Biomedical subjects

Stefan Dröse

Publications and source records attributed to Stefan Dröse.

14 recordsLinked to original sources

PubMed2006

Mitochondrial dysfunction, peroxidation damage and changes in glutathione metabolism in PARK6.

Oxidative stress and protein aggregation are biochemical hallmarks of Parkinson's disease (PD), a frequent sporadic late-onset degenerative disorder particularly of dopaminergic neurons in the substantia nigra, resulting in impaired spontaneous movement. PARK6 is a rare autosomal-recessively inherited disorder, mimicking the clinical picture of PD with earlier onset and slower progression. Genetic data demonstrated PARK6 to be caused by mutations in the protein PINK1, which is localized to mitochondria and has a serine-threonine kinase domain. To study the effect of PINK1 mutations on oxidative stress, we used primary fibroblasts and immortalized lymphoblasts from three patients homozygous for G309D-PINK1. Oxidative stress was evident from increases in lipid peroxidation and in antioxidant defenses by mitochondrial superoxide dismutase and glutathione. Elevated levels of glutathione reductase and glutathione-S-transferase were also observed. As a putative cause of oxidation, a mild decrease in complex I activity and a trend to superoxide elevation were detectable. These data indicate that PINK1 function is critical to prevent oxidative damage and that peripheral cells may be useful for studies of progression and therapy of PARK6.

Adult↗

PubMed2006

The proton pumping stoichiometry of purified mitochondrial complex I reconstituted into proteoliposomes.

NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme of aerobic electron transfer. The mechanism how it uses redox energy to pump protons across the bioenergetic membrane is still not understood. Here we determined the pumping stoichiometry of mitochondrial complex I from the strictly aerobic yeast Yarrowia lipolytica. With intact mitochondria, the measured value of 3.8H(+)/2e indicated that four protons are pumped per NADH oxidized. For purified complex I reconstituted into proteoliposomes we measured a very similar pumping stoichiometry of 3.6H(+)/2e . This is the first demonstration that the proton pump of complex I stayed fully functional after purification of the enzyme.

Aerobiosis↗

PubMed2006

Secondary mitochondrial dysfunction in propionic aciduria: a pathogenic role for endogenous mitochondrial toxins.

Mitochondrial dysfunction during acute metabolic crises is considered an important pathomechanism in inherited disorders of propionate metabolism, i.e. propionic and methylmalonic acidurias. Biochemically, these disorders are characterized by accumulation of propionyl-CoA and metabolites of alternative propionate oxidation. In the present study, we demonstrate uncompetitive inhibition of PDHc (pyruvate dehydrogenase complex) by propionyl-CoA in purified porcine enzyme and in submitochondrial particles from bovine heart being in the same range as the inhibition induced by acetyl-CoA, the physiological product and known inhibitor of PDHc. Evaluation of similar monocarboxylic CoA esters showed a chain-length specificity for PDHc inhibition. In contrast with CoA esters, non-esterified fatty acids did not inhibit PDHc activity. In addition to PDHc inhibition, analysis of respiratory chain and tricarboxylic acid cycle enzymes also revealed an inhibition by propionyl-CoA on respiratory chain complex III and alpha-ketoglutarate dehydrogenase complex. To test whether impairment of mitochondrial energy metabolism is involved in the pathogenesis of propionic aciduria, we performed a thorough bioenergetic analysis in muscle biopsy specimens of two patients. In line with the in vitro results, oxidative phosphorylation was severely compromised in both patients. Furthermore, expression of respiratory chain complexes I-IV and the amount of mitochondrial DNA were strongly decreased, and ultrastructural mitochondrial abnormalities were found, highlighting severe mitochondrial dysfunction. In conclusion, our results favour the hypothesis that toxic metabolites, in particular propionyl-CoA, are involved in the pathogenesis of inherited disorders of propionate metabolism, sharing mechanistic similarities with propionate toxicity in micro-organisms.

Acetyl Coenzyme A↗

PubMed2006

The Redox-Bohr group associated with iron-sulfur cluster N2 of complex I.

Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H(+)/2e(-). This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.

Electron Transport Complex I↗

PubMed2006

K+-independent actions of diazoxide question the role of inner membrane KATP channels in mitochondrial cytoprotective signaling.

