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。我们以前发现，尽管氧化后的信使核糖核酸与多聚体的结合常数与未氧化的信使核糖核酸相似，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。为此，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，例如氧化的RNA和细胞色素c之间的交联物。有趣的是，氧化的RNA和细胞色素c之间的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。已有研究表明，在AD和ALS中，某些mRNA被选择性氧化。为了了解这一观察的分子基础，我们开展了全基因组研究，以量化Neuro2a细胞中单个mRNA的氧化水平。初步结果表明，大量表达的mRNA似乎对氧化损伤具有更好的保护作用。 (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OmpB的甲基化与细菌的毒力有关。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，以及对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。它们是PKMT1和PKMT2。动力学和产物分析表明，PKMT1主要催化单甲基化，而PKMT2主要催化三甲基转移酶。罗氏杆菌的RP789和伤寒杆菌的RT0776为PKMT1，而普氏杆菌的RP027-028和伤寒杆菌的RT0101为PKMT2。用半定量的液相色谱-串联质谱仪对纯化的甲基转移酶和从立克次体中纯化的天然OmpB催化的甲基化rOmpB片段的位置、状态和水平进行了表征。我们的结果表明，在纯化的天然蛋白和甲基转移酶催化的rOmpB片段中，OmpB上的甲基化发生在多个位置。此外，体外三甲基化发生在OmpB中相对特定的位置，具有共同的基序KX(G/A/V/I)N和KT(I/L/F)，而单甲基化在OmpB中普遍存在。有趣的是，来自伤寒沙门氏菌的天然OmpB在与PKMT1和PKMT2催化的位置上含有单甲基和三甲基赖氨酸残基。我们发现，在强毒株的OmpB中存在高度三甲基化的赖氨酸残基簇，而在无毒株中没有。此外，高度三甲基化的赖氨酸簇的数量与菌株的毒力有关。

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 showed previously that oxidized mRNA causes a reduction of translation fidelity despite the fact that the oxidized mRNA shows a similar binding constant to polysomes as that of non-oxidized mRNA. 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. To this end, 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 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 generated cross-linking products, e.g. 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 it may also gain a protective signal to facilitate cellular apoptosis in response to oxidative stress. It has been reported that certain mRNA was selectively oxidized in AD and ALS. Our attempt to understand the molecular basis of this observation led us to carry out genome-wide study to quantify oxidation levels of individual mRNA in Neuro2a cells. Preliminary results indicate that the abundantly expressed mRNA appears to be more protective from oxidative damage. (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. Methylation of rickettsial OmpB has been implicated in bacteria virulence. 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. Synthesis 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. Kinetic and products analysis revealed that PKMT1 catalyzes primarily monomethylation while PKMT2 functions as trimethyltransferase. The proteins, RP789 from R. prowazekii and RT0776 from R. typhi were found to be PKMT1 while proteins RP027-028 from R. prowazekii and RT0101 from R. Typhi to be PKMT2. Semiquantitative integrated liquid chromatography-tandem mass spectrometer was used to characterize the location, state and level of methylated rOmpB fragments catalyzed by the purified methyltransferases as well as the native OmpB purified from Rickettsia. Our results revealed that methylation on OmpB occurs at multiple sites in both purified native proteins and methyltransferase-catalyzed rOmpB fragments. Furthermore, in vitro trimethylation, proceeds via a processive mechanism, occurs at relatively specific locations in OmpB with consensus motifs, KX(G/A/V/I)N and KT(I/L/F), while monomethylation is pervasive in OmpB. Interestingly, native OmpB from R. Typhi contains mono- and trimethllysine residues at location well correlated with those catalyzed by PKMT1 and PKMT2. We revealed that clusters of highly trimethylated lysine residues are present in OmpB from virulent strains but not in avirulent strain. Furthermore, the number of highly trimethylated lysine clusters is correlated with the virulent of the strains.