Subunit Of Mitochondrial Complex Research Articles

Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans

Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20°C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25°C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856>N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856>N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear–mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism.

Hydroxytyrosol prevents diet-induced metabolic syndrome and attenuates mitochondrial abnormalities in obese mice

A Mediterranean diet rich in olive oil has profound influence on health outcomes including metabolic syndrome. However, the active compound and detailed mechanisms still remain unclear. Hydroxytyrosol (HT), a major polyphenolic compound in virgin olive oil, has received increased attention for its antioxidative activity and regulation of mitochondrial function. Here, we investigated whether HT is the active compound in olive oil exerting a protective effect against metabolic syndrome. In this study, we show that HT could prevent high-fat-diet (HFD)-induced obesity, hyperglycemia, hyperlipidemia, and insulin resistance in C57BL/6J mice after 17 weeks supplementation. Within liver and skeletal muscle tissues, HT could decrease HFD-induced lipid deposits through inhibition of the SREBP-1c/FAS pathway, ameliorate HFD-induced oxidative stress by enhancing antioxidant enzyme activities, normalize expression of mitochondrial complex subunits and mitochondrial fission marker Drp1, and eventually inhibit apoptosis activation. Moreover, in muscle tissue, the levels of mitochondrial carbonyl protein were decreased and mitochondrial complex activities were significantly improved by HT supplementation. In db/db mice, HT significantly decreased fasting glucose, similar to metformin. Notably, HT decreased serum lipid, at which metformin failed. Also, HT was more effective at decreasing the oxidation levels of lipids and proteins in both liver and muscle tissue. Similar to the results in the HFD model, HT decreased muscle mitochondrial carbonyl protein levels and improved mitochondrial complex activities in db/db mice. Our study links the olive oil component HT to diabetes and metabolic disease through changes that are not limited to decreases in oxidative stress, suggesting a potential pharmaceutical or clinical use of HT in metabolic syndrome treatment.

Fifteen novel mutations in the mitochondrial NADH dehydrogenase subunit 1, 2, 3, 4, 4L, 5 and 6 genes from Iranian patients with Leber’s hereditary optic neuropathy (LHON)

Leber's hereditary optic neuropathy (LHON) is an optic nerve dysfunction resulting from mutations in mitochondrial DNA (mtDNA), which is transmitted in a maternal pattern of inheritance. It is caused by three primary point mutations: G11778A, G3460A and T14484C; in the mitochondrial genome. These mutations are sufficient to induce the disease, accounting for the majority of LHON cases, and affect genes that encode for the different subunits of mitochondrial complexes I and III of the mitochondrial respiratory chain. Other mutations are secondary mutations associated with the primary mutations. The purpose of this study was to determine MT-ND variations in Iranian patients with LHON. In order to determine the prevalence and distribution of mitochondrial mutations in the LHON patients, their DNA was studied using PCR and DNA sequencing analysis. Sequencing of MT-ND genes from 35 LHON patients revealed a total of 44 nucleotide variations, in which fifteen novel variations-A14020G, A13663G, C10399T, C4932A, C3893G, C10557A, C12012A, C13934T, G4596A, T12851A, T4539A, T4941A, T13255A, T14353C and del A 4513-were observed in 27 LHON patients. However, eight patients showed no variation in the ND genes. These mutations contribute to the current database of mtDNA polymorphisms in LHON patients and may facilitate the definition of disease-related mutations in human mtDNA. This research may help to understand the disease mechanism and open up new diagnostic opportunities for LHON.

Accessory subunits of mitochondrial complex I

Mitochondrial complex I has a molecular mass of almost 1 MDa and comprises more than 40 polypeptides. Fourteen central subunits harbour the bioenergetic core functions. We are only beginning to understand the significance of the numerous accessory subunits. The present review addresses the role of accessory subunits for assembly, stability and regulation of complex I and for cellular functions not directly associated with redox-linked proton translocation.

