Mitochondrial ATP Synthase Activity Research Articles

Estrogen promotes fetal skeletal muscle mitochondrial distribution and ATP synthase activity important for insulin sensitivity in offspring

PurposeWe previously showed that offspring delivered to baboons in which levels of estradiol (E2) were suppressed during the second half of gestation exhibit insulin resistance. Mitochondria are essential for the production of ATP as the main source of energy for intracellular metabolic pathways, and skeletal muscle of type 2 diabetics exhibit mitochondrial abnormalities. Mitochondria express estrogen receptor β and E2 enhances mitochondrial function in adults. Therefore, the current study ascertained whether exposure of the fetus to E2 is essential for mitochondrial development.MethodsLevels of ATP synthase and citrate synthase and the morphology of mitochondria were determined in fetal skeletal muscle obtained near term from baboons untreated or treated daily with the aromatase inhibitor letrozole or letrozole plus E2.ResultsSpecific activity and amount of ATP synthase were 2-fold lower (P < 0.05) in mitochondria from skeletal muscle of E2 suppressed letrozole-treated fetuses and restored to normal by treatment with letrozole plus E2. Immunocytochemistry showed that in contrast to the punctate formation of mitochondria in myocytes of untreated and letrozole plus E2 treated animals, mitochondria appeared to be diffuse in myocytes of estrogen-suppressed fetuses. However, citrate synthase activity and levels of proteins that control mitochondrial fission/fusion were similar in estrogen replete and suppressed animals.ConclusionWe suggest that estrogen is essential for fetal skeletal muscle mitochondrial development and thus glucose homeostasis in adulthood.

Discovery and in vivo evaluation of novel inhibitors of the hydrolytic activity of mitochondrial F1Fo ATP synthase to alleviate myocardial ischemia reperfusion injury

Abstract Background F1Fo ATP-synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, ATP synthase reverses its activity to hydrolyze ATP leading to energetic deficit and cardiac injury. We have previously discovered via in-silico virtual screening selective hydrolase inhibitors with pyrazole-pyridine scaffold and verified their inhibitory effect on hydrolysis on isolated murine mitochondria. Purpose In the present work we aimed to 1) synthesize novel hydrolase inhibitors, 2) select the best candidates to evaluate their effect against myocardial ischemia (I) reperfusion (R) injury in vivo and 3) investigate the underlying molecular mechanisms of cardioprotection. Methods The inhibitory effect on ATP hydrolysis for all compounds was evaluated on isolated murine heart mitochondria and at a cellular level on H9C2 cells in the presence of rotenone where their ability to maintain membrane potential was monitored. The previously described ATP hydrolase inhibitor BTB06584 (BTB) was used in vitro and in vivo as a reference and three novel compounds were selected for in vivo evaluation namely 1117, 1119 and STK-71. In the first cohort, C57Bl6 mice were randomized into 5 groups that received intravenously: 1) vehicle, 2) 1117, 3)1119, 4) STK-71 and 5) BTB. The administration was performed 10 min prior to 30 min of I and 2 hours of R and infarct size (IS) was determined. The dose of each compound was based on the effective concentration needed at a cellular level to inhibit the ATP hydrolysis. In a second cohort of mice, the best candidate and vehicle were given 10 min after the induction of I and IS was measured to test the intervention in a more translational setting. Finally, in the third cohort, the cardioprotective mechanism was investigated at the 10th min of R. Results We discovered three candidates that inhibit the ATP hydrolytic activity with comparable IC50 values to BTB. These three compounds inhibited ATP hydrolysis on the H9C2 cells at 50μΜ while BTB was inactive at the same concentration. In vivo, the three novel inhibitors reduced IS compared to the control group with STK-71 having the most remarkable cardioprotective effect while BTB did not result in attenuation of IS. STK-71 when given after the induction of I, also reduced IS indicating that its cardioprotective effect is pertained in a translational protocol. Regarding the mechanism of cardioprotection, STK-71 increased T173 phosphorylation and activation of PKA which led to the downstream activation of phospholamban. STK-71 also led to the significant activation of the RISK pathway, increased the expression of the antiapoptotic protein Bcl-Xl and reduced apoptosis. Conclusion We discovered a novel pyrazolopyridine derivative that acts as an inhibitor of ATP hydrolysis and reduces myocardial IS in vivo. Its cardioprotective mechanism involves the activation of PKA signaling and alleviation of apoptosis.

