Biliverdin Reductase-A integrates insulin signaling with mitochondrial metabolism through phosphorylation of GSK3ß.

Brain insulin resistance links the failure of energy metabolism with cognitive decline in both type 2 Diabetes Mellitus (T2D) and Alzheimer's disease (AD), although the molecular changes preceding overt brain insulin resistance remain unexplored. Abnormal biliverdin reductase-A (BVR-A) levels were observed in both T2D and AD and were associated with insulin resistance. Here, we demonstrate that reduced BVR-A levels alter insulin signaling and mitochondrial bioenergetics in the brain. Loss of BVR-A leads to IRS1 hyper-activation but dysregulates Akt-GSK3ß complex in response to insulin, hindering the accumulation of pGSK3ßS9 into the mitochondria. This event impairs oxidative phosphorylation and fosters the activation of the mitochondrial Unfolded Protein Response (UPRmt). Remarkably, we unveil that BVR-A is required to shuttle pGSK3ßS9 into the mitochondria. Our data sheds light on the intricate interplay between insulin signaling and mitochondrial metabolism in the brain unraveling potential targets for mitigating the development of brain insulin resistance and neurodegeneration.

Hydrogen sulfide production does not affect antibiotic resistance in Pseudomonas aeruginosa.

Hydrogen sulfide (H2S) has been proposed to protect bacteria from antibiotics, pointing to H2S-producing enzymes as possible targets for the development of antibiotic adjuvants. Here, MIC assays performed with Pseudomonas aeruginosa mutants producing altered H2S levels demonstrate that H2S does not affect antibiotic resistance in this bacterium. Moreover, correlation analyses in a large collection of P. aeruginosa cystic fibrosis isolates argue against the protective role of H2S from antibiotic activity during chronic lung infection.

Impact of Hydrogen Sulfide on Mitochondrial and Bacterial Bioenergetics.

This review focuses on the effects of hydrogen sulfide (H2S) on the unique bioenergetic molecular machines in mitochondria and bacteria-the protein complexes of electron transport chains and associated enzymes. H2S, along with nitric oxide and carbon monoxide, belongs to the class of endogenous gaseous signaling molecules. This compound plays critical roles in physiology and pathophysiology. Enzymes implicated in H2S metabolism and physiological actions are promising targets for novel pharmaceutical agents. The biological effects of H2S are biphasic, changing from cytoprotection to cytotoxicity through increasing the compound concentration. In mammals, H2S enhances the activity of FoF1-ATP (adenosine triphosphate) synthase and lactate dehydrogenase via their S-sulfhydration, thereby stimulating mitochondrial electron transport. H2S serves as an electron donor for the mitochondrial respiratory chain via sulfide quinone oxidoreductase and cytochrome c oxidase at low H2S levels. The latter enzyme is inhibited by high H2S concentrations, resulting in the reversible inhibition of electron transport and ATP production in mitochondria. In the branched respiratory chain of Escherichia coli, H2S inhibits the bo3 terminal oxidase but does not affect the alternative bd-type oxidases. Thus, in E. coli and presumably other bacteria, cytochrome bd permits respiration and cell growth in H2S-rich environments. A complete picture of the impact of H2S on bioenergetics is lacking, but this field is fast-moving, and active ongoing research on this topic will likely shed light on additional, yet unknown biological effects.

Bacterial Oxidases of the Cytochrome bd Family: Redox Enzymes of Unique Structure, Function, and Utility As Drug Targets.

