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

摘要 多种证据表明线粒体功能障碍和相关的细胞还原-氧化（氧化还原） 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+） 在细胞能量代谢中起核心作用，是支持线粒体氧化的重要辅助因子。 磷酸化（OXPHOS）。许多研究表明，衰老过程中细胞NAD+水平降低- 相关的代谢变化，但其确切作用目前仍有争议。这主要是因为NAD+ 及其磷酸化形式NADP+是数百种氧化还原反应的底物， 由不同细胞区室中发现的旁系同源酶进行。细胞区室化 代谢是复杂真核生物最基本的特性之一， 细胞功能许多代谢途径在空间上和时间上是分隔的。据我们所知， 还没有任何关于衰老的隔室特异性氧化还原代谢的全面研究， NAD+辅因子仅被视为“NAD+消耗”或信号传导酶的底物， 参与表观遗传修饰（sirtuins）和DNA修复（聚（ADP-核糖）聚合酶），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的工具，可以用来增加 哺乳动物细胞中胞质溶胶或线粒体中NAD+与NADH或NADP+与NADPH的比率。本申请中 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室化在衰老中的作用。 细胞核、胞浆、线粒体、内质网和过氧化物酶体 成纤维细胞和多细胞线虫C.优雅在这两个模型系统中，我们将探讨如何增加 在细胞衰老的不同区室中的NAD+与NADH或NADP+与NADPH的比率中， 抗压力和寿命。我们目前的方法，第一次，将使我们能够识别NAD-和 NADP偶联的氧化还原途径或机制，在衰老的调节中发挥关键作用。

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.