Defining and targeting the compartmentalization of redox metabolism in aging using novel genetically encoded tools

摘要 多条证据表明线粒体功能障碍和相关的细胞还原-氧化(Redox) 不平衡是衰老的标志之一。氧化还原辅因子烟酰胺腺嘌呤二核苷酸(NAD+)发挥作用 在细胞能量代谢中起中心作用，是支持线粒体氧化的重要辅因子 磷酸化(OXPHOS)。大量研究表明，细胞NAD+水平在衰老过程中降低- 相关的代谢变化，但其确切的作用目前仍有争议。这主要是因为NAD+ 和它的磷酸化形式NADP+是数百个氧化还原反应的底物，这些反应往往是 由在不同细胞隔间中发现的平行酶执行。细胞的区隔 新陈代谢是复杂真核生物最基本的特性之一，为了支持健康 细胞功能许多代谢途径在空间和时间上是分开的。据我们所知， 目前还没有任何关于衰老的特定脑室氧化还原代谢的全面研究。 过程中，NAD+辅因子仅被视为“NAD+消耗”或信号酶的底物， 参与表观遗传修饰(Sirtuins)和DNA修复(聚(ADP-核糖)聚合酶)，被广泛忽视 它在氧化还原反应中的作用。我们最近开发了基因编码工具，可以用来增加 哺乳动物细胞胞浆或线粒体中NAD+与NADH或NADP+与NADPH的比值。在此应用程序中 我们建议通过在不同的细胞中表达我们的工具来研究氧化还原区域化在衰老中的作用。 两种原代人的胞核、胞浆、线粒体、内质网和过氧化物体 成纤维细胞和多细胞线虫线虫。在这两个模型系统中，我们将探索如何提高 在不同隔室中的NAD+与NADH或NADP+与NADPH的比率中，随着细胞衰老而变化， 抗压性和寿命。我们目前的方法将首次使我们能够识别NAD-和 NADP偶联的氧化还原途径或机制在衰老调节中起关键作用。

Abstract Multiple lines of evidence designate mitochondrial dysfunction and related cellular reduction-oxidation (redox) imbalance as one of the hallmarks of aging. The redox cofactor nicotinamide adenine dinucleotide (NAD+) plays a central role in cellular energy metabolism, and it is an essential cofactor for supporting mitochondrial oxidative phosphorylation (OXPHOS). Numerous studies have implicated lowering cellular NAD+ levels in aging- associated metabolic changes, but its precise role at present remains contentious. This is mostly because NAD+ and its phosphorylated form NADP+ are substrates in hundreds of redox reactions which are often times performed by paralogous enzymes found in different cellular compartments. Compartmentalization of cellular metabolism is one of the most fundamental properties of complex eukaryotic life and in order to support healthy cellular functions many metabolic pathways are spatially and temporally compartmentalized. To our knowledge, there have not been any comprehensive studies of the compartment-specific redox metabolism of the aging process, and the NAD+ cofactor is viewed only as a substrate for “NAD+-consuming” or signaling enzymes which are involved in epigenetic modifications (sirtuins) and DNA repair (poly(ADP-ribose) polymerase), widely ignoring its role in redox reactions. We recently developed genetically encoded tools which can be used to increase the NAD+-to-NADH or NADP+-to-NADPH ratios in the cytosol or mitochondria in mammalian cells. In this application we propose to study the role of redox compartmentalization in aging by expressing our tools in different cellular compartments (nucleus, cytosol, mitochondria, endoplasmic reticulum and peroxisomes) of both human primary fibroblasts and the multicellular nematode C. elegans. In both model systems we will explore how an increase in the NAD+-to-NADH or NADP+-to-NADPH ratios in different compartments tracks with cellular senescence, stress resistance and lifespan. Our current approach, for the first time, will allow us to identify both NAD- and NADP-coupled redox pathways or mechanisms which play key roles in the regulation of aging.