Multiple proteins with essential mitochondrial functions have glycosylated isoforms

Nucleocytosolic and secreted proteins are commonly glycosylated. However, reports of glycosylated mitochondrial proteins are rare. Using lectin chromatography on bovine heart, we detected low-abundance glycoforms of nuclear-encoded proteins with well-established mitochondrial function: pyruvate dehydrogenase E1α, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, ADP/ATP translocase, ATP synthase d and oligomycin sensitivity-conferring protein. Notably, the latter two have been previously detected at the plasma membrane. Our findings indicate that glycosylation of classic mitochondrial proteins may be more common than previously appreciated. We discuss the implication that glycosylation could represent an unexplored mechanism for regulating these proteins' functions within mitochondria or at extra-mitochondrial locations.     

Local proteins associated with methamphetamine-induced nigrostriatal dopaminergic neurotoxicity

The present study aimed to examine the proteins involved in the methamphetamine (MA)-induced nigrostriatal dopaminergic toxicity. Infusion of anisomycin into striatum and substantia nigra both abolished the MA-induced striatal dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) depletions, indicating a critical role of local protein synthesis in determining such dopaminergic toxicity. Moreover, local protein synthesis blockade reversed this neurotoxicity via a temperature-independent mechanism. We then employed a proteomic approach, two-dimensional gel electrophoresis (2-DE) in conjunction with mass spectrometry analysis, to identify the protein candidates associated with the MA-induced neurotoxicity. In striatal samples, 2-DE analysis revealed that the intensities of nine protein spots were altered by MA treatment. Mass spectrometry analysis allowed us to identify five proteins, including an up-regulated protein, alpha-synuclein, and four down-regulated proteins, ATPase, F-actin capping protein beta subunit, ubiquitin carboxy-terminal hydrolase/PGP 9.5, and peroxidase. MA-altered expression levels of alpha-synuclein and ubiquitin carboxy-terminal hydrolase/PGP 9.5 in striata were confirmed by western blotting analysis. Taken together, these results suggest that local up-regulation of alpha-synuclein and down-regulation of ubiquitin carboxy-terminal hydrolase/PGP 9.5 could be linked to the MA-induced dopaminergic terminal toxicity.     

The iron-sulphur protein Ind1 is required for effective complex I assembly

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.     

Exposure to Far Infrared Ray Protects Methamphetamine-Induced Behavioral Sensitization in Glutathione Peroxidase-1 Knockout Mice via Attenuating Mitochondrial Burdens and Dopamine D1 Receptor Activation

Evidence indicates that stress conditions might lead to drug dependence. Recently, we have demonstrated that exposure to far infrared ray (FIR) attenuates acute restraint stress via induction of glutathione peroxidase-1 (GPx-1) gene. We investigated whether FIR affects methamphetamine (MA)-induced behavioral sensitization and whether FIR-mediated pharmacological activity requires interaction between dopamine receptor and GPx-1 gene. We observed that MA treatment significantly increased GPx-1 expression in the striatum of wild-type (WT) mice. Interestingly, exposure to FIR potentiated MA-induced increase in GPx-1 expression. This phenomenon was also observed in animals receiving MA with dopamine D1 receptor antagonist SCH23390. However, dopamine D2 receptor antagonist sulpiride did not affect MA-induced GPx-1 expression. FIR exposure or SCH23390, but not sulpiride, significantly attenuated MA-induced behavioral sensitization. Exposure to FIR significantly attenuated MA-induced dopamine D1 receptor expression, c-Fos induction and oxidative burdens. FIR-mediated antioxidant effects were also more pronounced in mitochondrial- than cytosolic-fraction. In addition, FIR significantly attenuated against MA-induced changes in mitochondrial superoxide dismutase and mitochondrial GPx activities, mitochondrial transmembrane potential, intramitochondrial Ca²⁺ level, mitochondrial complex-I activity, and mitochondrial oxidative burdens. The attenuation by FIR was paralleled that by SCH23390. Effects of FIR or SCH23390 were more sensitive to GPx-1 KO than WT mice, while SCH23390 treatment did not exhibit any additive effects on the protective activity mediated by FIR, indicating that dopamine D1 receptor constitutes a molecular target of FIR. Our result suggests that exposure to FIR ameliorates MA-induced behavioral sensitization via possible interaction between dopamine D1 receptor and GPx-1 gene.     

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide.

