Publications by authors named "Ryan J Mailloux"

67 Publications

An update on methods and approaches for interrogating mitochondrial reactive oxygen species production.

Authors:
Ryan J Mailloux

Redox Biol 2021 09 16;45:102044. Epub 2021 Jun 16.

The School of Human Nutrition, Faculty of Agricultural and Environmental Sciences, McGill University, Sainte-Anne-de-Bellevue, Canada. Electronic address:

The chief ROS formed by mitochondria are superoxide (O) and hydrogen peroxide (HO). Superoxide is converted rapidly to HO and therefore the latter is the chief ROS emitted by mitochondria into the cell. Once considered an unavoidable by-product of aerobic respiration, HO is now regarded as a central mitokine used in mitochondrial redox signaling. However, it has been postulated that O can also serve as a signal in mammalian cells. Progress in understanding the role of mitochondrial HO in signaling is due to significant advances in the development of methods and technologies for its detection. Unfortunately, the development of techniques to selectively measure basal O changes has been met with more significant hurdles due to its short half-life and the lack of specific probes. The development of sensitive techniques for the selective and real time measure of O and HO has come on two fronts: development of genetically encoded fluorescent proteins and small molecule reporters. In 2015, I published a detailed comprehensive review on the state of knowledge for mitochondrial ROS production and how it is controlled, which included an in-depth discussion of the up-to-date methods utilized for the detection of both superoxide (O) and HO. In the article, I presented the challenges associated with utilizing these probes and their significance in advancing our collective understanding of ROS signaling. Since then, many other authors in the field of Redox Biology have published articles on the challenges and developments detecting O and HO in various organisms [1-3]. There has been significant advances in this state of knowledge, including the development of novel genetically encoded fluorescent HO probes, several O sensors, and the establishment of a toolkit of inhibitors and substrates for the interrogation of mitochondrial HO production and the antioxidant defenses utilized to maintain the cellular HO steady-state. Here, I provide an update on these methods and their implementation in furthering our understanding of how mitochondria serve as cell ROS stabilizing devices for HO signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2021.102044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8220584PMC
September 2021

The glutathionylation agent disulfiram augments superoxide/hydrogen peroxide production when liver mitochondria are oxidizing ubiquinone pool-linked and branched chain amino acid substrates.

Free Radic Biol Med 2021 May 27;172:1-8. Epub 2021 May 27.

The School of Human Nutrition, Faculty of Agricultural and Environmental Sciences, McGill University, Ste.-Anne-de-Bellevue, Quebec, Canada. Electronic address:

Our group has previously observed that protein S-glutathionylation serves as an integral feedback inhibitor for the production of superoxide (O)/hydrogen peroxide (HO) by α-ketoglutarate dehydrogenase (KGDH), pyruvate dehydrogenase (PDH), and complex I in muscle and liver mitochondria, respectively. In the present study, we hypothesized that glutathionylation would fulfill a similar role for the O/HO sources sn-glycerol-3-phosphate dehydrogenase (G3PDH), proline dehydrogenase (PRODH), and branched chain keto acid dehydrogenase (BCKDH). Surprisingly, we found that inducing glutathionylation with disulfiram increased the production of O/HO by mitochondria oxidizing glycerol-3-phosphate (G3P), proline (Pro), or α-keto-β-methylvaleric acid (KMV). Treatment of mitochondria oxidizing G3P or Pro with rotenone or myxothiazol increased the rate of ROS production after incubating in 1000 nM disulfiram. Incubating mitochondria treated with disulfiram in both rotenone and myxothiazol prevented this increase in O/HO production. In addition, when adminstered together, ROS production decreased below control levels. Disulfiram-treated mitochondria displayed higher rates of ROS production when oxidizing succinate, which was inhibited by rotenone, myxothiazol, and malonate, respectively. Disulfiram also increased ROS production by mitocondria oxidizing KMV. Treatment of mitochondria oxidizing KMV with disulfiram and rotenone or myxothiazol did not alter the rate O/HO production further when compared to mitochondria treated with disulfiram only. Analysis of BCKDH activity following disulfiram treatment revealed that glutathionylation does not inhibit the enzyme complex, indicating this α-keto acid dehydrogenase is not a target for glutathione modification. However, treatment of mitochondria with rotenone and myxothiazol without disulfiram also augmented ROS production. Overall, we were able to demonstrate for the first time that glutathionylation augments ROS production by the respiratory chain during forward electron transfer (FET) and reverse electron transfer (RET) from the UQ pool. Additionally, we were able to show that BCKDH is not a target for glutathione modification and that glutathionylation can also increase ROS production in mitochondria oxidizing branched chain amino acids following the modification of enzymes upstream of BCKDH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2021.05.030DOI Listing
May 2021

An investigation into the impact of deleting one copy of the gene on diet-induced weight gain and the bioenergetics of muscle mitochondria in female mice fed a high fat diet.

Redox Rep 2020 Dec;25(1):87-94

Department of Biochemistry, Faculty of Science, Memorial University of Newfoundland, St. John's, Canada.

Our group recently documented that male mice containing a deletion for one copy of the gene were completely protected from developing diet-induced obesity (DIO). Here, we conducted a similar investigation but with female littermates. In comparison to our recent publication using male mice, exposure of WT and GRX2+/- female mice to a HFD from 3-to-10 weeks of age did not induce any changes in body mass, circulating blood glucose, food intake, hepatic glycogen levels, or abdominal fat pad mass. Examination of the bioenergetics of muscle mitochondria revealed no changes in the rate of superoxide ( )/hydrogen peroxide (HO) or O consumption under different states of respiration or alterations in lipid peroxidation adduct levels regardless of mouse strain or diet. Additionally, we measured the bioenergetics of mitochondria isolated from liver tissue and found that partial loss of GRX2 augmented respiration but did not alter ROS production. Overall, our findings demonstrate there are sex differences in the protection of female GRX2+/- mice from DIO, fat accretion, intrahepatic lipid accumulation, and the bioenergetics of mitochondria from muscle and liver tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/13510002.2020.1826750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580715PMC
December 2020

The GLP-1 Receptor Agonist Liraglutide Increases Myocardial Glucose Oxidation Rates via Indirect Mechanisms and Mitigates Experimental Diabetic Cardiomyopathy.

Can J Cardiol 2021 01 8;37(1):140-150. Epub 2020 Mar 8.

