Francis Sluse

Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism

HIF prolyl hydroxylases (PHD1–3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparα pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2α and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.

Mitochondria as a Source of Reactive Oxygen and Nitrogen Species: From Molecular Mechanisms to Human Health

Mitochondrially generated reactive oxygen species are involved in a myriad of signaling and damaging pathways in different tissues. In addition, mitochondria are an important target of reactive oxygen and nitrogen species. Here, we discuss basic mechanisms of mitochondrial oxidant generation and removal and the main factors affecting mitochondrial redox balance. We also discuss the interaction between mitochondrial reactive oxygen and nitrogen species, and the involvement of these oxidants in mitochondrial diseases, cancer, neurological, and cardiovascular disorders.

GENERATION OF SUPEROXIDE ANION BY MITOCHONDRIA AND IMPAIRMENT OF THEIR FUNCTIONS DURING ANOXIA AND REOXYGENATION IN VITRO

A small portion of the oxygen consumed by aerobic cells is converted to superoxide anion at the level of the mitochondrial respiratory chain. If produced in excess, this harmful radical is considered to impair cellular structures and functions. Damage at the level of mitochondria have been reported after ischemia and reperfusion of organs. However, the complexity of the in vivo system prevents from understanding and describing precise mechanisms and locations of mitochondrial impairment. An in vitro model of isolated-mitochondria anoxia-reoxygenation is used to investigate superoxide anion generation together with specific damage at the level of mitochondrial oxidative phosphorylation. Superoxide anion is detected by electron paramagnetic resonance spin trapping with POBN-ethanol. Mitochondrial respiratory parameters are calculated from oxygen consumption traces recorded with a Clark electrode. Respiring mitochondria produce superoxide anion in unstressed conditions, however, the production is raised during postanoxic reoxygenation. Several respiratory parameters are impaired after reoxygenation, as shown by decreases of phosphorylating and uncoupled respiration rates and of ADP/O ratio and by increase of resting respiration. Partial protection of mitochondrial function by POBN suggests that functional damage is related and secondary to superoxide anion production by the mitochondria in vitro.

Phylogenetic classification of the mitochondrial carrier family of Saccharomyces cerevisiae.

The screening of the open reading frames identified in the whole yeast genome has allowed us to discover 34 proteins belonging to the mitochondrial carrier family. By phylogenetic study, they can be divided into 27 subfamilies including ADP/ATP, phosphate and citrate carriers, putative oxoglutarate and GDC carriers and 22 new subfamilies. Topology predictions using the ‘positive inside rule’ approach have shown that the yeast carriers are similarly oriented with both extremities exposed to the cytosol. In each subfamily, a strict conservation of the charged residues in the six transmembrane α‐helices is observed, suggesting a functional role for these residues and the existence of 27 functionally distinct carriers.

Free fatty acids regulate the uncoupling protein and alternative oxidase activities in plant mitochondria

Two energy‐dissipating systems, an alternative oxidase and an uncoupling protein, are known to exist in plant mitochondria. In tomato fruit mitochondria linoleic acid, a substrate for the uncoupling protein, inhibited the alternative oxidase‐sustained respiration and decreased the ADP/O ratio to the same value regardless of the level of alternative oxidase activity. Experiments with varying concentrations of linoleic acid have shown that inhibition of the alternative oxidase is more sensitive to the linoleic acid concentration than the uncoupling protein activation. It can be proposed that these dissipating systems work sequentially during the life of the plant cell, since a high level of free fatty acid‐induced uncoupling protein activity excludes alternative oxidase activity.

Stress-induced premature senescence. Essence of life, evolution, stress, and aging.