Melatonin regulates mitochondrial enzymes and ascorbate–glutathione system during plant responses to drought stress through involving endogenous calcium

The study presented here was carried out to evaluate the effect of melatonin (Mel; N-acetyl-5-methoxytryptamine) on the arugula (Eruca sativa Mill.) plants grown under drought stress (DS). The results of the study show that DS significantly reduced the activity of mitochondrial ATP synthase and ATPase. Exposure of plants to DS caused a significant increase in the activity of NADPH oxidase and glycolate oxidase that resulted in the excess generation of superoxide (O2•−) and hydrogen peroxide (H2O2) radicals, respectively. Creation of oxidative stress caused substantial damage to membrane lipids, leakage of electrolytes and reduced the hydration level of the drought-stressed plants. The damaging effects of DS were probably carried out because of lower activity of ATP synthase and ATPase, weak performance of antioxidant defense system and insufficient accumulation of osmolytes. Nevertheless, supplementation of drought stressed plants with 30 µM Mel was remarkable in improving the activity of ATP synthase and ATPase with a concomitant activation of ascorbate–glutathione system, antioxidant enzymes, and accumulation of osmolytes. However, Mel inhibited the activity of NADPH oxidase and glycolate oxidase. This combined effect of events protected the plants from DS-induced impairments through reducing oxidative and osmotic stress as witnessed by a reduction in lipid peroxidation, electrolyte leakage and improvement in hydration status of the plants even under DS. On the contrary, inclusion of calcium (Ca2+) chelator overturned the positive effect of Mel on the stressed plants, that validates the involvement of endogenous Ca2+ during Mel-mediated plant adaptive responses to drought. The results of the study demonstrate that Mel involves Ca2+ in the orchestration of plant adaptive responses to DS by exerting regulatory control over the activity of mitochondrial enzymes and the function of the antioxidant defense system.

Cationic Proteins Rich in Lysine Residue Trigger Formation of Non-bilayer Lipid Phases in Model and Biological Membranes: Biophysical Methods of Study.

Cationic membrane-active toxins are the most abundant group of proteins in the venom of snakes and insects. Cationic proteins such as cobra venom cytotoxin and bee venom melittin are known for their pharmacological reactions including anticancer and antimicrobial effects which arise from the toxin-induced alteration in the dynamics and structure of plasma membranes and membranes of organelles. It has been established that these cationic toxins trigger the formation of non-bilayer lipid phase transitions in artificial and native mitochondrial membranes. Remarkably, the toxin-induced formation of non-bilayer lipid phase increases at certain conditions mitochondrial ATP synthase activity. This observation opens an intriguing avenue for using cationic toxins in the development of novel drugs for the treatment of cellular energy deficiency caused by aging and diseases. This observation also warrants a thorough investigation of the molecular mechanism(s) of lipid phase polymorphisms triggered by cationic proteins. This article presents a review on the application of powerful biophysical methods such as resonance spectroscopy (31P-, 1H-, 2H-nuclear magnetic resonance, and electron paramagnetic resonance), luminescence, and differential scanning microcalorimetry in studies of non-bilayer lipid phase transitions triggered by cationic proteins in artificial and biological membranes. A phenomenon of the triggered by cationic proteins the non-bilayer lipid phase transitions occurring within 10-2-10-11s is discussed in the context of potential pharmacological applications of cationic proteins. Next to the ATP dimer is an inverted micelle made of cardiolipin that serves as a vehicle for the transport of H+ ions from the intra-crista space to the matrix. It is proposed that such inverted micelles are triggered by the high density of H+ ions and the cationic proteins rich in lysine residue which compete with the conserved lysine residues of the ATP synthase rotor for binding to cardiolipin in the inner mitochondrial membrane and perturb the bilayer lipid packing of cristae. Phospholipids with a blue polar head represent cardiolipin and those with a red polar head represent other phospholipids found in the crista membrane.

