CYTOSOLIC SOURCES OF NADPH AS ANTIOXIDANTS

作为抗氧化剂的 NADPH 的细胞质来源

• 批准号: 6629856
• 负责人: Lee McAlister-Henn
• 金额: $ 24.71万
• 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 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6629856
• 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.

己糖单磷酸（或磷酸五磷酸五磷酸）途径和胞质异戊酸脱氢酶（IDP2）已被发现是过氧化氢酶功能的必不可少的辅助物，可预防脂肪酸脂肪酸在多氧化物中的β-氧化过程中产生的细胞内过氧化氢的细胞内积累。 NADPH的这两个胞质来源的缺失导致酵母细胞的致死性，该细胞的致死性转移到脂肪酸碳源的培养基中。这些观察结果是，过氧化物酶体代谢是致命的过氧化氢水平的来源，并且需要胞质NADPH预防致死性，这是细胞氧化应激区域的新观察结果。提出了对这些现象的进一步研究，因为对细胞大分子的氧化损害显然与退行性疾病和衰老过程有关。该提案的目的是：（a）将使用一系列用编码这些基因和其他酶与特定的抗氧化剂的基因和其他酶的基因中的破坏性组合的酿酒酵母突变体的集合来检查在线粒体呼吸过程中产生的氧化副产物的类似要求。 （b）将使用酵母突变菌株的遗传交叉评估对特定基因和细胞蛋白的遗传力，这些杂种菌株的遗传杂交由于内源性氧化损伤而表现出相似的生长表型。 （c）将通过用同源酵母同工酶和使用酵母两杂化测定法测试和筛选与其他细胞蛋白的特定相互作用来探索IDP2的独特抗氧化功能。 （d）将检查哺乳动物IDP2的表达和细胞定位模式，以阐明支持过氧化物酶体β-氧化和/或为抗氧化功能提供NADPH的作用。