The stress syndrome was discovered accidentally by Hans Selye while searching for new hormones in the placenta.1 After injecting rats with crude preparations, Selye found adrenal enlargements and involution of thymus and lymph nodes, which he thought were specific for a particular hormone. It occurred to Selye that these symptoms might represent a nonspecific response to noxious agents. Indeed, this was found to be the case when he injected rats with diverse agents. Selye defined the stress response as the “general adaptation syndrome.”2,3 According to this theory, the initial reaction to stress is shock, it is followed by a countershock phase, and gradually resistance develops to the stressor. This resistance may turn into exhaustion, however, if the stressor persists, and death may ensue. Both specific and nonspecific resistance develops during stress.4 In his last scientific book, Selye defined biologic stress as “the non-specific response of the body to any demand made upon it.”5 Beside the transfer of the word “stress” from physics to biology, Selye also coined the words corticosteroids, glucocorticoids, and mineralocorticoids.6 Nowadays, the concept of stress has invaded most fields of the biologic, medical, and social sciences. Cellular and molecular biology has become interested in the study of the stress response of human, animal, and plant cells, the consensus being that “any environmental factor potentially unfavourable to living organism” is stress.7 It is also generally agreed that “if the limits of tolerance are exceeded and the adaptive capacity is over-worked, the result may be permanent damage or even death.”8 Three phases of the stress response have been defined based on experimental observations: (1) the response phase of alarm reaction with deviation of functional norm, decline of vitality, and excess of catabolic processes over anabolism, (2) the restitution phase or stage of resistance with adaptation processes and repair processes, and (3) either the end phase, that stage of exhaustion or long-term response when stress intensity is too high, leading to overcharge of the adaptation capacity, damage, chronic dis-

Alternative oxidase in the branched mitochondrial respiratory network: an overview on structure, function, regulation, and role

Plants and some other organisms including protists possess a complex branched respiratory network in their mitochondria. Some pathways of this network are not energy-conserving and allow sites of energy conservation to be bypassed, leading to a decrease of the energy yield in the cells. It is a challenge to understand the regulation of the partitioning of electrons between the various energy-dissipating and -conserving pathways. This review is focused on the oxidase side of the respiratory chain that presents a cyanide-resistant energy-dissipating alternative oxidase (AOX) besides the cytochrome pathway. The known structural properties of AOX are described including transmembrane topology, dimerization, and active sites. Regulation of the alternative oxidase activity is presented in detail because of its complexity. The alternative oxidase activity is dependent on substrate availability: total ubiquinone concentration and its redox state in the membrane and O2 concentration in the cell. The alternative oxidase activity can be long-term regulated (gene expression) or short-term (post-translational modification, allosteric activation) regulated. Electron distribution (partitioning) between the alternative and cytochrome pathways during steady-state respiration is a crucial measurement to quantitatively analyze the effects of the various levels of regulation of the alternative oxidase. Three approaches are described with their specific domain of application and limitations: kinetic approach, oxygen isotope differential discrimination, and ADP/O method (thermokinetic approach). Lastly, the role of the alternative oxidase in non-thermogenic tissues is discussed in relation to the energy metabolism balance of the cell (supply in reducing equivalents/demand in energy and carbon) and with harmful reactive oxygen species formation.

Uncoupling proteins outside the animal and plant kingdoms: functional and evolutionary aspects

The appearance of intracellular oxidative phosphorylation at the time of acquisition of mitochondria in Eukarya was very soon accompanied by the emergence of uncoupling protein, a carrier specialized in free fatty acid‐mediated H+ recycling that can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis, thereby maintaining a balance between energy supply and demand in the cell and defending cells against damaging reactive oxygen species production when electron carriers of the respiratory chain become overreduced. The simultaneous occurrence of redox free energy‐dissipating oxidase, which has the same final effect, could be related to the functional interactions between both dissipative systems.

First evidence and characterization of an uncoupling protein in fungi kingdom: CpUCP of Candida parapsilosis.

An uncoupling protein (UCP) was identified in mitochondria from Candida parapsilosis (CpUCP), a non‐fermentative parasitic yeast. CpUCP was immunodetected using polyclonal antibodies raised against plant UCP. Activity of CpUCP, investigated in mitochondria depleted of free fatty acids, was stimulated by linoleic acid (LA) and inhibited by GTP. Activity of CpUCP enhanced state 4 respiration by decreasing ΔΨ and lowered the ADP/O ratio. Thus, it was able to divert energy from oxidative phosphorylation. The voltage dependence of electron flux indicated that LA had a pure protonophoretic effect. The discovery of CpUCP proves that UCP‐like proteins occur in the four eukaryotic kingdoms: animals, plants, fungi and protists.

Biochemical, genetic and molecular characterization of new respiratory-deficient mutants in Chlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.