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Mitochondrial Proteins (mitochondrial + protein)
Selected AbstractsProteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque depositionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2007Frank Gillardon Dr. Abstract Synaptic dysfunction is an early event in Alzheimer's disease patients and has also been detected in transgenic mouse models. In the present study, we analyzed proteomic changes in synaptosomal fractions from Tg2576 mice that overexpress mutant human amyloid precursor protein (K670N, M671L) and from their nontransgenic littermates. Cortical and hippocampal tissue was microdissected at the onset of cognitive impairment, but before deposition of amyloid plaques. Crude synaptosomal fractions were prepared by differential centrifugation, proteins were separated by 2-D DIGE and identified by MS/MS. Significant alterations were detected in mitochondrial heat shock protein 70 pointing to a mitochondrial stress response. Subsequently, synaptosomal versus nonsynaptic mitochondria were purified from Tg2576 mice brains by density gradient centrifugation. Mitochondrial proteins were separated by IEF or Blue-native gel electrophoresis in the first dimension and SDS-PAGE in the second dimension. Numerous changes in the protein subunit composition of the respiratory chain complexes I and III were identified. Levels of corresponding mRNAs remain unchanged as shown by Affymetrix oligonucleotide array analysis. Functional examination revealed impaired state 3 respiration and uncoupled respiration in brain mitochondria from young Tg2576 mice. By immunoblotting, amyloid-beta oligomers were detected in synaptosomal fractions from Tg2576 mice and reduced glucose metabolism was observed in Tg2576 mice brains by [14C]-2-deoxyglucose infusion. Taken together, we demonstrate alterations in the mitochondrial proteome and function that occur in Tg2576 mice brains before amyloid plaque deposition suggesting that mitochondria are early targets of amyloid-beta aggregates. [source] Ethanol-Induced Oxidative Stress and Mitochondrial Dysfunction in Rat Placenta: Relevance to Pregnancy LossALCOHOLISM, Issue 3 2010Fusun Gundogan Background:, Ethanol consumption during pregnancy increases the risk of early pregnancy loss and causes intrauterine growth restriction. We previously showed that chronic gestational exposure to ethanol impairs placentation, and that this effect is associated with inhibition of insulin and insulin growth factor signaling. Since ethanol also causes oxidative stress and DNA damage, we extended our investigations to assess the role of these pathological processes on placentation and placental gene expression. Methods:, Pregnant Long Evans rats were pair-fed liquid diets containing 0% or 24% ethanol by caloric content. Placentas harvested on gestation day 16 were used to examine DNA damage, lipid peroxidation, apoptosis, mitochondrial gene/protein and hormonal gene expression in relation to ethanol exposure. Results:, Gestational exposure to ethanol increased fetal resorption, and trophoblast apoptosis/necrosis, oxidative stress, DNA damage, and lipid peroxidation. These adverse effects of ethanol were associated with increased expression of pro-apoptotic (Bax and Bak) and reduced levels of the anti-apoptotic Bcl-2 protein. In addition, increased trophoblast apoptosis proneness was associated with p53-independent activation of p21, reduced mitochondrial gene and protein expression, and dysregulated expression of prolactin (PRL) family hormones that are required for implantation and pregnancy-related adaptations. Conclusions:, Chronic gestational exposure to ethanol increases fetal demise due to impaired survival and mitochondrial function, increased oxidative stress, DNA damage and lipid peroxidation, and dysregulated expression of prolactin family hormones in placental trophoblasts. [source] Apparent mitochondrial asymmetry in Xenopus eggsDEVELOPMENTAL DYNAMICS, Issue 4 2003Natalia Volodina Abstract Cell polarity is manifest along the animal/vegetal axis in eggs of the frog, Xenopus laevis. Along this axis, maternal cytoplasmic components are asymmetrically distributed and are thought to underlie specification of distinct cell fates. To ascertain the molecular identities of such cytoplasmic components, we have used a monoclonal antibody that specifically stains the vegetal hemisphere of Xenopus eggs. The antigenic protein Vp67 (vegetal protein of 67 kDa) was identified through purification and cloning as a Xenopus homolog of the mitochondrial protein dihydrolipoamide acetyltransferase, a component of the pyruvate dehydrogenase complex. The identification of Vp67 as a mitochondrial protein could indicate that populations of mitochondria are asymmetrically distributed in Xenopus eggs. Developmental Dynamics 226:654,662, © 2003 Wiley-Liss, Inc. [source] Interaction of a novel mitochondrial protein, 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1), with the amyloid precursor protein familyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2010Hemachand Tummala Abstract Amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein-1 and amyloid precursor-like protein-2, appear to have redundant but essential role(s) during development. To gain insights into the physiological and possibly pathophysiological functions of APP, we used a functional proteomic approach to identify proteins that interact with the highly conserved C-terminal region of APP family proteins. Previously, we characterized an interaction between APP and ubiquitous mitochondrial creatine kinase. Here, we describe an interaction between APP and a novel protein, 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1). The interaction between APP and NIPSNAP1 was confirmed both in transiently transfected COS7 cells and in the