Battling AIDS via Mechanistic Understanding of the tRNA Phe modification enzyme T

通过对 tRNA Phe 修饰酶 T 的机制理解来对抗艾滋病

• 批准号: 8229462
• 负责人: Lei Li
• 金额: $ 22.93万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8229462
• 关键词: Acquired Immunodeficiency Syndrome; Anabolism; Animals; Biochemical; Biochemical Process; Biological Assay; Catalysis; Cells; Characteristics; Chemicals; Chemistry; Cleaved cell; DNA Repair Enzymes; Development; Disease; Electrons; Enzymatic Biochemistry; Enzyme Kinetics; Enzymes; Epidemic; Family; Foundations; Future; Goals; Grant; Guanine; HIV; Investigation; Ions; Iron; Knowledge; Laboratories; Learning; Ligands; Light; Methods; Modification; Molecular; Nature; Oligonucleotides; Oxygen; Pathway interactions; Phenylalanine; Phenylalanine-Specific tRNA; Plants; Population; Positioning Attribute; Pyruvate; RNA; RNA-Directed DNA Polymerase; Reaction; Research; Resources; S-Adenosylmethionine; Societies; Structure; Subfamily lentivirinae; Sulfur; Techniques; Testing; Thymine Dimers; Time; Transfer RNA; Translations; Virus; Virus Replication; Y base; analog; chemical synthesis; design; enzyme mechanism; enzyme substrate; experience; fundamental research; microorganism; novel; novel strategies; oxidation; planetary Atmosphere; prevent; repaired; research study; spore photoproduct lyase; wybutosine

DESCRIPTION (provided by applicant): The overall goal of this research is to reveal the missing substrate "X" and shed light on the reaction mechanism for a phenylalanine transfer RNA (tRNAPhe) modification enzyme TYW1. TYW1 catalyzes the N1- methylguanine (m1G) modification to imG-14, a guanine derivative with a tricyclic aromatic ring. This reaction is the key step in wybutosine (base Y) biosynthetic pathway. Malfunction of TYW1 is suggested in HIV infected cells, which leads to the absence of base Y in tRNAPhe and causes -1 frame shifting to increase 400% during translation. This frame shifting is the ONLY way for HIV to produce reverse transcriptase, the key enzyme for virus replication. Thus, elucidating the mechanism of TYW1 via fundamental research is of great significance to the understanding of AIDS development and could potentially provide a novel approach in battling this deadly disease. TYW1 is found to possess a C-X3-C-X2-C motif, which is the characteristic feature of the radical SAM superfamily. The enzymes in this family utilize a unique [4Fe-4S] cluster to reductively cleave the S- adenosylmethionine, generating the 52-deoxyadenosyl (52-dA) radical. However, how this 52-dA radical catalyzes the N1-methylguanine modification in TYW1 is unclear due to the unknown nature of the second enzyme substrate "X". This proposal is therefore devoted to revealing the substrate "X" and removing the last obstacle in mechanistic elucidation of this important enzyme. Chemical, biochemical, spectroscopic, and enzyme kinetic methods will be employed in our experimental approach. The TYW1 enzyme expressed from different resources will be utilized in this study. In addition, tRNAPhe containing either m1G or m1G analog prepared via chemical synthesis will be employed in our investigation as well. These experiments will enable us to uncover the nature of substrate "X". The revealed structure of "X" could additionally shed light on the reaction mechanism of TYW1, which will be tested in this proposal as well as in the future investigations after the R21 grant period. PUBLIC HEALTH RELEVANCE: Malfunction of enzyme TYW1 results in the absence of base Y, a guanine derivative, at the position 37 of phenylalanine transfer RNA, which subsequently enables the HIV to produce reverse transcriptase, the key enzyme for virus replication. The mechanistic elucidation of TYW1 is hindered by an unknown substrate "X" and this project is devoted to revealing its nature and removing the major obstacle in studying TYW1 catalysis. A better understanding of TYW1 may eventually enable its induction and resume the Y biosynthesis in the HIV infected cells, providing a novel approach to battle AIDS.

描述(申请人提供)：这项研究的总体目标是揭示缺失的底物“X”，并阐明苯丙氨酸转移RNA(TRNAPhe)修饰酶TYW1的反应机理。TYW1催化N1-甲基鸟嘌呤(M1G)修饰成带有三环芳环的鸟嘌呤衍生物IMG-14。该反应是Wybuosine(碱基Y)生物合成途径中的关键步骤。在HIV感染的细胞中可能存在TYW1的故障，这导致tRNAPhe中没有Y碱基，并导致-1帧移位在翻译过程中增加了400%。这种框架转换是HIV产生逆转录酶的唯一途径，逆转录酶是病毒复制的关键酶。因此，通过基础研究阐明TYW1的作用机制对于理解艾滋病的发展具有重要意义，并可能为抗击这一致命疾病提供一种新的途径。TYW1具有C-X3-C-X2-C基序，这是自由基SAM超家族的特征。该家族中的酶利用独特的[4Fe-4S]簇还原裂解S-腺苷蛋氨酸，产生52-脱氧腺苷(52-da)自由基。然而，由于第二酶底物“X”的性质尚不清楚，这个52-da自由基如何催化TYW1中的N1-甲基鸟嘌呤修饰尚不清楚。因此，这项提议致力于揭示底物“X”，并消除这一重要酶的机制阐明的最后障碍。在我们的实验方法中，将使用化学、生化、光谱和酶动力学方法。本研究将利用不同来源表达的TYW1酶。此外，我们还将使用通过化学合成制备的含有m1G或m1G类似物的tRNAPhe进行研究。这些实验将使我们能够揭示底物“X”的本质。所揭示的“X”结构可以进一步阐明TYW1的反应机理，这将在本提案以及R21授权期后的未来调查中得到测试。 与公共卫生相关：TYW1酶的故障导致苯丙氨酸转移RNA的第37位缺少鸟嘌呤衍生物Y碱基，从而使艾滋病毒能够产生逆转录酶，这是病毒复制的关键酶。TYW1的作用机理受到一种未知底物“X”的阻碍，本项目致力于揭示其性质，消除TYW1催化研究的主要障碍。更好地了解TYW1基因可能最终使其在HIV感染细胞中被诱导并恢复Y的生物合成，从而为抗击艾滋病提供一种新的途径。