsia prowazekii, both the apo-form and in complex with its cofactors, S-adenosylmethionine or S-adenosylhomocysteine, and for PKMT2 from Rickettsia typhi. The structure of PKMT1 in complex with S-adenosylhomocysteine is solved to a resolution of 1.9 . Both enzymes are dimeric, with each monomer containing an S-adenosylmethionine binding domain with a seven-strand Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding and substrate interactions were examined using site-directed mutagenesis followed by steady-state kinetic analysis to ascertain their catalytic functions in solution. Together, our data provide new structural and mechanistic insights on how rickettsial methyltransferases catalyze OmpB methylation.

在这个计划中，我们重点关注了以下项目： (i)RNA氧化。 RNA 的氧化损伤相对较少受到关注，尽管越来越多的证据表明 mRNA 氧化与许多与年龄相关的神经退行性疾病（包括阿尔茨海默氏病）相关，并且这一发现表明 mRNA 氧化发生在肌萎缩侧索硬化症运动神经元退化的早期。我们之前已经证明，氧化的 mRNA 会导致翻译保真度降低。在另外的研究中，我们发现细胞色素 c (cyt c)/H2O2 或 Fe(II)/抗坏血酸/H2O2 系统催化的体外 RNA 氧化产生了不同的共价修饰的 RNA 衍生物。 RNA中的鸟苷是细胞色素c介导的氧化中主要被氧化的核糖核苷，而Fe(II)/抗坏血酸系统氧化所有核糖核苷，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而且还脱嘌呤形成脱碱基糖部分。无碱基位点上的醛部分与蛋白质中的氨基形成希夫碱，并导致交联产物的形成，例如氧化RNA和细胞色素之间c．这种交联产物的形成促进了细胞色素c从含心磷脂的脂质体中的释放，这可能代表细胞色素c从线粒体释放到胞质溶胶。因此，RNA 的氧化修饰（包括交联）不仅会损害 RNA 的正常功能，而且还可能获得保护信号，促进细胞凋亡以应对氧化应激。为了研究这一观察结果的分子基础，我们对 Neuro2a 细胞中氧化的 mRNA 种类进行了微阵列分析。我们的结果表明翻译依赖性 mRNA 氧化调节机制的参与，与 mRNA 监视机制的概念一致，例如。 g。 No-go介导的mRNA衰减途径，可能参与应对受损的mRNA。为了研究氧化 mRNA 的水平是否与核糖体相关，我们对 Neuro2 细胞的细胞裂解物进行蔗糖密度梯度离心，将 mRNA 分为三个部分：多核糖体、寡聚体至单核糖体和游离部分。在每个级分中分离 RNA，并分析它们的氧化水平。结果显示，在正常培养条件下，游离组分中的 mRNA 水平最高，而寡核糖体/单体组分中的 mRNA 水平最低（即，β-肌动蛋白中，多核糖体：0.22%，寡核糖体/单体：0.01%，游离：0.93%）。据信大量氧化的mRNA可能通过降解途径被消除。分析用 0.2 mM H2O2 处理 30 分钟后的细胞裂解物，结果显示三个组分中的氧化 mRNA 水平升高到不同的水平（即肌动蛋白内，多核糖体：0.96 %，寡核糖体/单体：0.59 %，游离：1.33 %）。寡核糖体/单体组分中的氧化水平被诱导，该组分中的mRNA总量增加了3倍，可能由于多核糖体的部分解离而源自多核糖体mRNA。请注意，尽管多核糖体级分中的总 mRNA 减少了（减少了 50%），但多核糖体级分中的氧化水平也大幅增加（4 倍）。有人提出，在急性氧化应激下，与多核糖体相关的氧化mRNA的一部分在多核糖体中积累而不是解离。然而，多核糖体中氧化的 mRNA 是否正在积极翻译或与多个停滞的核糖体相关，仍有待证明。这些观察结果明显表明氧化的 mRNA 与核糖体相关，这意味着氧化的 mRNA 通过翻译机制被代谢或降解。 (ii) 立克次体是一种强制性细胞内传染性革兰氏阴性菌，在没有疫苗或早期检测方法的情况下，是导致主要立克次体病的原因，其中立克次体病包括流行性斑疹伤寒、斑疹伤寒和恙虫病。立克次体的外膜主要由一种称为 OmpB 的外膜蛋白组成，其含量高达细胞总蛋白的 15%。已知 OmpB 参与细胞粘附、附着和侵袭。我们和其他人发现立克次体 OmpB 中赖氨酰残基的甲基化与细菌毒力相关。使用 LC-MS/MS 方法的甲基化谱分析显示，从细菌纯化的天然蛋白中检测到的甲基化位点与体外甲基化获得的甲基化位点之间存在高度相关性，但天然蛋白中的甲基化水平明显高于使用过表达 OmpB 片段作为底物的纯化甲基转移酶进行甲基化的蛋白。此外，从普瓦泽基立克次体无毒马德里E菌株中天然纯化的OmpB不含任何三甲基赖氨酸。观察结果与报道一致，即马德里 E 中编码三甲基转移酶 RP027-028 的基因已被移码突变中断，产生无活性的三甲基转移酶 RP027 和 RP028 片段。然而，来自高毒力普瓦泽基立克次体 Breinl 菌株的 OmpB 和 RP22 各自包含位置相对较近的三甲基赖氨酸簇。因此，OmpB 甲基化的状态和类型与立克次体致病性相关。为此，有关催​​化 OmpB 甲基化的酶和甲基化 OmpB 性质的知识可以为 OmpB 甲基化及其对毒力作用的作用提供新的见解。通过对立克次体基因组DNA序列的生物信息学分析，杨博士的实验室揭示了推定蛋白质赖氨酸甲基转移酶的五个潜在序列。这些基因的合成和表达，以及基因产物的纯化和表征揭示了两种不同类型的蛋白质赖氨酸甲基转移酶的存在。它们是 PKMT1 和 PKMT2，分别主要催化单甲基化和三甲基化。在已知的蛋白质赖氨酸甲基转移酶中，立克次体 PKMT1 和 PKMT2 的独特之处在于，它们的底物似乎仅限于 OmpB，并且两种甲基转移酶都能够以广泛的序列特异性甲基化多个赖氨酰残基。为了更好地了解 PKMT1 和 PKMT2 差异催化 OmpB 甲基化的机制，我们对来自普氏立克次体的 PKMT1（包括 apo 形式和与其辅因子 S-腺苷甲硫氨酸或 S-腺苷高半胱氨酸的复合物）以及来自伤寒立克次体的 PKMT2 进行了晶体结构分析。 PKMT1 与 S-腺苷高半胱氨酸复合物的结构解析度为 1.9。两种酶都是二聚体，每个单体都含有一个具有七链罗斯曼折叠的 S-腺苷甲硫氨酸结合结构域、一个二聚化结构域、一个中间结构域、一个 C 末端结构域和一个位于中心的开放空腔。根据晶体结构，使用定点诱变和稳态动力学分析来检查参与催化、辅因子结合和底物相互作用的残基，以确定它们在溶液中的催化功能。总之，我们的数据为立克次体甲基转移酶如何催化 OmpB 甲基化提供了新的结构和机制见解。