IF1 ablation prevents ATP synthase oligomerization, enhances mitochondrial ATP turnover and promotes an adenosine-mediated pro-inflammatory phenotype

ATPase Inhibitory Factor 1 (IF1) regulates the activity of mitochondrial ATP synthase. The expression of IF1 in differentiated human and mouse cells is highly variable. In intestinal cells, the overexpression of IF1 protects against colon inflammation. Herein, we have developed a conditional IF1-knockout mouse model in intestinal epithelium to investigate the role of IF1 in mitochondrial function and tissue homeostasis. The results show that IF1-ablated mice have increased ATP synthase/hydrolase activities, leading to profound mitochondrial dysfunction and a pro-inflammatory phenotype that impairs the permeability of the intestinal barrier compromising mouse survival upon inflammation. Deletion of IF1 prevents the formation of oligomeric assemblies of ATP synthase and alters cristae structure and the electron transport chain. Moreover, lack of IF1 promotes an intramitochondrial Ca2+ overload in vivo, minimizing the threshold to Ca2+-induced permeability transition (mPT). Removal of IF1 in cell lines also prevents the formation of oligomeric assemblies of ATP synthase, minimizing the threshold to Ca2+-induced mPT. Metabolomic analyses of mice serum and colon tissue highlight that IF1 ablation promotes the activation of de novo purine and salvage pathways. Mechanistically, lack of IF1 in cell lines increases ATP synthase/hydrolase activities and installs futile ATP hydrolysis in mitochondria, resulting in the activation of purine metabolism and in the accumulation of adenosine, both in culture medium and in mice serum. Adenosine, through ADORA2B receptors, promotes an autoimmune phenotype in mice, stressing the role of the IF1/ATP synthase axis in tissue immune responses. Overall, the results highlight that IF1 is required for ATP synthase oligomerization and that it acts as a brake to prevent ATP hydrolysis under in vivo phosphorylating conditions in intestinal cells.

PHB3 Is Required for the Assembly and Activity of Mitochondrial ATP Synthase in Arabidopsis.

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits β and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.

Naringenin mitigates cadmium-induced cell death, oxidative stress, mitochondrial dysfunction, and inflammation in KGN cells by regulating the expression of sirtuin-1

The objective of this study was to examine the potential protective role of naringenin against the harmful effects induced by cadmium in KGN cell line. Cell viability was evaluated by cell counting kit-8 assay. Caspase-3/-9 activities were determined by caspase-3/-9 activity assay kits, respectively. Intracellular reactive oxygen species (ROS) level was detected by ROS-Glo™ H2O2 Assay, antioxidant capacity was determined by a total antioxidant capacity assay kit. Mitochondrial membrane potential (MMP), ATP level, and ATP synthase activity were determined by JC-1, ATP assay kit, and ATP synthase activity assay kit, respectively. The mRNA expression was determined by qRT-PCR. Cadmium reduced cell viability and increased caspase-3/-9 activities in a concentration-dependent manner. Naringenin improved cell viability and reduced caspase-3/-9 activities in cadmium-stimulated KGN cells in a concentration-dependent manner. Cadmium diminished the antioxidant capacity, increased ROS production, and induced mitochondrial dysfunction in KGN cells. These effects were ameliorated by naringenin treatment in a concentration-dependent manner. Furthermore, naringenin reduced the levels of pro-inflammatory cytokines in KGN cells exposed to cadmium. SIRT1 knockdown downregulated its expression in KGN cells and compromised the protective effects of naringenin on cell viability and caspase-3/-9 activities in cadmium-stimulated KGN cells. Naringenin prevented the reduction of MMP, ATP levels, and ATP synthase activity in cadmium-stimulated KGN cells in a concentration-dependent manner. However, these protective effects were significantly reversed by SIRT1 knockdown. In conclusion, this study suggests that naringenin protects against cadmium-induced damage by regulating oxidative stress, mitochondrial function, and inflammation in KGN cells, with SIRT1 playing a potential mediating role.