Significance: Cytochrome bd is a ubiquinol:oxygen oxidoreductase of many prokaryotic respiratory chains with a unique structure and functional characteristics. Its primary role is to couple the reduction of molecular oxygen, even at submicromolar concentrations, to water with the generation of a proton motive force used for adenosine triphosphate production. Cytochrome bd is found in many bacterial pathogens and, surprisingly, in bacteria formally denoted as anaerobes. It endows bacteria with resistance to various stressors and is a potential drug target. Recent Advances: We summarize recent advances in the biochemistry, structure, and physiological functions of cytochrome bd in the light of exciting new three-dimensional structures of the oxidase. The newly discovered roles of cytochrome bd in contributing to bacterial protection against hydrogen peroxide, nitric oxide, peroxynitrite, and hydrogen sulfide are assessed. Critical Issues: Fundamental questions remain regarding the precise delineation of electron flow within this multihaem oxidase and how the extraordinarily high affinity for oxygen is accomplished, while endowing bacteria with resistance to other small ligands. Future Directions: It is clear that cytochrome bd is unique in its ability to confer resistance to toxic small molecules, a property that is significant for understanding the propensity of pathogens to possess this oxidase. Since cytochrome bd is a uniquely bacterial enzyme, future research should focus on harnessing fundamental knowledge of its structure and function to the development of novel and effective antibacterial agents.

The multifaceted roles of sulfane sulfur species in cancer-associated processes.

Sulfane sulfur species comprise a variety of biologically relevant hydrogen sulfide (H2S)-derived species, including per- and poly-sulfidated low molecular weight compounds and proteins. A growing body of evidence suggests that H2S, currently recognized as a key signaling molecule in human physiology and pathophysiology, plays an important role in cancer biology by modulating cell bioenergetics and contributing to metabolic reprogramming. This is accomplished through functional modulation of target proteins via H2S binding to heme iron centers or H2S-mediated reversible per- or poly-sulfidation of specific cysteine residues. Since sulfane sulfur species are increasingly viewed not only as a major source of H2S but also as key mediators of some of the biological effects commonly attributed to H2S, the multifaceted role of these species in cancer biology is reviewed here with reference to H2S, focusing on their metabolism, signaling function, impact on cell bioenergetics and anti-tumoral properties.

N-Acetylcysteine Serves as Substrate of 3-Mercaptopyruvate Sulfurtransferase and Stimulates Sulfide Metabolism in Colon Cancer Cells.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST.

Hydrogen Sulfide Oxidation: Adaptive Changes in Mitochondria of SW480 Colorectal Cancer Cells upon Exposure to Hypoxia.

Hydrogen sulfide (H2S), a known inhibitor of cytochrome c oxidase (CcOX), plays a key signaling role in human (patho)physiology. H2S is synthesized endogenously and mainly metabolized by a mitochondrial sulfide-oxidizing pathway including sulfide:quinone oxidoreductase (SQR), whereby H2S-derived electrons are injected into the respiratory chain stimulating O2 consumption and ATP synthesis. Under hypoxic conditions, H2S has higher stability and is synthesized at higher levels with protective effects for the cell. Herein, working on SW480 colon cancer cells, we evaluated the effect of hypoxia on the ability of cells to metabolize H2S. The sulfide-oxidizing activity was assessed by high-resolution respirometry, measuring the stimulatory effect of sulfide on rotenone-inhibited cell respiration in the absence or presence of antimycin A. Compared to cells grown under normoxic conditions (air O2), cells exposed for 24 h to hypoxia (1% O2) displayed a 1.3-fold reduction in maximal sulfide-oxidizing activity and 2.7-fold lower basal O2 respiration. Based on citrate synthase activity assays, mitochondria of hypoxia-treated cells were 1.8-fold less abundant and displayed 1.4-fold higher maximal sulfide-oxidizing activity and 2.6-fold enrichment in SQR as evaluated by immunoblotting. We speculate that under hypoxic conditions mitochondria undergo these adaptive changes to protect cell respiration from H2S poisoning.

Nitrosative stress defences of the enterohepatic pathogenic bacterium Helicobacter pullorum.

