Erich B. Tahara

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release.

Higher Respiratory Activity Decreases Mitochondrial Reactive Oxygen Release and Increases Life Span in Saccharomyces cerevisiae

Increased replicative longevity in Saccharomyces cerevisiae because of calorie restriction has been linked to enhanced mitochondrial respiratory activity. Here we have further investigated how mitochondrial respiration affects yeast life span. We found that calorie restriction by growth in low glucose increased respiration but decreased mitochondrial reactive oxygen species production relative to oxygen consumption. Calorie restriction also enhanced chronological life span. The beneficial effects of calorie restriction on mitochondrial respiration, reactive oxygen species release, and replicative and chronological life span could be mimicked by uncoupling agents such as dinitrophenol. Conversely, chronological life span decreased in cells treated with antimycin (which strongly increases mitochondrial reactive oxygen species generation) or in yeast mutants null for mitochondrial superoxide dismutase (which removes superoxide radicals) and for RTG2 (which participates in retrograde feedback signaling between mitochondria and the nucleus). These results suggest that yeast aging is linked to changes in mitochondrial metabolism and oxidative stress and that mild mitochondrial uncoupling can increase both chronological and replicative life span.

Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging

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 (pnc1Δ, npt1Δ, and bna6Δ) 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 npt1Δ and bna6Δ mutants. Furthermore, pyruvate and α‐ketoglutarate, substrates for dihydrolipoyl dehydrogenasecontaining 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. Tahara, E. B., Barros, M. H., Oliveira, G. A., Netto, L. E. S., Kowaltowski, A. J. Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging. FASEB J. 21, 274–283 (2007)

Increased aerobic metabolism is essential for the beneficial effects of caloric restriction on yeast life span

Calorie restriction is a dietary regimen capable of extending life span in a variety of multicellular organisms. A yeast model of calorie restriction has been developed in which limiting the concentration of glucose in the growth media of Saccharomyces cerevisiae leads to enhanced replicative and chronological longevity. Since S. cerevisiae are Crabtree-positive cells that present repression of aerobic catabolism when grown in high glucose concentrations, we investigated if this phenomenon participates in life span regulation in yeast. S. cerevisiae only exhibited an increase in chronological life span when incubated in limited concentrations of glucose. Limitation of galactose, raffinose or glycerol plus ethanol as substrates did not enhance life span. Furthermore, in Kluyveromyces lactis, a Crabtree-negative yeast, glucose limitation did not promote an enhancement of respiratory capacity nor a decrease in reactive oxygen species formation, as is characteristic of conditions of caloric restriction in S. cerevisiae. In addition, K. lactis did not present an increase in longevity when incubated in lower glucose concentrations. Altogether, our results indicate that release from repression of aerobic catabolism is essential for the beneficial effects of glucose limitation in the yeast calorie restriction model. Potential parallels between these changes in yeast and hormonal regulation of respiratory rates in animals are discussed.

Yeast as a model to study mitochondrial mechanisms in ageing

Despite the fact that ageing necessarily displays unique aspects in a single-cell organism, yeast, in particular Saccharomyces cerevisiae, are useful as model organisms to study ageing. Here we review mitochondrial characteristics involved in yeast longevity, including biogenesis, autophagy, respiration and oxidative phosphorylation, nutrient sensing, mitochondria-nuclear signaling, redox state and mitochondrial DNA integrity. Altogether, the yeast model unearths a rich and complex network involving many mitochondrial functions in ageing, and uncovers physiological and genetic mechanisms capable of extending lifespan in this model which may be shared with more complex organisms.

Proline Dehydrogenase Regulates Redox State and Respiratory Metabolism in Trypanosoma cruzi

Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ1-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle.

Saccharomyces cerevisiae coq10 null mutants are responsive to antimycin A

Deletion of COQ10 in Saccharomyces cerevisiae elicits a respiratory defect characterized by the absence of cytochrome c reduction, which is correctable by the addition of exogenous diffusible coenzyme Q2. Unlike other coq mutants with hampered coenzyme Q6 (Q6) synthesis, coq10 mutants have near wild‐type concentrations of Q6. In the present study, we used Q‐cycle inhibitors of the coenzyme QH2–cytochrome c reductase complex to assess the electron transfer properties of coq10 cells. Our results show that coq10 mutants respond to antimycin A, indicating an active Q‐cycle in these mutants, even though they are unable to transport electrons through cytochrome c and are not responsive to myxothiazol. EPR spectroscopic analysis also suggests that wild‐type and coq10 mitochondria accumulate similar amounts of Q6 semiquinone, despite a lower steady‐state level of coenzyme QH2–cytochrome c reductase complex in the coq10 cells. Confirming the reduced respiratory chain state in coq10 cells, we found that the expression of the Aspergillus fumigatus alternative oxidase in these cells leads to a decrease in antimycin‐dependent H2O2 release and improves their respiratory growth.