Nitric oxide quenches the fire in heart mitochondria

During resuscitation after cardiac arrest, a burst of reactive oxygen species (ROS) generated in mitochondria triggers a lethal cascade of events. Nitric oxide is known to be protective, but the mechanism is unknown. A new study shows that a mitochondria-targeted nitric oxide donor S-nitrosates the ND3 subunit of mitochondrial complex I, limiting its ability to generate ROS and protecting the heart against injury (pages 753–759 ).

Mitochondrial UQCRB regulates VEGFR2 signaling in endothelial cells

Vascular endothelial growth factor (VEGF) signal transduction is involved in tumor angiogenesis, and the inhibition of this pathway is considered to be a powerful strategy for cancer therapy. We previously showed that small molecules targeting the ubiquinol-cytochrome c reductase binding protein (UQCRB), a subunit of mitochondrial complex III, in tumor cells suppress hypoxia-induced tumor angiogenesis. However, the mechanism by which UQCRB functioned remained unknown. In the present study, we demonstrate in endothelial cells (ECs) that UQCRB enhances VEGF receptor type 2 (VEGFR2) signaling by increasing the levels of mitochondrial reactive oxygen species (ROS). By contrast, terpestacin, a UQCRB targeting small molecule, blocked mitochondrial ROS-mediated VEGFR2 signaling pathways in ECs, thereby suppressing VEGF-dependent angiogenesis in vitro and in vivo. Furthermore, treatment with both terpestacin and bevacizumab, a VEGF signaling inhibitor, resulted in additive inhibition of tumor-induced angiogenesis both in vitro and in vivo. These data demonstrate that mitochondrial UQCRB positively regulates VEGFR2 signaling in ECs and the UQCRB targeting agent could be applied in new therapeutic approaches for human cancer. Inhibiting angiogenesis has been a focus for anti-cancer strategies. Mitochondrial UQCRB enhances VEGFR2 signaling by increasing ROS in endothelial cells. UQCRB inhibitor blocks angiogenesis by suppressing mitochondrial ROS. Findings may provide a new therapeutic approach for human cancer.

Suppression of mitochondrial complex I influences cell metastatic properties.

Despite the fact that mitochondrial dysfunction has an important role in tumorigenesis and metastasis, the underlying mechanism remains to be elucidated. Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) is the first and the largest protein complex of the mitochondrial electron-transport chain (ETC),which has an essential role in maintaining mitochondrial function and integrity. In this study, we separately knocked down two subunits of mitochondrial complex I, GRIM-19 or NDUFS3, and investigated their effects on metastatic behaviors and explored the possible mechanisms. Our data showed that stable down-modulation of GRIM-19 or NDUFS3 decreased complex I activity and reactive oxygen species (ROS) production; led to enhanced cell adhesion, migration, invasion, and spheroid formation; and influenced the expressions of extracellular matrix (ECM) molecules and its related proteins. We also observed that the expressions of GRIM-19, NDUFS3, and ECM elements were correlated with invasive capabilities of breast cancer cell lines. These results suggest that inhibition of complex I affects metastatic properties of cancer cells, and mitochondrial ROS might play a crucial role in these processes by regulating ECM.

Functional inhibition of UQCRB suppresses angiogenesis in zebrafish

As a subunit of mitochondrial complex III, UQCRB plays an important role in complex III stability, electron transport, and cellular oxygen sensing. Herein, we report UQCRB function regarding angiogenesis in vivo with the zebrafish (Danio rerio). UQCRB knockdown inhibited angiogenesis in zebrafish leading to the suppression of VEGF expression. Moreover, the UQCRB-targeting small molecule terpestacin also inhibited angiogenesis and VEGF levels in zebrafish, supporting the role of UQCRB in angiogenesis. Collectively, UQCRB loss of function by either genetic and pharmacological means inhibited angiogenesis, indicating that UQCRB plays a key role in this process and can be a prognostic marker of angiogenesis- and mitochondria-related diseases.

