Tahara Erich B, Barros Mario H, Oliveira Graciele A, Netto Luis E S, Kowaltowski Alicia J
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, 05508-900, Brazil.
FASEB J. 2007 Jan;21(1):274-83. doi: 10.1096/fj.06-6686com. Epub 2006 Nov 16.
Replicative life span in Saccharomyces cerevisiae is increased by glucose (Glc) limitation [calorie restriction (CR)] and by augmented NAD+. Increased survival promoted by CR was attributed previously to the NAD+-dependent histone deacetylase activity of sirtuin family protein Sir2p but not to changes in redox state. Here we show that strains defective in NAD+ synthesis and salvage pathways (pnc1delta, npt1delta, and bna6delta) exhibit decreased oxygen consumption and increased mitochondrial H2O2 release, reversed over time by CR. These null mutant strains also present decreased chronological longevity in a manner rescued by CR. Furthermore, we observed that changes in mitochondrial H2O2 release alter cellular redox state, as attested by measurements of total, oxidized, and reduced glutathione. Surprisingly, our results indicate that matrix-soluble dihydrolipoyl-dehydrogenases are an important source of CR-preventable mitochondrial reactive oxygen species (ROS). Indeed, deletion of the LPD1 gene prevented oxidative stress in npt1delta and bna6delta mutants. Furthermore, pyruvate and alpha-ketoglutarate, substrates for dihydrolipoyl dehydrogenase-containing enzymes, promoted pronounced reactive oxygen release in permeabilized wild-type mitochondria. Altogether, these results substantiate the concept that mitochondrial ROS can be limited by caloric restriction and play an important role in S. cerevisiae senescence. Furthermore, these findings uncover dihydrolipoyl dehydrogenase as an important and novel source of ROS leading to life span limitation.
在酿酒酵母中，复制寿命可通过葡萄糖（Glc）限制[热量限制（CR）]和增加烟酰胺腺嘌呤二核苷酸（NAD⁺）来延长。此前，CR促进的存活率提高归因于沉默调节蛋白家族蛋白Sir2p的NAD⁺依赖性组蛋白脱乙酰酶活性，而非氧化还原状态的变化。在此，我们表明，在NAD⁺合成和补救途径中存在缺陷的菌株（pnc1δ、npt1δ和bna6δ）表现出氧气消耗减少和线粒体过氧化氢释放增加，随着时间的推移，CR可使其逆转。这些基因敲除突变菌株的时序寿命也以CR可挽救的方式降低。此外，我们观察到线粒体过氧化氢释放的变化会改变细胞氧化还原状态，这通过对总谷胱甘肽（GSH）、氧化型谷胱甘肽（GSSG）和还原型谷胱甘肽（GSH）的测量得到证实。令人惊讶的是，我们的结果表明，线粒体可溶性二氢硫辛酰胺脱氢酶是CR可预防的线粒体活性氧（ROS）的重要来源。事实上，缺失LPD1基因可防止npt1δ和bna6δ突变体中的氧化应激。此外，丙酮酸和α-酮戊二酸，即含二氢硫辛酰胺脱氢酶的酶的底物，在透化的野生型线粒体中促进了显著的活性氧释放。总之，这些结果证实了线粒体ROS可受热量限制的概念，并且在线粒体衰老中起重要作用。此外，这些发现揭示了二氢硫辛酰胺脱氢酶是导致寿命限制的ROS的重要且新的来源。
