Anne Devin

Mitochondrial ROS Generation and Its Regulation: Mechanisms Involved in H2O2 Signaling

Mitochondria are the main source of reactive oxygen species in the cell. These reactive oxygen species have long been known as being involved in oxidative stress. This is a review of the mechanisms involved in reactive oxygen species generation by the respiratory chain and some of the dehydrogenases and the control by thermodynamic and kinetic constraints. Mitochondrial ROS produced at the level of the bc1 complex as well at the level of complex I are discussed. It was recognized more than a decade ago that they can also function as signaling molecules. This signaling role will be developed both in terms of mechanism and in terms of mitochondrial ROS signaling. The notion that hydrogen peroxide acts not only as a damaging oxidant but also as a signaling molecule was proposed more than a decade ago. Hydrogen peroxide signaling can be either direct (oxidation of its target) or indirect (involving peroxiredoxins, for example). The consequences of ROS signaling on crucial biologic processes such as cell proliferation and differentiation are discussed.

The role of the death-domain kinase RIP in tumour-necrosis-factor-induced activation of mitogen-activated protein kinases.

The death‐domain kinase RIP (receptor‐interacting protein) is an important effector of tumour necrosis factor (TNF) signalling and is essential for TNF‐induced nuclear factor‐κB activation. However, the function of RIP in the TNF‐induced activation of mitogen‐activated protein kinases (MAPKs) has not been fully investigated. In this report, using Rip null (Rip−/−) mouse fibroblast cells, we investigated whether RIP is required for TNF‐induced activation of the MAPKs extracellular‐signal‐related kinase (ERK), p38 and c‐Jun amino‐terminal kinase (JNK). We found that TNF‐induced activation of ERK, p38 and JNK is decreased in Rip−/− cells. The activation of these kinases by interleukin‐1 is normal in Rip−/− cells. More importantly, we showed that the kinase activity of RIP is needed for ERK activation.

Mitochondrial Oxidative Phosphorylation Is Regulated by Fructose 1,6-Bisphosphate A POSSIBLE ROLE IN CRABTREE EFFECT INDUCTION?

In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the “Crabtree effect.” We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into “Crabtree-positive,” where the Crabtree effect occurs, and “Crabtree-negative,” where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect.

Effect of polyunsaturated fatty acids deficiency on oxidative phosphorylation in rat liver mitochondria

Liver mitochondria isolated from controls or polyunsaturated fatty acid (PUFA) deficient rats were studied for oxidative phosphorylation. A PUFA-deficient diet led to a dramatic change in the fatty acid composition of mitochondrial lipid content, similar to that reported in the literature. Besides the changes in lipid composition, mitochondrial volume was enlarged (+45% in state 4 and two-fold in state 3). State 4 respiration was increased together with a decrease in protonmotive force. The non-ohmicity of the relationship between non-phosphorylating respiration and protonmotive force was more pronounced in the PUFA-deficient group. State 3 oxygen consumption as well as the rate of ATP synthesis showed no difference between the two groups, whereas the protonmotive force decreased substantially in mitochondria from PUFA-deficient animals. In contrast, ATP/O ratios were decreased in the PUFA-deficient group when determined at subsaturating ADP concentration. Taken together, these results are in agreement with both an increased non-ohmic proton leak and an increased redox slipping. The relative importance of these two effects on the overall efficiency of oxidative phosphorylation depends on both the rate of oxidative phosphorylation and the maintained protonmotive force. Hence, in isolated mitochondria the respective role of each effect may vary between state 4 and state 3.

A yeast model for amyloid-β aggregation exemplifies the role of membrane trafficking and PICALM in cytotoxicity

SUMMARY Alzheimer’s disease is the most common neurodegenerative disease, associated with aggregation of amyloid-β (Aβ) peptides. The exact mechanism of neuronal cell dysfunction in Alzheimer’s disease is poorly understood and numerous models have been used to decipher the mechanisms leading to cellular death. Yeast cells might be a good model to understand the intracellular toxicity triggered by Aβ peptides. Indeed, yeast has been used as a model to examine protein functions or cellular pathways that mediate the secretion, aggregation and subsequent toxicity of proteins associated with human neurodegenerative disorders. In the present study, we use the yeast Saccharomyces cerevisiae as a model system to study the effects of intracellular Aβ in fusion with green fluorescent protein. We sent this fusion protein into the secretory pathway and showed that intracellular traffic pathways are necessary for the generation of toxic species. Yeast PICALM orthologs are involved in cellular toxicity, indicating conservation of the mechanisms of toxicity from mammals to yeast. Finally, our model demonstrates the capacity for intracellular Aβ to cross intracellular membranes and target mitochondrial organelles.