mouse brain, where NIPSNAP1 is expressed at a high level. We demonstrate that NIPSNAP1 is targeted to the mitochondria via its N-terminal targeting sequence, and interacts with mitochondrial chaperone translocase of the outer membrane 22. Mitochondrial localization of NIPSNAP1 appears to be critical for its interaction with APP, and overexpression of APP appeared to disrupt NIPSNAP1 mitochondrial localization. Moreover, APP overexpression resulted in downregulation of NIPSNAP1 levels in cultured cells. Our data suggest that APP may affect mitochondrial function through a direct interaction with NIPSNAP1 as well as with other mitochondrial proteins. [source] Mitochondrial affinity for ADP is twofold lower in creatine kinase knock-out musclesFEBS JOURNAL, Issue 4 2005Possible role in rescuing cellular energy homeostasis Adaptations of the kinetic properties of mitochondria in striated muscle lacking cytosolic (M) and/or mitochondrial (Mi) creatine kinase (CK) isoforms in comparison to wild-type (WT) were investigated in vitro. Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains (cytosolic (M-CK,/,), mitochondrial (Mi-CK,/,) and double knock-out (MiM-CK,/,), respectively). Maximal ADP-stimulated oxygen consumption flux (State3 Vmax; nmol O2·mg mitochondrial protein,1·min,1) and ADP affinity (; µm) were determined by respirometry. State 3 Vmax and of M-CK,/, and MiM-CK,/, gastrocnemius mitochondria were twofold higher than those of WT, but were unchanged for Mi-CK,/,. For mutant cardiac mitochondria, only the of mitochondria isolated from the MiM-CK,/, phenotype was different (i.e. twofold higher) than that of WT. The implications of these adaptations for striated muscle function were explored by constructing force-flow relations of skeletal muscle respiration. It was found that the identified shift in affinity towards higher ADP concentrations in MiM-CK,/, muscle genotypes may contribute to linear mitochondrial control of the reduced cytosolic ATP free energy potentials in these phenotypes. [source] Purification and cDNA cloning of nitric oxide reductase cytochrome P450nor (CYP55A4) from Trichosporon cutaneumFEBS JOURNAL, Issue 11 2001Li Zhang Cytochrome P450nor is involved in fungal denitrification as nitric oxide (NO) reductase. Although the heme protein has been known to occur in restricted species of fungi that belong to ascomycotina, we have previously suggested that it would also occur in the yeast Trichosporon cutaneum, which is phylogenetically far from those P450nor-producing ascomycetous fungi. Here we isolated and characterized the heme protein from the basidiomycetous yeast T. cutaneum. P450nor of the yeast (TcP450nor) exhibited properties in terms of catalysis, absorption spectrum and molecular mass that are almost identical to those of its counterparts in ascomycetous fungi. We also isolated and sequenced its cDNA. The predicted primary structure of TcP450nor showed high sequence identities (around 65%) to those of other P450nors, indicating that they belong to the same family. TcP450nor protein cofractionated with cytochrome c oxidase by subcellular fractionation and its predicted primary structure contained an extension on its amino terminus that is characteristic of a mitochondrial-targeting signal, indicating that it is a mitochondrial protein like some of the isoforms of other fungi. On the other hand, TcP450nor was unique in that inducers such as nitrate, nitrite, or NO were not required for its production in the cells. The occurrence of P450nor across the subdivisions of eumycota suggests that P450nor and denitrification are distributed more universally among fungi than was previously thought. [source] A respiratory-deficient mutation associated with high salt sensitivity in Kluyveromyces lactisFEMS YEAST RESEARCH, Issue 2 2007Paola Goffrini Abstract A salt-sensitive mutant of Kluyveromyces lactis was isolated that was unable to grow in high-salt media. This mutant was also respiratory-deficient and temperature-sensitive for growth. The mutation mapped in a single nuclear gene that is the ortholog of BCS1 of Saccharomyces cerevisiae. The BCS1 product is a mitochondrial protein required for the assembly of respiratory complex III. The bcs1 mutation of S. cerevisiae leads to a loss of respiration, but, unlike in K. lactis, it is not accompanied by salt sensitivity. All the respiratory-deficient K. lactis mutants tested were found to be salt-sensitive compared to their isogenic wild-type strains. In the presence of the respiratory inhibitor antimycin A, the wild-type strain also became salt-sensitive. By contrast, none of the S. cerevisiae respiratory-deficient mutants tested showed increased salt sensitivity. The salt sensitivity of the Klbcs1 mutant, but not its respiratory deficiency, was suppressed by the multicopy KlVMA13 gene, a homolog of the S. cerevisiae VMA13 gene encoding a subunit of the vacuolar H+ -ATPase. These results suggest that cellular salt homeostasis in K. lactis is strongly dependent on mitochondrial respiratory activity, and/or that the ion homeostasis of mitochondria themselves could be a primary target of salt stress. [source] slowmo is required for Drosophila germline proliferationGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 2 2007Simon Reeve Abstract Null mutations in the Drosophila gene, slowmo (slmo), result in reduced mobility and lethality in first-instar larvae. Slowmo encodes a mitochondrial protein of unknown function, as do the two other homologs found in Drosophila. Here, we have studied a hypomorphic P-element allele of slmo demonstrating its effects on germline divisions in both testes and ovaries. Using in situ studies, enhancer-trap activity, and promoter fusions, we have shown that slmo expression in testes is found in the somatic cyst cells (SCC). The hypomorphic allele for Slmo