Mitochondrial Haemoglobin Is Upregulated with Hypoxia in Skeletal Muscle and Has a Conserved Interaction with ATP Synthase and Inhibitory Factor 1

The globin protein superfamily has diverse functions. Haemoglobin has been found in non-erythroid locations, including within the mitochondria. Using co-immunoprecipitation and in silico methods, we investigated the interaction of mitochondrial haemoglobin with ATP synthase and its associated proteins, including inhibitory factor 1 (IF1). We measured the expression of mitochondrial haemoglobin in response to hypoxia. In vitro and in silico evidence of interactions between mitochondrial haemoglobin and ATP synthase were found, and we report upregulated mitochondrial haemoglobin expression in response to hypoxia within skeletal muscle tissue. Our observations indicate that mitochondrial pH and ATP synthase activity are implicated in the mitochondrial haemoglobin response to hypoxia.

Plasma level of ATPase inhibitory factor 1 (IF1) is associated with type 2 diabetes risk in humans: A prospective cohort study

AimMitochondrial dysfunction is associated with the development of type 2 diabetes mellitus (T2DM). It is thus of clinical relevance to identify plasma biomarkers of mitochondrial dysfunction associated with the risk of T2DM.ATPase inhibitory factor 1 (IF1) endogenously inhibits mitochondrial ATP synthase activity. Here, we analyzed association of the plasma IF1 level with markers of glucose homeostasis and with the conversion to new-onset diabetes (NOD) in individuals with prediabetes. MethodsIn the IT-DIAB prospective study, the baseline plasma level of IF1 was measured in 307 participants with prediabetes. The primary outcome was the incidence of NOD within five years of follow-up. Cross-sectional analysis of the IF1 level was also done in two independent interventional studies. Correlations between plasma IF1 and metabolic parameters at baseline were assessed by Spearman's correlation coefficients, and the association with the risk of NOD was determined using Cox proportional‐hazards models. ResultsIn IT-DIAB, the mean IF1 plasma level was lower in participants who developed NOD than in those who did not (537 ± 248 versus 621 ± 313 ng/mL, P = 0.01). The plasma IF1 level negatively correlated with clinical variables associated with obesity and insulin resistance, including the body mass index (r = −0.20, P = 0.0005) and homeostasis model assessment of insulin resistance (HOMA-IR). (r = −0.37, P < 0.0001). Conversely, IF1 was positively associated with plasma markers of cardiometabolic health, such as HDL-C (r = 0.63, P < 0.0001) and apoA-I (r = 0.33, P < 0.0001). These correlations were confirmed in cross-sectional analyses. In IT-DIAB, the IF1 level was significantly associated with a lower risk of T2DM after adjustment for age, sex, and fasting plasma glucose (HR [95% CI] per 1 SD = 0.76 [0.62; 0.94], P = 0.012). ConclusionWe identified for the first time the mitochondrial-related biomarker IF1 as being associated with the risk of T2DM.

Impact of ATP synthase/coupling factor 6 in hypoxic pulmonary arterial hypertension: An experimental rat model.

Hypoxia-induced pulmonary arterial hypertension (PAH) is characterized by prostacyclin (PGI2 ) disorder, which manifests in the same manner as in monocrotaline (MCT)-induced PAH. Endogenous PGI2 inhibitor coupling factor 6 (CF6) is involved in MCT-induced PAH. This study aimed to explore the presence or absence of a correlation between hypoxia-induced PAH and CF6. This study was conducted between January 2019 and June 2020. A total of 135 male Wistar rats (aged 8 weeks and weighing 200-250 g) were randomly divided into five groups: (A) control, (B) 1 week of hypoxia, (C) 2 weeks of hypoxia, (D) 3 weeks of hypoxia, and (E) 4 weeks of hypoxia. CF6 expression in both lung tissue and blood samples from the lung vasculature and tail vein was measured by western blotting, immunohistochemistry, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay. Hemodynamic and morphological changes in hypoxia-induced rats indicated PAH development. The results showed the presence of a correlation between the mRNA and protein levels of CF6 in lung tissue, activity of mitochondrial ATP synthase, and hypoxia time, and there was a significant increment in the group exposed to hypoxia for 4 weeks compared to the control group. The decrement expression of ATPase inhibitory factor 1 (IF 1) mRNA was consistent with the outcomes of ATP synthase activity in lung tissue in the 4 weeks of hypoxia group compared with the control group. However, the levels of CF6 and ATP synthase activity did not differ between blood samples from the lung vasculature and tail vein. : In hypoxia-induced PAH, CF6 showed downregulated expression in lung tissue, but not in pulmonary vasculature and circulation. Therefore, we speculated that CF6 and ATP synthase may play important roles in hypoxia-induced PAH.