Mitochondrial and nuclear genes as the cause of complex I deficiency

Multiple sclerosis (MS) is an immunological inflammatory disease of the central nervous system. The pathogenesis of MS is incompletely understood, but various studies have suggested that mitochondrial dysfunction is associated with the disease. Mitochondria are among the main cellular sources of reactive oxygen and nitrogen species, and they play a pivotal role in many neuro-pathological conditions. The mitochondrial nuclear subunit of complex I gene in mitochondria may play a role in MS, and understanding this role may provide rationale for novel approaches to treatment of the disease and the development of novel therapies. We designed a molecular study to demonstrate biochemical defects in complex I activity and found some novel nucleotide substitutions in mitochondrial DNA that might be involved in the pathogenesis of MS. The mitochondrial complex subunit I sequence was amplified and sequenced in MS patients. Although no reported pathogenic mutations were found in these patients, other studies have clearly indicated that the mitochondrial nuclear complex subunit I gene plays a significant role in MS pathogenesis.

Abstract 19466: Metabolomic and Gene Expression Changes in End-Stage Heart Failure are Reversed by Left Ventricular Assist Device

Introduction: Heart muscle tissue in advanced heart failure (HF) is reported to be ‘energy-depleted’ and inflexible to metabolic changes. Left ventricular assist devices (LVAD) and ventricular unloading with LVAD is known to regress cardiac hypertrophy, reduce fibrosis and partially improve function. The metabolic alterations with HF and LVAD support are not fully understood. We hypothesize that HF is associated with metabolic changes that are reversible with LVAD placement. Methods: We analyzed gene and protein expression and performed metabolomics analyses in failing left ventricular (LV) tissue obtained at the time of heart explant or LVAD placement (HF, n = 13) and removal (post-LVAD, n = 7). Control LV tissue was procured from patients undergoing heart-lung transplantation for severe right HF (control, n = 4). P<0.05 was considered significant. Results: In HF samples, expression levels of mitochondrial transcription factors and coactivators were decreased, while LVAD increased expression of PGC1α, PGC1β, ERRγ and TFAM to levels of control samples. Gene and protein expression of mitochondrial complex subunits NDUFS6, SDHA, UQCRC1, Cox5a, ATP5a1 were reduced in HF, but increased after LVAD. LVAD rescued reduced expression of fatty acid oxidation genes CPT2, ACADvl, ACADm, HADHA and PPARα, as well as pyruvate and glucose metabolism genes ME3, MCT1, PDHB, GPT and PFK. Targeted metabolomics analysis showed a reduction in short- and medium-chain acylcarnitines (C2-C10) in failing LV indicating decreased substrate catabolism, with these analytes universally restored to normal post-LVAD. Pyruvate levels tended to increase (P=0.07), whereas all measured Krebs cycle intermediates including citrate, α-ketoglutarate, succinate, fumarate and malate were reduced in the HF samples compared to normal controls. LVAD normalized pyruvate levels, and normalized the levels of all Krebs cycle intermediates. Conclusion: Both the gene expression and metabolomics profiles for mitochondrial, fatty acid, pyruvate and glucose metabolism pathways indicate that HF is associated with a global defect in substrate utilization. Complete reversal by ventricular unloading demonstrates the reversibility of metabolic defects.

