Electron Transport Chain Enzymes Research Articles

Disruption of testis-enriched cytochrome c oxidase subunit COX6B2 but not COX8C leads to subfertility.

Mammalian sperm flagellum contains the midpiece characterized by a mitochondrial sheath that packs tightly around the axoneme and outer dense fibers. Mitochondria are known as the "powerhouse" of the cell, and produce ATP through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). However, the contribution of the TCA cycle and OXPHOS to sperm motility and male fertility is less clear. Cytochrome c oxidase (COX) is an oligomeric complex localized within the mitochondrial inner membrane, and the terminal enzyme of the mitochondrial electron transport chain in eukaryotes. Both COX6B2 and COX8C are testis-enriched COX subunits whose functions in vivo are poorly studied. Here, we generated Cox6b2 and Cox8c knockout (KO) mice using the CRISPR/Cas9 system. We examined their fertility and sperm mitochondrial function to determine the significance of testis-enriched COX subunits in male fertility. The mating test revealed that disrupting COX6B2 induces male subfertility, while disrupting COX8C does not affect male fertility. Cox6b2 KO spermatozoa showed low sperm motility, but mitochondrial function was normal according to oxygen consumption rates. Therefore, low sperm motility seems to cause subfertility in Cox6b2 KO male mice. These results also indicate that testis-enriched COX, COX6B2 and COX8C, are not essential for OXPHOS in mouse spermatozoa.

Cardioprotective Action of a Novel Synthetic 19,20-EDP Analog Is Sirt Dependent.

Mounting evidence suggests that cytochrome P450 epoxygenase-derived metabolites of docosahexaenoic acid, called epoxydocosapentaenoic acids (EDPs), limit mitochondrial damage after cardiac injury. In particular, the 19,20-EDP regioisomer has demonstrated potent cardioprotective action. Thus, we investigated our novel synthetic 19,20-EDP analog SA-22 for protection against cardiac ischemia-reperfusion (IR) injury. Isolated C57BL/6J mouse hearts were perfused through Langendorff apparatus for 20 minutes to obtain baseline function, followed by 30 minutes of global ischemia. Hearts were then treated with vehicle, 19,20-EDP, SA-22, or SA-22 with the pan-sirtuin inhibitor nicotinamide or the SIRT3-selective inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) at the start of 40 minutes reperfusion (N = 5-8). We assessed IR injury-induced changes in recovery of myocardial function, using left ventricular developed pressure and systolic and diastolic pressure change. Tissues were assessed for electron transport chain function, SIRT1 and SIRT3, optic atrophy type 1, and caspase-1. We also used H9c2 cells in an in vitro model of hypoxia/reoxygenation injury (N = 3-6). Hearts perfused with SA-22 had significantly improved postischemic left ventricular developed pressure, systolic and diastolic recovery (64% of baseline), compared with vehicle control (15% of baseline). In addition, treatment with SA-22 led to better catalytic function observed in electron transport chain and SIRT enzymes. The protective action of SA-22 resulted in reduced activation of pyroptosis in both hearts and cells after injury. Interestingly, although nicotinamide cotreatment worsened functional outcomes, cell survival, and attenuated sirtuin activity, it failed to completely attenuate SA-22-induced protection against pyroptosis, possibly indicating EDPs exert cytoprotection through pleiotropic mechanisms. In short, these data demonstrate the potential of our novel synthetic 19,20-EDP analog, SA-22, against IR/hypoxia-reoxygenation injury and justify further development of therapeutic agents based on 19,20-EDP.

Alteration of pulmonary mitochondrial oxidation status, inflammation, energy metabolizing enzymes, oncogenic and apoptotic markers in mice model of diisononyl phthalate-induced asthma

