Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP ؉ -dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.Cell damage induced by oxidative stress and reactive oxygen species (ROS) 1 has been implicated in several human diseases including aging, alcohol-mediated organ damage, neurodegenerative diseases, many types of cancers, cardiovascular diseases, and UV-mediated skin disorders (1). As one of the major sources of ROS (2), mitochondria are highly susceptible to oxidative damage. ROS can damage mitochondrial enzymes directly (3), and they can cause mutation in mitochondrial DNAs (4). At the same time, ROS can change the mitochondrial transmembrane potential (⌬m), which is indicative of mitochondrial membrane integrity (5) and precedes cell death induced by various toxic compounds and cytokines (6). Recent reports indicate that mitochondrial ROS cause apoptosis (7, 8) by activating various apoptotic effectors such as cytochrome c release, procaspase-2, procaspase-9, procaspase-3, and latent apoptosis-inducing factor, which is released from the mitochondria during apoptosis (9 -11). Another report also suggested that mitochondrial ROS directly caused apoptosis of T cells (12). It was also reported that tumor necrosis factor ␣ causes a rapid production of mitochondrial ROS (13) and that ceramide, an apoptotic stimulus, also plays a crucial role in tumor necrosis factor ␣-induced mitochondrial ROS generation (14). Furthermore, several other investigators demonstrated that ROS are involved in the signaling pathway of certain growth factors (15) and cytokines (16). In addition, mitochondrial ROS, under hypoxic conditions, activate the transcription of the genes for glycolytic enzymes as well as erythropoietin and vascular endothelial growth factor by upregulating a transcriptional factor, hypoxia-inducible factor 1 (17), suggesting that mitochondrial ROS mediate cross-talk b...

To understand the interactions and functional role of each of the three mitochondrial NAD؉ -dependent isocitrate dehydrogenase (IDH) subunits (␣, ␤, and ␥), we have characterized human cDNAs encoding two ␤ isoforms (␤ 1 and ␤ 2 ) and the ␥ subunit. Analysis of deduced amino acid sequences revealed that ␤ 1 and ␤ 2 encode 349 and 354 amino acids, respectively, and the two isoforms only differ in the most carboxyl 28 amino acids. The ␥ cDNA encodes 354 amino acids and is almost identical to monkey IDH␥. Northern analyses revealed that the smaller ␤ 2 transcript (1.3 kilobases) is primarily expressed in heart and skeletal muscle, whereas the larger ␤ 1 mRNA (1.6 kilobases) is prevalent in nonmuscle tissues. Sequence analysis of the IDH␤ gene indicates that the difference in the C-terminal 28 amino acids between ␤ 1 and ␤ 2 proteins results from alternative splicing of a single transcript. Among the various combinations of human IDH subunits co-expressed in bacteria, ␣␤␥, ␣␤, and ␣␥ combinations exhibited significant amounts of IDH activity, whereas subunits produced alone and ␤␥ showed no detectable activity. These data suggest that the ␣ is the catalytic subunit and that at least one of the other two subunits plays an essential supporting role for activity. Substitution of ␤ 1 with ␤ 2 in the co-expression system lowered the pH optimum for IDH activity from 8.0 to 7.6. This difference in optimal pH was analogous to what was observed in mouse kidney and brain (␤ 1 prevalent; optimal pH 8.0) versus heart (␤ 2 prevalent; pH 7.6) mitochondria. Experiments with a specially designed splicing reporter construct stably transfected into HT1080 cells indicate that acidic conditions favor a splicing pattern responsible for the muscle-and heartspecific ␤ 2 isoform. Taken together, these data indicate a regulatory role of IDH␤ isoforms in determining the pH optimum for IDH activity through the tissue-specific alternative splicing.

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The reactive oxygen species (ROS) include superoxide anion, the hydroxyl radical, and the singlet oxygen. The mitochondria are not only a major site where a large amount of cellular ROS is generated, but also they are a main target of ROS; thus, various pathological states including aging have been reported to involve the mitochondrial dysfunctions and ROS generation. However, a catalase, a major enzyme that removes hydrogen peroxide, has been reported to be absent in most mitochondria of mammalian cells. Therefore, mitochondrial GSH is important to the mitochondrial defenses against ROS because it is a prerequisite to the roles of mitochondrial GPx and phospholipid hydrogenperoxide glutathione peroxidase (PHGPx), which play key roles in removing the peroxides in mitochondria. In addition to the role of reducing agent for the regeneration of GSH, NADPH is also necessary to the antioxidant function of the peroxiredoxin family in mitochondria. We have previously reported the isolation and molecular characterization of bovine mitochondrial NADP + -dependent isocitrate dehydrogenase (IDPm). Here, we first report that expression of cloned mouse IDPm cDNA reduces oxidative stress. Conversely, reduction of IDPm in mitochondria by the expression of antisense mouse IDPm cDNA increases spontaneous generation of ROS by stimulating mitochondrial injury. Our findings further support that IDPm is a major producer of mitochondrial NADPH by which the mitochondrial GSH pool required in defense against cellular oxidative stress is replenished.