Circulating Inhibitory Factor 1 levels in adult patients with Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a rare genetic syndrome characterized by hyperphagia and early development of morbid obesity. Cardiovascular disease (CVD) and metabolic syndrome (MetS) are major comorbidities in these patients leading to premature death. Inhibitory factor 1 (IF1) works as a regulatory protein, inhibiting the ATP hydrolase activity of mitochondrial ATP synthase and likely playing a role in lipid metabolism. We aimed to assay IF1 in adult patients with PWS evaluating any relationship with clinical, genetic and biochemical parameters. We recruited 35 adult patients with genetically confirmed PWS. IF1 serum concentration displayed a normal distribution with an average value of 70.7±22.6pg/mL, a median value of 66.1pg/mL. It was above the reference range only in one patient. All parameters were compared from both sides of IF1 median without displaying any significant differences. Patients with normal or low HDL-cholesterol did not present any difference as regards IF1 levels, which were not different between patients with and without MetS. Non-esterified fatty acids (NEFA) serum levels (r=0.623; p<0.001) showed a statistically significant correlation with IF1. Cholesterol and its fractions did not present any correlation with IF1. In this study we do not confirm that HDL-cholesterol and IF1 are correlated, but we show that in adult PWS patients, NEFA are correlated with serum IF1. This protein could play a role to some extent in determining the complex metabolic alterations in PWS patients.

Neural cell-derived plasma exosome protein abnormalities implicate mitochondrial impairment in first episodes of psychosis.

Neuroprotective and other functional proteins of mitochondria were quantified in extracts of plasma neural-derived exosomes from ten first-episode psychosis (FP) patients and ten matched psychiatrically normal controls (ctls). Astrocyte-derived extracellular vesicles (ADEVs) and neuron-derived extracellular vesicles (NDEVs) were immunoabsorbed separately from physically precipitated plasma total EVs. Extracted mitochondrial ATP synthase was specifically immunofixed to plastic wells for quantification of catalytic activity based on conversion of NADH to NAD+ . Other extracted mitochondrial functional proteins were quantified by ELISAs. All protein levels were normalized with EV content of the CD81 exosome marker. FP patient ADEV level but not NDEV level of mitochondrial ATP synthase activity was significantly lower than that of ctls. FP patient ADEV and NDEV levels of the functionally critical mitochondrial proteins mitofusin 2 and cyclophilin D, but not of transcription factor A of mitochondria, and of the mitochondrial short open-reading frame neuroprotective and metabolic regulatory peptides humanin and MOTS-c were significantly lower than those of ctls. In contrast, FP patient NDEV, but not ADEV, level of the mitochondrial-tethering protein syntaphilin, but not of myosin VI, was significantly higher than that of ctls. The distinctively different neural levels of some mitochondrial proteins in FP patients than ctls now should be correlated with diverse clinical characteristics. Drugs that increase depressed levels of proteins and mimetics of deficient short open-reading frame peptides may be of therapeutic value in early phases of schizophrenia.

Hydrolytic activity of mitochondrial F1Fo ATP synthase as a target for myocardial ischemia: discovery and in vitro evaluation of novel inhibitors