Primary plasticizer used in polyvinyl chloride is diisononyl phthalate (DiNP) and exposure to DiNP has been associated with the development of asthma and allergies. In the current study, how DiNP alters pulmonary antioxidant status, inflammation, energy metabolizing enzymes, oncogenic and apoptotic markers in DiNP-induced asthmatic mice was examined. Male BALB/c mice (n=20, 20-30 g) were divided into 2 groups of 10 mice each: group 1 (control) received saline (0.2ml/kg) orally for 23 days, and group 2 (DiNP) received 50 mg/kg DiNP (Intraperitoneal and intranasal) once per day. After the last administration, mice were sacrificed, lungs were removed and used for biochemical and histopathological analysis. DiNP treated mice experienced alterations in their lung histoarchitecture, levels of oncogenic and apoptotic factors, glycolytic, tricarboxylic acid cycle (TCA), and electron transport chain enzymes (ETC), antioxidant status, and inflammatory biomarkers. DiNP decreased the lungs levels of reduced glutathione and ascorbic acid, and the activities of superoxide dismutase, catalase, and glutathione-s-transferase. In the lungs of DiNP-treated mice compared to the control group, malondialdehyde and inflammatory biomarkers (nitric oxide and myeloperoxidase) were significantly greater (p<0.05). Furthermore, the activities of glycolytic enzymes hexokinase, aldolase, lactate dehydrogenase were downregulated with a concomitant increase in NADase (77%). TCA enzymes and ETC enzymes were significantly reduced as well. CAS-3, p53, Bax, c-MYC, K-Ras increased by 65%, 51%, 70%, 59% and 82% respectively while BCL-2 decreased by 74%. Histopathological analysis revealed distortion of the airway structure characterized by inflammatory cell infiltration, oedema, hemorrhage, and constricted alveoli space. Exposure to DiNP caused oxidative stress which promotes lung inflammation via depletion of antioxidants, pulmonary energy transduction enzymes, levels of oncogenic and apoptotic factors were impaired as well, suggesting that the lungs may not be able to perform its morphological and physiological functions effectively.

Why Is Iron Deficiency/Anemia Linked to Alzheimer's Disease and Its Comorbidities, and How Is It Prevented?

Impaired iron metabolism has been increasingly observed in many diseases, but a deeper, mechanistic understanding of the cellular impact of altered iron metabolism is still lacking. In addition, deficits in neuronal energy metabolism due to reduced glucose import were described for Alzheimer's disease (AD) and its comorbidities like obesity, depression, cardiovascular disease, and type 2 diabetes mellitus. The aim of this review is to present the molecular link between both observations. Insufficient cellular glucose uptake triggers increased ferritin expression, leading to depletion of the cellular free iron pool and stabilization of the hypoxia-induced factor (HIF) 1α. This transcription factor induces the expression of the glucose transporters (Glut) 1 and 3 and shifts the cellular metabolism towards glycolysis. If this first line of defense is not adequate for sufficient glucose supply, further reduction of the intracellular iron pool affects the enzymes of the mitochondrial electron transport chain and activates the AMP-activated kinase (AMPK). This enzyme triggers the translocation of Glut4 to the plasma membrane as well as the autophagic recycling of cell components in order to mobilize energy resources. Moreover, AMPK activates the autophagic process of ferritinophagy, which provides free iron urgently needed as a cofactor for the synthesis of heme- and iron-sulfur proteins. Excessive activation of this pathway ends in ferroptosis, a special iron-dependent form of cell death, while hampered AMPK activation steadily reduces the iron pools, leading to hypoferremia with iron sequestration in the spleen and liver. Long-lasting iron depletion affects erythropoiesis and results in anemia of chronic disease, a common condition in patients with AD and its comorbidities. Instead of iron supplementation, drugs, diet, or phytochemicals that improve energy supply and cellular glucose uptake should be administered to counteract hypoferremia and anemia of chronic disease.

Histochemical mapping of the duration of action of photobiomodulation on cytochrome c oxidase in the rat brain.

This is the first study mapping the duration of action of in vivo photobiomodulation (PBM) on cytochrome-c-oxidase (CCO). In cellular bioenergetics, CCO is the terminal rate-limiting enzyme in the mitochondrial electron transport chain, which catalyzes oxygen utilization for aerobic energy production. PBM using transcranial infrared laser stimulation (TILS) is a promising intervention for non-invasively modulating CCO in the brain. TILS of the human prefrontal cortex directly causes CCO photo-oxidation, which is associated with increased cerebral oxygenation and improved cognition. This experiment aimed to map the duration of action of in vivo PBM on CCO activity in discrete neuroanatomic locations within rat brains up to 4 weeks after a single TILS session (50 s, 1064 nm CW, 250 mW/cm2). Control brains from rats treated with a sham session without TILS (laser off) were compared to brains from TILS-treated rats that were collected 1 day, 2 weeks, or 4 weeks post-TILS. Cryostat sections of the 36 collected brains were processed using quantitative enzyme histochemistry and digitally imaged. Densitometric readings of 28 regions of interest were recorded and converted to CCO activity units of oxygen utilization using calibration standards. Data analysis (ANCOVA) compared each laser-treated group to sham with whole-brain average as a covariate. The prefrontal infralimbic cortex showed the earliest significant increase in CCO activity between 1-day post-TILS and sham groups, which continued elevated for 2-4 weeks post-TILS. Significant differences in CCO activity between 2-weeks and sham groups were also found in the lateral septum, accumbens core, CA3 of the hippocampus, and the molecular layer of the hippocampus. The medial amygdala showed a significant decrease in CCO activity between 4-weeks and sham. Further analyses showed significant inter-regional CCO activity correlations among the brain regions as the result of TILS, with the most pronounced changes at 4-weeks post-stimulation. The time course of changes in CCO activity and network connectivity suggested that TILS caused different neuroplasticity types of bioenergetic changes at different time scales, depending on brain region and its depth from the cortex. In conclusion, this controlled CCO histochemical study demonstrated a long-lasting duration of action of PBM in the rat brain.