Reactive oxygen species mediated regulation of mitochondrial biogenesis in the yeast Saccharomyces cerevisiae

Mitochondrial biogenesis is a complex process. It necessitates the participation of both the nuclear and the mitochondrial genomes. This process is highly regulated, and mitochondrial content within a cell varies according to energy demand. In the yeast Saccharomyces cerevisiae, the cAMP pathway is involved in the regulation of mitochondrial biogenesis. An overactivation of this pathway leads to an increase in mitochondrial enzymatic content. Of the three yeast cAMP protein kinases, we have previously shown that Tpk3p is the one involved in the regulation of mitochondrial biogenesis. In this paper, we investigated the molecular mechanisms that govern this process. We show that in the absence of Tpk3p, mitochondria produce large amounts of reactive oxygen species that signal to the HAP2/3/4/5 nuclear transcription factors involved in mitochondrial biogenesis. We establish that an increase in mitochondrial reactive oxygen species production down-regulates mitochondrial biogenesis. It is the first time that a redox sensitivity of the transcription factors involved in yeast mitochondrial biogenesis is shown. Such a process could be seen as a mitochondria quality control process.

Reactive Oxygen Species-Mediated Control of Mitochondrial Biogenesis

Mitochondrial biogenesis is a complex process. It necessitates the contribution of both the nuclear and the mitochondrial genomes and therefore crosstalk between the nucleus and mitochondria. It is now well established that cellular mitochondrial content can vary according to a number of stimuli and physiological states in eukaryotes. The knowledge of the actors and signals regulating the mitochondrial biogenesis is thus of high importance. The cellular redox state has been considered for a long time as a key element in the regulation of various processes. In this paper, we report the involvement of the oxidative stress in the regulation of some actors of mitochondrial biogenesis.

Dependence of flux size and efficiency of oxidative phosphorylation on external osmolarity in isolated rat liver mitochondria: role of adenine nucleotide carrier

The aim of this work was a thermodynamic and kinetic study of the influence of varying external osmolarity on overall oxidative phosphorylations in isolated rat liver mitochondria. When external osmolarity is increased from 100 to 400 mosM by using a non-penetrant sugar: (i) matrix volume diminishes, (ii) state 3 respiratory rate decreases when state 4 slightly varies, (iii) states 3 and 4 protonmotive force and NAD(P)H level increase, whereas oxidative phosphorylation efficiency (ATP/O) decreases. Indeed, respiratory flux versus protonmotive force relationships depend on the osmolarity considered: the lower the external osmolarity, the higher the span of overall driving force necessary for the same respiratory rate. To further investigate the mechanism of the decrease in respiratory and ATP synthesis flux leading to a lowering in oxidative phosphorylation efficiency, we determined the adenine nucleotide carrier control coefficient on respiratory and ATP synthesis rates respectively. The main result is that the adenine nucleotide carrier control coefficient on respiratory rate decreases, and conversely that adenine nucleotide carrier control on ATP synthesis rate increases, from iso- to hyperosmolarity. Furthermore, whatever the osmolarity, when state 3 respiratory rate is titrated with carboxyatractyloside, the same relationship is observed between ATP/O ratio and respiratory flux. From many previous studies, it has been shown that an increase in external osmolarity and a consequent decrease in matrix volume inhibits almost all mitochondrial proton pumps (coupling site 1 and 2 of respiratory chain, ATPase) in different ways. In this work, we show that in phosphorylating mitochondria, the adenine nucleotide carrier plays a key role: its inhibition as the external osmolarity increases lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency.

Chronic ethanol ingestion increases efficiency of oxidative phosphorylation in rat liver mitochondria

The efficiency of oxidative phosphorylation was compared between rats chronically fed with ethanol and controls. (i) Results showed that the liver mitochondria state 4 respiratory rate was strongly inhibited, while the corresponding proton‐motive force was not affected; (ii) the cytochrome oxidase content and activity were decreased and (iii) the oxidative‐phosphorylation yield was increased in the ethanol exposed group. Furthermore, oxidative phosphorylation at coupling site II was not affected by ethanol. Cytochrome oxidase inhibition by sodium‐azide mimicked the effects of ethanol intoxication in control mitochondria. This indicates that the decrease in cytochrome oxidase activity induced by ethanol intoxication directly increases the efficiency of oxidative phosphorylation.

Cytosolic NAD+ content strictly depends on ATP concentration in isolated liver cells

By focusing on the question of the thermodynamic relationships involved in the regulation of biological energy conversion, bioenergetic studies usually consider the free pyridine and adenine nucleotide rather than their total pools, in either cytosol or mitochondria. In this study, we report a new observation that, at steady state, nicotinamide nucleotide content is increased by a rise in the ATP content of the whole cell under physiological conditions. It is a straight line relationship when only NAD+ and ATP are considered. When regarding the compartmentation of this phenomenon, it appears that the linear relationship between [NAD+] and [ATP] occurs only in the cytosol. Such a dependence could be a supplementary mechanism of regulation between various metabolic pathways in the liver cell.