Abstract Background/Introduction F1Fo ATP synthase is the mitochondrial complex responsible for ATP production. During myocardial ischemia, ATP synthase reverses its activity to hydrolyse ATP leading to cellular energetic deficit, contracture and lethal cardiomyocyte injury. Therefore, inhibition of ATP hydrolase is of major significance and the discovery of selective hydrolase inhibitors could be of particular interest in terms of cardiac response to ischemia. Purpose We performed an in vitro screening in order to identify and evaluate novel specific inhibitors of the hydrolytic activity of ATP synthase. Methods Initially, we identified inhibitors of the hydrolytic activity of ATP synthase using virtual screening methods. Docking-scoring calculations were performed targeting the three major inhibition sites of a holistic model of ATP synthase of mus musculus that was modelled for the first time. In silico ligand-based virtual screening was carried out using known binders such as BMS199264. The workflow was implemented on our in-house library of 2000 compounds, Pharmalab, plus 266,151 compounds of the National Cancer Institute (NCI) database. The best candidates were evaluated in vitro on isolated murine heart mitochondria to verify their inhibitory effect on ATP synthase hydrolytic activity. Moreover, we confirmed their action at a cellular level. H9C2 cells were treated with the three best candidates in the presence of rotenone and their ability to maintain membrane potential was monitored. The experiment was repeated in absence of rotenone in order to detect toxic or non-specific effects (n=5 independent experiments). Finally, the compounds were tested using an assay in which primary adult rat ventricular cardiomyocytes (ARVCs) were challenged with the mitochondrial uncoupler CCCP to induce reverse ATPase activity, and time until contracture was determined. For the in vitro experiments, oligomycin, a non-selective ATPase inhibitor and BTB06584 a selective hydrolase inhibitor were used as positive controls. Results Different steps of filtering through the virtual screening provided 38 molecules from Pharmalab and 15 candidates form NCI database. From the 53 candidates in total, five compounds displayed in vitro inhibitory activity at 200μM on isolated mitochondria and their IC50 values were determined. Among them, three synthetic derivatives possessing a central pyrazolopyridine core displayed the best IC50 values (81.7±1.3 μM, 99.8±1.2 μM, 144.8±1.3μM) and were evaluated on the H9C2 cells. They exhibited significant inhibitory activity at 50μM (p&amp;lt;0.01 compared to vehicle) while the BTB06584 inhibitor was inactive at the same concentration and two of them were selective. Finally, the final novel inhibitors significantly extended (p&amp;lt;0.001) the time until shortening of the ARVCs. Conclusion We have discovered two novel agents that act as selective inhibitors of ATP hydrolase and can be tested against myocardial ischemia. Funding Acknowledgement Type of funding source: None

Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis.

Inhibitors of mitochondrial respiration and ATP synthesis may promote the selective killing of respiration-competent cancer cells that are critical for tumor progression. We previously reported that CADD522, a small molecule inhibitor of the RUNX2 transcription factor, has potential for breast cancer treatment. In the current study, we show that CADD522 inhibits mitochondrial oxidative phosphorylation by decreasing the mitochondrial oxygen consumption rate (OCR) and ATP production in human breast cancer cells in a RUNX2-independent manner. The enzyme activity of mitochondrial ATP synthase was inhibited by CADD522 treatment. Importantly, results from cellular thermal shift assays that detect drug-induced protein stabilization revealed that CADD522 interacts with both α and β subunits of the F1-ATP synthase complex. Differential scanning fluorimetry also demonstrated interaction of α subunits of the F1-ATP synthase to CADD522. These results suggest that CADD522 might target the enzymatic F1 subunits in the ATP synthase complex. CADD522 increased the levels of intracellular reactive oxygen species (ROS), which was prevented by MitoQ, a mitochondria-targeted antioxidant, suggesting that cancer cells exposed to CADD522 may elevate ROS from mitochondria. CADD522-increased mitochondrial ROS levels were enhanced by exogenously added pro-oxidants such as hydrogen peroxide or tert-butyl hydroperoxide. Conversely, CADD522-mediated cell growth inhibition was blocked by N-acetyl-l-cysteine, a general ROS scavenger. Therefore, CADD522 may exert its antitumor activity by increasing mitochondrial driven cellular ROS levels. Collectively, our data suggest in vitro proof-of-concept that supports inhibition of mitochondrial ATP synthase and ROS generation as contributors to the effectiveness of CADD522 in suppression of tumor growth.