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.     

A protective function of superoxide dismutase during respiratory chain activity.

(1) Aerobic incubation of heart muscle submitochondrial particles in phosphate buffer after treatment with NADH causes a progressive and substantial inhibition of the NADH oxidation system. Succinate oxidation remains almost unaffected by NADH treatment. (2) The loss of NADH oxidase activity is due to an inhibition of the respiratory chain-linked NADH dehydrogenase. This inhibition of the enzyme is very similar to that caused by combination of the organic mercurial mersalyl with NADH dehydrogenase. (3) The inhibition of NADH oxidation is largely prevented by compounds that are known to react with superoxide ions (02-.), including superoxide dismutase, cytochrome c, tiron and Mn2+. EDTA also has a protective effect, but a number of other metal chelating agents, and several proteins, including catalase, are without effect. (4) It is concluded that the inhibition of NADH oxidation of NADH oxidation by superoxide ions or by mersalyl is reversible and is therefore not due to the loss of oxidoreduction components from the respiratory chain or to an irreversible change in protein conformation. (6) The function of mitochondrial superxide dismutase is discussed in relation to the key role of NADH dehydrogenase in energy-conserving reactions and the formation of hydrogen peroxide during mitochondrial oxidations.     

Association of MT-ND5 gene variation with mitochondrial respiratory control ratio and NADH dehydrogenase activity in Tibet chicken embryos

NADH dehydrogenase (complex I) couples the oxidation of NADH for the reduction of ubiquinone with the generation of a proton gradient that can be used for the synthesis of ATP. We have found a missense mutation in the MT-ND5 subunit of NADH dehydrogenase in the Tibet chicken breed. In the present study, the mitochondrial respiratory control ratio (RCR) and NADH dehydrogenase activity in Tibet chicken embryonic brain with different genotypes were measured. Significant differences between animals carrying mitochondria with the EF493865.1:m.1627A vs. EF493865.1:m.1627C alleles were observed for RCR and enzyme activity.     

Proteomics reveals that redox regulation is disrupted in patients with ethylmalonic encephalopathy

Deficiency of the sulfide metabolizing protein ETHE1 is the cause of ethylmalonic encephalopathy (EE), an inherited and severe metabolic disorder. To study the molecular effects of EE, we performed a proteomics study on mitochondria from cultured patient fibroblast cells. Samples from six patients were analyzed and revealed seven differentially regulated proteins compared with healthy controls. Two proteins involved in pathways of detoxification and oxidative/reductive stress were underrepresented in EE patient samples: mitochondrial superoxide dismutase (SOD2) and aldehyde dehydrogenase X (ALDH1B). Sulfide:quinone oxidoreductase (SQRDL), which takes part in the same sulfide pathway as ETHE1, was also underrepresented in EE patients. The other differentially regulated proteins were apoptosis inducing factor (AIFM1), lactate dehydrogenase (LDHB), chloride intracellular channel (CLIC4) and dimethylarginine dimethylaminohydrolase 1 (DDAH1). These proteins have been reported to be involved in encephalopathy, energy metabolism, ion transport, and nitric oxide regulation, respectively. Interestingly, oxidoreductase activity was overrepresented among the regulated proteins indicating that redox perturbation plays an important role in the molecular mechanism of EE. This observation may explain the wide range of symptoms associated with the disease, and highlights the potency of the novel gaseous mediator sulfide.     

Ribonucleoprotein Y-box-binding protein-1 regulates mitochondrial oxidative phosphorylation (OXPHOS) protein expression after serum stimulation through binding to OXPHOS mRNA

Mitochondria play key roles in essential cellular functions, such as energy production, metabolic pathways and aging. Growth factor-mediated expression of the mitochondrial OXPHOS (oxidative phosphorylation) complex proteins has been proposed to play a fundamental role in metabolic homoeostasis. Although protein translation is affected by general RNA-binding proteins, very little is known about the mechanism involved in mitochondrial OXPHOS protein translation. In the present study, serum stimulation induced nuclear-encoded OXPHOS protein expression, such as NDUFA9 [NADH dehydrogenase (ubiquinone) 1α subcomplex, 9, 39 kDa], NDUFB8 [NADH dehydrogenase (ubiquinone) 1β subcomplex, 8, 19 kDa], SDHB [succinate dehydrogenase complex, subunit B, iron sulfur (Ip)] and UQCRFS1 (ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1), and mitochondrial ATP production, in a translation-dependent manner. We also observed that the major ribonucleoprotein YB-1 (Y-box-binding protein-1) preferentially bound to these OXPHOS mRNAs and regulated the recruitment of mRNAs from inactive mRNPs (messenger ribonucleoprotein particles) to active polysomes. YB-1 depletion led to up-regulation of mitochondrial function through induction of OXPHOS protein translation from inactive mRNP release. In contrast, YB-1 overexpression suppressed the translation of these OXPHOS mRNAs through reduced polysome formation, suggesting that YB-1 regulated the translation of mitochondrial OXPHOS mRNAs through mRNA binding. Taken together, our findings suggest that YB-1 is a critical factor for translation that may control OXPHOS activity.     