Helicobacter pullorum is an avian bacterium that causes gastroenteritis, intestinal bowel and hepatobiliary diseases in humans. Although H. pullorum has been shown to activate the mammalian innate immunity with release of nitric oxide (NO), the proteins that afford protection against NO and reactive nitrogen species (RNS) remain unknown. Here several protein candidates of H. pullorum, namely a truncated (TrHb) and a single domain haemoglobin (SdHb), and three peroxiredoxin-like proteins (Prx1, Prx2 and Prx3) were investigated. We report that the two haemoglobin genes are induced by RNS, and that SdHb confers resistance to nitrosative stress both in vitro and in macrophages. For peroxiredoxins, the prx2 and prx3 expression is enhanced by peroxynitrite and hydrogen peroxide, respectively. Mutation of prx1 does not alter the resistance to these stresses, while the single ∆prx2 and double ∆prx1∆prx2 mutants have decreased viability. To corroborate the physiological data, the biochemical analysis of the five recombinant enzymes was done, namely by stopped-flow spectrophotometry. It is shown that H. pullorum SdHb reacts with NO much more quickly than TrHb, and that the three Prxs react promptly with peroxynitrite, Prx3 displaying the highest reactivity. Altogether, the results unveil SdHb and Prx3 as major protective systems of H. pullorum against nitrosative stress.

Bioenergetic Impairment in Animal and Cellular Models of Alzheimer's Disease: PARP-1 Inhibition Rescues Metabolic Dysfunctions.

Amyloid-beta peptide accumulation in the brain is one of the main hallmarks of Alzheimer's disease. The amyloid aggregation process is associated with the generation of free radical species responsible for mitochondrial impairment and DNA damage that in turn activates poly(ADP-ribose)polymerase 1 (PARP-1). PARP-1 catalyzes the poly(ADP-ribosylation), a post-translational modification of proteins, cleaving the substrate NAD+ and transferring the ADP-ribose moieties to the enzyme itself or to an acceptor protein to form branched polymers of ADP-ribose. In this paper, we demonstrate that a mitochondrial dysfunction occurs in Alzheimer's transgenic mice TgCRND8, in SH-SY5Y treated with amyloid-beta and in 7PA2 cells. Moreover, PARP-1 activation contributes to the functional energetic decline affecting cytochrome oxidase IV protein levels, oxygen consumption rates, and membrane potential, resulting in cellular bioenergetic deficit. We also observed, for the first time, an increase of pyruvate kinase 2 expression, suggesting a modulation of the glycolytic pathway by PARP-1. PARP-1 inhibitors are able to restore both mitochondrial impairment and pyruvate kinase 2 expression. The overall data here presented indicate a pivotal role for this enzyme in the bioenergetic network of neuronal cells and open new perspectives for investigating molecular mechanisms underlying energy charge decline in Alzheimer's disease. In this scenario, PARP-1 inhibitors might represent a novel therapeutic intervention to rescue cellular energetic metabolism.

Bioenergetic relevance of hydrogen sulfide and the interplay between gasotransmitters at human cystathionine ß-synthase.

Merely considered as a toxic gas in the past, hydrogen sulfide (H2S) is currently viewed as the third 'gasotransmitter' in addition to nitric oxide (NO) and carbon monoxide (CO), playing a key signalling role in human (patho)physiology. H2S can either act as a substrate or, similarly to CO and NO, an inhibitor of mitochondrial respiration, in the latter case by targeting cytochrome c oxidase (CcOX). The impact of H(2)S on mitochondrial energy metabolism crucially depends on the bioavailability of this gaseous molecule and its interplay with the other two gasotransmitters. The H(2)S-producing human enzyme cystathionine ß-synthase (CBS), sustaining cellular bioenergetics in colorectal cancer cells, plays a role in the interplay between gasotransmitters. The enzyme was indeed recently shown to be negatively modulated by physiological concentrations of CO and NO, particularly in the presence of its allosteric activator S-adenosyl-l-methionine (AdoMet). These newly discovered regulatory mechanisms are herein reviewed. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Evidence for Detrimental Cross Interactions between Reactive Oxygen and Nitrogen Species in Leber's Hereditary Optic Neuropathy Cells.