Model animals for the study of oxidative stress from complex II

Mitochondria play a role of energy production and produce intracellular reactive oxygen species (ROS), especially superoxide anion (O2−) as a byproduct of energy metabolism at the same time. O2− is converted from oxygen and is overproduced by excessive electron leakage from the mitochondrial respiratory chain. It is well known that mitochondrial complexes I and III in the electron transport system are the major endogenous ROS sources. We have previously demonstrated that mutations in complex II can result in excessive ROS (specifically in SDHC: G71E in Caenorhabditis elegans, I71E in Drosophila and V69E in mouse). Moreover, this results in premature death in C. elegans and Drosophila as well as tumorigenesis in mouse embryonic fibroblast cells. In humans, it has been reported that mutations in SDHB, SDHC or SDHD, which are the subunits of mitochondrial complex II, often result in inherited head and neck paragangliomas (PGLs). Recently, we established Tet-mev-1 conditional transgenic mice using our uniquely developed Tet-On/Off system, which can induce the mutated SDHC gene to be equally and competitively expressed compared to the endogenous wild-type SDHC gene. These mice experienced mitochondrial respiratory chain dysfunction that resulted in oxidative stress. The mitochondrial oxidative stress caused excessive apoptosis in several tissues leading to low-birth-weight infants and growth retardation during neonatal developmental phase in Tet-mev-1 mice. Tet-mev-1 mice also displayed precocious age-dependent corneal physiological changes, delayed corneal epithelialization, decreased corneal endothelial cells, thickened Descemet's membrane and thinning of parenchyma with corneal pathological dysfunctions such as keratitis, Fuchs' corneal dystrophy (FCD) and probably keratoconus after the normal development and growth phase. Here, we review the relationships between mitochondrial oxidative stress and phenomena in mev-1 animal models with mitochondrial complex II SDHC mutations. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease.

Journal-related Activities and Other Special Activities at the 2012 American Society of Anesthesiologists Annual Meeting

Journal-related Activities and Other Special Activities at the 2012 American Society of Anesthesiologists Annual Meeting

Function of DJ-1 in Mitochondria

Parkinson's disease is a degenerative disorder of the central nervous system caused by selective dopamine-generating cell death, and oxidative stress and mitochondrial dysfunction are thought to be responsible for the onset of Parkinson's disease. While most cases of Parkinson's disease are idiopathic, 5-10% of cases are attributed to genetic factors. DJ-1 was first identified as an activated ras-dependent oncogene and later found to be a causative gene for a familial form of Parkinson's disease, PARK7. We and others found that DJ-1 plays roles in transcriptional regulation and anti-oxidative stress function, and loss of its function is thought to affect the onset of Parkinson's disease. DJ-1 is mainly located in the cytoplasma and nucleus and partially in mitochondria. When mice or mouse cells were treated with bisphenol A, an endocrine disruptor and inducer of reactive oxygen species, DJ-1 was translocated into mitochondria to maintain mitochondrial complex I activity. We also found that DJ-1 directly bound to and was co-localized with NDUFA4 and ND1, nuclear and mitochondrial DNA-encoding subunits of mitochondrial complex I, respectively, and that these associations were enhanced by oxidative stress. Furthermore, complex I activity was reduced in two types of DJ-1-knockdown NIH3T3 and HEK293 cells. These findings suggest that DJ-1 is an integral mitochondrial protein and maintains mitochondrial complex I activity to regulate mitochondrial homeostasis.

Specific hypersensitivity to volatile anesthetics in a mouse lacking Ndufs4, a subunit of mitochondrial complex I

Specific hypersensitivity to volatile anesthetics in a mouse lacking Ndufs4, a subunit of mitochondrial complex I

Altered Anesthetic Sensitivity of Mice Lacking Ndufs4, a Subunit of Mitochondrial Complex I

Anesthetics are in routine use, yet the mechanisms underlying their function are incompletely understood. Studies in vitro demonstrate that both GABAA and NMDA receptors are modulated by anesthetics, but whole animal models have not supported the role of these receptors as sole effectors of general anesthesia. Findings in C. elegans and in children reveal that defects in mitochondrial complex I can cause hypersensitivity to volatile anesthetics. Here, we tested a knockout (KO) mouse with reduced complex I function due to inactivation of the Ndufs4 gene, which encodes one of the subunits of complex I. We tested these KO mice with two volatile and two non-volatile anesthetics. KO and wild-type (WT) mice were anesthetized with isoflurane, halothane, propofol or ketamine at post-natal (PN) days 23 to 27, and tested for loss of response to tail clamp (isoflurane and halothane) or loss of righting reflex (propofol and ketamine). KO mice were 2.5 - to 3-fold more sensitive to isoflurane and halothane than WT mice. KO mice were 2-fold more sensitive to propofol but resistant to ketamine. These changes in anesthetic sensitivity are the largest recorded in a mammal.

Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p Paralogs Results in Catalytically Active Succinate Dehydrogenase Isoenzymes

Succinate dehydrogenase (SDH), also known as complex II, is required for respiratory growth; it couples the oxidation of succinate to the reduction of ubiquinone. The enzyme is composed of two domains. A membrane-extrinsic catalytic domain composed of the Sdh1p and Sdh2p subunits harbors the flavin and iron-sulfur cluster cofactors. A membrane-intrinsic domain composed of the Sdh3p and Sdh4p subunits interacts with ubiquinone and may coordinate a b-type heme. In many organisms, including Saccharomyces cerevisiae, possible alternative SDH subunits have been identified in the genome. S. cerevisiae contains one paralog of the Sdh3p subunit, Shh3p (YMR118c), and two paralogs of the Sdh4p subunit, Shh4p (YLR164w) and Tim18p (YOR297c). We cloned and expressed these alternative subunits. Shh3p and Shh4p were able to complement Δsdh3 and Δsdh4 deletion mutants, respectively, and support respiratory growth. Tim18p was unable to do so. Microarray and proteomics data indicate that the paralogs are expressed under respiratory and other more restrictive growth conditions. Strains expressing hybrid SDH enzymes have distinct metabolic profiles that we distinguished by (1)H NMR analysis of metabolites. Surprisingly, the Sdh3p subunit can form SDH isoenzymes with Sdh4p or with Shh4p as well as be a subunit of the TIM22 mitochondrial protein import complex.

Uncovering the biological function of UQCRB, a terpestacin‐binding mitochondrial protein, implies its pro‐angiogenic activity in vitro and in vivo

Recently, we identified ubiquinol‐cytochrome c reductase binding protein (UQCRB), one of subunits of mitochondrial Complex III, as a target protein of the anti‐angiogenic natural small molecule, terpestacin. The Complex III of mitochondrial respiratory chain has been considered as a key regulator in hypoxia‐induced angiogenesis through mitochondria‐derived reactive oxygen species (ROS) involved in O2 sensing. Here, cell permeable UQCRB protein is generated using protein transduction domain (PTD), a small peptide transferring its binding complex into the cell, to reveal the biological function of UQCRB. Consequently, intracellular delivery of PTD‐UQCRB fusion protein enhances generation of mitochondrial ROS and HIF‐1α stability. Also, PTD‐UQCRB transduction induces vascular endothelial growth factor (VEGF) expression and invasion of human umbilical vascular endothelial cells (HUVECs) in vitro. Furthermore, trans‐membrane delivery of PTD‐UQCRB enhances wound healing in vivo. These results provide new insights into the function of PTD‐UQCRB in angiogenesis via mitochondria mediated ROS generation and also open new basis on application of PTD‐UQCRB as a pro‐angiogenic agent via regulating mitochondrial function. This study is supported by self‐research funding.

A Single Nucleotide Polymorphism Associates With the Response of Muscle ATP Synthesis to Long-Term Exercise Training in Relatives of Type 2 Diabetic Humans

