Assembly defects induce oxidative stress in inherited mitochondrial complex I deficiency

Complex I (CI) deficiency is the most common respiratory chain defect representing more than 30% of mitochondrial diseases. CI is an L-shaped multi-subunit complex with a peripheral arm protruding into the mitochondrial matrix and a membrane arm. CI sequentially assembled into main assembly intermediates: the P (pumping), Q (Quinone) and N (NADH dehydrogenase) modules. In this study, we analyzed 11 fibroblast cell lines derived from patients with inherited CI deficiency resulting from mutations in the nuclear or mitochondrial DNA and impacting these different modules. In patient cells carrying a mutation located in the matrix arm of CI, blue native-polyacrylamide gel electrophoresis (BN-PAGE) revealed a significant reduction of fully assembled CI enzyme and an accumulation of intermediates of the N module. In these cell lines with an assembly defect, NADH dehydrogenase activity was partly functional, even though CI was not fully assembled. We further demonstrated that this functional N module was responsible for ROS production through the reduced flavin mononucleotide. Due to the assembly defect, the FMN site was not re-oxidized leading to a significant oxidative stress in cell lines with an assembly defect. These findings not only highlight the relationship between CI assembly and oxidative stress, but also show the suitability of BN-PAGE analysis in evaluating the consequences of CI dysfunction. Moreover, these data suggest that the use of antioxidants may be particularly relevant for patients displaying a CI assembly defect.     

Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts

Respiratory chain complex I (NADH:ubiquinone oxidoreductase) deficiency is one of the most frequent causes of mitochondrial disease in humans. The activity of this complex can be confidently measured in most tissue samples, but not in cultured skin fibroblasts or circulating lymphocytes. Highly contaminating non-mitochondrial NADH-quinone oxidoreductase activity in fibroblasts and the limited access of substrates to complex I in lymphocytes hinder its measurement in permeabilized cells. Complex I assay in these cells requires the isolation of mitochondria, which in turn necessitates large quantities of cells and is not feasible when studying circulating lymphocytes. Here we report a simple method to measure complex I activity in a minute amount of either cell type. The procedure strongly reduces contaminating NADH:quinone oxidoreductase activity and permits measuring high rates of rotenone-sensitive complex I activity thanks to effective cell permeabilization.     

Human mitochondrial complex I assembly: A dynamic and versatile process

One can but admire the intricate way in which biomolecular structures are formed and cooperate to allow proper cellular function. A prominent example of such intricacy is the assembly of the five inner membrane embedded enzymatic complexes of the mitochondrial oxidative phosphorylation (OXPHOS) system, which involves the stepwise combination of > 80 subunits and prosthetic groups encoded by both the mitochondrial and nuclear genomes. This review will focus on the assembly of the most complicated OXPHOS structure: complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3). Recent studies into complex I assembly in human cells have resulted in several models elucidating a thus far enigmatic process. In this review, special attention will be given to the overlap between the various assembly models proposed in different organisms. Complex I being a complicated structure, its assembly must be prone to some form of coordination. This is where chaperone proteins come into play, some of which may relate complex I assembly to processes such as apoptosis and even immunity.     

Isoniazid-induced cell death is precipitated by underlying mitochondrial complex I dysfunction in mouse hepatocytes