Activation by diazoxide and inhibition by 5-hydroxydecanoate are the hallmarks of mitochondrial ATP-sensitive K+ (K(ATP)) channels. Opening of these channels is thought to trigger cytoprotection (preconditioning) through the generation of reactive oxygen species. However, we found that diazoxide-induced oxidation of the widely used reactive oxygen species indicator 2',7'-dichlorodihydrofluorescein in isolated liver and heart mitochondria was observed in the absence of ATP or K+ and therefore independent of K(ATP) channels. The response was blocked by stigmatellin, implying a role for the cytochrome bc1 complex (complex III). Diazoxide, though, did not increase hydrogen peroxide (H2O2) production (quantitatively measured with Amplex Red) in intact mitochondria, submitochondrial particles, or purified cytochrome bc1 complex. We confirmed that diazoxide inhibited succinate oxidation, but it also weakly stimulated state 4 respiration even in K+-free buffer, excluding a role for K(ATP) channels. Furthermore, we have shown previously that 5-hydroxydecanoate is partially metabolized, and we hypothesized that fatty acid metabolism may explain the ability of this putative mitochondrial K(ATP) channel blocker to inhibit diazoxide-induced flavoprotein fluorescence, commonly used as an assay of K(ATP) channel activity. Indeed, consistent with our hypothesis, we found that decanoate inhibited diazoxide-induced flavoprotein oxidation. Taken together, our data question the "mitochondrial K(ATP) channel" hypothesis of preconditioning. Diazoxide did not evoke superoxide (which dismutates to H2O2) from the respiratory chain by a direct mechanism, and the stimulatory effects of this compound on mitochondrial respiration and 2',7'-dichlorodihydrofluorescein oxidation were not due to the opening of K(ATP) channels.

Animals↗

PubMed2006

Heterocyclic analogues of squamocin as inhibitors of mitochondrial complex I. On the role of the terminal lactone of annonaceous acetogenins.

Heterocyclic analogues of squamocin have been semisynthesized by condensation reactions between squamocin-derived alpha-keto esters and heterodinucleophiles. The strong complex I inhibitory potency of squamocin-benzimidazole, a hybrid derivative, illustrates for the first time the functional analogy existing between the terminal butenolide of annonaceous acetogenins and heteroaromatic substructures of classic inhibitors of the enzyme. This finding supports the categorization of this atypical group of inhibitors as antagonists of the ubiquinone substrates. In addition, competition experiments of squamocin-benzimidazole versus squamocin and rolliniastatin-2 suggest that the binding of this hybrid inhibitor is responsible for a negative allosteric effect at the level of the first ubiquinone-binding site (A site) of mitochondrial complex I. This result supports the existence of a large cooperatively regulated inhibitor/ubiquinone-binding pocket located within the catalytic core of the enzyme, consisting of the association of the previously defined affinity sites A and B.

Acetogenins↗

PubMed2005

Proton pumping by complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica reconstituted into proteoliposomes.

The mechanism of energy converting NADH:ubiquinone oxidoreductase (complex I) is still unknown. A current controversy centers around the question whether electron transport of complex I is always linked to vectorial proton translocation or whether in some organisms the enzyme pumps sodium ions instead. To develop better experimental tools to elucidate its mechanism, we have reconstituted the affinity purified enzyme into proteoliposomes and monitored the generation of DeltapH and Deltapsi. We tested several detergents to solubilize the asolectin used for liposome formation. Tightly coupled proteoliposomes containing highly active complex I were obtained by detergent removal with BioBeads after total solubilization of the phospholipids with n-octyl-beta-D-glucopyranoside. We have used dyes to monitor the formation of the two components of the proton motive force,DeltapH and Deltapsi, across the liposomal membrane, and analyzed the effects of inhibitors, uncouplers and ionophores on this process. We show that electron transfer of complex I of the lower eukaryote Y. lipolytica is clearly linked to proton translocation. While this study was not specifically designed to demonstrate possible additional sodium translocating properties of complex I, we did not find indications for primary or secondary Na+ translocation by Y. lipolytica complex I.

Electron Transport Complex I↗

PubMed2005

Proteomic and functional analyses reveal a mitochondrial dysfunction in P301L tau transgenic mice.