Two modes of irreversible inactivation of the mitochondrial electron-transfer system by tetradecanoic acid

Bovine heart submitochondrial particles were incubated for 2-6 h at 37 degrees C with various concentrations of tetradecanoic acid, and the effects on the activities, the total acid-labile sulphide content and EPR spectra of the electron transfer system were studied. Two distinct time-dependent processes of the slow irreversible inactivation of the electron-transfer system were found. They differ in the concentration of tetradecanoic acid required. The more specific effect, induced by 100-400 nmol tetradecanoic acid per mg protein, consists of a selective blockage of electron transfer between the Fe-S clusters of the NADH dehydrogenase and ubiquinone, without damage to any of the Fe-S clusters. Higher concentrations of tetradecanoic acid caused gradual destruction of all Fe-S clusters of NADH dehydrogenase and of the 3-Fe cluster of succinate dehydrogenase, leading to complete inactivation of both NADH and succinate oxidation.     

One- and two-electron reduction of ubiquinone homologs by NADH- dehydrogenase preparations from the mitochondrial respiratory chain

The mechanism of ubiquinone homologs reduction by different preparations of mitochondrial NADH dehydrogenase: complex I within submitochondrial particles, isolated NADH-ubiquinone oxidoreductase and soluble low molecular weight NADH dehydrogenase, has been investigated. It has been shown that NADH oxidation via the rotenone-insensitive reaction is associated with one-electron reduction of low molecular weight ubiquinone homologs (Q0, Q1, Q2) to semiquinone with subsequent fast oxidation of the latter by atmospheric oxygen to form a superoxide radical. The two-electron ubiquinone reduction to quinol in the rotenone-sensitive reaction is unaccompanied by the semiquinone release from the enzyme active center into the surrounding solution.     

Oxidation-reduction respiratory chains and ATP synthase complex are localized in detergent-resistant lipid rafts

In order to detect and identify ubiquitous lipid raft marker proteins, we isolated lipid rafts from different mouse organs, including the liver, lung, large brain, and kidney, and analyzed their proteins via 2-DE. Many protein spots were determined to be ubiquitous in all of the lipid rafts, and were annotated via LC and MS/MS. Twelve proteins were identified as ubiquitous raft proteins, and most of these were determined to be mitochondrial proteins, including mortalin, prohibitin, voltage-dependent anion channel, ATP synthase, NADH dehydrogenase, and ubiquinol-cytochrome c reductase. Via immunoblotting, these proteins were shown to exist in detergent-resistant lipid rafts prepared using different organ tissues. Since these oxidation-reduction respiratory chains and ATP synthase complex were detected in detergent-resistant lipid raft fractions which had been isolated from the plasma membrane but not from the mitochondria, and found in the cell surface when determined by immunofluoresence and immunohistochemistry, we conclude that plasma membrane lipid rafts might contain oxidation-reduction respiratory chains and ATP synthase complex.     

Up-regulation of protein tyrosine nitration in methamphetamine-induced neurotoxicity through DDAH/ADMA/NOS pathway

