tRNA editing by deamination: Balancing affinity and specificty

通过脱氨基进行 tRNA 编辑：平衡亲和力和特异性

• 批准号: 8321155
• 负责人: Juan D Alfonzo
• 金额: $ 32.88万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321155
• 关键词: Accounting; Adenosine; Affect; Affinity; Affinity Chromatography; Amino Acids; Anticodon; Binding; Biochemical; Biochemical Reaction; Biological; Biological Assay; Catalysis; Cell Line; Cell Nucleus; Cell physiology; Cells; Cellular biology; Chemicals; Codon Nucleotides; Complex; Coupled; Coupling; Cytidine Deaminase; Cytoplasm; Cytosine; Deaminase; Deamination; Disease; Enzymes; Epigenetic Process; Equilibrium; Eukaryota; Event; Face; Family member; Future; Gene Expression; Gene Mutation; Genetic; Genome; Genomics; Human; Inosine; Introns; Knowledge; Leishmania; Link; Mammals; Medical; Metabolic Diseases; Metabolism; Methylation; Methyltransferase; Mitochondria; Modeling; Modification; Molecular; Nature; Nuclear; Nucleotides; Organism; Outcome; Parasites; Pathway interactions; Play; Positioning Attribute; Printing; Process; Protein Biosynthesis; Protein Family; Proteins; RNA Interference; RNA Splicing; RNA-Protein Interaction; Reaction; Recombinants; Regulation; Reporter; Research; Role; Series; Site; Specific qualifier value; Specificity; Structure; Substrate Specificity; System; Testing; Therapeutic Intervention; Threonine-Specific tRNA; Transfer RNA; Trypanosoma; Trypanosoma brucei brucei; Uridine; Zinc; base; crosslink; diprotin B; drug development; flexibility; foot; in vitro activity; in vivo; insight; leucyl-alanine; member; mutant; novel; pathogen; reconstitution

DESCRIPTION (provided by applicant): Sequence alterations that change the meaning of a tRNA in decoding are part of a growing number of post-transcriptional changes collectively known as tRNA editing. Editing can be influenced by the structural context of an editing site and in the case of tRNA can be modulated by posttranscriptional modifications. In trypanosomatids, tRNAs are transcribed in the nucleus, exported to the cytoplasm, and later a subset of cytoplasmic tRNAs is actively imported into the mitochondria. However, before tRNAs can be rendered functional in any cellular compartment, they face many enzymatic reactions including end trimming, intron splicing, tRNA editing, and chemical modification. Some of these processes, for example those involved in trimming of extraneous sequences at the tRNA ends, occur in the nucleus, usually preceding cytoplasmic export. Others, like editing and modification, may occur at any point in the tRNA maturation pathway and in any of the tRNA-containing compartments. Despite much progress made in last few years, it is still not clear how editing and modification pathways are integrated both at the molecular and cellular levels. We have proposed that tRNA editing and modification events can be highly coupled and exploited by some organisms to control gene expression at the level of tRNA specificity. This is especially important in single-cell eukaryotes like trypanosomatid parasites where is well accepted that the bulk of the genetic regulation occurs post-transcriptionally. In this proposal, we have continued our studies on the very unique tRNA editing enzyme of T. brucei but now prompted by our newly solved crystal structure we ask new questions of what makes this enzyme so unique. We also focus on a newly discovered set of methyltransferases, which surprisingly target the editing site providing a wonderful testing ground for our hypothesis of the interrelation of editing and modification and what this coupling may mean in terms of cellular function. Significantly, reconstitution of methylation activity in vitro requires addition of the recombinant editing enzyme a finding that is without precedent in the tRNA editing and modification field. As essential steps in tRNA maturation in trypanosomatids (Leishmania and Trypanosoma), these types of editing and modification events also provide very attractive targets for therapeutic intervention against parasites of very major medical importance. Given the link between tRNA maturation and disease, these studies will further expand our knowledge of the role tRNA takes as a central player in cellular metabolism. ! PUBLIC HEALTH RELEVANCE: Members of the genus Leishmania and Trypanosoma infect millions of people worldwide. In these organisms, tRNAs undergo post-transcriptional modifications and editing changes that are unique to this system. The enzyme responsible for tRNA editing changes in trypanosomatids possesses substrate specificities that are not shared with any other member of this family of proteins. We have now discovered two new enzymes (tRNA methylases) whose functions are tightly coupled to the previously described editing deaminases. The propose studies will determine the basis for substrate specificity of the T. brucei enzymes which in the future may open doors towards the development of drugs against this unique and essential activities. These studies will also provide functional and evolutionary insights into important members of the cytidine deaminase (CDA) superfamily.

描述（由申请人提供）：在解码中改变 tRNA 含义的序列改变是越来越多统称为 tRNA 编辑的转录后变化的一部分。编辑可能会受到编辑位点结构背景的影响，并且在 tRNA 的情况下，可以通过转录后修饰进行调节。在锥虫中，tRNA 在细胞核中转录，输出到细胞质，随后细胞质 tRNA 的一个子集被主动输入线粒体中。然而，在 tRNA 在任何细胞区室中发挥功能之前，它们面临许多酶促反应，包括末端修剪、内含子剪接、tRNA 编辑和化学修饰。其中一些过程，例如涉及 tRNA 末端外来序列修剪的过程，发生在细胞核中，通常发生在细胞质输出之前。其他的，如编辑和修饰，可能发生在 tRNA 成熟途径中的任何点以及任何含有 tRNA 的区室中。尽管过去几年取得了很大进展，但仍不清楚编辑和修饰途径如何在分子和细胞水平上整合。我们提出，tRNA 编辑和修饰事件可以高度耦合，并被一些生物体利用，在 tRNA 特异性水平上控制基因表达。这对于像锥虫寄生虫这样的单细胞真核生物尤其重要，人们普遍认为大部分遗传调控发生在转录后。在这项提案中，我们继续研究布氏锥虫非常独特的 tRNA 编辑酶，但现在在我们新解决的晶体结构的推动下，我们提出了新的问题：是什么让这种酶如此独特。我们还关注一组新发现的甲基转移酶，它们令人惊讶地以编辑位点为目标，为我们关于编辑和修饰的相互关系的假设以及这种耦合对细胞功能的意义提供了一个极好的测试基础。值得注意的是，体外甲基化活性的重建需要添加重组编辑酶，这一发现在 tRNA 编辑和修饰领域是史无前例的。作为锥虫类动物（利什曼原虫和锥虫）tRNA 成熟的重要步骤，这些类型的编辑和修饰事件也为针对具有重大医学重要性的寄生虫的治疗干预提供了非常有吸引力的目标。鉴于 tRNA 成熟与疾病之间的联系，这些研究将进一步扩展我们对 tRNA 作为细胞代谢核心角色的认识。 ！ 公共卫生相关性：利什曼原虫属和锥虫属成员感染全世界数百万人。在这些生物体中，tRNA 会经历该系统特有的转录后修饰和编辑变化。锥虫中负责 tRNA 编辑变化的酶具有与该蛋白质家族的任何其他成员不共享的底物特异性。我们现在发现了两种新的酶（tRNA 甲基化酶），其功能与之前描述的编辑脱氨酶紧密耦合。拟议的研究将确定布氏锥虫酶的底物特异性的基础，这在未来可能为开发针对这种独特且重要的活性的药物打开大门。这些研究还将为胞苷脱氨酶（CDA）超家族的重要成员提供功能和进化方面的见解。