revealed apoptotic loss of germline cells in the larval germline, culminating in a complete absence of the germline in adult flies. In females, a similar degeneration of the germarium is observed, while reporter gene expression is found in both germline and somatic cells. Using a null mutation in female germline clones, we find slmo is dispensable from the germline cells. Our results suggest that Slowmo is not required in germline cells directly, but is required in SCCs responsible for maintaining germline survival in both sexes. genesis 45:66,75, 2007. © 2007 Wiley-Liss, Inc. [source] Genetic variability in the mitochondrial serine protease HTRA2 contributes to risk for Parkinson disease,HUMAN MUTATION, Issue 6 2008Veerle Bogaerts Abstract In one genetic study, the high temperature requirement A2 (HTRA2) mitochondrial protein has been associated with increased risk for sporadic Parkinson disease (PD). One missense mutation, p.Gly399Ser, in its C-terminal PDZ domain (from the initial letters of the postsynaptic density 95, PSD-95; discs large; and zonula occludens-1, ZO-1 proteins [Kennedy, 1995]) resulted in defective protease activation, and induced mitochondrial dysfunction when overexpressed in stably transfected cells. Here we examined the contribution of genetic variability in HTRA2 to PD risk in an extended series of 266 Belgian PD patients and 273 control individuals. Mutation analysis identified a novel p.Arg404Trp mutation within the PDZ domain predicted to freeze HTRA2 in an inactive form. Moreover, we identified six patient-specific variants in 5, and 3, regulatory regions that might affect HTRA2 expression as supported by data of luciferase reporter gene analyses. Our study confirms a role of the HTRA2 mitochondrial protein in PD susceptibility through mutations in its functional PDZ domain. In addition, it extends the HTRA2 mutation spectrum to functional variants possibly affecting transcriptional activity. The latter underpins a previously unrecognized role for altered HTRA2 expression as a risk factor relevant to parkinsonian neurodegeneration. Hum Mutat 29(6), 832,840, 2008. © 2008 Wiley-Liss, Inc. [source] Ancestral roles of eukaryotic frataxin: mitochondrial frataxin function and heterologous expression of hydrogenosomal Trichomonas homologues in trypanosomesMOLECULAR MICROBIOLOGY, Issue 1 2008Shaojun Long Summary Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe,S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin-depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe,S cluster biogenesis and protection from reactive oxygen species. [source] Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thalianaMOLECULAR MICROBIOLOGY, Issue 2 2004Patrice Hamel Summary Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner-membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant ,mir1 ,pic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic-like proteins are also able to complement the double mutant ,mir1 ,pic2. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions. [source] Apoptosis-inducing factor deficiency sensitizes dopaminergic neurons to parkinsonian neurotoxinsANNALS OF NEUROLOGY, Issue 2 2010Celine Perier PhD Objective Mitochondrial complex I deficits have long been associated with Parkinson disease (PD). However, it remains unknown whether such defects represent a primary event in dopaminergic neurodegeneration. Methods Apoptosis-inducing factor (AIF) is a mitochondrial protein that, independently of its proapoptotic properties, plays an essential physiologic role in maintaining a fully functional complex I. We used AIF-deficient harlequin (Hq) mice, which exhibit structural deficits in assembled complex I, to determine whether primary complex I defects linked to AIF depletion may cause dopaminergic neurodegeneration. Results Despite marked reductions in mitochondrial complex I protein levels, Hq mice did not display apparent alterations in the dopaminergic nigrostriatal system. However, these animals were much more susceptible to exogenous parkinsonian complex I inhibitors, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Subtoxic doses of MPTP, unable to cause damage to wild-type animals, produced marked nigrostriatal dopaminergic degeneration in Hq mice. This effect was associated with exacerbated complex I inhibition and increased production of mitochondrial-derived reactive oxygen species (ROS) in Hq brain mitochondria. The antioxidant superoxide dismutase-mimetic compound tempol was able to reverse the increased susceptibility of Hq mice to MPTP. Supporting an instrumental role for mitochondrial-derived ROS in PD-related neurodegeneration, transgenic mice overexpressing mitochondrially targeted catalase exhibited an attenuation of MPTP-induced mitochondrial ROS and dopaminergic cell death. Interpretation Structural complex I alterations linked to AIF deficiency do not cause dopaminergic neurodegeneration but increase the susceptibility of dopaminergic neurons to exogenous parkinsonian neurotoxins, reinforcing the concept that genetic and environmental factors may interact in a common molecular pathway to trigger PD. ANN NEUROL 2010;68:184,192 [source] Functional studies of frataxinACTA PAEDIATRICA, Issue 2004G Isaya Mitochondria generate adenosine triphosphate (ATP) but also dangerous reactive oxygen species (ROS). One-electron reduction of dioxygen in the early stages of the electron transport chain yields a superoxide radical that is detoxified by mitochondrial superoxide dismutase to give hydrogen peroxide. The hydroxyl radical is derived from decomposition of hydrogen peroxide via the Fenton reaction, catalyzed by Fe2+ ions. Mitochondria require a constant supply of Fe2+ for heme and iron-sulfur cluster biosyntheses and therefore are particularly susceptible to ROS attack. Two main antioxidant defenses