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada; Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada; Cardiovascular Research Centre, University of Alberta, Edmonton, Alberta, Canada. Electronic address:

Background: Type 2 diabetes (T2D) increases risk for cardiovascular disease. Of interest, liraglutide, a therapy for T2D that activates the glucagon-like peptide-1 receptor to augment insulin secretion, reduces cardiovascular-related death in people with T2D, though it remains unknown how liraglutide produces these actions. Notably, the glucagon-like peptide-1 receptor is not expressed in ventricular cardiac myocytes, making it likely that ventricular myocardium-independent actions are involved. We hypothesized that augmented insulin secretion may explain how liraglutide indirectly mediates cardioprotection, which thereby increases myocardial glucose oxidation.

Methods: C57BL/6J male mice were fed either a low-fat diet (lean) or were subjected to experimental T2D and treated with either saline or liraglutide 3× over a 24-hour period. Mice were subsequently euthanized and had their hearts perfused in the working mode to assess energy metabolism. A separate cohort of mice with T2D were treated with either vehicle control or liraglutide for 2 weeks for the assessment of cardiac function via ultrasound echocardiography.

Results: Treatment of lean mice with liraglutide increased myocardial glucose oxidation without affecting glycolysis. Conversely, direct treatment of the isolated working heart with liraglutide had no effect on glucose oxidation. These findings were recapitulated in mice with T2D and associated with increased circulating insulin levels. Furthermore, liraglutide treatment alleviated diastolic dysfunction in mice with T2D, which was associated with enhanced pyruvate dehydrogenase activity, the rate-limiting enzyme of glucose oxidation.

Conclusions: Our data demonstrate that liraglutide augments myocardial glucose oxidation via indirect mechanisms, which may contribute to how liraglutide improves cardiovascular outcomes in people with T2D.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cjca.2020.02.098DOI Listing
January 2021

An Update on Mitochondrial Reactive Oxygen Species Production.

Authors:
Ryan J Mailloux

Antioxidants (Basel) 2020 Jun 2;9(6). Epub 2020 Jun 2.

The School of Human Nutrition, Faculty of Agricultural and Environmental Sciences, McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada.

Mitochondria are quantifiably the most important sources of superoxide (O) and hydrogen peroxide (HO) in mammalian cells. The overproduction of these molecules has been studied mostly in the contexts of the pathogenesis of human diseases and aging. However, controlled bursts in mitochondrial ROS production, most notably HO, also plays a vital role in the transmission of cellular information. Striking a balance between utilizing HO in second messaging whilst avoiding its deleterious effects requires the use of sophisticated feedback control and HO degrading mechanisms. Mitochondria are enriched with HO degrading enzymes to desensitize redox signals. These organelles also use a series of negative feedback loops, such as proton leaks or protein -glutathionylation, to inhibit HO production. Understanding how mitochondria produce ROS is also important for comprehending how these organelles use HO in eustress signaling. Indeed, twelve different enzymes associated with nutrient metabolism and oxidative phosphorylation (OXPHOS) can serve as important ROS sources. This includes several flavoproteins and respiratory complexes I-III. Progress in understanding how mitochondria generate HO for signaling must also account for critical physiological factors that strongly influence ROS production, such as sex differences and genetic variances in genes encoding antioxidants and proteins involved in mitochondrial bioenergetics. In the present review, I provide an updated view on how mitochondria budget cellular HO production. These discussions will focus on the potential addition of two acyl-CoA dehydrogenases to the list of ROS generators and the impact of important phenotypic and physiological factors such as tissue type, mouse strain, and sex on production by these individual sites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/antiox9060472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346187PMC
June 2020

Protein S-glutathionylation reactions as a global inhibitor of cell metabolism for the desensitization of hydrogen peroxide signals.

Authors:
Ryan J Mailloux

Redox Biol 2020 05 7;32:101472. Epub 2020 Mar 7.

School of Human Nutrition, McGill University, Ste. Anne de Bellevue, Quebec, Canada. Electronic address:

The pathogenesis of many human diseases has been attributed to the over production of reactive oxygen species (ROS), particularly superoxide (O) and hydrogen peroxide (HO), by-products of metabolism that are generated by the premature reaction of electrons with molecular oxygen (O) before they reach complex IV of the respiratory chain. To date, there are 32 known ROS generators in mammalian cells, 16 of which reside inside mitochondria. Importantly, although these ROS are deleterious at high levels, controlled and temporary bursts in HO production is beneficial to mammalian cells. Mammalian cells use sophisticated systems to take advantage of the second messaging properties of HO. This includes controlling its availability using antioxidant systems and negative feedback loops that inhibit the genesis of ROS at sites of production. At its core, ROS production depends on fuel metabolism. Therefore, desensitizing HO signals would also require the temporary inhibition of fuel combustion and fluxes through metabolic pathways that promote ROS production. Additionally, this would also demand the diversion of fuels and nutrients into pathways that generate NADPH and other molecules required to maintain cellular redox buffering capacity. Therefore, fuel selection and metabolic flux plays an integral role in dictating the strength and duration of cellular redox signals. In the present review I provide an updated view on the function of protein S-glutathionylation, a ubiquitous redox sensitive modification involving the formation of a disulfide between the small molecular antioxidant glutathione and a cysteine residue, in the regulation of cellular metabolism on a global scale. To date, these concepts have mostly been reviewed at the level of mitochondrial bioenergetics in the contexts of health and disease. Careful examination of the literature revealed that glutathionylation is a temporary inhibitor of most metabolic pathways including glycolysis, the Krebs cycle, oxidative phosphorylation, amino acid metabolism, and fatty acid combustion, resulting in the diversion of fuels towards NADPH-producing pathways and the inhibition of ROS production. Armed with this information, I propose that protein S-glutathionylation reactions desensitize HO signals emanating from catabolic pathways using a three-pronged regulatory mechanism; 1) inhibition of metabolic flux through pathways that promote ROS production, 2) diversion of metabolites towards pathways that support antioxidant defenses, and 3) direct inhibition of ROS-generating enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2020.101472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076094PMC
May 2020

C57BL/6J mice upregulate catalase to maintain the hydrogen peroxide buffering capacity of liver mitochondria.

Free Radic Biol Med 2020 01 19;146:59-69. Epub 2019 Oct 19.

Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada; The School of Human Nutrition, Faculty of Agricultural and Environmental Sciences, McGill University, Ste.-Anne-de-Bellevue, Quebec, Canada. Electronic address:

Here, we demonstrate that the upregulation of catalase is required to compensate for the loss of nicotinamide nucleotide transhydrogenase (NNT) to maintain hydrogen peroxide (HO) steady-state levels in C57BL/6J liver mitochondria. Our investigations using the closely related mouse strains C57BL/6NJ (6NJ; +NNT) and C57BL/6J (6J; -NNT) revealed that NNT is required for the provision of NADPH and that the upregulation of isocitrate dehydrogenase-2 (IDH2) activity is not enough to compensate for the absence of NNT, which is consistent with previous observations. Intriguingly, despite the absence of NNT, 6J mitochondria had rates of HO production (58.56 ± 3.79 pmol mg min) that were similar to samples collected from 6NJ mice (72.75 ± 14.26 pmol mg min) when pyruvate served as the substrate. However, 6NJ mitochondria energized with succinate produced significantly less HO (59.95 ± 2.13 pmol mg min) when compared to samples from 6J mice (116.39 ± 20.74 pmol mg min), an effect that was attributed to the presence of NNT. Further investigations into the HO eliminating capacities of these mitochondria led to the novel observation that 6J mitochondria compensate for the loss of NNT by upregulating catalase. Indeed, 6NJ and 6J mitochondria energized with pyruvate or succinate displayed similar rates for HO elimination, quenching ~84% and ~86% of the HO, respectively, in the surrounding medium within 30 s. However, inclusion of palmitoyl-CoA, an NNT inhibitor, significantly limited HO degradation by 6NJ mitochondria only (~55% of HO eliminated in 30 s). Liver mitochondria from 6J mice treated with palmitoyl-CoA still cleared ~80% of the HO from the surrounding environment. Inhibition of catalase with triazole compromised the capacity of 6J mitochondria to maintain HO steady-state levels. By contrast, disabling NADPH-dependent antioxidant systems had a limited effect on the HO clearing capacity of 6J mitochondria. Liver mitochondria collected from 6NJ mice, on the other hand, were more reliant on the GSH and TRX systems to clear exogenously added HO. However, catalase still played an integral in eliminating HO in 6NJ liver mitochondria. Immunoblot analyses demonstrated that catalase protein levels were ~7.7-fold higher in 6J mitochondria. Collectively, our findings demonstrate for the first time that 6J liver mitochondria compensate for the loss of NNT by increasing catalase levels for the maintenance of HO steady-state levels. In general, our observations reveal that catalase is an integral arm of the antioxidant response in liver mitochondria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2019.10.409DOI Listing
January 2020

Lactate dehydrogenase supports lactate oxidation in mitochondria isolated from different mouse tissues.

Redox Biol 2020 01 5;28:101339. Epub 2019 Oct 5.

Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada; The School of Human Nutrition, Faculty of Agricultural and Environmental Sciences, McGill University, Ste.-Anne-de-Bellevue, Quebec, Canada. Electronic address:

Research over the past seventy years has established that mitochondrial-l-lactate dehydrogenase (m-L-LDH) is vital for mitochondrial bioenergetics. However, in recent report, Fulghum et al. concluded that lactate is a poor fuel for mitochondrial respiration [1]. In the present study, we have followed up on these findings and conducted an independent investigation to determine if lactate can support mitochondrial bioenergetics. We demonstrate herein that lactate can fuel the bioenergetics of heart, muscle, and liver mitochondria. Lactate was just as effective as pyruvate at stimulating mitochondrial coupling efficiency. Inclusion of LDH (sodium oxamate or GSK 2837808A) and pyruvate dehydrogenase (PDH; CPI-613) inhibitors abolished respiration in mitochondria energized with lactate. Lactate also fueled mitochondrial ROS generation and was just as effective as pyruvate at stimulating HO production. Additionally, lactate-induced ROS production was inhibited by both LDH and PDH inhibitors. Enzyme activity measurements conducted on permeabilized mitochondria revealed that LDH is localized in mitochondria. In aggregate, we can conclude that mitochondrial LDH fuels bioenergetics in several tissues by oxidizing lactate.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2019.101339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812140PMC
January 2020

Cysteine Switches and the Regulation of Mitochondrial Bioenergetics and ROS Production.

Authors:
Ryan J Mailloux

Adv Exp Med Biol 2019 ;1158:197-216

Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL, Canada.

Mitochondria are dynamic organelles that perform a number of interconnected tasks that are elegantly intertwined with the regulation of cell functions. This includes the provision of ATP, reactive oxygen species (ROS), and building blocks for the biosynthesis of macromolecules while also serving as signaling platforms for the cell. Although the functions executed by mitochondria are complex, at its core these roles are, to a certain degree, fulfilled by electron transfer reactions and the establishment of a protonmotive force (PMF). Indeed, mitochondria are energy conserving organelles that extract electrons from nutrients to establish a PMF, which is then used to drive ATP and NADPH production, solute import, and many other functions including the propagation of cell signals. These same electrons extracted from nutrients are also used to produce ROS, pro-oxidants that can have potentially damaging effects at high levels, but also serve as secondary messengers at low amounts. Mitochondria are also enriched with antioxidant defenses, which are required to buffer cellular ROS. These same redox buffering networks also fulfill another important role; regulation of proteins through the reversible oxidation of cysteine switches. The modification of cysteine switches with the antioxidant glutathione, a process called protein S-glutathionylation, has been found to play an integral role in controlling various mitochondrial functions. In addition, recent findings have demonstrated that disrupting mitochondrial protein S-glutathionylation reactions can have some dire pathological consequences. Accordingly, this chapter focuses on the role of mitochondrial cysteine switches in the modulation of different physiological functions and how defects in these pathways contribute to the development of disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-981-13-8367-0_11DOI Listing
September 2019

Sex-dependent Differences in the Bioenergetics of Liver and Muscle Mitochondria from Mice Containing a Deletion for .

Antioxidants (Basel) 2019 Jul 26;8(8). Epub 2019 Jul 26.

Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL A1B 3X7, Canada.

Our group recently published a study demonstrating that deleting the gene encoding the matrix thiol oxidoreductase, glutaredoxin-2 (GRX2), alters the bioenergetics of mitochondria isolated from male C57BL/6N mice. Here, we conducted a similar study, examining HO production and respiration in mitochondria isolated from female mice heterozygous (GRX2+/-) or homozygous (GRX2-/-) for glutaredoxin-2. First, we observed that deleting the gene does not alter the rate of HO production in liver and muscle mitochondria oxidizing pyruvate, α-ketoglutarate, or succinate. Examination of the rates of HO release from liver mitochondria isolated from male and female mice revealed that (1) sex has an impact on the rate of ROS production by liver and muscle mitochondria and (2) loss of GRX2 only altered ROS release in mitochondria collected from male mice. Assessment of the bioenergetics of these mitochondria revealed that loss of GRX2 increased proton leak-dependent and phosphorylating respiration in liver mitochondria isolated from female mice but did not alter rates of respiration in liver mitochondria from male mice. Furthermore, we found that deleting the gene did not alter rates of respiration in muscle mitochondria collected from female mice. This contrasts with male mice where loss of GRX2 substantially augmented proton leaks and ADP-stimulated respiration. Our findings indicate that some fundamental sexual dimorphisms exist between GRX2-deficient male and female rodents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/antiox8080245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6720827PMC
July 2019

Deletion of the Glutaredoxin-2 Gene Protects Mice from Diet-Induced Weight Gain, Which Correlates with Increased Mitochondrial Respiration and Proton Leaks in Skeletal Muscle.