Protein tyrosine nitration is an important post-translational modification mediated by nitric oxide (NO) associated oxidative stress, occurring in a variety of neurodegenerative diseases. In our previous study, an elevated level of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein was observed in different brain regions of acute methamphetamine (METH) treated rats, indicating the possibility of an enhanced expression of protein nitration that is mediated by excess NO through the DDAH1/ADMA (Asymmetric Dimethylated l-arginine)/NOS (Nitric Oxide Synthase) pathway. In the present study, proteomic methods, including stable isotope labeling with amino acids in cell culture (SILAC) and two dimensional electrophoresis, were used to determine the relationship between protein nitration and METH induced neurotoxicity in acute METH treated rats and PC12 cells. We found that acute METH administration evokes a positive activation of DDAH1/ADMA/NOS pathway and results in an over-production of NO in different brain regions of rat and PC12 cells, whereas the whole signaling could be repressed by DDAH1 inhibitor N^ω-(2-methoxyethyl)-arginine (l-257). In addition, enhanced expressions of 3 nitroproteins were identified in rat striatum and increased levels of 27 nitroproteins were observed in PC12 cells. These nitrated proteins are key factors for Cdk5 activation, cytoskeletal structure, ribosomes function, etc. l-257 also displayed significant protective effects against METH-induced protein nitration, apoptosis and cell death. The overall results illustrate that protein nitration plays a significant role in the acute METH induced neurotoxicity via the activation of DDAH1/ADMA/NOS pathway.     

Regulation of NADH/CoQ Oxidoreductase: Do Phosphorylation Events Affect Activity?

We had previously suggested that phosphorylation of proteins by mitochondrial kinases regulate the activity of NADH/CoQ oxidoreductase. Initial data showed that pyruvate dehydrogenase kinase (PDK) and cAMP-dependent protein kinase A (PKA) phosphorylate mitochondrial membrane proteins. Upon phosphorylation with crude PDK, mitochondria appeared to be deficient in NADH/cytochrome c reductase activity associated with increased superoxide production. Conversely, phosphorylation by PKA resulted in increased NADH/cytochrome c reductase activity and decreased superoxide formation. Current data confirms PKA involvement in regulating Complex I activity through phosphorylation of an 18 kDa subunit. Beef heart NADH/ cytochrome c reductase activity increases to 150% of control upon incubation with PKA and ATP-gamma-S. We have cloned the four human isoforms of PDK and purified beef heart Complex I. Incubation of mitochondria with PDK isoforms and ATP did not alter Complex I activity or superoxide production. Radiolabeling of mitochondria and purified Complex I with PDK failed to reveal phosphorylated proteins.     

Pretreatment with Bacopa monnieri extract offsets 3-nitropropionic acid induced mitochondrial oxidative stress and dysfunctions in the striatum of prepubertal mouse brain

The present investigation was designed to determine the efficacy of Bacopa monnieri (Brahmi; BM) to offset 3-nitropropionic acid (3-NPA) induced oxidative stress and mitochondrial dysfunction in dopaminergic (N27) cells and prepubertal mouse brain. Pretreatment of N27 cells with BM ethanolic extract (BME) significantly attenuated 3-NPA-induced cytotoxicity. Further, we determined the degree of oxidative stress induction, redox status, enzymic antioxidants, and protein oxidation in the striatal mitochondria of mice given BME prophylaxis followed by 3-NPA challenge. While 3-NPA-induced marked oxidative stress in the mitochondria of the striatum, BME prophylaxis markedly prevented 3-NPA-induced oxidative dysfunctions and depletion of reduced glutathione and thiol levels. The activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase, glutathione reductase, thioredoxin reductase), Na(+),K(+)-ATPase, and citric acid cycle enzymes in the striatum discernible among 3-NPA mice were significantly restored with BME prophylaxis. Interestingly, BME offered protection against 3-NPA-induced mitochondrial dysfunctions as evidenced by the restoration of the activities of ETC enzymes (NADH:ubiquinone oxidoreductase, NADH:cytochrome c reductase, succinate-ubiquinone oxidoreductase, and cytochrome c oxidase) and mitochondrial viability. We hypothesize that the neuroprotective effects of BME may be wholly or in part related to its propensity to scavenge free radicals, maintain redox status, and upregulate antioxidant machinery in striatal mitochondria.     