Maternal Obesity Programs the Premature Aging of Rat Offspring Liver Mitochondrial Electron Transport Chain Genes in a Sex-Dependent Manner.

We investigated whether maternal obesity affects the hepatic mitochondrial electron transport chain (ETC), sirtuins, and antioxidant enzymes in young (110 postnatal days (PND)) and old (650PND) male and female offspring in a sex- and age-related manner. Female Wistar rats ate a control (C) or high-fat (MO) diet from weaning, through pregnancy and lactation. After weaning, the offspring ate the C diet and were euthanized at 110 and 650PND. The livers were collected for RNA-seq and immunohistochemistry. Male offspring livers had more differentially expressed genes (DEGs) down-regulated by both MO and natural aging than females. C-650PND vs. C-110PND and MO-110PND vs. C-110PND comparisons revealed 1477 DEGs in common for males (premature aging by MO) and 35 DEGs for females. Analysis to identify KEGG pathways enriched from genes in common showed changes in 511 and 3 KEGG pathways in the male and female livers, respectively. Mitochondrial function pathways showed ETC-related gene down-regulation. All ETC complexes, sirtuin2, sirtuin3, sod-1, and catalase, exhibited gene down-regulation and decreased protein expression at young and old ages in MO males vs. C males; meanwhile, MO females down-regulated only at 650PND. Conclusions: MO accelerates the age-associated down-regulation of ETC pathway gene expression in male offspring livers, thereby causing sex-dependent oxidative stress, premature aging, and metabolic dysfunction.

Inorganic polyphosphate's role in energy production and mitochondrial permeability transition pore opening in tick mitochondria.

Inorganic polyphosphate (polyP) is a biopolymer composed of phosphoanhydride-linked orthophosphate molecules. PolyP is engaged in a variety of cellular functions, including mitochondrial metabolism. Here, we examined the effects of polyP on electron transport chain enzymes and F1 Fo ATP synthase in tick embryos during embryonic development. The study found that polyPs containing medium and long chains (polyP15 and polyP65 ) enhanced the activity of complex I, complex II, complex III, and F1 Fo ATP synthase, while short polyP chains (polyP3 ) had no effect. The study also examined the activity of exopolyphosphatases (PPX) in various energy-demand situations. PPX activity was stimulated when ADP concentrations are high, characterizing a low-energy context. When complexes I-III and F1 Fo ATP synthase inhibitors were added in energized mitochondria, PPX activity decreased, whereas the mitochondrial uncoupler FCCP had no impact on PPX activity. Additionally, the study investigated the effect of polyP on mitochondrial swelling, finding that polyP causes mitochondrial swelling by increasing calcium effects on the mitochondrial permeability transition pore. The findings presented here to increase our understanding of the function of polyP in mitochondrial metabolism and its relationship to mitochondrial permeability transition pore opening in an arthropod model.

Abstract B027: Elevated mitochondrial reactive oxygen species dysregulate the tumor microenvironment in prostate cancer of African American men