Calorie restriction hysteretically primes aging Saccharomyces cerevisiae toward more effective oxidative metabolism.

Calorie restriction (CR) is an intervention known to extend the lifespan of a wide variety of organisms. In S. cerevisiae, chronological lifespan is prolonged by decreasing glucose availability in the culture media, a model for CR. The mechanism has been proposed to involve an increase in the oxidative (versus fermentative) metabolism of glucose. Here, we measured wild-type and respiratory incompetent (ρ0) S. cerevisiae biomass formation, pH, oxygen and glucose consumption, and the evolution of ethanol, glycerol, acetate, pyruvate and succinate levels during the course of 28 days of chronological aging, aiming to identify metabolic changes responsible for the effects of CR. The concomitant and quantitative measurements allowed for calculations of conversion factors between different pairs of substrates and products, maximum specific substrate consumption and product formation rates and maximum specific growth rates. Interestingly, we found that the limitation of glucose availability in CR S. cerevisiae cultures hysteretically increases oxygen consumption rates many hours after the complete exhaustion of glucose from the media. Surprisingly, glucose-to-ethanol conversion and cellular growth supported by glucose were not quantitatively altered by CR. Instead, we found that CR primed the cells for earlier, faster and more efficient metabolism of respiratory substrates, especially ethanol. Since lifespan-enhancing effects of CR are absent in respiratory incompetent ρ0 cells, we propose that the hysteretic effect of glucose limitation on oxidative metabolism is central toward chronological lifespan extension by CR in this yeast.

nde1 deletion improves mitochondrial DNA maintenance in Saccharomyces cerevisiae coenzyme Q mutants

Saccharomyces cerevisiae has three distinct inner mitochondrial membrane NADH dehydrogenases mediating the transfer of electrons from NADH to CoQ (coenzyme Q): Nde1p, Nde2p and Ndi1p. The active site of Ndi1p faces the matrix side, whereas the enzymatic activities of Nde1p and Nde2p are restricted to the intermembrane space side, where they are responsible for cytosolic NADH oxidation. In the present study we genetically manipulated yeast strains in order to alter the redox state of CoQ and NADH dehydrogenases to evaluate the consequences on mtDNA (mitochondrial DNA) maintenance. Interestingly, nde1 deletion was protective for mtDNA in strains defective in CoQ function. Additionally, the absence of functional Nde1p promoted a decrease in the rate of H2O2 release in isolated mitochondria from different yeast strains. On the other hand, overexpression of the predominant NADH dehydrogenase NDE1 elevated the rate of mtDNA loss and was toxic to coq10 and coq4 mutants. Increased CoQ synthesis through COQ8 overexpression also demonstrated that there is a correlation between CoQ respiratory function and mtDNA loss: supraphysiological CoQ levels were protective against mtDNA loss in the presence of oxidative imbalance generated by Nde1p excess or exogenous H2O2. Altogether, our results indicate that impairment in the oxidation of cytosolic NADH by Nde1p is deleterious towards mitochondrial biogenesis due to an increase in reactive oxygen species release.

Respiratory and TCA cycle activities affect S. cerevisiae lifespan, response to caloric restriction and mtDNA stability.

We studied the importance of respiratory fitness in S. cerevisiae lifespan, response to caloric restriction (CR) and mtDNA stability. Mutants harboring mtDNA instability and electron transport defects do not respond to CR, while tricarboxylic acid cycle mutants presented extended lifespans due to CR. Interestingly, mtDNA is unstable in cells lacking dihydrolipoyl dehydrogenase under CR conditions, and cells lacking aconitase under standard conditions (both enzymes are components of the TCA and mitochondrial nucleoid). Altogether, our data indicate that respiratory integrity is required for lifespan extension by CR and that mtDNA stability is regulated by nucleoid proteins in a glucose-sensitive manner.