CYTOSOLIC SOURCES OF NADPH AS ANTIOXIDANTS

作为抗氧化剂的 NADPH 的细胞质来源

• 批准号: 6873336
• 负责人: Lee McAlister-Henn
• 金额: $ 25.35万
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873336
• 关键词: DNA damage; NAD(H) phosphate; Saccharomyces cerevisiae; aging; animal tissue; antioxidants; cellular respiration; cytoplasm; genetic strain; isocitrate dehydrogenase; longevity; macromolecule; mass spectrometry; oxidative stress; peroxisome; protein localization; protein protein interaction; two dimensional gel electrophoresis; yeast two hybrid system

DESCRIPTION (provided by applicant): Two cytosolic sources of NADPH, the hexose monophosphate pathway and cytosolic NADP+-specific isocitrate dehydrogenase (IDP2), have been found to be essential in preventing the accumulation of deleterious oxidative byproducts of endogenous metabolic pathways including peroxisomal beta-oxidation and mitochondrial respiration. Loss of glucose-6-phosphate dehydrogenase (ZWF1, the first enzyme in the hexose monophosphate pathway) and of IDP2 results in a rapid loss in viability of yeast cells transferred to medium with oleate or acetate as the carbon source. In contrast, loss of major cellular peroxidative enzymes has no effect on growth with these carbon sources. The lethality resulting from endogenous metabolic byproducts, and the specific requirement for cytosolic sources of NADPH to protect from this 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 associated with numerous degenerative diseases and with the process of aging. Primary aims of this proposal include the following: (A) Proteins and DNA from strains lacking cytosolic sources of NADPH will be examined to identify specific cellular and organellar macromolecular targets of deleterious oxidants generated by normal metabolic pathways. (B) We will assess the importance of cytosolic sources and of levels of NADPH as determinants of longevity, using yeast strains lacking or overexpressing these key antioxidant enzymes for life-span analysis. (C) We will initiate biochemical analyses of mammalian IDP2, and examine the physiological relevance of co-localization of this enzyme in the cytosol and in peroxisomes using both yeast and mouse models. In addition, we will continue to examine the structural basis for the unique physiological functions of yeast IDP2. (D) Finally, we will assess global changes in pools of central metabolites and reducing equivalents to directly test fundamental hypotheses related to loss or replacement of cytosolic sources of NADPH.

描述（由申请人提供）：NADPH 的两种胞质来源，即单磷酸己糖途径和胞质 NADP+ 特异性异柠檬酸脱氢酶 (IDP2)，已被发现对于防止内源代谢途径（包括过氧化物酶体 β 氧化和线粒体呼吸）有害氧化副产物的积累至关重要。葡萄糖-6-磷酸脱氢酶（ZWF1，单磷酸己糖途径中的第一种酶）和 IDP2 的损失会导致转移到以油酸盐或乙酸盐作为碳源的培养基中的酵母细胞活力迅速丧失。相反，主要细胞过氧化酶的损失对这些碳源的生长没有影响。内源性代谢副产物导致的致死性，以及为防止这种致死性而对细胞质 NADPH 来源的特定要求，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许多退行性疾病和衰老过程有关，因此建议对这些现象进行进一步研究。该提案的主要目标包括以下内容： (A) 将检查缺乏 NADPH 胞质来源的菌株的蛋白质和 DNA，以确定正常代谢途径产生的有害氧化剂的特定细胞和细胞器大分子靶标。 (B) 我们将使用缺乏或过度表达这些关键抗氧化酶的酵母菌株进行寿命分析，评估细胞质来源和 NADPH 水平作为寿命决定因素的重要性。 (C) 我们将启动哺乳动物 IDP2 的生化分析，并使用酵母和小鼠模型检查该酶在细胞质和过氧化物酶体中共定位的生理相关性。此外，我们将继续研究酵母IDP2独特生理功能的结构基础。 (D) 最后，我们将评估中心代谢物池和还原当量的整体变化，以直接检验与 NADPH 胞质来源的损失或替代相关的基本假设。