Antioxid Redox Signal 2019 12 14;31(17):1272-1288. Epub 2019 Aug 14.

Department of Biochemistry, Faculty of Science, Memorial University of Newfoundland, St. John's, Canada.

The aim of this study was to determine whether deleting the gene encoding glutaredoxin-2 (GRX2) could protect mice from diet-induced weight gain. Subjecting wild-type littermates to a high fat diet (HFD) induced a significant increase in overall body mass, white adipose tissue hypertrophy, lipid droplet accumulation in hepatocytes, and higher circulating insulin and triglyceride levels. In contrast, GRX2 heterozygotes (GRX2) fed an HFD had a body mass, white adipose tissue weight, and hepatic and circulating lipid and insulin levels similar to littermates fed a control diet. Examination of the bioenergetics of muscle mitochondria revealed that this protective effect was associated with an increase in respiration and proton leaks. Muscle mitochondria from GRX2 mice had a ∼2- to 3-fold increase in state 3 (phosphorylating) respiration when pyruvate/malate or succinate served as substrates and a ∼4-fold increase when palmitoyl-carnitine was being oxidized. Proton leaks were ∼2- to 3-fold higher in GRX2 muscle mitochondria. Treatment of mitochondria with either guanosine diphosphate, genipin, or octanoyl-carnitine revealed that the higher rate of O consumption under state 4 conditions was associated with increased leaks through uncoupling protein-3 and adenine nucleotide translocase. GRX2 mitochondria also had better protection from oxidative distress. For the first time, we demonstrate that deleting the gene can protect from diet-induced weight gain and the development of obesity-related disorders. Deleting the gene protects mice from diet-induced weight gain. This effect was related to an increase in muscle fuel combustion, mitochondrial respiration, proton leaks, and reactive oxygen species handling. 31, 1272-1288.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ars.2018.7715DOI Listing
December 2019

Estimation of the hydrogen peroxide producing capacities of liver and cardiac mitochondria isolated from C57BL/6N and C57BL/6J mice.

Free Radic Biol Med 2019 05 19;135:15-27. Epub 2019 Feb 19.

Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada. Electronic address:

Here, we examined the hydrogen peroxide (HO) producing capacities of pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), proline dehydrogenase (PRODH), glycerol-3-phosphate dehydrogenase (G3PDH), succinate dehydrogenase (SDH; complex II), and branched-chain keto acid dehydrogenase (BCKDH), in cardiac and liver mitochondria isolated from C57BL/6N (6N) and C57BL/6J (6J) mice. Various inhibitor combinations were used to suppress ROS production by complexes I, II, and III and estimate the native rates of HO production for these enzymes. Overall, liver mitochondria from 6N mice produced ∼2-fold more ROS than samples enriched from 6J mice. This was attributed, in part, to the higher levels of glutathione peroxidase-1 (GPX1) and catalase (CAT) in 6J mitochondria. Intriguingly, PDH, KGDH, and SDH comprised up to ∼95% of the ROS generating capacity of permeabilized 6N liver mitochondria, with PRODH, G3PDH, and BCKDH making minor contributions. By contrast, BCKDH accounted for ∼34% of the production in permeabilized 6J mitochondria with KGDH and PRODH accounting for ∼23% and ∼19%. G3PDH produced high amounts of ROS, accounting for ∼52% and ∼39% of the total HO generating capacity in 6N and 6J heart mitochondria. PRODH was also an important ROS source in 6J mitochondria, accounting for ∼43% of the total HO formed. In addition, 6J cardiac mitochondria produced significantly more ROS than 6N mitochondria. Taken together, our findings demonstrate that these other generators can also serve as important sources of HO. Additionally, we found that mouse strain influences the rate of production from the individual sites that were studied.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2019.02.012DOI Listing
May 2019

Protein S-glutathionylation: The linchpin for the transmission of regulatory information on redox buffering capacity in mitochondria.

Chem Biol Interact 2019 Feb 8;299:151-162. Epub 2018 Dec 8.

Department of Biochemistry, Faculty of Science, Memorial University of Newfoundland, St. John's, NL, Canada. Electronic address:

Protein S-glutathionylation reactions are a ubiquitous oxidative modification required to control protein function in response to changes in redox buffering capacity. These reactions are rapid and reversible and are, for the most part, enzymatically mediated by glutaredoxins (GRX) and glutathione S-transferases (GST). Protein S-glutathionylation has been found to control a range of cell functions in response to different physiological cues. Although these reactions occur throughout the cell, mitochondrial proteins seem to be highly susceptible to reversible S-glutathionylation, a feature attributed to the unique physical properties of this organelle. Indeed, mitochondria contain a number of S-glutathionylation targets which includes proteins involved in energy metabolism, solute transport, reactive oxygen species (ROS) production, proton leaks, apoptosis, antioxidant defense, and mitochondrial fission and fusion. Moreover, it has been found that conjugation and removal of glutathione from proteins in mitochondria fulfills a number of important physiological roles and defects in these reactions can have some dire pathological consequences. Here, we provide an updated overview on mitochondrial protein S-glutathionylation reactions and their importance in cell functions and physiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbi.2018.12.003DOI Listing
February 2019

Mitochondrial Antioxidants and the Maintenance of Cellular Hydrogen Peroxide Levels.

Authors:
Ryan J Mailloux

Oxid Med Cell Longev 2018 2;2018:7857251. Epub 2018 Jul 2.

Department of Biochemistry, Memorial University of Newfoundland, St. John's, NL, Canada.

