Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.5.3 (complex I)
8,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096-26101). Complex I is a approximately 980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.
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PMID:Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line. 1559 92

Oxidative stress and mitochondrial dysfunction signify two important biochemical events associated with the loss of dopaminergic neurons in Parkinson's disease (PD). Studies using in vitro and in vivo PD models and in affected tissues from the disease itself have demonstrated a selective inhibition of mitochondrial complex I activity that appears to affect normal mitochondrial physiology leading to neuronal cell death. Earlier experiments from our laboratory have demonstrated that induced depletion of glutathione (GSH + GSSG) in cultured dopaminergic cells resulted in increased oxidative stress and a decrease in mitochondrial function. Furthermore, this dysfunction was linked to a selective decrease in mitochondrial complex I activity that appears to be due to oxidation of this complex. Glutathione depletion is the earliest detectable biochemical event during PD progression and occurs prior to complex I inhibition. Recent observations have also indicated that oxidative damage to complex I via naturally occurring free radicals such as peroxynitrite leads to modification of tyrosine and/or cysteine residues resulting in complex I inhibition. Using the sucrose gradient method, we detected in complex I-enriched fractions from a glutathione-depleted dopaminergic cell line two bands corresponding to approximately 25-kDa and approximately 30-kDa polypeptides that demonstrate anti-nitrotyrosine immunoreactivity, suggesting the possible involvement of protein nitration by peroxynitrite in glutathione depletion-mediated complex I inhibition.
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PMID:Glutathione depletion in a midbrain-derived immortalized dopaminergic cell line results in limited tyrosine nitration of mitochondrial complex I subunits: implications for Parkinson's disease. 1599 45

Mitochondrial superoxide (O(2)(.)) production is an important mediator of oxidative cellular injury. While NADH dehydrogenase (NDH) is a critical site of this O(2)(.) production; its mechanism of O(2)(.) generation is not known. Therefore, the catalytic function of NDH in the mediation of O(2)(.) generation was investigated by EPR spin-trapping. In the presence of NADH, O(2)(.) generation from NDH was observed and was inhibited by diphenyleneiodinium chloride (DPI), indicating involvement of the FMN-binding site of NDH. Addition of FMN increased O(2)(.) production. Destruction of the cysteine ligands of iron-sulfur clusters decreased O(2)(.) generation, suggesting a secondary role of this site. This inhibitory effect was reversed by addition of FMN. However, FMN addition could not reverse the inhibition of NDH by either DPI or heat denaturation, demonstrating involvement of both FMN and its FMN-binding protein moiety in the catalysis of O(2)(.) generation. O(2)(.) production by NDH also induced self-inactivation. Immunospin-trapping with anti-DMPO antibody and subsequent mass spectrometry was used to define the sites of oxidative damage of NDH. A DMPO adduct was detected on the 51-kDa subunit and was O(2)(.)-dependent. Alkylation of the cysteine residues of NDH significantly inhibited NDH-DMPO spin adduct formation, indicating involvement of protein thiyl radicals. LC/MS/MS analysis of a tryptic digest of the 51-kDa polypeptide revealed that cysteine (Cys(206)) and tyrosine (Tyr(177)) were specific sites of NDH-derived protein radical formation. Thus, two domains of the 51-kDa subunit, Gly(200)-Ala-Gly-Ala-Tyr-Ile-Cys(206)-Gly-Glu-Glu-Thr-Ala-Leu-Ile-Glu-Ser-Ile-Glu-Gly-Lys(219) and Ala(176)-Tyr(177)-Glu-Ala-Gly-Leu-Ile-Gly-Lys(184), were demonstrated to be susceptible to oxidative attack, and their oxidative modification results in decreased electron transfer activity.
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PMID:Superoxide generation from mitochondrial NADH dehydrogenase induces self-inactivation with specific protein radical formation. 1615 Jul 35