Isoniazid (INH) is an antituberculosis drug that has been associated with idiosyncratic liver injury in susceptible patients. The underlying mechanisms are still unclear, but there is growing evidence that INH and/or its major metabolite, hydrazine, may interfere with mitochondrial function. However, hepatic mitochondria have a large reserve capacity, and minor disruption of energy homeostasis does not necessarily induce cell death. We explored whether pharmacologic or genetic impairment of mitochondrial complex I may amplify mitochondrial dysfunction and precipitate INH-induced hepatocellular injury. We found that INH (≤3000 μM) did not induce cell injury in cultured mouse hepatocytes, although it decreased hepatocellular respiration and ATP levels in a concentration-dependent fashion. However, coexposure of hepatocytes to INH and nontoxic concentrations of the complex I inhibitors rotenone (3 μM) or piericidin A (30 nM) resulted in massive ATP depletion and cell death. Although both rotenone and piericidin A increased MitoSox-reactive fluorescence, Mito-TEMPO or N-acetylcysteine did not attenuate the extent of cytotoxicity. However, preincubation of cells with the acylamidase inhibitor bis-p-nitrophenol phosphate provided protection from hepatocyte injury induced by rotenone/INH (but not rotenone/hydrazine), suggesting that hydrazine was the cell-damaging species. Indeed, we found that hydrazine directly inhibited the activity of solubilized complex II. Hepatocytes isolated from mutant Ndufs4^+/− mice, although featuring moderately lower protein expression levels of this complex I subunit in liver mitochondria, exhibited unchanged hepatic complex I activity and were therefore not sensitized to INH. These data indicate that underlying inhibition of complex I, which alone is not acutely toxic, can trigger INH-induced hepatocellular injury.     

Mass spectrometric identification of a novel phosphorylation site in subunit NDUFA10 of bovine mitochondrial complex I

Mitochondrial Complex I (NADH:ubiquinone oxidoreductase) consists of at least 46 subunits. Phosphorylation of the 42-kDa subunit NDUFA10 was recently reported using a novel phosphoprotein stain [Schulenberg et al. (2003) Analysis of steady-state protein phosphorylation in mitochondria using a novel fluorescent phosphosensor dye. J. Biol. Chem. 278, 27251]. Two smaller Complex I phosphoproteins, ESSS and MWFE, and their sites of modification, have since been determined [Chen et al. (2004) The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. 279, 26036]. Here we identify the site of phosphorylation in NDUFA10 from bovine heart mitochondria by tandem mass spectrometry. A single phosphopeptide spanning residues 47-60 was identified and confirmed by synthesis to be (47)LITVDGNICSGKpSK(60), establishing serine-59 as the site of phosphorylation.     

Proteomic approach to characterize mitochondrial complex I from plants

Mitochondrial NADH dehydrogenase complex (complex I) is by far the largest protein complex of the respiratory chain. It is best characterized for bovine mitochondria and known to consist of 45 different subunits in this species. Proteomic analyses recently allowed for the first time to systematically explore complex I from plants. The enzyme is especially large and includes numerous extra subunits. Upon subunit separation by various gel electrophoresis procedures and protein identifications by mass spectrometry, overall 47 distinct types of proteins were found to form part of Arabidopsis complex I. An additional subunit, ND4L, is present but could not be detected by the procedures employed due to its extreme biochemical properties. Seven of the 48 subunits occur in pairs of isoforms, six of which were experimentally proven. Fifteen subunits of complex I from Arabidopsis are specific for plants. Some of these resemble enzymes of known functions, e.g. carbonic anhydrases and l-galactono-1,4-lactone dehydrogenase (GLDH), which catalyzes the last step of ascorbate biosynthesis. This article aims to review proteomic data on the protein composition of complex I in plants. Furthermore, a proteomic re-evaluation on its protein constituents is presented.     

Generator-specific targets of mitochondrial reactive oxygen species

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for “signaling” and “damaging” ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc₁ complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H₂O₂) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.     

Redox cycling of adrenaline and adrenochrome catalysed by mitochondrial Complex I

Complex I in bovine heart submitochondrial particles catalyses the NADH-supported generation of superoxide anion; adrenaline is oxidised by superoxide to adrenochrome that, on its hand, is reduced by Complex I, thus establishing a redox cycle that amplifies the superoxide production. The routes in Complex I for superoxide formation and for adrenochrome reduction appear to be different, since they have a different sensitivity to Complex I inhibitors. The results are discussed in terms of current assays for superoxide detection and of pathologies linked to catecholamine oxidation.     