Abstract African American men (AAM) are more than twice as likely to die of prostate cancer (PCa) compared to European American men (EAM). While socioeconomics, access to optimal care and life-style factors contribute towards these disparities, underlying biologic differences in AAM remain important drivers of PCa disparities. In the current work, we aimed to explore the interplay between oxidative stress and inflammatory signaling in promoting progression of PCa in AAM. Oxidative stress results from excessive reactive oxygen species (ROS) generated mainly via membrane bound oxidases, the mitochondrial electron transport chain (ETC) or peroxisomes. We performed a robust comparative genomics to determine whether there is a race dependent expression of genes related to ROS in publicly available PCa datasets. We then prospectively examined the differential gene expression of these genesets in PCa from clinically matched AAM and EAM in VANDAAM, a validation study of Decipher genomic testing in 243 men with localized PCa. We then combined computational and experimental approaches to explore the molecular mechanisms and consequences of exacerbated oxidative stress. We found that ETC complex I, III related genes are the most differentially expressed in AAM compared with EAM. To examine whether AAM vs EAM cell lines exhibits differential sensitivity to ETC modulation, we blocked key ETC enzymes using complex I inhibitor, rotenone, and complex III inhibitor, antimycin A. We used 2 types of mitochondria-specific labels that distinguish respiring and ROS-generating mitochondria. While the relative levels of respiring mitochondria remain constant, we found that antimycin A induced higher level of mitochondrial ROS in AAM PCa cell lines compared with EAM PCa cell lines. To understand the interplay between mitochondrial ETC complex I & III and immune microenvironment, we computed the correlational discordance of ETC complex I & III expression scores and expression of >1200 immune-related genes between AAM vs EAM in the VANDAAM cohort. These analyses suggest that ETC complex I & III correlate with several immunosuppressive genes including the macrophage markers CD68 and CD163 and T cell exhaustion markers. In addition, intersecting discordant genes from several datasets suggests enrichment of inflammatory cytokines and type I interferon signature which we then validated using ex vivo culture of PCa tissues. In summary, we discovered a role of mitochondrial electron transport chain in regulating inflammatory circuits in PCa of AAM. Unraveling roles of mitochondria in promoting immune suppression will have significant impact on understanding the intersection between social pressures and biological stress responses in minority populations. Citation Format: Asmaa El-Kenawi, Ryan Putney, Shivanshu Awasthi, Amparo Serna, Corrado Caslini, Jasreman Dhillon, Kosj Yamoah. Elevated mitochondrial reactive oxygen species dysregulate the tumor microenvironment in prostate cancer of African American men [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B027.

Hypoxia-induced reprogramming of glucose-dependent metabolic pathways maintains the stemness of human bone marrow-derived endothelial progenitor cells

The benefits of hypoxia for maintaining the stemness of cultured human bone marrow-derived endothelial progenitor cells (BM EPCs) have previously been demonstrated but the mechanisms responsible remain unclear. Growing evidences suggest that cellular metabolism plays an important role in regulating stem cell fate and self-renewal. Here we aimed to detect the changes of glucose metabolism and to explore its role on maintaining the stemness of BM EPCs under hypoxia. We identified the metabolic status of BM EPCs by using extracellular flux analysis, LC–MS/MS, and 13C tracing HPLC-QE-MS, and found that hypoxia induced glucose metabolic reprogramming, which manifested as increased glycolysis and pentose phosphate pathway (PPP), decreased tricarboxylic acid (TCA) and mitochondrial respiration. We further pharmacologically altered the metabolic status of cells by employing various of inhibitors of key enzymes of glycolysis, PPP, TCA cycle and mitochondria electron transport chain (ETC). We found that inhibiting glycolysis or PPP impaired cell proliferation either under normoxia or hypoxia. On the contrary, inhibiting pyruvate oxidation, TCA or ETC promoted cell proliferation under normoxia mimicking hypoxic conditions. Moreover, promoting pyruvate oxidation reverses the maintenance effect of hypoxia on cell stemness. Taken together, our data suggest that hypoxia induced glucose metabolic reprogramming maintains the stemness of BM EPCs, and artificial manipulation of cell metabolism can be an effective way for regulating the stemness of BM EPCs, thereby improving the efficiency of cell expansion in vitro.

Alleviative effects of pinostrobin against cadmium-induced renal toxicity in rats by reducing oxidative stress, apoptosis, inflammation, and mitochondrial dysfunction.

