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。尽管越来越多的证据表明，RNA的氧化损伤与包括阿尔茨海默病在内的许多与年龄相关的神经退行性疾病有关，但对RNA的氧化损伤却相对较少受到关注，这一发现表明，在肌萎缩侧索硬化症的运动神经元退化过程中，mRNA的氧化发生在早期。在另一项研究中，我们发现在体外，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氧化会产生不同的共价修饰的RNA衍生物。在细胞色素c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。碱性位点上的醛部分与蛋白质中的氨基形成席夫碱，并导致交联物的形成，例如在氧化的RNA和细胞色素c之间。这种交联物的形成促进了含有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，不仅会损害RNA的正常功能，而且还可能获得一种保护性信号，促进细胞在氧化应激下的凋亡。为了研究这一观察的分子基础，我们对Neuro2a细胞中氧化的mRNA物种进行了微阵列分析。这些数据表明，无论有没有过氧化氢处理，mRNA的氧化都是有选择性的。此外，我们还发现，氧化水平与mRNA的半衰期没有相关性，这表明mRNA的氧化是受调控的。为了研究这种调节机制，在放线菌素或嘌呤霉素存在的情况下，用过氧化氢处理细胞。结果表明，翻译抑制剂的加入导致氧化的mRNA水平增加。此外，用过氧化氢处理细胞20min，然后转移到新鲜的培养液中，发现1h内升高的mRNA氧化水平降至50%。在培养液中加入放线菌酮可阻断氧化后mRNA的还原。这些结果表明参与了翻译依赖的mRNA氧化调节机制，这一观察结果与以下观点一致：mRNA监测机制，如NO-GO介导的mRNA衰变途径，可能参与处理受损的mRNA。 (Ii)立克次体是细胞内必需的革兰氏阴性感染性细菌，可导致重大立克次体病，包括流行性斑疹伤寒、斑疹伤寒和丛林斑疹伤寒，目前尚无疫苗或早期检测方法。立克次体的外膜主要由一种名为OmpB的外膜蛋白组成，占细胞总蛋白的15%。OmpB是一种毒力因子，参与细胞的黏附、附着和侵袭。已证明它在赖氨酸残基上发生甲基化，甲基化似乎与立克次体致病性有关。我们以前的研究发现，来自立克次体强毒株的OmpB蛋白含有多种三甲基化赖氨酸残基，而无毒立克次体株的OmpB蛋白主要含有单甲基化赖氨酸残基。因此，了解催化ompB甲基化的酶(S)和甲基化的ompB的性质可以为ompB-甲基化及其在毒力效应中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五种可能的蛋白质赖氨酸甲基转移酶序列。这些基因的合成和表达，以及基因产物的纯化和鉴定表明，存在两种不同类型的蛋白质赖氨酸甲基转移酶。它们分别是PKMT1和PKMT2，它们分别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是独一无二的，因为它们的底物似乎仅限于OmpB，而且这两种甲基转移酶都能够甲基化多个赖氨酸残基，具有广泛的序列特异性。为了更好地了解PKMT1和PKMT2不同地催化OmpB甲基化的机制，我们对来自普罗瓦泽克杆菌的PKMT1和来自伤寒杆菌的PKMT2的晶体结构进行了分析。我们的数据表明，这两种酶都是二聚体，每个单体都含有一个Adobe Met结合结构域，其中有一个核心Rossmann折叠、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，采用定点突变和稳态动力学分析的方法研究了参与催化、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方面的见解。

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 showed previously that oxidized mRNA causes a reduction of translation fidelity.In additional study we showed 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 lead to the formation of cross-linking products, e.g. between oxidized RNA and cyt c. The formation of this cross-linking product 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 it may 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. The data indicate that mRNA oxidation was selective either with or without hydrogen peroxide treatment. Furthermore, we found that the oxidation levels were not co-related with the mRNA half-life, suggesting that mRNA oxidation is regulated. To investigate the regulatory mechanism, the cells were treated with hydrogen peroxide in the presence of cycloheximide or puromycin. The results indicate that the addition of translation inhibitors led to an increase in the levels of oxidized mRNA. In addition, when cells were treated with hydrogen peroxide for 20 min then transferred to fresh medium, the elevated mRNA oxidation was found reduced to 50% within 1h. Addition of cycloheximide in the medium blocked the reduction of oxidized mRNA. These results suggest the involvement of a translation-dependent mRNA oxidation regulatory mechanism, an observation consistents with the notion that mRNA surveilliance mechanisms, e. g. No-go mediated mRNA decay pathway, may participate to cope with damaged mRNA. (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 total cellular proteins. OmpB is a virulence factor that is involved in cell adhesion, attachment, and invasion. It has been shown to be methylated at lysine residues and methylation appears to associate with rickettsial pathogenicity. Our previous studies revealed that OmpB proteins from virulent rickettsial strains contain multiple trimethylated lysyl residues while avirulent strains contain predominantly monomethylated lysyl residues. Thus, 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 R. prowazekii, both the apo-form and in complex with its cofactors AdoMet and AdoHcy, and for PKMT2 from R. typhi. Our data reveal that both enzymes are dimeric, with each monomer containing an AdoMet binding domain with a core 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.