Changes in physiology and protein abundance in salt-stressed wheat chloroplasts

Leaves are the final site of salinity perception through the roots. To better understand how wheat chloroplasts proteins respond to salt stress, the study aimed to the physiochemical and comparative proteomics analysis. Seedlings (12-days-old) were exposed to 150 mM NaCl for 1, 2, or 3 days. Na(+) ions were rapid and excessively increase in roots, stems and leaves. Photosynthesis and transpiration rate, stomatal conductance, and relative water content decreased whereas the level of proline increased. Statistically significant positive correlations were found among the content of hydrogen peroxide, activity of catalase, and superoxide dismutase under salt stress in wheat. Protein abundance within the chloroplasts was examined by two-dimensional electrophoresis. More than 100 protein spots were reproducibly detected on each gel, 21 protein spots were differentially expressed during salt treatment. Using linear quadruple trap-Fourier transform ion cyclotron resonance (LTQ-FTICR) hybrid mass spectrometry, 65 unique proteins assigned in the differentially abundant spots. Most proteins were up-regulated at 2 and 3 days after being down-regulated at 1 day. Others showed only slight responses after 3 days of treatment, including Rubisco, glutamate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase, photosystem I, and pyridoxal biosynthesis protein PDX1.2 and PDX1.3. The ATP synthase (α, β, and γ) and V-type proton ATPase subunits were down-regulated resulting showed negative impact by Na(+) on the photosynthetic machinery. This ephemeral increase and subsequent decrease in protein contents may demonstrate a counterbalancing influence of identified proteins. Several proteins such as cytochrome b6-f (Cyt b6-f), germin-like-protein, the γ-subunit of ATP synthase, glutamine synthetase, fructose-bisphosphate aldolase, S-adenosylmethionine synthase, carbonic anhydrase were gradually up-regulated during the period of treatment, which can be identified as marker proteins.     

Aberrant neuronal and mitochondrial proteins in hippocampus of transgenic mice overexpressing human Cu/Zn superoxide dismutase 1

Mutations of Cu/Zn superoxide dismutase 1 (SOD1), a metalloenzyme catalyzing the conversion of superoxide anion to hydrogen peroxide (H(2)O(2)), are linked to motor neuron degeneration. Transgenic mouse strains overexpressing wild-type human SOD1 (Tg-SOD1) were shown to have mitochondrial swelling, vacuolization, or learning and memory deficits and are widely used for biochemical, genetic, and cognitive studies; this, along with the advent of advanced proteomic methods, made us investigate protein expression in hippocampus. Hippocampal tissues of wild-type, hemizygous, and homozygous Tg-SOD1 mice were isolated and used for two-dimensional gel electrophoresis with subsequent matrix-assisted laser desorption/ionization-time of flight identification. We identified several synaptosomal, neuronal, antioxidant, and mitochondrial proteins in hippocampus, and expression levels of syntaxin-binding protein 1, N-ethylmaleimide-sensitive factor, synaptosomal-associated protein 25, dynamin-1, neurofilament triplet L protein, neurofilament triplet M protein, neuronal tropomodulin, and neuronal protein 25 were significantly decreased in Tg-SOD1. None of the antioxidant proteins were altered except mouse SOD1. Mitochondrial ATP synthase alpha/beta chain and elongation factor Tu were aberrant in Tg-SOD1. We conclude that derangement of neuronal and mitochondrial proteins may indicate synaptosomal and neuronal loss in Tg-SOD1 hippocampus, already reported in morphological terms. This observation is of relevance to understanding brain deficits in Down syndrome, as SOD1 is encoded on chromosome 21.     

Effect of the myosin light chain kinase inhibitor ML-7 on the proteome of hearts subjected to ischemia-reperfusion injury

In the development of ischemia/reperfusion (I/R) injury, the role of the myosin light chain (MLC) phosphorylation has been given increased consideration. ML-7, a MLC kinase inhibitor, has been shown to protect cardiac function from I/R, however the exact mechanism remains unclear. Isolated rat hearts were perfused under aerobic conditions (controls) or subjected to I/R in the presence or absence of ML-7. Continuous administration of ML-7 (5 μM) from 10 min before onset of ischemia to the first 10 min of reperfusion resulted in significant recovery of heart contractility. Analysis of gels from two-dimensional electrophoresis revealed eight proteins with decreased levels in I/R hearts. Six proteins are involved in energy metabolism:ATP synthase beta subunit, cytochrome b-c1 complex subunit 1, 24-kDa mitochondrial NADH dehydrogenase, NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, cytochrome c oxidase subunit, and succinyl-CoA ligase subunit. The other two proteins with decreased levels in I/R hearts are: peroxiredoxin-2 and tubulin. Administration of ML-7 increased level of succinyl-CoA ligase, key enzyme involved in the citric acid cycle. The increased level of succinyl-CoA ligase in I/R hearts perfused with ML-7 suggests that the cardioprotective effect of ML-7, at least partially, also may involve increase of energy production.