are known in mitochondria: enzymes that catalytically remove ROS, e.g. superoxide dismutase and glutathione peroxidase, and low molecular weight agents that scavenge ROS, including coenzyme Q, glutathione, and vitamins E and C. An effective defensive system, however, should also involve means to control the availability of pro-oxidants such as Fe2+ ions. There is increasing evidence that this function may be carried out by the mitochondrial protein frataxin. Frataxin deficiency is the primary cause of Friedreich's ataxia (FRDA), an autosomal recessive degenerative disease. Frataxin is a highly conserved mitochondrial protein that plays a critical role in iron homeostasis. Respiratory deficits, abnormal cellular iron distribution and increased oxidative damage are associated with frataxin defects in yeast and mouse models of FRDA. The mechanism by which frataxin regulates iron metabolism is unknown. The yeast frataxin homologue (mYfhlp) is activated by Fe(II) in the presence of oxygen and assembles stepwise into a 48-subunit multimer (,48) that sequesters <2000 atoms of iron in a ferrihydrite mineral core. Assembly of mYfhlp is driven by two sequential iron oxidation reactions: a fast ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (,,3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding ,48. Depending on the ionic environment, stepwise assembly is associated with the sequestration of 50,75 Fe(II)/subunit. This Fe(II) is initially loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. However, as iron oxidation and mineralization proceed, Fe(III) becomes progressively inaccessible and a stable iron-protein complex is produced. In conclusion, by coupling iron oxidation with stepwise assembly, frataxin can successively function as an iron chaperon or an iron store. Reduced iron availability and solubility and increased oxidative damage may therefore explain the pathogenesis of FRDA. [source] Regulation of skeletal muscle mitochondrial function: genes to proteinsACTA PHYSIOLOGICA, Issue 4 2010I. R. Lanza Abstract The impact of ageing on mitochondrial function and the deterministic role of mitochondria on senescence continue to be topics of vigorous debate. Many studies report that skeletal muscle mitochondrial content and function are reduced with ageing and metabolic diseases associated with insulin resistance. However, an accumulating body of literature suggests that physical inactivity typical of ageing may be a more important determinant of mitochondrial function than chronological age, per se. Reports of age-related declines in mitochondrial function have spawned a vast body of literature devoted to understanding the underlying mechanisms. These mechanisms include decreased abundance of mtDNA, reduced mRNA levels, as well as decreased synthesis and expression of mitochondrial proteins, ultimately resulting in decreased function of the whole organelle. Effective therapies to prevent, reverse or delay the onset of the aforementioned mitochondrial changes, regardless of their inevitability or precise underlying causes, require an intimate understanding of the processes that regulate mitochondrial biogenesis, which necessitates the coordinated regulation of nuclear and mitochondrial genomes. Herein we review the current thinking on regulation of mitochondrial biogenesis by transcription factors and transcriptional co-activators and the role of hormones and exercise in initiating this process. We review how exercise may help preserve mitochondrial content and functionality across the lifespan, and how physical inactivity is emerging as a major determinant of many age-associated changes at the level of the mitochondrion. We also review evidence that some mitochondrial changes with ageing are independent of exercise or physical activity and appear to be inevitable consequences of old age. [source] Muscle type-specific response of PGC-1, and oxidative enzymes during voluntary wheel running in mouse skeletal muscleACTA PHYSIOLOGICA, Issue 3-4 2006S. Ikeda Abstract Aim:, It is generally accepted that endurance exercise increases the expression of peroxisome proliferator-activated receptor , coactivator-1, (PGC-1,), which governs the expression of oxidative metabolic enzymes. A previous report demonstrated that the regulation of mitochondrial protein expression in skeletal muscles in response to cold exposure depends on muscle fibre type. Cold exposure and endurance exercise are both metabolic challenges that require adjustments in mitochondrial energy metabolism, we hypothesized that the exercise-induced increase in oxidative enzymes and PGC-1, expression is higher in fast-type than in slow-type muscle. Methods:, Female ICR mice were individually housed in cages equipped with running wheel for 1, 2, 4, 6 or 8 weeks. The soleus, plantaris (PLA) and tibialis anterior (TA) muscles were then prepared from each mouse. The expression levels of PGC-1,, mitochondrial proteins and GLUT4 were evaluated by Western blotting. Results:, The expression level of PGC-1, was increased only in the PLA muscle. Furthermore, the expression levels of all mitochondrial proteins and GLUT4 in the PLA muscle were increased. In the TA muscle, although there was no increase in PGC-1, expression, the expression levels of mitochondrial proteins and GLUT4 were increased. Conclusions:, These results suggest that muscle type-specific responses occur during endurance exercise, and that the increase in PGC-1, expression is not the only factor that promotes oxidative capacity as a result of endurance exercise. [source] Interaction of a novel mitochondrial protein, 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1), with the amyloid precursor protein familyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2010Hemachand Tummala Abstract Amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein-1 