Cadmium (Cd) is a highly toxic heavy metal that can be found everywhere in the environment and can have harmful effects on both human and animal health. Pinostrobin (PSB) is a bioactive natural flavonoid isolated from Boesenbergia rotunda with several pharmacological properties, such as antiinflammatory, anticancer, antioxidant, and antiviral. This investigation was intended to assess the therapeutic potential of PSB against Cd-induced kidney damage in rats. In total, 48 Sprague Dawley rats were divided into four groups: a control, a Cd (5 mg/kg), a Cd + PSB group (5 mg/kg Cd and 10 mg/kg PSB), and a PSB group (10 mg/kg) that received supplementation for 30 days. Exposure to Cd led to a decrease in the activities of catalase (CAT), glutathione reductase (GSR), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX), whereas levels of reactive oxygen species (ROS) and malondialdehyde (MDA) increased. Cd exposure also caused a substantial increase in urea, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and creatinine levels. Moreover, a noticeable decline was noticed in creatinine clearance. Moreover, Cd exposure considerably increased the levels of inflammatory indices, including interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), nuclear factor kappa-B (NF-kB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) activity. Cd treatment decreased the expression of the antiapoptotic markers (Bcl-2) while increasing the expression of apoptotic markers (Bax and Caspase-3). Furthermore, Cd treatment substantially reduced the TCA cycle enzyme activity, such as alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase. Moreover, mitochondrial electron transport chain enzymes, succinatedehydrogenase, NADH dehydrogenase, cytochrome c-oxidase, and coenzyme Q-cytochrome reductase activities were also decreased following Cd exposure. PSB administration substantially reduced the mitochondrial membrane potential while inducing significant histological damage. However, PSB treatment significantly reduced Cd-mediated renal damage in rats. Thus, the present investigation discovered that PSB has ameliorative potential against Cd-induced renal dysfunction in rats.

Abstract 1956: Stress-mediated reactive oxygen species from mitochondria dysregulates the tumor microenvironment in prostate cancer of African American men

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in men in United States. African American men (AAM) have higher rates of PCa incidence and mortality compared with European American men (EAM). While socioeconomics, access to optimal care and life-style factors contribute towards these disparities, underlying biologic differences in AAM remain important drivers of PCa disparities. In the current work, we aimed to explore the interplay between oxidative stress and inflammatory signaling in promoting progression of PCa in AAM. Oxidative stress results from excessive reactive oxygen species (ROS) generated mainly via membrane bound oxidases, peroxisomes or the mitochondrial electron transport chain (ETC). We performed a robust comparative genomics to determine whether there is a race dependent expression of genes related to ROS in publicly available PCa datasets. We then prospectively examined the differential gene expression of these genesets in PCa from clinically matched AAM and EAM in the VANDAAM prospective study. VANDAAM is a validation study of DecipherTM genomic testing in 243 men with localized PCa. We then combined computational and experimental approaches to explore the molecular mechanisms and consequences of exacerbated oxidative stress. We found that ETC complex I, III related genes are the most differentially expressed in AAM compared with EAM. To examine whether AAM vs EAM cell lines exhibits differential sensitivity to ETC modulation, we blocked key ETC enzymes using complex I inhibitor, rotenone, and complex III inhibitor, antimycin A. We used 2 types of mitochondria-specific labels that distinguish respiring and ROS-generating mitochondria. While the relative levels of respiring mitochondria remain constant, we found that antimycin A induced higher level of mitochondrial ROS in AAM PCa cell lines compared with EAM PCa cell lines. To understand the interplay between mitochondrial ETC complex I & III and immune microenvironment, we computed the correlational discordance of ETC complex I & III expression scores and expression of >1200 immune-related genes between AAM vs EAM in VANDAAM cohort. These analyses suggest that ETC complex I & III correlate with several immunosuppressive genes including the macrophage markers CD68 and CD163 and T cell exhaustion markers. In addition, intersecting discordant genes from several datasets suggests enrichment of inflammatory cytokines and type I interferon signature which we then validated using ex vivo culture of PCa tissues. In summary, we discovered a role of mitochondrial electron transport chain in regulating inflammatory circuits in PCa of AAM. Unraveling roles of mitochondria in promoting immune suppression will have significant impact on understanding the intersection between social pressures and biological stress responses. Citation Format: Asmaa El-Kenawi, Ryan Putney, Shivanshu Awasthi, Amparo Serna, Corrado Caslini, Jasreman Dhillon, Kosj Yamoah. Stress-mediated reactive oxygen species from mitochondria dysregulates the tumor microenvironment in prostate cancer of African American men [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1956.

Switching ubiquitous and muscle-specific isoforms of mitochondrial respiratory complex IV in skeletal muscle fine-tunes complex IV activity.