Xanthohumol induces generation of reactive oxygen species and triggers apoptosis through inhibition of mitochondrial electron transfer chain complex I

Xanthohumol is a prenylflavonoid extracted from hops (Humulus lupulus). It possesses anti-cancer and anti-inflammatory activities in vitro and in vivo, and offers therapeutic benefits for treatment of metabolic syndromes. However, the precise mechanisms underlying its pharmacological effects remain to be elucidated, together with its cellular target. Here, we provide evidence that xanthohumol directly interacts with the mitochondrial electron transfer chain complex I (NADH dehydrogenase), inhibits the oxidative phosphorylation, triggers the production of reactive oxygen species, and induces apoptosis. In addition, we show that as a result of the inhibition of the mitochondrial oxidative phosphorylation, xanthohumol exposure causes a rapid decrease of mitochondrial transmembrane potential. Furthermore, we showed that xanthohumol up-regulates the glycolytic capacity in cells, and thus compensates cellular ATP generation. Dissection of the multiple steps of aerobic respiration by extracellular flux assays revealed that xanthohumol specifically inhibits the activity of mitochondrial complex I, but had little effect on that of complex II, III and IV. Inhibition of complex I by xanthohumol caused the overproduction of reactive oxygen species, which are responsible for the induction of apoptosis in cancer cells. We also found that isoxanthohumol, the structural isomer of xanthohumol, is inactive to cells, suggesting that the reactive 2-hydroxyl group of xanthohumol is crucial for its targeting to the mitochondrial complex I. Together, the remodeling of cell metabolism revealed here has therapeutic potential for the use of xanthohumol.     

Gamma carbonic anhydrase like complex interact with plant mitochondrial complex I

We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtCAL1 and AtCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtCA and AtCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtCAs. All plant species analyzed contain both AtCA and AtCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtCAL sequences show a deviation of functionally important active site residues with respect to CAs but could form active interfaces in the interaction with AtCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.     

L-galactono-1,4-lactone dehydrogenase (GLDH) forms part of three subcomplexes of mitochondrial complex I in Arabidopsis thaliana

L-galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the terminal step of the Smirnoff-Wheeler pathway for vitamin C (l-ascorbate) biosynthesis in plants. A GLDH in gel activity assay was developed to biochemically investigate GLDH localization in plant mitochondria. It previously has been shown that GLDH forms part of an 850-kDa complex that represents a minor form of the respiratory NADH dehydrogenase complex (complex I). Because accumulation of complex I is disturbed in the absence of GLDH, a role of this enzyme in complex I assembly has been proposed. Here we report that GLDH is associated with two further protein complexes. Using native gel electrophoresis procedures in combination with the in gel GLDH activity assay and immunoblotting, two mitochondrial complexes of 470 and 420 kDa were identified. Both complexes are of very low abundance. Protein identifications by mass spectrometry revealed that they include subunits of complex I. Finally, the 850-kDa complex was further investigated and shown to include the complete "peripheral arm" of complex I. GLDH is attached to a membrane domain, which represents a major fragment of the "membrane arm" of complex I. Taken together, our data further support a role of GLDH during complex I formation, which is based on its binding to specific assembly intermediates.     

Dopamine modulates mitochondrial function in viable SH-SY5Y cells possibly via its interaction with complex I: Relevance to dopamine pathology in schizophrenia

Deleterious effects of dopamine (DA) involving mitochondrial dysfunction have an important role in DA-associated neuronal disorders, including schizophrenia and Parkinson's disease. DA detrimental effects have been attributed to its ability to be auto-oxidized to toxic reactive oxygen species. Since, unlike Parkinson's disease, schizophrenia does not involve neurodegenerative processes, we suggest a novel mechanism by which DA impairs mitochondrial function without affecting cell viability. DA significantly dissipated mitochondrial membrane potential (Δψ_m) in SH-SY5Y cells. Bypassing complex I prevented the DA-induced depolarization. Moreover, DA inhibited complex I but not complex II activity in disrupted mitochondria, suggesting complex I participation in DA-induced mitochondrial dysfunction. We further demonstrated that intact mitochondria can accumulate DA in a saturated manner, with an apparent K_m = 122.1 ± 28.6 nM and V_max = 1.41 ± 0.15 pmol/mg protein/min, thereby enabling the interaction between DA and complex I. DA accumulation was an energy and Na⁺-dependent process. The pharmacological profile of mitochondrial DA uptake differed from that of other characterized DA transporters. Finally, relevance to schizophrenia is demonstrated by an abnormal interaction between DA and complex I in schizophrenic patients. These results suggest a non-lethal interaction between DA and mitochondria possibly via complex I, which can better explain DA-related pathological processes observed in non-degenerative disorders, such as schizophrenia.     