and amyloid precursor-like protein-2, appear to have redundant but essential role(s) during development. To gain insights into the physiological and possibly pathophysiological functions of APP, we used a functional proteomic approach to identify proteins that interact with the highly conserved C-terminal region of APP family proteins. Previously, we characterized an interaction between APP and ubiquitous mitochondrial creatine kinase. Here, we describe an interaction between APP and a novel protein, 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1). The interaction between APP and NIPSNAP1 was confirmed both in transiently transfected COS7 cells and in the mouse brain, where NIPSNAP1 is expressed at a high level. We demonstrate that NIPSNAP1 is targeted to the mitochondria via its N-terminal targeting sequence, and interacts with mitochondrial chaperone translocase of the outer membrane 22. Mitochondrial localization of NIPSNAP1 appears to be critical for its interaction with APP, and overexpression of APP appeared to disrupt NIPSNAP1 mitochondrial localization. Moreover, APP overexpression resulted in downregulation of NIPSNAP1 levels in cultured cells. Our data suggest that APP may affect mitochondrial function through a direct interaction with NIPSNAP1 as well as with other mitochondrial proteins. [source] Hypoxia induces complex I inhibition and ultrastructural damage by increasing mitochondrial nitric oxide in developing CNSEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Sebastián Giusti Abstract NO-mediated toxicity contributes to neuronal damage after hypoxia; however, the molecular mechanisms involved are still a matter of controversy. Since mitochondria play a key role in signalling neuronal death, we aimed to determine the role of nitrative stress in hypoxia-induced mitochondrial damage. Therefore, we analysed the biochemical and ultrastructural impairment of these organelles in the optic lobe of chick embryos after in vivo hypoxia,reoxygenation. Also, we studied the NO-dependence of damage and examined modulation of mitochondrial nitric oxide synthase (mtNOS) after the hypoxic event. A transient but substantial increase in mtNOS content and activity was observed at 0,2 h posthypoxia, resulting in accumulation of nitrated mitochondrial proteins measured by immunoblotting. However, no variations in nNOS content were observed in the homogenates, suggesting an increased translocation to mitochondria and not a general de novo synthesis. In parallel with mtNOS kinetics, mitochondria exhibited prolonged inhibition of maximal complex I activity and ultrastructural phenotypes associated with swelling, namely, fading of cristae, intracristal dilations and membrane disruption. Administration of the selective nNOS inhibitor 7-nitroindazole 20 min before hypoxia prevented complex I inhibition and most ultrastructural damage. In conclusion, we show here for the first time that hypoxia induces NO-dependent complex I inhibition and ultrastructural damage by increasing mitochondrial NO in the developing brain. [source] Involvement of caspase 1 and its activator Ipaf upstream of mitochondrial events in apoptosisFEBS JOURNAL, Issue 12 2006Subhash Thalappilly PTP-S2/TC45 is a nuclear protein tyrosine phosphatase that activates p53 and induces caspase 1-dependent apoptosis. We analyzed the role of ICE protease-activating factor (Ipaf), an activator of caspase 1 in p53-dependent apoptosis. We also determined the sequence of events that lead to apoptosis upon caspase 1 activation by Ipaf. PTP-S2 expression induced Ipaf mRNA in MCF-7 cells which was dependent on p53. PTP-S2-induced apoptosis was inhibited by a dominant-negative mutant of Ipaf and also by an Ipaf-directed short-hairpin RNA. Doxorubicin-induced apoptosis was potentiated by the expression of caspase 1 (but not by a catalytic mutant of caspase 1) and required endogenous Ipaf. Doxorubicin treatment of MCF-7 cells resulted in activation of exogenous caspase 1, which was partly dependent on endogenous Ipaf. An activated form of Ipaf induced caspase 1-dependent apoptosis that was inhibited by Bcl2 and also by a dominant inhibitor of caspase 9 (caspase 9s). Caspase 1-dependent apoptosis induced by doxorubicin was also inhibited by Bcl2 and caspase 9s, but caspase 1 activation by activated Ipaf was not inhibited by Bcl2. Mitochondrial membrane permeabilization was induced by caspase 1 and activated Ipaf, which was inhibited by Bcl2, but not by caspase 9s. Expression of caspase 1 with activated Ipaf resulted in the activation of Bax at mitochondria. Our results suggest that Ipaf is involved in PTP-S2-induced apoptosis and that caspase 1, when activated by Ipaf, causes release of mitochondrial proteins (cytochrome c and Omi) through Bax activation, thereby functioning as an initiator caspase. [source] Parvalbumin deficiency in fast-twitch muscles leads to increased ,slow-twitch type' mitochondria, but does not affect the expression of fiber specific proteinsFEBS JOURNAL, Issue 1 2006Peter Racay Parvalbumin (PV), a small cytosolic protein belonging to the family of EF-hand calcium-binding proteins, is highly expressed in mammalian fast-twitch muscle fibers. By acting as a ,slow-onset' Ca2+ buffer, PV does not affect the rapid contraction phase, but significantly contributes to increase the rate of relaxation, as demonstrated in PV,/, mice. Unexpectedly, PV,/, fast-twitch muscles were considerably more resistant to fatigue than the wild-type fast-twitch muscles. This effect was attributed mainly to the increased fractional volume of mitochondria in PV,/, fast-twitch muscle, extensor digitorum longus, similar to levels observed in the slow-twitch muscle, soleus. Quantitative analysis of selected mitochondrial proteins, mitochondrial DNA-encoded cytochrome oxidase c subunit I and nuclear