Respiratory complex IV (CIV, cytochrome c oxidase) is the terminal enzyme of the mitochondrial electron transport chain. Some CIV subunits have two or more isoforms, which are ubiquitously expressed or are expressed in specific tissues like the lung, muscle, and testis. Among the tissue-specific CIV isoforms, the muscle-specific isoforms are expressed in adult cardiac and skeletal muscles. To date, the physiological and biochemical association between the muscle-specific CIV isoforms and aerobic respiration in muscles remains unclear. In this study, we profiled the CIV organization and expression pattern of muscle-specific CIV isoforms in different mouse muscle tissues. We found extensive CIV-containing supramolecular organization in murine musculature at advanced developmental stages, while a switch in the expression from ubiquitous to muscle-specific isoforms of CIV was also detected. Such a switch was confirmed during the in vitro differentiation of mouse C2C12 myoblasts. Unexpectedly, a CIV expression decrease was observed during C2C12 differentiation, which was probably due to a small increase in the expression of muscle-specific isoforms coupled with a dramatic decrease in the ubiquitous isoforms. We also found that the enzymatic activity of CIV containing the muscle-specific isoform COX6A2 was higher than that with COX6A1 in engineered HEK293T cells. Overall, our results indicate that switching the expression from ubiquitous to muscle-specific CIV isoforms is indispensable for optimized oxidative phosphorylation in mature skeletal muscles. We also note that the in vitro C2C12 differentiation model is not suitable for the study of muscular aerobic respiration due to insufficient expression of muscle-specific CIV isoforms.

Ripretinib induced skeletal muscle toxicity through mitochondrial impairment in C2C12 myotubes

Ripretinib is a multikinase inhibitor drug approved in 2020 by the FDA and in 2021 by EMA for use in the treatment of advanced gastrointestinal stromal tumors (GIST) which have not adequately responded to previous treatments with kinase inhibitors. The most common side effects of the drug are myalgia and fatigue, which likely causes interruption of the treatment or reduction of the dose. Skeletal muscle cells highly depend on ATP to perform their functions and mitochondrial damage may play a role in skeletal muscle toxicity induced by kinase inhibitors. However, the molecular mechanism has not been clearly identified in the literature yet. In this study, it has been aimed to elucidate the role of mitochondria in the toxic effect of ripretinib on skeletal muscle using the mouse C2C12 myoblast-derived myotubes. The myotubes were exposed to ripretinib at the range of 1–20 μM concentrations for 24 h. To determine the potential role of mitochondrial impairment in ripretinib-induced skeletal muscle toxicity, intracellular ATP level, mitochondrial membrane potential (MMP), mitochondrial ROS production (mtROS), mitochondrial DNA (mtDNA) copy number, and mitochondrial mass were examined after ripretinib treatment. Furthermore, changes in PGC 1α/NRF 1/NRF 2 expression levels that play a role in mitochondrial biogenesis and mitophagy were investigated. Additionally, the mitochondrial electron transport chain (ETC) enzyme activities were evaluated. Lastly, a molecular docking study was done to see ripretinib’s possible interaction with DNA polymerase gamma (POLG) which is important for DNA replication in the mitochondria. According to the findings, ripretinib decreases the ATP level and mtDNA copy number, induces loss of MMP, and reduces mitochondrial mass. The activities of the ETC complexes were inhibited with ripretinib exposure which is in line with the observed ATP depletion and MMP loss. The molecular docking study revealed that ripretinib has inhibitory potential against POLG which supports the observed inhibition of mtDNA. The expression of PGC 1α was reduced in the nuclear fraction indicating that PGC-1α was not activated since the NRF 1 expression was reduced and NRF 2 level did not show significant change. Consequently, mtROS production increased in all treatment groups and mitophagy-related gene expressions and Parkin protein expression level were up-regulated at high doses. In conclusion, mitochondrial damage/loss can be one of the underlying causes of ripretinib-induced skeletal muscle toxicity. However, further studies are needed to confirm the results in vivo.

Microarrays, Enzymatic Assays, and MALDI-MS for Determining Specific Alterations to Mitochondrial Electron Transport Chain Activity, ROS Formation, and Lipid Composition in a Monkey Model of Parkinson’s Disease

Multiple evidences suggest that mitochondrial dysfunction is implicated in the pathogenesis of Parkinson's disease via the selective cell death of dopaminergic neurons, such as that which occurs after prolonged exposure to the mitochondrial electron transport chain (ETC) complex I inhibitor, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP). However, the effects of chronic MPTP on the ETC complexes and on enzymes of lipid metabolism have not yet been thoroughly determined. To face these questions, the enzymatic activities of ETC complexes and the lipidomic profile of MPTP-treated non-human primate samples were determined using cell membrane microarrays from different brain areas and tissues. MPTP treatment induced an increase in complex II activity in the olfactory bulb, putamen, caudate, and substantia nigra, where a decrease in complex IV activity was observed. The lipidomic profile was also altered in these areas, with a reduction in the phosphatidylserine (38:1) content being especially relevant. Thus, MPTP treatment not only modulates ETC enzymes, but also seems to alter other mitochondrial enzymes that regulate the lipid metabolism. Moreover, these results show that a combination of cell membrane microarrays, enzymatic assays, and MALDI-MS provides a powerful tool for identifying and validating new therapeutic targets that might accelerate the drug discovery process.