OBJECTIVEMyocellular ATP synthesis (fATP) associates with insulin sensitivity in first-degree relatives of subjects with type 2 diabetes. Short-term endurance training can modify their fATP and insulin sensitivity. This study examines the effects of moderate long-term exercise using endurance or resistance training in this cohort.RESEARCH DESIGN AND METHODSA randomized, parallel-group trial tested 16 glucose-tolerant nonobese relatives (8 subjects in the endurance training group and 8 subjects in the resistance training group) before and after 26 weeks of endurance or resistance training. Exercise performance was assessed from power output and oxygen uptake (Vo2) during incremental tests and from maximal torque of knee flexors (MaxTflex) and extensors (MaxText) using isokinetic dynamometry. fATP and ectopic lipids were measured with 1H/31P magnetic resonance spectroscopy.RESULTSEndurance training increased power output and Vo2 by 44 and 30%, respectively (both P < 0.001), whereas resistance training increased MaxText and MaxTflex by 23 and 40%, respectively (both P < 0.001). Across all groups, insulin sensitivity (382 ± 90 vs. 389 ± 40 mL ⋅ min−1 ⋅ m−2) and ectopic lipid contents were comparable after exercise training. However, 8 of 16 relatives had 26% greater fATP, increasing from 9.5 ± 2.3 to 11.9 ± 2.4 μmol ⋅ mL−1 ⋅ m−1 (P < 0.05). Six of eight responders were carriers of the G/G single nucleotide polymorphism rs540467 of the NDUFB6 gene (P = 0.019), which encodes a subunit of mitochondrial complex I.CONCLUSIONSModerate exercise training for 6 months does not necessarily improve insulin sensitivity but may increase ATP synthase flux. Genetic predisposition can modify the individual response of the ATP synthase flux independently of insulin sensitivity.

GRIM-19 Mediated Translocation of STAT3 to Mitochondria is Necessary for TNF Induced Necroptosis.

Tumor necrosis factor (TNF) can induce necroptosis, wherein inhibition of caspase activity prevents apoptosis but initiates an alternative programmed necrosis. The activity of receptor-interacting serine/threonine-protein kinase 1 (RIPK-1) is required for necroptosis to proceed, with suppression of RIPK-1 expression or inhibition of RIPK-1 activity with necrostatin-1 preventing TNF-induced necroptosis. Downstream from the TNF receptor, the generation of reactive oxygen species at the mitochondria has been identified as necessary for the execution of necroptosis; with antioxidants and inhibitors of mitochondrial complex I preventing TNF-induced cytotoxicity. However, components of the signaling pathway that lie between activated RIPK-1 and the mitochondria are unknown. In the study reported here we demonstrate that during TNF-induced necroptosis, STAT3 is phosphorylated on serine 727, which is dependent on RIPK-1 expression or activity. The phosphorylation of STAT3 induces interaction with GRIM-19, a subunit of mitochondrial complex I, with a resultant translocation of STAT3 to the mitochondria, where it induces an increase in reactive oxygen species production and cell death.

Mitochondrial and liver oxidative stress alterations induced by N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine: relevance for hepatotoxicity

The most significant toxicological effect of nitrosamines like N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is their carcinogenic activity, which may result from exposure to a single large dose or from chronic exposure to relatively small doses. However, its effects on mitochondrial liver bioenergetics were never investigated. Liver is the principal organ responsible for BBN metabolic activation, and mitochondria have a central function in cellular energy production, participating in multiple metabolic pathways. Therefore any negative effect on mitochondrial function may affect cell viability. In the present work, ICR male mice were given 0.05% of BBN in drinking water for a period of 12 weeks and were sacrificed one week later. Mitochondrial physiology was characterized in BBN- and control-treated mice. Transmembrane electric potential developed by mitochondria was significantly affected when pyruvate-malate was used, with an increase in state 4 respiration observed for pyruvate-malate (46%) and succinate (38%). A decrease in the contents of one subunit of mitochondrial complex I and in one subunit of mitochondrial complex IV was also observed. In addition, the activity of both complexes I and II was also decreased by BBN treatment. The treatment with BBN increases the susceptibility of liver mitochondria to the opening of the mitochondrial permeability transition pore. This susceptibility could be related with the increase in the production of H2 O2 by mitochondria and increased oxidative stress confirmed by augmented susceptibility to lipid peroxidation. These results lead to the conclusion that hepatic mitochondria are one primary target for BBN toxic action during liver metabolism.