DNA-encoded cytochrome oxidase c subunit Vb and F1-ATPase subunit , revealed the PV,/,tibialis anterior mitochondria composition to be almost identical to that in wild-type soleus, but not in wild-type fast-twitch muscles. Northern and western blot analyses of the same proteins in different muscle types and in liver are indicative of a complex regulation, probably also at the post-transcriptional level. Besides the function in energy metabolism, mitochondria in both fast- and slow-twitch muscles act as temporary Ca2+ stores and are thus involved in the shaping of Ca2+ transients in these cells. Previously observed altered spatio-temporal aspects of Ca2+ transients in PV,/, muscles are sufficient to up-regulate mitochondria biogenesis through the probable involvement of both calcineurin- and Ca2+/calmodulin-dependent kinase II-dependent pathways. We propose that ,slow-twitch type' mitochondria in PV,/, fast muscles are aimed to functionally replace the slow-onset buffer PV based on similar kinetic properties of Ca2+ removal. [source] Mitochondrial preprotein translocases as dynamic molecular machinesFEMS YEAST RESEARCH, Issue 6 2006Martin Van Der Laan Abstract Proteomic studies have demonstrated that yeast mitochondria contain roughly 1000 different proteins. Only eight of these proteins are encoded by the mitochondrial genome and are synthesized on mitochondrial ribosomes. The remaining 99% of mitochondrial precursors are encoded within the nuclear genome and after their synthesis on cytosolic ribosomes must be imported into the organelle. Targeting of these proteins to mitochondria and their import into one of the four mitochondrial subcompartments , outer membrane, intermembrane space (IMS), inner membrane and matrix , requires various membrane-embedded protein translocases, as well as numerous chaperones and cochaperones in the aqueous compartments. During the last years, several novel protein components involved in the import and assembly of mitochondrial proteins have been identified. The picture that emerges from these exciting new findings is that of highly dynamic import machineries, rather than of regulated, but static protein complexes. In this review, we will give an overview on the recent progress in our understanding of mitochondrial protein import. We will focus on the presequence translocase of the inner mitochondrial membrane, the TIM23 complex and the presequence translocase-associated motor, the PAM complex. These two molecular machineries mediate the multistep import of preproteins with cleavable N-terminal signal sequences into the matrix or inner membrane of mitochondria. [source] Mass spectrometric analysis of microtubule co-sedimented proteins from rat brainGENES TO CELLS, Issue 4 2008Tatsuhiko Sakamoto Microtubules (MTs) play crucial roles in a variety of cell functions, such as mitosis, vesicle transport and cell motility. MTs also compose specialized structures, such as centrosomes, spindles and cilia. However, molecular mechanisms of these MT-based functions and structures are not fully understood. Here, we analyzed MT co-sedimented proteins from rat brain by tandem mass spectrometry (MS) upon ion exchange column chromatography. We identified a total of 391 proteins. These proteins were grouped into 12 categories: 57 MT cytoskeletal proteins, including MT-associated proteins (MAPs) and motor proteins; 66 other cytoskeletal proteins; 4 centrosomal proteins; 10 chaperons; 5 Golgi proteins; 7 mitochondrial proteins; 62 nucleic acid-binding proteins; 14 nuclear proteins; 13 ribosomal proteins; 28 vesicle transport proteins; 83 proteins with diverse function and/or localization; and 42 uncharacterized proteins. Of these uncharacterized proteins, six proteins were expressed in cultured cells, resulting in the identification of three novel components of centrosomes and cilia. Our present method is not specific for MAPs, but is useful for identifying low abundant novel MAPs and components of MT-based structures. Our analysis provides an extensive list of potential candidates for future study of the molecular mechanisms of MT-based functions and structures. [source] Inactivation of oxidized and S -nitrosylated mitochondrial proteins in alcoholic fatty liver of rats,HEPATOLOGY, Issue 5 2006Kwan-Hoon Moon Increased oxidative/nitrosative stress is a major contributing factor to alcohol-mediated mitochondrial dysfunction. However, which mitochondrial proteins are oxidatively modified under alcohol-induced oxidative/nitrosative stress is poorly understood. The aim of this study was to systematically investigate oxidized and/or S -nitrosylated mitochondrial proteins and to use a biotin- N -maleimide probe to evaluate their inactivation in alcoholic fatty livers of rats. Binge or chronic alcohol exposure significantly elevated nitric oxide, inducible nitric oxide synthase, and ethanol-inducible CYP2E1. The biotin- N -maleimide-labeled oxidized and/or S -nitrosylated mitochondrial proteins from pair-fed controls or alcohol-fed rat livers were subsequently purified with streptavidin-agarose. The overall patterns of oxidized and/or S -nitrosylated proteins resolved by 2-dimensional polyacrylamide gel electrophoresis were very similar in the chronic and binge alcohol treatment groups. Seventy-nine proteins that displayed differential spot intensities from those of control rats were identified by mass spectrometry. These include mitochondrial aldehyde dehydrogenase 2 (ALDH2), ATP synthase, acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and many proteins involved in chaperone activity, mitochondrial electron transfer, and ion transport. The activity of 3-ketoacyl-CoA thiolase involved in mitochondrial ,-oxidation of fatty acids was significantly inhibited in alcohol-exposed rat livers, consistent with hepatic fat accumulation, as determined