Recessive pathogenic variants in MCAT cause combined oxidative phosphorylation deficiency.

Malonyl-CoA-acyl carrier protein transacylase (MCAT) is an enzyme involved in mitochondrial fatty acid synthesis (mtFAS) and catalyzes the transfer of the malonyl moiety of malonyl-CoA to the mitochondrial acyl carrier protein (ACP). Previously, we showed that loss-of-function of mtFAS genes, including Mcat, is associated with severe loss of electron transport chain (ETC) complexes in mouse immortalized skeletal myoblasts (Nowinski et al., 2020). Here, we report a proband presenting with hypotonia, failure to thrive, nystagmus, and abnormal brain MRI findings. Using whole exome sequencing, we identified biallelic variants in MCAT. Protein levels for NDUFB8 and COXII, subunits of complex I and IV respectively, were markedly reduced in lymphoblasts and fibroblasts, as well as SDHB for complex II in fibroblasts. ETC enzyme activities were decreased in parallel. Re-expression of wild-type MCAT rescued the phenotype in patient fibroblasts. This is the first report of a patient with MCAT pathogenic variants and combined oxidative phosphorylation deficiency.

Mycobacterium tuberculosis protein MoxR1 enhances virulence by inhibiting host cell death pathways and disrupting cellular bioenergetics

ABSTRACT Mycobacterium tuberculosis (M. tb) utilizes the multifunctionality of its protein factors to deceive the host. The unabated global incidence and prevalence of tuberculosis (TB) and the emergence of multidrug-resistant strains warrant the discovery of novel drug targets that can be exploited to manage TB. This study reports the role of M. tb AAA+ family protein MoxR1 in regulating host-pathogen interaction and immune system functions. We report that MoxR1 binds to TLR4 in macrophage cells and further reveal how this signal the release of proinflammatory cytokines. We show that MoxR1 activates the PI3K-AKT-MTOR signalling cascade by inhibiting the autophagy-regulating kinase ULK1 by potentiating its phosphorylation at serine 757, leading to its suppression. Using autophagy-activating and repressing agents such as rapamycin and bafilomycin A1 suggested that MoxR1 inhibits autophagy flux by inhibiting autophagy initiation. MoxR1 also inhibits apoptosis by suppressing the expression of MAPK JNK1/2 and cFOS, which play critical roles in apoptosis induction. Intriguingly, MoxR1 also induced robust disruption of cellular bioenergetics by metabolic reprogramming to rewire the citric acid cycle intermediates, as evidenced by the lower levels of citric acid and electron transport chain enzymes (ETC) to dampen host defence. These results point to a multifunctional role of M. tb MoxR1 in dampening host defences by inhibiting autophagy, apoptosis, and inducing metabolic reprogramming. These mechanistic insights can be utilized to devise strategies to combat TB and better understand survival tactics by intracellular pathogens.

VDAC1 Knockout Affects Mitochondrial Oxygen Consumption Triggering a Rearrangement of ETC by Impacting on Complex I Activity.

Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1) is the most abundant isoform of the outer mitochondrial membrane (OMM) porins and the principal gate for ions and metabolites to and from the organelle. VDAC1 is also involved in a number of additional functions, such as the regulation of apoptosis. Although the protein is not directly involved in mitochondrial respiration, its deletion in yeast triggers a complete rewiring of the whole cell metabolism, with the inactivation of the main mitochondrial functions. In this work, we analyzed in detail the impact of VDAC1 knockout on mitochondrial respiration in the near-haploid human cell line HAP1. Results indicate that, despite the presence of other VDAC isoforms in the cell, the inactivation of VDAC1 correlates with a dramatic impairment in oxygen consumption and a re-organization of the relative contributions of the electron transport chain (ETC) enzymes. Precisely, in VDAC1 knockout HAP1 cells, the complex I-linked respiration (N-pathway) is increased by drawing resources from respiratory reserves. Overall, the data reported here strengthen the key role of VDAC1 as a general regulator of mitochondrial metabolism.

