Diphthamide biosynthesis

二邻苯二甲酰胺生物合成

• 批准号: 8120752
• 负责人: STEVEN E EALICK
• 金额: $ 30.99万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8120752
• 关键词: ADP ribosylation; Adenosine Diphosphate Ribose; Amino Acid Sequence; Anabolism; Archaea; Bacterial Infections; Bacterial Toxins; Binding; Biochemical; Biological Process; Biology; Cell Death; Cells; Chemistry; Complex; Cytosol; Data; Development; Diphtheria Toxin; Electron Spin Resonance Spectroscopy; Enzymatic Biochemistry; Enzymes; Eukaryota; Eukaryotic Cell; Exotoxins; Genes; Genetic; Goals; Health; Histidine; Human; Imidazole; In Vitro; Iron; Knowledge; Lead; Learning; Link; Literature; Malignant Neoplasms; Malignant neoplasm of ovary; Methods; Methyltransferase; Modification; Mossbauer Spectroscopy; Mutation; Names; Nicotinamide adenine dinucleotide; Nitrogen; Oncogenes; Pathway interactions; Peptide Elongation Factor 2; Peptide Sequence Determination; Post-Translational Protein Processing; Protein Biosynthesis; Proteins; Pseudomonas aeruginosa toxA protein; Reaction; Regulation; Reporting; S-Adenosylmethionine; Structure; Sulfur; Testing; Toxin; Translations; Tumor Suppression; Tumor Suppressor Proteins; Ursidae Family; amidation; base; in vitro Assay; in vivo; insight; interest; malignant breast neoplasm; pathogen; prevent; tumor

DESCRIPTION (provided by applicant): Diphthamide is a post-translationally modified histidine residue found in translation elongation factor 2 (eEF-2) in all eukaryotic cells. This single modification concentrates a lot of interesting chemistry and biology. This modification involves a unique C-C bond formation reaction that links the C2 of the imidazole ring and the 3-amino-3-carboxyl propyl group derived from S-adenosylmethionine (SAM). This C-C bond formation reaction is followed by a trimethylation step and an amidation step to give the final diphthamide modification. During certain bacterial infections, this modified residue (but not the unmodified histidine residue) is specifically recognized by bacterial toxins, including diphtheria toxin and Pseudomonas exotoxin A. These toxins catalyze the transfer of an adenosine diphosphate ribose (ADP-ribose) from nicotinamide adenine dinucleotide (NAD) to one of the nitrogen atoms in the imidazole ring. The ADP-ribosylation by bacterial toxins inhibits the function of eEF-2 and thus protein synthesis, leading to cell death. Based on genetic studies, the first step of the biosynthesis, the formation of the C-C bond, requires four proteins in eukaryotes, Dph1, Dph2, Dph3, and Dph4. However, how these four proteins catalyze the reaction and why so many proteins are involved in one reaction is unknown. Heterozygous deletions of dph1 and dph4 genes are associated with several human cancers, particularly ovarian and breast cancers, raising interesting questions about the biological function of diphthamide and the tumor suppression mechanism of Dph1 and Dph4. In this proposal, we aim to study how Dph1, Dph2, Dph3, and Dph4 catalyze the first step of diphthamide biosynthesis. Understanding the biosynthesis will uncover some very interesting enzymology, as predicted by our preliminary results, and provide insights into how the biosynthesis is regulated. Understanding its biosynthesis and regulation will help to understand the biological function of this modification and the tumor suppression mechanism of Dph1 and Dph4, and may ultimately lead to the development of new ways to treat or prevent cancer. PUBLIC HEALTH RELEVANCE: Diphthamide is a unique post-translational modification that occurs in all eukaryotes. The biosynthesis of diphthamide requires multiple proteins, and mutations in several of them have been connected to cancer. This proposal aims to study the mechanism of diphthamide biosynthesis and the function of each protein required for the biosynthesis. Understanding the biosynthesis will provide important insight on the function of diphthamide, the regulation of diphthamide biosynthesis, and the mechanism of tumor formation in the absence of diphthamide biosynthesis, possibly leading to new ways to treat or prevent cancer.

描述（由申请人提供）：Diphthamide 是在所有真核细胞的翻译延伸因子 2 (eEF-2) 中发现的翻译后修饰的组氨酸残基。这种单一的修饰集中了许多有趣的化学和生物学知识。这种修饰涉及独特的 C-C 键形成反应，该反应将咪唑环的 C2 与源自 S-腺苷甲硫氨酸 (SAM) 的 3-氨基-3-羧基丙基连接起来。该C-C键形成反应之后是三甲基化步骤和酰胺化步骤，得到最终的二邻苯二甲酰胺修饰。在某些细菌感染期间，这种修饰残基（但不是未修饰的组氨酸残基）被细菌毒素特异性识别，包括白喉毒素和假单胞菌外毒素 A。这些毒素催化烟酰胺腺嘌呤二核苷酸转移腺苷二磷酸核糖 (ADP-核糖) (NAD)连接至咪唑环中的氮原子之一。细菌毒素的 ADP-核糖基化会抑制 eEF-2 的功能，从而抑制蛋白质合成，导致细胞死亡。根据遗传学研究，生物合成的第一步，即C-C键的形成，需要真核生物中的四种蛋白质：Dph1、Dph2、Dph3和Dph4。然而，这四种蛋白质如何催化该反应以及为什么这么多蛋白质参与一个反应尚不清楚。 dph1 和 dph4 基因的杂合缺失与多种人类癌症有关，特别是卵巢癌和乳腺癌，这引发了关于二苯胺的生物学功能以及 Dph1 和 Dph4 的肿瘤抑制机制的有趣问题。在本提案中，我们的目标是研究 Dph1、Dph2、Dph3 和 Dph4 如何催化二邻苯二甲酰胺生物合成的第一步。正如我们的初步结果所预测的那样，了解生物合成将揭示一些非常有趣的酶学，并提供有关生物合成如何调节的见解。了解其生物合成和调控将有助于了解这种修饰的生物学功能以及Dph1和Dph4的肿瘤抑制机制，并可能最终导致开发治疗或预防癌症的新方法。公共健康相关性：二邻苯二甲酰胺是一种独特的翻译后修饰，存在于所有真核生物中。二苯甲酰胺的生物合成需要多种蛋白质，其中几种蛋白质的突变与癌症有关。本提案旨在研究二邻苯二甲酰胺的生物合成机制以及生物合成所需的每种蛋白质的功能。了解生物合成将为了解二邻苯二甲酰胺的功能、二邻苯二甲酰胺生物合成的调节以及二邻苯二甲酰胺生物合成缺失下肿瘤形成的机制提供重要见解，并可能带来治疗或预防癌症的新方法。