by biochemical and histological analyses. Measurement of activity and immunoblot results showed that ALDH2 and ATP synthase were also inhibited through oxidative modification of their cysteine or tyrosine residues in alcoholic fatty livers of rats. In conclusion, our results help to explain the underlying mechanism for mitochondrial dysfunction and increased susceptibility to alcohol-mediated liver damage. (HEPATOLOGY 2006;44:1218,1230.) [source] The mitochondrial genome of the Mediterranean fruit fly, Ceratitis capitataINSECT MOLECULAR BIOLOGY, Issue 2 2000L. Spanos Abstract The complete sequence of the mitochondrial genome of Ceratitis capitata has been determined. The circular genome is 15 980 bp long and contains a standard gene complement, i.e. the large and small ribosomal RNA subunits, twenty-two transfer RNA (tRNA) genes and thirteen genes encoding mitochondrial proteins. When comparing the sequence to fragments previously sequenced from other isolates it becomes apparent that interstrain polymorphisms are not rare. These differences are potentially useful for the development of diagnostic tools for population analysis applications, such as determining the source of recent introductions. Moreover, they could help obtain a solution to the long-lasting controversy on the possible eradication of the Medfly from certain locations. [source] Involvement of Ca2+ and ROS in ,-tocopheryl succinate-induced mitochondrial permeabilizationINTERNATIONAL JOURNAL OF CANCER, Issue 8 2010Vladimir Gogvadze Abstract Release of mitochondrial proteins such as cytochrome c, AIF, Smac/Diablo etc., plays a crucial role in apoptosis induction. A redox-silent analog of vitamin E, ,-tocopheryl succinate (,-TOS), was shown to stimulate cytochrome c release via production of reactive oxygen species (ROS) and Bax-mediated permeabilization of the outer mitochondrial membrane. Here we show that ,-TOS facilitates mitochondrial permeability transition (MPT) in isolated rat liver mitochondria, Tet21N neuroblastoma cells and Jurkat T-lymphocytes. In particular, in addition to ROS production, ,-TOS stimulates rapid Ca2+ entry into the cells with subsequent accumulation of Ca2+ in mitochondria,a prerequisite step for MPT induction. Alteration of mitochondrial Ca2+ buffering capacity was observed as early as 8 hr after incubation with ,-TOS, when no activation of Bax was yet detected. Ca2+ accumulation in mitochondria was important for apoptosis progression, since inhibition of mitochondrial Ca2+ uptake significantly mitigated the apoptotic response. Importantly, Ca2+ -induced mitochondrial destabilization might cooperate with Bax-mediated mitochondrial outer membrane permeabilization to induce cytochrome c release from mitochondria. [source] Protein Import Into MitochondriaIUBMB LIFE, Issue 3-5 2001Stefan A. Paschen Abstract Most mitochondrial proteins are encoded by the nuclear genome and thus have to be imported into mitochondria from the cytosol. Protein translocation across and into the mitochondrial membranes is a multistep process facilitated by the coordinated action of at least four specialized translocation systems in the outer and inner membranes of mitochondria. The outer membrane contains one general translocase, the TOM complex, whereas three distinct translocases are located in the inner membrane, which facilitates translocation of different classes of preproteins. The TIM23 complex mediates import of matrix-targeted preproteins with N -terminal presequences, whereas hydrophobic preproteins with internal targeting signals are inserted into the inner membrane via the TIM22 complex. The OXA translocase mediates the insertion of preproteins from the matrix space into the inner membrane. This review focuses on the structural organization and function of the import machinery of the model organisms of Saccharomyces cerevisiae and Neurospora crassa . In addition, the molecular basis of a new human mitochondrial disorder is discussed, the Mohr-Tranebjaerg syndrome. This is the first known disease, which is caused by an impaired mitochondrial protein import machinery leading to progressive neurodegeneration. [source] Methylene blue-mediated photodynamic therapy induces mitochondria-dependent apoptosis in HeLa CellJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008Yan Lu Abstract Methylene blue (MB), a widely studied reagent, is investigated in this work for its usage in photodynamic therapy (PDT). PDT has been proved to be highly effective in the treatment of different types of cancers. Previous studies showed MB has both high affinity for mitochondria and high photodynamic efficiency. To elucidate the effects of MB in PDT, we analyzed PDT-induced apoptosis in HeLa cells by introducing different doses of MB into the culture media. Our data showed that MB-mediated PDT triggered intense apoptotic cell death through a series of steps, beginning with photochemical generation of reactive oxygen species. The release of cytochrome c and activation of caspase-3 indicated that MB-PDT-mediated apoptosis in HeLa cells was executed by the mitochondria-dependent apoptotic pathway. Importantly, proteomic studies confirmed that expression levels of several mitochondrial proteins were altered in MB-PDT-induced apoptosis, including TRAP1, mitochondrial elongation factor Tu and peroxiredoxin 3 isoform b. Western blot data showed that phosphorylation of ERK1/2 and PKA were reduced in MB-PDT treated cells, indicating several signal molecules participating in this apoptotic cascade. Moreover, MB-PDT induced an increase in the strength of interaction between Bcl-xL and dephosphorylated Bad. This led to loss of the pro-survival function of Bcl-xL and resulted in mitochondria-mediated apoptosis. This study provides solid evidence of a strong induction by MB-PDT of a mitochondria-dependent apoptosis cascade in HeLa cells. J. Cell. Biochem. 