Constitutive Expression of Hif2α Confers Acute O2 Sensitivity to Carotid Body Glomus Cells.

Acute oxygen (O2) sensing and adaptation to hypoxia are essential for physiological homeostasis. The prototypical acute O2 sensing organ is the carotid body, which contains chemosensory glomus cells expressing O2-sensitive K+ channels. Inhibition of these channels during hypoxia leads to cell depolarization, transmitter release, and activation of afferent sensory fibers terminating in the brain stem respiratory and autonomic centers. Focusing on recent data, here we discuss the special sensitivity of glomus cell mitochondria to changes in O2 tension due to Hif2α-dependent expression of several atypical mitochondrial electron transport chain subunits and enzymes. These are responsible for an accelerated oxidative metabolism and the strict dependence of mitochondrial complex IV activity on O2 availability. We report that ablation of Epas1 (the gene coding Hif2α) causes a selective downregulation of the atypical mitochondrial genes and a strong inhibition of glomus cell acute responsiveness to hypoxia. Our observations indicate that Hif2α expression is required for the characteristic metabolic profile of glomus cells and provide a mechanistic explanation for the acute O2 regulation of breathing.

Inhibition of hepatic energy metabolizing enzymes in murine model exposed to diisononyl phthalate

Background and objectives: Diisononyl phthalate (DINP) is a class of phthalates and phthalates are known to be metabolism disrupting chemicals (MDCs). Numerous MDCs, to which humans are exposed, have an effect on every aspect of energy transduction. They affect the liver by impairing insulin secretion in pancreatic cells and altering the liver’s insulin-dependent glucose metabolism. Methods: For this study, eighteen male albino rats weighing 200±20g were randomly assigned to three groups (of six rats each) and followed for a 14-days period. The groups were: group A or control which was given Tween-80 orally, group B or DINP1 group which was given 20 mg/kg b.wt. DINP, and Group C or DINP2 group which received 200 mg/kg b.wt. DINP. The rats were then sacrificed, their livers were removed, and the glycolytic and oxidative phosphorylation enzyme activities were evaluated. Results: Activities of the glycolytic, tricarboxylic acid cycle and electron transport chain enzymes under investigation were significantly down-regulated with severity observed in decreased activities of hepatic oxidative phosphorylation enzymes when compared with control (P<0.05). Hepatic tissue sections of 20 and 200mg/kg DiNP group revealed distorted cytoarchitecture of hepatocytes ranging from histocellular disarrangement to vaocular changes suggestive of loss of liver integrity or fibrosis. Conclusions: Finally, DINP exposure impairs hepatic energy transduction enzymes as evident in down-regulation of the various enzymes of energy metabolism under investigation and this may invariably be a good tool for the diagnosis of hepatic energy impairment as seen in some disease conditions.

Effect of geranylated dihydrochalcone from Artocarpus altilis leaves extract on Plasmodium falciparum ultrastructural changes and mitochondrial malate: Quinone oxidoreductase

Nearly half of the world's population is at risk of being infected by Plasmodium falciparum, the pathogen of malaria. Increasing resistance to common antimalarial drugs has encouraged investigations to find compounds with different scaffolds. Extracts of Artocarpus altilis leaves have previously been reported to exhibit in vitro antimalarial activity against P. falciparum and in vivo activity against P. berghei. Despite these initial promising results, the active compound from A. altilis is yet to be identified. Here, we have identified 2-geranyl-2′, 4′, 3, 4-tetrahydroxy-dihydrochalcone (1) from A. altilis leaves as the active constituent of its antimalarial activity. Since natural chalcones have been reported to inhibit food vacuole and mitochondrial electron transport chain (ETC), the morphological changes in food vacuole and biochemical inhibition of ETC enzymes of (1) were investigated. In the presence of (1), intraerythrocytic asexual development was impaired, and according to the TEM analysis, this clearly affected the ultrastructure of food vacuoles. Amongst the ETC enzymes, (1) inhibited the mitochondrial malate: quinone oxidoreductase (PfMQO), and no inhibition could be observed on dihydroorotate dehydrogenase (DHODH) as well as bc1 complex activities. Our study suggests that (1) has a dual mechanism of action affecting the food vacuole and inhibition of PfMQO-related pathways in mitochondria.