For over 40 years, mitochondrial reactive oxygen species (ROS) production and balance has been studied in the context of oxidative distress and tissue damage. However, research over the past decade has demonstrated that the mitochondria have a more complicated relationship with ROS. Superoxide (O) and hydrogen peroxide (HO) are the proximal ROS formed by the mitochondria, and the latter molecule is used as a secondary messenger to coordinate oxidative metabolism with changes in cell physiology. Like any other secondary messenger, HO levels need to be regulated through its production and degradation and the mitochondria are enriched with the antioxidant defenses required to degrade ROS formed by nutrient oxidation and respiration. Recent work has also demonstrated that these antioxidant systems also carry the capacity to clear HO formed outside of mitochondria. These observations led to the development of the postulate that the mitochondria serve as "ROS stabilizing devices" that buffer cellular HO levels. Here, I provide an updated view on mitochondrial ROS homeostasis and discuss the "ROS stabilizing" function of the mitochondria in mammalian cells. This will be followed by a hypothetical discussion on the potential function of the mitochondria and proton motive force in degrading cellular HO signals emanating from cytosolic enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2018/7857251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051038PMC
October 2018

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases.

J Vis Exp 2018 02 24(132). Epub 2018 Feb 24.

Department of Biochemistry, Memorial University of Newfoundland.

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems and side reactions that also form O2/H2O2. Use of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/56975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931358PMC
February 2018

Partial loss of complex I due to NDUFS4 deficiency augments myocardial reperfusion damage by increasing mitochondrial superoxide/hydrogen peroxide production.

Biochem Biophys Res Commun 2018 03 1;498(1):214-220. Epub 2018 Mar 1.

Department of Biochemistry, Faculty of Science, Memorial University of Newfoundland, St. John's, Newfoundland and Labrador, Canada. Electronic address:

Recent work has found that complex I is the sole source of reactive oxygen species (ROS) during myocardial ischemia-reperfusion (IR) injury. However, it has also been reported that heart mitochondria can also generate ROS from other sources in the respiratory chain and Krebs cycle. This study examined the impact of partial complex I deficiency due to selective loss of the Ndufs4 gene on IR injury to heart tissue. Mice heterozygous for NDUFS4 (NDUFS4+/-) did not display any significant changes in overall body or organ weight when compared to wild-type (WT) littermates. There were no changes in superoxide (O)/hydrogen peroxide (HO) release from cardiac or liver mitochondria isolated from NDUFS4 ± mice. Using selective ROS release inhibitors, we found that complex III is a major source of ROS in WT and NDUFS4 ± cardiac mitochondria respiring under state 4 conditions. Subjecting hearts from NDUFS4 ± mice to reperfusion injury revealed that the partial loss of complex I decreases contractile recovery and increases myocardial infarct size. These results correlated with a significant increase in O/HO release rates in mitochondria isolated from NDUFS4 ± hearts subjected to an IR challenge. Taken together, these results demonstrate that the partial absence of complex I sensitizes the myocardium towards IR injury and that the main source of ROS following reperfusion is complex III.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2018.02.208DOI Listing
March 2018

Protein S-glutathionylation lowers superoxide/hydrogen peroxide release from skeletal muscle mitochondria through modification of complex I and inhibition of pyruvate uptake.

PLoS One 2018 14;13(2):e0192801. Epub 2018 Feb 14.

Memorial University of Newfoundland, Department of Biochemistry, St. John's, Newfoundland, Canada.

Protein S-glutathionylation is a reversible redox modification that regulates mitochondrial metabolism and reactive oxygen species (ROS) production in liver and cardiac tissue. However, whether or not it controls ROS release from skeletal muscle mitochondria has not been explored. In the present study, we examined if chemically-induced protein S-glutathionylation could alter superoxide (O2●-)/hydrogen peroxide (H2O2) release from isolated muscle mitochondria. Disulfiram, a powerful chemical S-glutathionylation catalyst, was used to S-glutathionylate mitochondrial proteins and ascertain if it can alter ROS production. It was found that O2●-/H2O2 release rates from permeabilized muscle mitochondria decreased with increasing doses of disulfiram (100-500 μM). This effect was highest in mitochondria oxidizing succinate or palmitoyl-carnitine, where a ~80-90% decrease in the rate of ROS release was observed. Similar effects were detected in intact mitochondria respiring under state 4 conditions. Incubation of disulfiram-treated mitochondria with DTT (2 mM) restored ROS release confirming that these effects were associated with protein S-glutathionylation. Disulfiram treatment also inhibited phosphorylating and proton leak-dependent respiration. Radiolabelled substrate uptake experiments demonstrated that disulfiram inhibited pyruvate import but had no effect on carnitine uptake. Immunoblot analysis of complex I revealed that it contained several protein S-glutathionylation targets including NDUSF1, a subunit required for NADH oxidation. Taken together, these results demonstrate that O2●-/H2O2 release from muscle mitochondria can be altered by protein S-glutathionylation. We attribute these changes to the protein S-glutathionylation complex I and inhibition of mitochondrial pyruvate carrier.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192801PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5812644PMC
April 2018

Characterization of the impact of glutaredoxin-2 (GRX2) deficiency on superoxide/hydrogen peroxide release from cardiac and liver mitochondria.

Redox Biol 2018 05 14;15:216-227. Epub 2017 Dec 14.

Memorial University of Newfoundland, Department of Biochemistry, St. John's, Newfoundland, Canada. Electronic address:

Mitochondria are critical sources of hydrogen peroxide (HO), an important secondary messenger in mammalian cells. Recent work has shown that O/HO emission from individual sites of production in mitochondria is regulated by protein S-glutathionylation. Here, we conducted the first examination of O/HO release rates from cardiac and liver mitochondria isolated from mice deficient for glutaredoxin-2 (GRX2), a matrix-associated thiol oxidoreductase that facilitates the S-glutathionylation and deglutathionylation of proteins. Liver mitochondria isolated from mice heterozygous (GRX2+/-) and homozygous (GRX2-/-) for glutaredoxin-2 displayed a significant decrease in O/HO release when oxidizing pyruvate or 2-oxoglutarate. The genetic deletion of the Grx2 gene was associated with increased protein expression of pyruvate dehydrogenase (PDH) but not 2-oxoglutarate dehydrogenase (OGDH). By contrast, O/HO production was augmented in cardiac mitochondria from GRX2+/- and GRX2-/- mice metabolizing pyruvate or 2-oxoglutarate which was associated with decreased PDH and OGDH protein levels. ROS production was augmented in liver and cardiac mitochondria metabolizing succinate. Inhibitor studies revealed that OGDH and Complex III served as high capacity ROS release sites in liver mitochondria. By contrast, Complex I and Complex III were found to be the chief O/HO emitters in cardiac mitochondria. These findings identify an essential role for GRX2 in regulating O/HO release from mitochondria in liver and cardiac tissue. Our results demonstrate that the GRX2-mediated regulation of O/HO release through the S-glutathionylation of mitochondrial proteins may play an integral role in controlling cellular ROS signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2017.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773472PMC
May 2018

Physiological levels of formate activate mitochondrial superoxide/hydrogen peroxide release from mouse liver mitochondria.