Oxidation of Fe2+, ascorbic acid, propyl gallate, tiron, L-cysteine, and glutathione by Acidithiobacillus ferrooxidans was studied with respect to the effect of electron transport inhibitors and uncouplers on the rate of oxidation. All the oxidations were sensitive to inhibitors of cytochrome c oxidase, KCN, and NaN3. They were also partially inhibited by inhibitors of complex I and complex III of the electron transport system. Uncouplers at low concentrations stimulated the oxidation and inhibited it at higher concentrations. The oxidation rates of Fe2+ and L-cysteine inhibited by complex I and complex III inhibitors (amytal, rotenone, antimycin A, myxothiazol, and HQNO) were stimulated more extensively by uncouplers than the control rates. Atabrine, a flavin antagonist, was an exception, and atabrine-inhibited oxidation activities of all these compounds were further inhibited by uncouplers. A model for the electron transport pathways of A. ferrooxidans is proposed to account for these results. In the model these organic substrates reduce ferric iron on the surface of cells to ferrous iron, which is oxidized back to ferric iron through the Fe2+ oxidation pathway, leading to cytochrome oxidase to O2. Some of electrons enter the uphill (energy-requiring) electron transport pathway to reduce NAD+. Uncouplers at low concentrations stimulate Fe2+ oxidation by stimulating cytochrome oxidase by uncoupling. Higher concentrations lower deltap to the level insufficient to overcome the potentially uphill reaction at rusticyanin-cytochrome c4. Inhibition of uphill reactions at complex I and complex III leads to deltap accumulation and inhibition of cytochrome oxidase. Uncouplers remove the inhibition of deltap and stimulate the oxidation. Atabrine inhibition is not released by uncouplers, which implies a possibility of atabrine inhibition at a site other than complex I, but a site somehow involved in the Fe2+ oxidation pathway.
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PMID:Effects of electron transport inhibitors and uncouplers on the oxidation of ferrous iron and compounds interacting with ferric iron in Acidithiobacillus ferrooxidans. 1623 67

The time course and critical determinants of mitochondrial dysfunction and oxidative stress following limbic status epilepticus (SE) were investigated in hippocampal sub-regions of an electrical stimulation model in rats, at time points 4-44h after status. Mitochondrial and cytosolic enzyme activities were measured spectrophotometrically, and reduced glutathione (GSH) concentrations by HPLC, and compared to results from sham controls. The earliest change in any sub-region was a fall in GSH, appearing as early as 4h in CA3 (-13%, p<0.05), and persisting at all time points. This was followed by a transient fall in complex I activity (CA3, 16h, -13%, p<0.05), and later changes in aconitase (CA1,-18% and CA3, -22% at 44h, p<0.05). The activity of the cytosolic enzyme glyceraldehyde-3-phosphate-dehydrogenase was unaffected at all time points. It is known that GSH levels are dependent both on redox status, and on the availability of the precursor cysteine, in turn dependent on the cysteine/glutamate antiporter, for which extracellular glutamate concentrations are rate limiting. Both mechanisms are likely to contribute indirectly to GSH depletion following seizures. That a relative deficiency in GSH precedes later changes in the activities of complex I and aconitase in vulnerable hippocampal sub-regions, occurring within a clinically relevant therapeutic time window, suggests that strategies to boost GSH levels and/or otherwise reduce oxidative stress following seizures, deserve further study, both in terms of preventing the biochemical consequences of SE and the neuronal dysfunction and clinical consequences.
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PMID:Depletion of reduced glutathione precedes inactivation of mitochondrial enzymes following limbic status epilepticus in the rat hippocampus. 1629 Mar 21

NO* (nitric oxide) is a pleiotropic signalling molecule, with many of its effects on cell function being elicited at the level of the mitochondrion. In addition to the well-characterized binding of NO* to the Cu(B)/haem-a3 site in mitochondrial complex IV, it has been proposed by several laboratories that complex I can be inhibited by S-nitrosation of a cysteine. However, direct molecular evidence for this is lacking. In this investigation we have combined separation techniques for complex I (blue-native gel electrophoresis, Superose 6 column chromatography) with sensitive detection methods for S-nitrosothiols (chemiluminescence, biotin-switch assay), to show that the 75 kDa subunit of complex I is S-nitrosated in mitochondria treated with S-nitrosoglutathione (10 microM-1 mM). The stoichiometry of S-nitrosation was 7:1 (i.e. 7 mol of S-nitrosothiols per mol of complex I) and this resulted in significant inhibition of the complex. Furthermore, S-nitrosothiols were detected in mitochondria isolated from hearts subjected to ischaemic preconditioning. The implications of these results for the physiological regulation of respiration, for reactive oxygen species generation and for a potential role of S-nitrosation in cardioprotection are discussed.
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PMID:Direct evidence for S-nitrosation of mitochondrial complex I. 1846 11

We have proposed that the age-associated increase of reactive oxygen species (ROS) by electron transport chain (ETC) dysfunction may cause the elevated basal level of p38 MAPK stress response pathway activity. However, the mechanism by which ROS activates this pathway is not clear. Here we propose that activation of the p38 MAPK pathway by complex I (CI) generated ROS, in response to rotenone (ROT) treatment, is based on the ability of reduced Trx to bind to and inhibit ASK 1 and its release from the complex upon oxidation. This balance of free vs. bound ASK1 regulates the level of p38 MAPK pathway activity. To support this mechanism we demonstrate that the production of ROS by ROT treated AML12 hepatocyte cells dissociates the Trx-ASK1 complex, thereby increasing p38 MAPK pathway activity. This mechanism is supported by the ability of N-acetyl cysteine (NAC) to prevent dissociation of Trx-ASK1 and activation of the p38 MAPK pathway. We also demonstrated that the ratio of ASK1/Trx-ASK1 increases in aged mouse livers and that this correlates with the increased basal activity of the p38 MAPK pathway. The longevity of Snell dwarf mice has been attributed to their resistance to oxidative stress. A comparison of the levels of Trx-ASK1 in young and aged dwarfs showed a higher abundance of the complex than in their age-matched controls. These results, which are indicative of a decreased level of oxidative stress, suggest that increased ROS production in aged liver may alter the ratio of ASK1 and Trx-ASK1, thereby increasing the age-associated basal level of p38 MAPK pathway activity.
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PMID:Thioredoxin-ASK1 complex levels regulate ROS-mediated p38 MAPK pathway activity in livers of aged and long-lived Snell dwarf mice. 1644 98