Carvedilol inhibits mitochondrial complex I and induces resistance to H2O2-mediated oxidative insult in H9C2 myocardial cells

Carvedilol, a β-adrenoreceptor antagonist with strong antioxidant activity, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Although growing evidences suggest specific effects on mitochondrial metabolism, how carvedilol would exert its overall activity has not been completely disclosed. In the present work we have investigated the impact of carvedilol-treatment on mitochondrial bioenergetic functions and ROS metabolism in H9C2 cells. This analysis has revealed a dose-dependent decrease in respiratory fluxes by NAD-dependent substrates associated with a consistent decline of mitochondrial complex I activity. These changes were associated with an increase in mitochondrial H₂O₂ production, total glutathione and protein thiols content. To evaluate the antioxidant activity of carvedilol, the effect of the exposure of control and carvedilol-pretreated H9C2 cells to H₂O₂ were investigated. The H₂O₂-mediated oxidative insult resulted in a significant decrease of mitochondrial respiration, glutathione and protein thiol content and in an increased level of GSSG. These changes were prevented by carvedilol-pretreatment. A similar protective effect on mitochondrial respiration could be obtained by pre-treatment of the cells with a sub-saturating amount of rotenone, a complex I inhibitor.We therefore suggest that carvedilol exerts its protective antioxidant action both by a direct antioxidant effect and by a preconditioning-like mechanism, via inhibition of mitochondrial complex I.     

Immunocytochemical characterization of the mitochondrially encoded ND1 subunit of complex I (NADH : ubiquinone oxidoreductase) in rat brain

In Parkinson's disease, there is a selective defect in complex I of the electron transfer chain. To better understand complex I and its involvement in neurodegenerative disease, we raised an antibody against a conserved epitope of the human mitochondrially encoded subunit 1 of complex I (ND1). Antibodies were affinity purified and assessed by ELISA, immunoblotting, and immunocytochemistry. Immunoblots of brain homogenates from mouse, rat, and monkey brain showed a single 33-kDa band consistent with the predicted molecular mass of the protein. Subcellular fractionation showed the protein to be enriched in mitochondria. Immunocytochemistry in rat brain revealed punctate labeling in cell bodies and processes of neurons. Immunoreactively generally co-localized with subunit IV of complex IV. In striatum, ND1 immunoreactively was greatly enriched in large cholinergic neurons and neurons containing nitric oxide synthase, two cell populations that are resistant to excitotoxic and metabolic insults. In substantia nigra, many dopaminergic neurons had little ND1 immunoreactivity, which may help to explain their sensitivity to complex I inhibitors. In spinal cord, ND1 immunoreactively was enriched in motor neurons. We conclude that complex I is differentially distributed across brain regions, between neurons and glia, and between types of neurons. This antibody should provide a valuable tool for assessing complex I in normal and pathological conditions.     