105: 1451,1460, 2008. © 2008 Wiley-Liss, Inc. [source] Maintenance of mitochondrial DNA copy number and expression are essential for preservation of mitochondrial function and cell growthJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Jaan-Yeh Jeng Abstract To examine whether a reduction in the mtDNA level will compromise mitochondrial biogenesis and mitochondrial function, we created a cell model with depleted mtDNA. Stable transfection of small interfering (si)RNA of mitochondrial transcription factor A (Tfam) was used to interfere with Tfam gene expression. Selected stable clones showed 60,95% reduction in Tfam gene expression and 50,90% reduction in cytochrome b (Cyt b) gene expression. Tfam gene knockdown clones also showed decreased mtDNA-encoded cytochrome c oxidase subunit I (COX I) protein expression. However, no significant differences in protein expression were observed in nuclear DNA (nDNA)-encoded mitochondrial respiratory enzyme subunits. The cell morphology changed from a rhombus-like to a spindle-like form as determined in clones with decreased expressions of Tfam, mtRNA, and mitochondrial proteins. The mitochondrial respiratory enzyme activities and ATP production in such clones were significantly lower. The proportions of mtDNA mutations including 8-hydroxy-2,-deoxyguanosine (8-OHdG), a 4,977-bp deletion, and a 3,243-point mutation were also examined in these clones. No obvious increase in mtDNA mutations was observed in mitochondrial dysfunctional cell clones. The mitochondrial respiratory activity and ATP production ability recovered in cells with increased mtDNA levels after removal of the specific siRNA treatment. These experimental results provide direct evidence to substantiate that downregulation of mtDNA copy number and expression may compromise mitochondrial function and subsequent cell growth and morphology. J. Cell. Biochem. 103: 347,357, 2008. © 2007 Wiley-Liss, Inc. [source] Protein modification and replicative senescence of WI-38 human embryonic fibroblastsAGING CELL, Issue 2 2010Emad K. Ahmed Summary Oxidized proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro. To better understand the mechanisms by which these damaged proteins build up and potentially affect cellular function during replicative senescence of WI-38 fibroblasts, proteins targeted by these modifications have been identified using a bidimensional gel electrophoresis-based proteomic approach coupled with immunodetection of HNE-, AGE-modified and carbonylated proteins. Thirty-seven proteins targeted for either one of these modifications were identified by mass spectrometry and are involved in different cellular functions such as protein quality control, energy metabolism and cytoskeleton. Almost half of the identified proteins were found to be mitochondrial, which reflects a preferential accumulation of damaged proteins within the mitochondria during cellular senescence. Accumulation of AGE-modified proteins could be explained by the senescence-associated decreased activity of glyoxalase-I, the major enzyme involved in the detoxification of the glycating agents methylglyoxal and glyoxal, in both cytosol and mitochondria. This finding suggests a role of detoxification systems in the age-related build-up of damaged proteins. Moreover, the oxidized protein repair system methionine sulfoxide reductase was more affected in the mitochondria than in the cytosol during cellular senescence. Finally, in contrast to the proteasome, the activity of which is decreased in senescent fibroblasts, the mitochondrial matrix ATP-stimulated Lon-like proteolytic activity is increased in senescent cells but does not seem to be sufficient to cope with the increased load of modified mitochondrial proteins. [source] Calorie restriction alters mitochondrial protein acetylationAGING CELL, Issue 5 2009Bjoern Schwer Summary Calorie restriction (CR) increases lifespan in organisms ranging from budding yeast through mammals. Mitochondrial adaptation represents a key component of the response to CR. Molecular mechanisms underlying this adaptation are largely unknown. Here we show that lysine acetylation of mitochondrial proteins is altered during CR in a tissue-specific fashion. Via large-scale mass spectrometry screening, we identify 72 candidate proteins involved in a variety of metabolic pathways with altered acetylation during CR. Mitochondrial acetylation changes may play an important role in the pro-longevity CR response. [source] p53/CEP-1 increases or decreases lifespan, depending on level of mitochondrial bioenergetic stressAGING CELL, Issue 4 2009Natascia Ventura Summary Mitochondrial pathologies underlie a number of life-shortening diseases in humans. In the nematode Caenorhabditis elegans, severely reduced expression of mitochondrial proteins involved in electron transport chain-mediated energy production also leads to pathological phenotypes, including arrested development and/or shorter life; in sharp contrast, mild suppression of these same proteins extends lifespan. In this study, we show that the C. elegans p53 ortholog cep-1 mediates these opposite effects. We found that cep-1 is required to extend longevity in response to mild suppression of several bioenergetically relevant mitochondrial proteins, including frataxin , the protein defective in patients with Friedreich's Ataxia. Importantly, we show that cep-1 also mediates both the developmental arrest and life shortening induced by severe mitochondrial stress. These findings support an evolutionarily conserved function for p53 in modulating organismal responses to mitochondrial dysfunction and suggest that metabolic checkpoint responses may play a role in longevity control and in human mitochondrial-associated diseases. [source] | |||||||||||||||||||||||||||||||