Characterization of the molecular network for the maintenance of nucleotide pools in plants

植物中维持核苷酸库的分子网络的表征

• 批准号: 471841140
• 负责人: Dr. Marco Herde
• 金额: --
• 依托单位: Institut für Pflanzenernährung
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/471841140?language=en
• 关键词: Characterization; molecular; network; maintenance; nucleotide

A number of proteins with currently unknown functions are thought to be involved in the elimination of metabolites resulting from chemical or enzymatic "accidents." The characterization of these enzymes in vivo presents an analytical challenge because the damaged metabolites involved are rare, and their detection is hampered, particularly by a complex plant matrix. However, since many of these metabolites are more abundant as a result of abiotic stresses, such as heat or drought, mechanisms for their removal may contribute to stress resistance and are therefore of great interest.We have recently developed a method for the highly sensitive analysis of nucleotides that allows to detect and quantify damaged nucleotides in plants. Using this method, we intend to investigate the molecular mechanisms that contribute to the removal of damaged nucleotides.Two different metabolic pathways will be investigated comprehensively and the experience gained will help to identify novel damaged nucleotides that occur under stress conditions. In the first subproject, we will investigate the removal of inosine (deoxy)-triphosphate ((d)ITP), which, after incorporation into RNA or DNA, impairs translation or the precision of replication. We will study an enzyme that converts (d)ITP to the canonical metabolite inosine monophosphate and observe changes in DNA and RNA from mutants lacking this enzyme. Furthermore, we will identify and characterize another enzyme that removes erroneously incorporated dITP from DNA. In addition, the metabolic fate of the product from this enzymatic reaction will be investigated.In another subproject, the complete catabolism of an oxidation product formed from (deoxy)guanosine triphosphate will be investigated. Here we hypothesize that enzymes already characterized in the catabolism of canonical metabolites are multifunctional and also convert an oxidized version of the canonical metabolite. We will use a combination of genetic analysis, biochemistry, and metabolite measurements to fully characterize the metabolism of this damaged nucleotide, including the metabolic fate of the nucleobase released as a consequence of DNA repair mechanisms.In the final part of this project, we will establish different techniques to enrich damaged nucleotides and nucleosides in plant extracts and improve their detection by mass spectrometry. Here, strategies such as isotope labeling and bioinformatic techniques (network analysis) will be used to expand the space of metabolites known in plants, especially with respect to nucleotides and nucleosides.

许多目前功能未知的蛋白质被认为参与消除由化学或酶促“事故”产生的代谢物。“这些酶在体内的表征提出了一个分析挑战，因为所涉及的受损代谢物是罕见的，它们的检测受到阻碍，特别是复杂的植物基质。然而，由于许多这些代谢物是更丰富的非生物胁迫的结果，如热或干旱，其去除机制可能有助于胁迫抗性，因此极大的interests.We最近开发了一种方法，允许检测和量化受损的核苷酸在植物中的核苷酸的高灵敏度分析。利用这种方法，我们打算研究有助于去除受损核苷酸的分子机制。两种不同的代谢途径将被全面研究，所获得的经验将有助于识别在胁迫条件下发生的新的受损核苷酸。在第一个子项目中，我们将研究肌苷（脱氧）-三磷酸（（d）ITP）的去除，它在掺入RNA或DNA后会损害翻译或复制的精确性。我们将研究一种将（d）ITP转化为典型代谢物肌苷一磷酸的酶，并观察缺乏这种酶的突变体的DNA和RNA的变化。此外，我们将确定和表征另一种酶，从DNA中删除错误纳入dITP。此外，还将研究这种酶促反应产物的代谢命运，另一个子项目将研究由（脱氧）鸟苷三磷酸形成的氧化产物的完全催化。在这里，我们假设，已经在典型代谢物的催化剂的特点是酶是多功能的，也转换的典型代谢物的氧化版本。我们将使用遗传分析、生物化学和代谢物测量的组合来充分表征这种受损核苷酸的代谢，包括作为DNA修复机制的结果释放的核碱基的代谢命运。在本项目的最后部分，我们将建立不同的技术来富集植物提取物中的受损核苷酸和核苷，并通过质谱法提高其检测。在这里，战略，如同位素标记和生物信息学技术（网络分析）将被用来扩大空间的代谢物在植物中已知的，特别是相对于核苷酸和核苷。