Electrostatics of the FeS clusters in respiratory complex I

Respiratory complex I couples the transfer of electrons from NADH to ubiquinone and the translocation of protons across the mitochondrial membrane. A detailed understanding of the midpoint reduction potentials (E_m) of each redox center and the factors which influence those potentials are critical in the elucidation of the mechanism of electron transfer in this enzyme. We present accurate electrostatic interaction energies for the iron-sulfur (FeS) clusters of complex I to facilitate the development of models and the interpretation of experiments in connection to electron transfer (ET) in this enzyme. To calculate redox titration curves for the FeS clusters it is necessary to include interactions between clusters, which in turn can be used to refine E_m values and validate spectroscopic assignments of each cluster. Calculated titration curves for clusters N4, N5, and N6a are discussed. Furthermore, we present some initial findings on the electrostatics of the redox centers of complex I under the influence of externally applied membrane potentials. A means of determining the location of the FeS cofactors within the holo-complex based on electrostatic arguments is proposed. A simple electrostatic model of the protein/membrane system is examined to illustrate the viability of our hypothesis.     

Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria

A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD+/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD+ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD+ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I.     

Modulation of autophagy by the novel mitochondrial complex I inhibitor Authipyrin

Autophagy ensures cellular homeostasis by the degradation of long-lived proteins, damaged organelles and pathogens. This catabolic process provides essential cellular building blocks upon nutrient deprivation. Cellular metabolism, especially mitochondrial respiration, has a significant influence on autophagic flux, and complex I function is required for maximal autophagy. In Parkinson’s disease mitochondrial function is frequently impaired and autophagic flux is altered. Thus, dysfunctional organelles and protein aggregates accumulate and cause cellular damage. In order to investigate the interdependency between mitochondrial function and autophagy, novel tool compounds are required. Herein, we report the discovery of a structurally novel autophagy inhibitor (Authipyrin) using a high content screening approach. Target identification and validation led to the discovery that Authipyrin targets mitochondrial complex I directly, leading to the potent inhibition of mitochondrial respiration as well as autophagy.     

A mitochondrial DNA variant associated with left ventricular hypertrophy in diabetes

Diabetes was reported to be associated with a mitochondrial (mt) DNA mutation at 3243 and variants at 1310, 1438, 3290, 3316, 3394, 12,026, 15,927, and 16,189. Among these mtDNA abnormalities, those at 3243, 3316, 15,927, and 16,189 were also suggested to cause cardiomyopathies. We investigated the prevalence of such mtDNA abnormalities in 68 diabetic patients with LV hypertrophy (LVH), 100 without LVH, and 100 controls. Among the 9 mtDNA abnormalities, those at 3243, 3316, and 15,927 tended to be more prevalent in diabetic patients with LVH than in those without LVH (1%, 1%, and 4% vs. 0%, 0%, and 0%). Notably, the variant at 16,189 was more prevalent in diabetic patients with LVH than without LVH (46% vs. 24%, [Formula: see text] ). The odds ratio for LVH was 3.0 (95% CI, 1.5-6.1) for the 16,189 variant. A common mtDNA variant at 16,189 was found to be associated with LVH in diabetic patients.     

The NDUFB6 subunit of the mitochondrial respiratory chain complex I is required for electron transfer activity: A proof of principle study on stable and controlled RNA interference in human cell lines

Molecular bases of inherited deficiencies of mitochondrial respiratory chain complex I are still unknown in a high proportion of patients. Among 45 subunits making up this large complex, more than half has unknown function(s). Understanding the function of these subunits would contribute to our knowledge on mitochondrial physiology but might also reveal that some of these subunits are not required for the catalytic activity of the complex. A direct consequence of this finding would be the reduction of the number of candidate genes to be sequenced in patients with decreased complex I activity. In this study, we tested two different methods to stably extinct complex I subunits in cultured cells. We first found that lentivirus-mediated shRNA expression frequently resulted in the unpredicted extinction of additional gene(s) beside targeted ones. This can be ascribed to uncontrolled genetic material insertions in the genome of the host cell. This approach thus appeared inappropriate to study unknown functions of a gene. Next, we found it possible to specifically extinct a CI subunit gene by direct insertion of a miR targeting CI subunits in a Flp site (HEK293 Flp-In cells). By using this strategy we unambiguously demonstrated that the NDUFB6 subunit is required for complex I activity, and defined conditions suitable to undertake a systematic and stable extinction of the different supernumerary subunits in human cells.