Biochemical Mechanisms of Enzyme Action and Cellular Regulation

酶作用和细胞调节的生化机制

• 批准号: 8939747
• 负责人: P. BOON Chock
• 金额: $ 25.85万
• 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/8939747
• 关键词: Aging; Aldehydes; Apoptosis; Bacteria; Binding; Biochemical; Bioinformatics; Biological Process; Cardiolipins; Cells; Consensus; Cytosol; DNA Sequence; Deterioration; Disease; Early Diagnosis; Enzymes; Epidemic; Etiology; Fever; Free Radicals; Gene Expression; Genomic DNA; Gram-Negative Bacteria; Guanine; Guanosine; Histone-Lysine N-Methyltransferase; Hydrogen Peroxide; In Vitro; Individual; Kinetics; Knowledge; Liposomes; Liquid Chromatography; Location; Lysine; Mediating; Membrane Proteins; Messenger RNA; Methods; Methylation; Methyltransferase; Mitochondria; Modification; Molecular; Mono-S; Motor Neurons; Nature; Neurodegenerative Disorders; Oxidative Stress; Physiological; Polyribosomes; Protein Methyltransferases; Proteins; RNA; Reactive Oxygen Species; Reporting; Research; Ribonucleosides; Rickettsia; Rickettsia Infections; Role; Schiff Bases; Scrub Typhus; Signal Transduction; Site; Spottings; System; Translations; Typhus; Vaccines; Virulence; Virulent; age related; amino group; ascorbate; base; cell growth regulation; crosslink; cytochrome c; enzyme mechanism; genome wide association study; insight; liquid chromatography mass spectrometry; mass spectrometer; oxidation; oxidative damage; preference; programs; response; 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 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.

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显示出类似的结合常数的非氧化的mRNA的多核糖体。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。为此，在RNA中的鸟苷是细胞色素c（cyt c）介导的氧化中氧化的主要核糖核苷，而Fe（II）/抗坏血酸盐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。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，例如在氧化的RNA和cyt c之间。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，而且还可能获得保护性信号，以促进细胞对氧化应激的凋亡。已有研究表明，AD和ALS中某些mRNA被选择性氧化。我们试图了解这一观察结果的分子基础，这使我们进行了全基因组研究，以量化Neuro 2a细胞中单个mRNA的氧化水平。初步结果表明，大量表达的mRNA似乎对氧化损伤更具保护作用。 (ii)立克次体（Rickettsiae）是一种专性的细胞内革兰氏阴性菌，可引起包括流行性斑疹伤寒、斑点热和恙虫病在内的主要立克次体病，目前尚无疫苗或早期检测方法。 立克次体OmpB的甲基化与细菌毒力有关。因此，关于催化OmpB甲基化的酶和甲基化OmpB的性质的知识可以提供关于OmpB甲基化及其在毒性效应中的作用的新见解。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个潜在的蛋白质赖氨酸甲基转移酶序列。这些基因的合成和表达，随后的纯化和基因产物的表征揭示了存在两种不同类型的蛋白质赖氨酸甲基转移酶。它们是PKMT 1和PKMT 2。动力学和产物分析表明，PKMT 1主要催化单甲基化，而PKMT 2的功能作为三甲基转移酶。从R. prowazekii和R.伤寒沙门氏菌蛋白RP 027 -028为PKMT 1，而伤寒沙门氏菌蛋白RP 027 -028为PKMT 1。prowazekii和R.伤寒为PKMT 2。采用半定量积分液相色谱-串联质谱仪对纯化的甲基转移酶催化的甲基化rOmpB片段以及从立克次体纯化的天然OmpB片段的位置、状态和水平进行表征。我们的研究结果表明，OmpB上的甲基化发生在多个位点，在纯化的天然蛋白质和甲基转移酶催化的rOmpB片段。此外，在体外三甲基化，通过进行性机制，发生在相对特定的位置在OmpB的共识基序，KX（G/A/V/I）N和KT（I/L/F），而单甲基化是普遍的OmpB。有趣的是，R.伤寒杆菌在与PKMT 1和PKMT 2催化的位置相关的位置含有单甲基赖氨酸和三甲基赖氨酸残基。我们发现，集群的高度三甲基化的赖氨酸残基存在于OmpB从毒力菌株，但不是在无毒力菌株。此外，高度三甲基化赖氨酸簇的数量与菌株的毒性相关。