FEBS Lett 2017 08 15;591(16):2426-2438. Epub 2017 Aug 15.

Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.

Here, we found that formate, an essential one-carbon metabolite, activates superoxide (O2·-)/hydrogen peroxide (H O ) release from mitochondria. Sodium formate (30 μm) induces a significant increase in O2·-/H O production in liver mitochondria metabolizing pyruvate (50 μm). At concentrations deemed to be toxic, formate does not increase O2·-/H O production further. It was observed that the formate-mediated increase in O2·-/H O production is not associated with cytochrome c oxidase (COX) inhibition or changes in membrane potential and NAD(P)H levels. Sodium formate supplementation increases phosphorylating respiration without altering proton leaks. Finally, it was observed that the 2-oxoglutarate dehydrogenase (OGDH) inhibitors 3-methyl-2-oxovaleric acid (KMV) and CPI-613 inhibit the formate-induced increase in pyruvate-driven ROS production. The importance of these findings in one-carbon metabolism and physiology are discussed herein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12777DOI Listing
August 2017

Progress in understanding the molecular oxygen paradox - function of mitochondrial reactive oxygen species in cell signaling.

Biol Chem 2017 10;398(11):1209-1227

.

The molecular oxygen (O2) paradox was coined to describe its essential nature and toxicity. The latter characteristic of O2 is associated with the formation of reactive oxygen species (ROS), which can damage structures vital for cellular function. Mammals are equipped with antioxidant systems to fend off the potentially damaging effects of ROS. However, under certain circumstances antioxidant systems can become overwhelmed leading to oxidative stress and damage. Over the past few decades, it has become evident that ROS, specifically H2O2, are integral signaling molecules complicating the previous logos that oxyradicals were unfortunate by-products of oxygen metabolism that indiscriminately damage cell structures. To avoid its potential toxicity whilst taking advantage of its signaling properties, it is vital for mitochondria to control ROS production and degradation. H2O2 elimination pathways are well characterized in mitochondria. However, less is known about how H2O2 production is controlled. The present review examines the importance of mitochondrial H2O2 in controlling various cellular programs and emerging evidence for how production is regulated. Recently published studies showing how mitochondrial H2O2 can be used as a secondary messenger will be discussed in detail. This will be followed with a description of how mitochondria use S-glutathionylation to control H2O2 production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1515/hsz-2017-0160DOI Listing
October 2017

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria.

Biochim Biophys Acta Gen Subj 2017 Aug 12;1861(8):1960-1969. Epub 2017 May 12.

Memorial University of Newfoundland, Department of Biochemistry, St. John's, Newfoundland, Canada. Electronic address:

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (HO). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O/HO formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O/HO formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial HO. In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O/HO production by KGDHC, induced a ~86% and ~84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O/HO formation by ~45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O/HO production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial HO.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2017.05.010DOI Listing
August 2017

Protein S-glutathionylation alters superoxide/hydrogen peroxide emission from pyruvate dehydrogenase complex.

Free Radic Biol Med 2017 05 27;106:302-314. Epub 2017 Feb 27.

Department of Biochemistry, Memorial University of Newfoundland, 230 Elizabeth Ave, St. John's, Newfoundland, Canada A1B 3X9. Electronic address:

Pyruvate dehydrogenase (Pdh) is a vital source of reactive oxygen species (ROS) in several different tissues. Pdh has also been suggested to serve as a mitochondrial redox sensor. Here, we report that O/ HO emission from pyruvate dehydrogenase (Pdh) is altered by S-glutathionylation. Glutathione disulfide (GSSG) amplified O/ HO production by purified Pdh during reverse electron transfer (RET) from NADH. Thiol oxidoreductase glutaredoxin-2 (Grx2) reversed these effects confirming that Pdh is a target for S-glutathionylation. S-glutathionylation had the opposite effect during forward electron transfer (FET) from pyruvate to NAD lowering O/ HO production. Immunoblotting for protein glutathione mixed disulfides (PSSG) following diamide treatment confirmed that purified Pdh can be S-glutathionylated. Similar observations were made with mouse liver mitochondria. S-glutathionylation catalysts diamide and disulfiram significantly reduced pyruvate or 2-oxoglutarate driven O/ HO production in liver mitochondria, results that were confirmed using various Pdh, 2-oxoglutarate dehydrogenase (Ogdh), and respiratory chain inhibitors. Immunoprecipitation of Pdh and Ogdh confirmed that either protein can be S-glutathionylated by diamide and disulfiram. Collectively, our results demonstrate that the S -glutathionylation of Pdh alters the amount of ROS formed by the enzyme complex. We also confirmed that Ogdh is controlled in a similar manner. Taken together, our results indicate that the redox sensing and ROS forming properties of Pdh and Ogdh are linked to S-glutathionylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.046DOI Listing
May 2017

Application of Mitochondria-Targeted Pharmaceuticals for the Treatment of Heart Disease.

Authors:
Ryan J Mailloux

Curr Pharm Des 2016 ;22(31):4763-4779

Memorial University of Newfoundland, Department of Biochemistry, St. John's, Newfoundland, Canada.

Background: Mitochondria fulfill the massive energy demands of the human heart through oxidative phosphorylation (OXPHOS) which couples nutrient oxidation and the reduction of molecular oxygen (O2) to the phosphorylation of ADP. Reactive oxygen species (ROS) are also generated during OXPHOS which can be damaging at high levels but serve as secondary messengers when produced in a controlled manner.

Methods: Here, I review how disruption of control over mitochondrial ROS production can lead to the pathogenesis of a range of cardiovascular diseases (CVD) including decompensated left ventricular hypertrophy, alcoholic and diabetic hypertrophy, myocardial infarction (MI), ischemic-reperfusion injury (IR), and heart failure. In particular I focus on the function of protein S-glutathionylation (PGlu) reactions, a rapid and reversible redox signaling mechanism that involves the conjugation and removal of glutathione from cysteine switches, in the modulation of ROS production in myocardial mitochondria and how these reactions become deregulated in heart disease. I also discuss the use of mitochondria penetrating antioxidants in the treatment of heart disease.

Results: I propose that heart disease related to deregulated PGlu reactions can be treated with a novel and hypothetical class of mitochondria penetrating reduced glutathione (GSH) molecules called MitoGSH. This synthetic form of GSH can be tagged with either SS peptides or triphenylphosphonium ions to ensure accumulation in mitochondria which could restore glutathione levels and preserve redox buffering networks.