After reduction with nicotinamide adenine dinucleotide (NADH), NADH:ubiquinone oxidoreductase (complex I) of the strictly aerobic yeast Yarrowia lipolytica shows clear signals from five different paramagnetic iron-sulfur (FeS) clusters (N1-N5) which can be detected using electron paramagnetic resonance (EPR) spectroscopy. The ligand environment and the assignment of several FeS clusters to specific binding motifs found in several subunits of the complex are still under debate. In order to characterize the hyperfine interaction of the surrounding nuclei with FeS cluster N1, one- and two-dimensional electron spin echo envelope modulation experiments were performed at a temperature of 30 K. At this temperature only cluster N1 contributes to the overall signal in a pulsed EPR experiment. The hyperfine and quadrupole tensors of a nitrogen nucleus and the isotropic and dipolar hyperfine couplings of two sets of protons could be determined by numerical simulation of the one- and two-dimensional spectra. The values obtained are in perfect agreement with a ferredoxin-like binding structure by four cysteine amino acid residues and allow the assignment of the nitrogen couplings to a backbone nitrogen nucleus and the proton couplings to the beta-protons of the bound cysteine residues.
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PMID:Cluster N1 of complex I from Yarrowia lipolytica studied by pulsed EPR spectroscopy. 1650 21

Up-regulation of activity of gamma-glutamyl transpeptidase (GGT) has been reported to occur in the Parkinsonian substantia nigra, the area of the brain affected by the disease. Increased GGT activity has been hypothesized to play a role in subsequent mitochondrial complex I (CI) inhibition by increasing cysteine as substrate for cellular uptake. Intracellular cysteine has been proposed to form toxic adducts with dopamine which can be metabolized to compounds which inhibit CI activity. We have demonstrated that in addition to CI inhibition, GGT activity is up-regulated in dopaminergic cells as a consequence of glutathione depletion. Inhibition of GGT rather than resulting in increased CI inhibition results in exacerbation of this inhibitory effect. This suggests that increased GGT activity is likely an adaptive response to the loss of glutathione to conserve intracellular glutathione content and results in a compensatory effect on CI activity rather than in its inhibition as has been previously widely hypothesized.
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PMID:Up-regulation of gamma-glutamyl transpeptidase activity following glutathione depletion has a compensatory rather than an inhibitory effect on mitochondrial complex I activity: implications for Parkinson's disease. 1663 16

In prokaryotes and yeast, the general mechanism of biogenesis of iron-sulfur (Fe-S) clusters involves activities of several proteins among which IscS and Nfs1p provide, through cysteine desulfuration, elemental sulfide for Fe-S core formation. Although these proteins have been well characterized, the role of their mammalian homolog in Fe-S cluster biogenesis has never been evaluated. We report here the first functional study that implicates the putative cysteine desulfurase m-Nfs1 in the biogenesis of both mitochondrial and cytosolic mammalian Fe-S proteins. Depletion of m-Nfs1 in cultured fibroblasts through small interfering RNA-based gene silencing significantly inhibited the activities of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II) of the respiratory chain, as well as aconitase of the Krebs cycle, with no alteration in their protein levels. Activity of cytosolic xanthine oxidase, which holds a [2Fe-2S] cluster, was also specifically reduced, and iron-regulatory protein-1 was converted from its [4Fe-4S] aconitase form to its apo- or RNA-binding form. Reduction of Fe-S enzyme activities occurred earlier and more markedly in the cytosol than in mitochondria, suggesting that there is a mechanism that primarily dedicates m-Nfs1 to the biogenesis of mitochondrial Fe-S clusters in order to maintain cell survival. Finally, depletion of m-Nfs1, which conferred on apo-IRP-1 a high affinity for ferritin mRNA, was associated with the down-regulation of the iron storage protein ferritin.
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PMID:RNA silencing of mitochondrial m-Nfs1 reduces Fe-S enzyme activity both in mitochondria and cytosol of mammalian cells. 1678 28


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