Dysfunctional oxidative phosphorylation shunts branched‐chain amino acid catabolism onto lipogenesis in skeletal muscle

It is controversial whether mitochondrial dysfunction in skeletal muscle is the cause or consequence of metabolic disorders. Herein, we demonstrate that in vivo inhibition of mitochondrial ATP synthase in muscle alters whole‐body lipid homeostasis. Mice with restrained mitochondrial ATP synthase activity presented intrafiber lipid droplets, dysregulation of acyl‐glycerides, and higher visceral adipose tissue deposits, poising these animals to insulin resistance. This mitochondrial energy crisis increases lactate production, prevents fatty acid β‐oxidation, and forces the catabolism of branched‐chain amino acids (BCAA) to provide acetyl‐CoA for de novo lipid synthesis. In turn, muscle accumulation of acetyl‐CoA leads to acetylation‐dependent inhibition of mitochondrial respiratory complex II enhancing oxidative phosphorylation dysfunction which results in augmented ROS production. By screening 702 FDA‐approved drugs, we identified edaravone as a potent mitochondrial antioxidant and enhancer. Edaravone administration restored ROS and lipid homeostasis in skeletal muscle and reinstated insulin sensitivity. Our results suggest that muscular mitochondrial perturbations are causative of metabolic disorders and that edaravone is a potential treatment for these diseases.

Abstract 801: Dual inhibition of distinct metabolic features targets osteosarcoma stem cells

Abstract [Objective] Mitochondria are the places for the energy production of the cells, while reactive oxygen species (ROS) are also produced alongside. In recent years, it has been reported that cancer stem cells metabolize predominantly through oxidative phosphorylation (OXPHOS) rather than glycolysis in certain cancer cells. Targeting OXPHOS achieved by suppression of ATP synthesis through mitochondrial ATP synthase could be a potential therapeutic option against cancer stem cells. In the current study, we have identified the mitochondria metabolism as the potential therapeutic target in osteosarcoma (OS) stem cells, presenting the synergistic effects of combination of OXPHOS inhibition by pterostilebene (PTE) with c-Myc inhibitor, which target both OXPHOS-dominant cancer stem cells and glycolysis-dominant non-cancer stem cells as a ‘two hit’ or ‘dual inhibition’ of metabolic pathways, OXPHOS and glycolysis. [Materials &amp; Methods] Using human OS cell lines of SaOS2, U2OS and MG63 cells, cell survival and the ability of sphere formation was assessed with or without PTE, and the expression of stem cell markers mRNA such as Oct3, NS, CD44 was examined by RT-PCR. Next, the activity of mitochondrial ATP synthase, mitochondrial respiration capacity of oxygen consumption rate, and the amount of ATP as well as ROS production were measured under the treatment of PTE. Furthermore, we examined the synergistic effect of PTE with cMyc transcription inhibitors of JQ1 or Honokiol (HNK). [Results] PTE treatment on human OS cell lines reduced the viabilities of all cell lines in dose-dependent manner and expression of stem cell marker and the ability of sphere formation were also decrease in terms of sphere number and size. PTE reduced the activity of F0F1-ATP synthase; Complex V predominantly, and the mitochondrial oxygen consumption rates and synthetic amount of ATP were also decreased in spheroid condition. These results suggest that PTE possibly targets stem cell population which preferably relies on OXPHOS, suppressing ATP synthesis via F0F1-ATP synthase inhibition as well as increased ROS production in OS cells and changes metabolic flax to glycolysis dependent feature. The dual inhibition of OXPHOS by PTE and c-Myc inhibition by HNK or JQ1 showed the synergistically inhibition of OS cell growth in a dose-dependent manner. [Discussion] Prognosis of the patients with osteosarcoma has reached to plateau without any breakthroughs over the last quarter century, and nearly 30% of patients still have to face very severe poor prognosis, especially with metastatic disease. Current study suggests that modulation of metabolic flux by c-Myc and OXPHOS inhibitors showed a greater synergistic effect with ‘two metabolic hit’ or ‘dual metabolic inhibition’ of distinct metabolic features and it could be a novel therapeutic strategy against osteosarcoma, possibly targeting both stem-like cell population and general tumor cell population. Citation Format: Shingo Kishi, Kanya Honoki, Shinji Tsukamoto, Hiromasa Fujii, Yumiko Kondo, Yasuhito Tanaka, Hiroki Kuniyasu. Dual inhibition of distinct metabolic features targets osteosarcoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 801.

Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation

Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free trans-cyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid (k ~2000 M−1 s−1), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F1-Fo sub-unit alpha (ATP-α-F1) oxidation—an established molecular correlate of impaired enzyme activity—in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F1 oxidation at evolutionary conserved cysteine residues (i.e., C244 and C294) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50–60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intermolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F1-Fo ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease.

FUS interacts with ATP synthase beta subunit and induces mitochondrial unfolded protein response in cellular and animal models

FUS (fused in sarcoma) proteinopathy is a group of neurodegenerative diseases characterized by the formation of inclusion bodies containing the FUS protein, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Previous studies show that mitochondrial damage is an important aspect of FUS proteinopathy. However, the molecular mechanisms by which FUS induces mitochondrial damage remain to be elucidated. Our biochemical and genetic experiments demonstrate that FUS interacts with the catalytic subunit of mitochondrial ATP synthase (ATP5B), disrupts the formation of ATP synthase complexes, and inhibits mitochondrial ATP synthesis. FUS expression activates the mitochondrial unfolded protein response (UPRmt). Importantly, down-regulating expression of ATP5B or UPRmt genes in FUS transgenic flies ameliorates neurodegenerative phenotypes. Our data show that mitochondrial impairment is a critical early event in FUS proteinopathy, and provide insights into the pathogenic mechanism of FUS-induced neurodegeneration.

Dexpramipexole improves bioenergetics and outcome in experimental stroke.

Dexpramipexole, a drug recently tested in patients with amyotrophic lateral sclerosis (ALS,) is able to bind F1Fo ATP synthase and increase mitochondrial ATP production. Here, we have investigated its effects on experimental ischaemic brain injury. The effects of dexpramipexole on bioenergetics, Ca2+ fluxes, electrophysiological functions and death were evaluated in primary neural cultures and hippocampal slices exposed to oxygen-glucose deprivation (OGD). Effects on infarct volumes and neurological functions were also evaluated in mice following proximal or distal middle cerebral artery occlusion (MCAo). Distribution of dexpramipexole within the ischaemic brain was evaluated by means of mass spectrometry imaging. Dexpramipexole increased mitochondrial ATP production in cultured neurons or glia and reduces energy failure, prevents intracellular Ca2+ overload and affords cytoprotection when cultures are exposed to OGD. This compound also counteracted ATP depletion, mitochondrial swelling, anoxic depolarization, loss of synaptic activity and neuronal death in hippocampal slices subjected to OGD. Post-ischaemic treatment with dexpramipexole, at doses consistent with those already used in ALS patients, reduced brain infarct size and ameliorated neuroscore in mice subjected to transient or permanent MCAo. Notably, the concentrations of dexpramipexole reached within the ischaemic penumbra equalled those found neuroprotective in vitro. Dexpramipexole, a compound able to increase mitochondrial F1Fo ATP-synthase activity reduced ischaemic brain injury. These findings, together with the excellent brain penetration and favourable safety profile in humans, make dexpramipexole a drug with realistic translational potential for the treatment of stroke. This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Stimulation of mitochondrial ATP synthase activity - a new diazoxide-mediated mechanism of cardioprotection.

Pharmacological preconditioning by diazoxide and a model of experimental streptozotocin-induced acute diabetes mellitus (STZ-DM) provided similar levels of cardioprotection assessed as limiting myocardial infarct size. The aim was to explore the possibility of existence of another in vitro mechanism, which could be contributory to cardioprotection mediated by diazoxide treatment. Mitochondrial membrane fluidity and ATP synthase activity in isolated heart mitochondria were determined under the influence of two factors, STZ-DM condition and treatment with diazoxide. Both factors independently increased the ATP synthase activity (p<0.05), as no interaction effect was observed upon the combination of STZ-DM with diazoxide. On the other hand, the mitochondrial membrane fluidity was significantly increased by STZ-DM only; no significant main effect for diazoxide was found. Based on the results from measurements of enzyme kinetics, we assume a direct interaction of diazoxide with the molecule of ATP synthase stimulated its activity by noncompetitive activation. Our present work revealed, for the first time, that cardioprotection induced by diazoxide may not be caused exclusively by mitochondrial K(ATP) opening, but presumably also by a direct interaction of diazoxide with ATP synthase, although the mechanisms for achieving this activation cannot be fully delineated.