Conclusion: Mitochondria penetrating antioxidants have been shown to be efficient at restoring mitochondrial antioxidant defense in CVD. However, CVD and various other disorders are associated with a depletion of GSH pools. Use of mitochondria-targeted GSH analogs could serve as a more efficient means of treating heart disease since it would allow for the direct restoration of GSH levels and preserve mitochondrial redox buffering and signaling capacity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1381612822666160629070914DOI Listing
November 2017

Choline and dimethylglycine produce superoxide/hydrogen peroxide from the electron transport chain in liver mitochondria.

FEBS Lett 2016 Dec 9;590(23):4318-4328. Epub 2016 Nov 9.

Department of Biochemistry, Memorial University of Newfoundland, St. John's, Canada.

Here, we report that choline and dimethylglycine can stimulate reactive oxygen species (ROS) production in liver mitochondria. Choline stimulated O ˙ /H O formation at a concentration of 5 μm. We also observed that Complex II and III inhibitors, atpenin A5 and myxothiazol, collectively induced a 95% decrease in O ˙ /H O production indicating both sites serve as the main sources of ROS during choline oxidation. Dimethylglycine, an intermediate of choline oxidation, was a more effective ROS generator. Rates of production were ~ 43% higher than choline-mediated O ˙ /H O production. The main site for dimethylglycine-mediated ROS production was via reverse electron transfer to Complex I. Our results demonstrate that metabolism of essential metabolites involved in methionine and folic acid biosynthesis can stimulate mitochondrial ROS production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1873-3468.12461DOI Listing
December 2016

Bisphenol A exposure alters release of immune and developmental modulators and expression of estrogen receptors in human fetal lung fibroblasts.

J Environ Sci (China) 2016 Oct 29;48:11-23. Epub 2016 Apr 29.

Regulatory Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, HPFB, Health Canada, Ottawa, Ontario, Canada. Electronic address:

Bisphenol A (BPA) has been shown to exert biological effects through estrogen receptor (ER)-dependent and ER-independent mechanisms. Recent studies suggest that prenatal exposure to BPA may increase the risk of childhood asthma. To investigate the underlying mechanisms in the actions of BPA, human fetal lung fibroblasts (hFLFs) were exposed to varying doses of BPA in culture for 24hr. Effects of BPA on localization and uptake of BPA, cell viability, release of immune and developmental modulators, cellular localization and expression of ERα, ERβ and G-protein coupled estrogen receptor 30 (GPR30), and effects of ERs antagonists on BPA-induced changes in endothelin-1 (ET-1) release were examined. BPA at 0.01-100μmol/L caused no changes in cell viability after 24hr of exposure. hFLFs expresses all three ERs. BPA had no effects on either cellular distribution or protein expression of ERα, however, at 100μmol/L (or 23μmol/L intracellular BPA) increased ERβ protein levels in the cytoplasmic fractions and GPR30 protein levels in the nuclear fractions. These paralleled with increased release of growth differentiation factor-15, decreased phosphorylation of nuclear factor kappa B p65 at serine 536, and decreased release of ET-1, interleukin-6, and interferon gamma-induced protein 10. ERs antagonists had no effects on BPA-induced decrease in ET-1 release. These data suggest that BPA at 100μmol/L altered the release of immune and developmental modulators in hFLFs, which may negatively influence fetal lung development, maturation, and susceptibility to environmental stressors, although the role of BPA in childhood asthma remains to be confirmed in in vivo studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jes.2016.02.013DOI Listing
October 2016

2-Oxoglutarate dehydrogenase is a more significant source of O2(·-)/H2O2 than pyruvate dehydrogenase in cardiac and liver tissue.

Free Radic Biol Med 2016 08 6;97:501-512. Epub 2016 Jul 6.

Department of Biochemistry, Memorial University of Newfoundland, 230 Elizabeth Ave, St. John's, Newfoundland, Canada A1B 3×9.

Pyruvate dehydrogenase (Pdh) and 2-oxoglutarate dehydrogenase (Ogdh) are vital for Krebs cycle metabolism and sources of reactive oxygen species (ROS). O2(·-)/H2O2 formation by Pdh and Ogdh from porcine heart were compared when operating under forward or reverse electron transfer conditions. Comparisons were also conducted with liver and cardiac mitochondria. During reverse electron transfer (RET) from NADH, purified Ogdh generated ~3-3.5× more O2(·-)/H2O2 in comparison to Pdh when metabolizing 0.5-10µM NADH. Under forward electron transfer (FET) conditions Ogdh generated ~2-4× more O2(·-)/H2O2 than Pdh. In both liver and cardiac mitochondria, Ogdh displayed significantly higher rates of ROS formation when compared to Pdh. Ogdh was also a significant source of ROS in liver mitochondria metabolizing 50µM and 500µM pyruvate or succinate. Finally, we also observed that DTT directly stimulated O2(·-)/H2O2 formation by purified Pdh and Ogdh and in cardiac or liver mitochondria in the absence of substrates and cofactors. Taken together, Ogdh is a more potent source of ROS than Pdh in liver and cardiac tissue. Ogdh is also an important ROS generator regardless of whether pyruvate or succinate serve as the sole source of carbon. Our observations provide insight into the ROS generating capacity of either complex in cardiac and liver tissue. The evidence presented herein also indicates DTT, a reductant that is routinely added to biological samples, should be avoided when assessing mitochondrial O2(·-)/H2O2 production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2016.06.014DOI Listing
August 2016

Methylmercury alters glutathione homeostasis by inhibiting glutaredoxin 1 and enhancing glutathione biosynthesis in cultured human astrocytoma cells.

Toxicol Lett 2016 Aug 11;256:1-10. Epub 2016 May 11.

