CYTOSOLIC SOURCES OF NADPH AS ANTIOXIDANTS

作为抗氧化剂的 NADPH 的细胞质来源

• 批准号: 6028103
• 负责人: Lee McAlister-Henn
• 金额: $ 20.67万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6028103
• 关键词: DNA damage; NAD(H) phosphate; Saccharomyces cerevisiae; antioxidants; cellular respiration; genetic strain; isocitrate dehydrogenase; oxidative stress; peroxisome; protein localization; protein protein interaction; yeast two hybrid system

The hexose monophosphate (or pentose phosphate) pathway and cytosolic isocitrate dehydrogenase (IDP2) have been found to be essential adjuncts to catalase function in preventing intracellular accumulation of the hydrogen peroxide generated during beta-oxidation of fatty acids in peroxisomes. The absence of these two cytosolic sources of NADPH results in lethality of yeast cells transferred to media with fatty acid carbon sources. These observations, peroxisomal metabolism as a source of lethal levels of hydrogen peroxide and a requirement for cytosolic NADPH to prevent lethality, are novel observations in the area of cellular oxidative stress. Further investigation of these phenomena is proposed because oxidative damage to cellular macromolecules has been clearly associated with degenerative diseases and with the process of aging. Aims of this proposal are: (A) Similar requirements for cytosolic NADPH in removal of oxidative byproducts generated during mitochondrial respiration will be examined using a collection of Saccharomyces cerevisiae mutants with combinations of disruptions in genes encoding these and other enzymes with specific antioxidant functions. (B) The heritability of damage to specific genes and cellular proteins will be assessed using genetic crosses of yeast mutant strains that exhibit similar growth phenotypes as a result of endogenous oxidative damage. (C) The unique antioxidant functions of IDP2 will be explored by replacement with homologous yeast isozymes and by using yeast two-hybrid assays to test and screen for specific interactions with other cellular proteins. (D) Patterns of expression and cellular localization of mammalian IDP2 will be examined to clarify possible roles in support of peroxisomal beta- oxidation and/or in provision of NADPH for antioxidant functions.

已发现单磷酸己糖（或磷酸戊糖）途径和胞质异柠檬酸脱氢酶（IDP2）是过氧化氢酶功能的重要辅助物，可防止过氧化物酶体中脂肪酸β-氧化过程中产生的过氧化氢在细胞内积聚。这两种 NADPH 胞质来源的缺失会导致转移到含有脂肪酸碳源的培养基中的酵母细胞死亡。这些观察结果表明，过氧化物酶体代谢是致死水平过氧化氢的来源，并且是细胞质 NADPH 防止致死的必要条件，是细胞氧化应激领域的新观察结果。由于细胞大分子的氧化损伤与退行性疾病和衰老过程明显相关，因此建议对这些现象进行进一步研究。该提案的目的是：（A）将使用一系列酿酒酵母突变体以及编码这些酶和其他具有特定抗氧化功能的酶的基因破坏的组合来检查在去除线粒体呼吸过程中产生的氧化副产物时对胞质 NADPH 的类似要求。 (B) 将使用因内源性氧化损伤而表现出相似生长表型的酵母突变株的遗传杂交来评估对特定基因和细胞蛋白质的损伤的遗传性。 (C) 将通过用同源酵母同工酶替代并使用酵母双杂交测定来测试和筛选与其他细胞蛋白的特异性相互作用来探索 IDP2 独特的抗氧化功能。 (D)将检查哺乳动物IDP2的表达模式和细胞定位，以阐明支持过氧化物酶体β-氧化和/或提供NADPH以实现抗氧化功能的可能作用。