Here we have collected evidence suggesting that chronic changes in the NO homeostasis and the rise of reactive oxygen species bioavailability can contribute to cell dysfunction in Leber's hereditary optic neuropathy (LHON) patients. We report that peripheral blood mononuclear cells (PBMCs), derived from a female LHON patient with bilateral reduced vision and carrying the pathogenic mutation 11778/ND4, display increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as revealed by flow cytometry, fluorometric measurements of nitrite/nitrate, and 3-nitrotyrosine immunodetection. Moreover, viability assays with the tetrazolium dye MTT showed that lymphoblasts from the same patient are more sensitive to prolonged NO exposure, leading to cell death. Taken together these findings suggest that oxidative and nitrosative stress cooperatively play an important role in driving LHON pathology when excess NO remains available over time in the cell environment.

Mitochondria and nitric oxide: chemistry and pathophysiology.

Cell respiration is controlled by nitric oxide (NO) reacting with respiratory chain complexes, particularly with Complex I and IV. The functional implication of these reactions is different owing to involvement of different mechanisms. Inhibition of complex IV is rapid (milliseconds) and reversible, and occurs at nanomolar NO concentrations, whereas inhibition of complex I occurs after a prolonged exposure to higher NO concentrations. The inhibition of Complex I involves the reversible S-nitrosation of a key cysteine residue on the ND3 subunit. The reaction of NO with cytochrome c oxidase (CcOX) directly involves the active site of the enzyme: two mechanisms have been described leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) or a more labile nitrite-derivative (CcOX-NO (2) (-) ). Both adducts are inhibited, though with different K(I); one mechanism prevails on the other depending on the turnover conditions and availability of substrates, cytochrome c and O(2). SH-SY5Y neuroblastoma cells or lymphoid cells, cultured under standard O(2) tension, proved to follow the mechanism leading to degradation of NO to nitrite. Formation of CcOX-NO occurred upon rising the electron flux level at this site, artificially or in the presence of higher amounts of endogenous reduced cytochrome c. Taken together, the observations suggest that the expression level of mitochondrial cytochrome c may be crucial to determine the respiratory chain NO inhibition pathway prevailing in vivo under nitrosative stress conditions. The putative patho-physiological relevance of the interaction between NO and the respiratory complexes is addressed.

The characterization of Thermotoga maritima ferritin reveals an unusual subunit dissociation behavior and efficient DNA protection from iron-mediated oxidative stress.

Ferritin from the hyperthermophilic anaerobe Thermotoga maritima, a bacterium of ancient phylogenetic origin, is structurally similar to known bacterial and eukaryotic ferritins: 24 identical subunits assemble into a shell having octahedral symmetry and a Mr of about 460 kDa. T. maritima ferritin (TmFtn), purified to homogeneity as a recombinant protein, contains approximately 2-3 iron atoms and can incorporate efficiently up to 3,500 atoms in the form of a ferric oxy-hydroxide mineral at 80°C, the optimal growth temperature of the bacterium. The 24-mer unexpectedly dissociates reversibly into dimers at low ionic strengths. In turn, dimers re-associate into the native 24-mer assembly at high protein concentrations and upon incorporation of iron micelles containing at least 500 Fe(III). TmFtn uses O(2) as efficient iron oxidant. The reaction stoichiometry is 3-4 O(2):Fe(II) as in all bacterial ferritins. Accordingly no H(2)O(2) is released into solution, a feature reflected in the in vitro ability of TmFtn to reduce significantly iron-mediated oxidative damage to DNA at 80°C. A similar TmFtn-mediated ROS detoxifying role likely occurs in the bacterium which lacks the SOD/catalase defense systems of the aerobic world.

Nitric oxide and mitochondrial complex IV.

Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl- [a3(2+) -NO] or a nitrite- [a3(3+) -NO2-] derivative of the enzyme. In the presence of O2, the nitrosyl adduct recovers activity slowly, following NO displacement at k' approximately equal to 0.01 s(-1) (37 degrees C); the recovery of the nitrite adduct is much faster. Relevant to pathophysiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one.