University of Ottawa, Department of Biology, Center for Advanced Research in Environmental Genomics, Ottawa K1N 6N5, ON, Canada. Electronic address:

Methylmercury (MeHg) is a neurotoxin that binds strongly to thiol residues on protein and low molecular weight molecules like reduced glutathione (GSH). The mechanism of its effects on GSH homeostasis particularly at environmentally relevant low doses is not fully known. We hypothesized that exposure to MeHg would lead to a depletion of reduced glutathione (GSH) and an accumulation of glutathione disulfide (GSSG) leading to alterations in S-glutathionylation of proteins. Our results showed exposure to low concentrations of MeHg (1μM) did not significantly alter GSH levels but increased GSSG levels by ∼12-fold. This effect was associated with a significant increase in total cellular glutathione content and a decrease in GSH/GSSG. Immunoblot analyses revealed that proteins involved in glutathione synthesis were upregulated accounting for the increase in cellular glutathione. This was associated an increase in cellular Nrf2 protein levels which is required to induce the expression of antioxidant genes in response to cellular stress. Intriguingly, we noted that a key enzyme involved in reversing protein S-glutathionylation and maintaining glutathione homeostasis, glutaredoxin-1 (Grx1), was inhibited by ∼50%. MeHg treatment also increased the S-glutathionylation of a high molecular weight protein. This observation is consistent with the inhibition of Grx1 and elevated H2O2 production however; contrary to our original hypothesis we found few S-glutathionylated proteins in the astrocytoma cells. Collectively, MeHg affects multiple arms of glutathione homeostasis ranging from pool management to protein S-glutathionylation and Grx1 activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.toxlet.2016.05.013DOI Listing
August 2016

Induction of mitochondrial reactive oxygen species production by GSH mediated S-glutathionylation of 2-oxoglutarate dehydrogenase.

Redox Biol 2016 08 17;8:285-97. Epub 2016 Feb 17.

Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada.

2-Oxoglutarate dehydrogenase (Ogdh) is an important mitochondria redox sensor that can undergo S-glutathionylation following an increase in H2O2 levels. Although S-glutathionylation is required to protect Ogdh from irreversible oxidation while simultaneously modulating its activity it remains unknown if glutathione can also modulate reactive oxygen species (ROS) production by the complex. We report that reduced (GSH) and oxidized (GSSG) glutathione control O2(∙-)/H2O2 formation by Ogdh through protein S-glutathionylation reactions. GSSG (1mM) induced a modest decrease in Ogdh activity which was associated with a significant decrease in O2(∙-)/H2O2 formation. GSH had the opposite effect, amplifying O2(∙-)/H2O2 formation by Ogdh. Incubation of purified Ogdh in 2.5mM GSH led to significant increase in O2(∙-)/H2O2 formation which also lowered NADH production. Inclusion of enzymatically active glutaredoxin-2 (Grx2) in reaction mixtures reversed the GSH-mediated amplification of O2(∙-)/H2O2 formation. Similarly pre-incubation of permeabilized liver mitochondria from mouse depleted of GSH showed an approximately ~3.5-fold increase in Ogdh-mediated O2(∙-)/H2O2 production that was matched by a significant decrease in NADH formation which could be reversed by Grx2. Taken together, our results demonstrate GSH and GSSG modulate ROS production by Ogdh through S-glutathionylation of different subunits. This is also the first demonstration that GSH can work in the opposite direction in mitochondria-amplifying ROS formation instead of quenching it. We propose that this regulatory mechanism is required to modulate ROS emission from Ogdh in response to variations in glutathione redox buffering capacity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2016.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776629PMC
August 2016

Protein S-glutathionlyation links energy metabolism to redox signaling in mitochondria.

Redox Biol 2016 08 31;8:110-8. Epub 2015 Dec 31.

University of Manitoba, Department of Biological Sciences, Winnipeg, Manitoba, Canada; Department of Human Nutritional Sciences, Winnipeg, Manitoba, Canada; Centre on Aging, Winnipeg, Manitoba, Canada.

At its core mitochondrial function relies on redox reactions. Electrons stripped from nutrients are used to form NADH and NADPH, electron carriers that are similar in structure but support different functions. NADH supports ATP production but also generates reactive oxygen species (ROS), superoxide (O2(·-)) and hydrogen peroxide (H2O2). NADH-driven ROS production is counterbalanced by NADPH which maintains antioxidants in an active state. Mitochondria rely on a redox buffering network composed of reduced glutathione (GSH) and peroxiredoxins (Prx) to quench ROS generated by nutrient metabolism. As H2O2 is quenched, NADPH is expended to reactivate antioxidant networks and reset the redox environment. Thus, the mitochondrial redox environment is in a constant state of flux reflecting changes in nutrient and ROS metabolism. Changes in redox environment can modulate protein function through oxidation of protein cysteine thiols. Typically cysteine oxidation is considered to be mediated by H2O2 which oxidizes protein thiols (SH) forming sulfenic acid (SOH). However, problems begin to emerge when one critically evaluates the regulatory function of SOH. Indeed SOH formation is slow, non-specific, and once formed SOH reacts rapidly with a variety of molecules. By contrast, protein S-glutathionylation (PGlu) reactions involve the conjugation and removal of glutathione moieties from modifiable cysteine residues. PGlu reactions are driven by fluctuations in the availability of GSH and oxidized glutathione (GSSG) and thus should be exquisitely sensitive to changes ROS flux due to shifts in the glutathione pool in response to varying H2O2 availability. Here, we propose that energy metabolism-linked redox signals originating from mitochondria are mediated indirectly by H2O2 through the GSH redox buffering network in and outside mitochondria. This proposal is based on several observations that have shown that unlike other redox modifications PGlu reactions fulfill the requisite criteria to serve as an effective posttranslational modification that controls protein function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2015.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731959PMC
August 2016

Superoxide produced in the matrix of mitochondria enhances methylmercury toxicity in human neuroblastoma cells.

Toxicol Appl Pharmacol 2015 Dec 7;289(3):371-80. Epub 2015 Nov 7.

University of Ottawa, Department of Biology, Center for Advanced Research in Environmental Genomics, Ottawa, ON, K1N 6N5, Canada. Electronic address:

The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O2(-)), the proximal reactive oxygen species (ROS) generated by mitochondria, is responsible for MeHg demethylation. Here, we investigated the impact of different mitochondrial respiratory inhibitors, namely rotenone and antimycin A, on the O2(-)mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O2(-) in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O2(-) production by mitochondria. Both rotenone and PQ, which increase O2(-) in the mitochondrial matrix at a dose-dependent manner, enhanced the conversion of MeHg to inorganic mercury (iHg). Surprisingly, antimycin A, which prompts emission of O2(-) into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O2(-) emission. Rotenone and PQ also enhanced the toxicity of sub-toxic doses (0.1 μM) MeHg which correlated with the accumulation of iHg in mitochondria and depletion of mitochondrial protein thiols. Taken together, our results demonstrate that MeHg degradation is mediated by mitochondrial O2(-), specifically within the matrix of mitochondria when O2(-) is in adequate supply. Our results also show that O2(-) amplifies MeHg toxicity specifically through its conversion to iHg and subsequent interaction with protein cysteine thiols (R-SH). The implications of our findings in mercury neurotoxicity are discussed herein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.taap.2015.11.001DOI Listing
December 2015
-->