Transgenic mice overexpressing the P301L mutant human tau protein exhibit an accumulation of hyperphosphorylated tau and develop neurofibrillary tangles. The consequences of tau pathology were investigated here by proteomics followed by functional analysis. Mainly metabolism-related proteins including mitochondrial respiratory chain complex components, antioxidant enzymes, and synaptic proteins were identified as modified in the proteome pattern of P301L tau mice. Significantly, the reduction in mitochondrial complex V levels in the P301L tau mice revealed using proteomics was also confirmed as decreased in human P301L FTDP-17 (frontotemporal dementia with parkinsonism linked to chromosome 17) brains. Functional analysis demonstrated a mitochondrial dysfunction in P301L tau mice together with reduced NADH-ubiquinone oxidoreductase activity and, with age, impaired mitochondrial respiration and ATP synthesis. Mitochondrial dys-function was associated with higher levels of reactive oxygen species in aged transgenic mice. Increased tau pathology as in aged homozygous P301L tau mice revealed modified lipid peroxidation levels and the up-regulation of antioxidant enzymes in response to oxidative stress. Furthermore, P301L tau mitochondria displayed increased vulnerability toward beta-amyloid (Abeta) peptide insult, suggesting a synergistic action of tau and Abeta pathology on the mitochondria. Taken together, we conclude that tau pathology involves a mitochondrial and oxidative stress disorder possibly distinct from that caused by Abeta.

Alzheimer Disease↗

PubMed2004

5-Hydroxydecanoate is metabolised in mitochondria and creates a rate-limiting bottleneck for beta-oxidation of fatty acids.

5-Hydroxydecanoate (5-HD) blocks pharmacological and ischaemic preconditioning, and has been postulated to be a specific inhibitor of mitochondrial ATP-sensitive K(+) (K(ATP)) channels. However, recent work has shown that 5-HD is activated to 5-hydroxydecanoyl-CoA (5-HD-CoA), which is a substrate for the first step of beta-oxidation. We have now analysed the complete beta-oxidation of 5-HD-CoA using specially synthesised (and purified) substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared it with the metabolism of the physiological substrate decanoyl-CoA. At the second step of beta-oxidation, catalysed by enoyl-CoA hydratase, enzyme kinetics were similar using either decenoyl-CoA or 5-hydroxydecenoyl-CoA as substrate. The last two steps were investigated using l-3-hydroxyacyl-CoA dehydrogenase (HAD) coupled to 3-ketoacyl-CoA thiolase. V(max) for the metabolite of 5-HD (3,5-dihydroxydecanoyl-CoA) was fivefold slower than for the corresponding metabolite of decanoate (l-3-hydroxydecanoyl-CoA). The slower kinetics were not due to accumulation of d-3-hydroxyoctanoyl-CoA since this enantiomer did not inhibit HAD. Molecular modelling of HAD complexed with 3,5-dihydroxydecanoyl-CoA suggested that the 5-hydroxyl group could decrease HAD turnover rate by interacting with critical side chains. Consistent with the kinetic data, 5-hydroxydecanoyl-CoA alone acted as a weak substrate in isolated mitochondria, whereas addition of 100 mum 5-HD-CoA inhibited the metabolism of decanoyl-CoA or lauryl-carnitine. In conclusion, 5-HD is activated, transported into mitochondria and metabolised via beta-oxidation, albeit with rate-limiting kinetics at the penultimate step. This creates a bottleneck for beta-oxidation of fatty acids. The complex metabolic effects of 5-HD invalidate the use of 5-HD as a blocker of mitochondrial K(ATP) channels in studies of preconditioning.

Animals↗

PubMed2004

Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species.

Dihydrocalcein (H2-calcein) is recommended as a superior probe for intracellular radical (ROS) detection as different to dichlorodihydrofluorescein (H2-DCF), its oxidation product calcein is thought not to leak out of cells. We determined whether H2-calcein is a useful tool to measure ROS in vascular smooth muscle cells. In vitro, both compounds were oxidized by peroxynitrite, hydroxyl radicals and peroxidase, but not hydrogen peroxide or nitric oxide. The intracellular half-life of calcein was several hours whereas that of DCF was approximately 5 min. Intracellular ROS, as generated by the angiotensin II (Ang II)-activated NADPH oxidase, did not increase the oxidation of H2-calcein but increased the oxidation of H2-DCF by approximately 50%. Similar changes were detected using electron spin resonance spectroscopy. Inhibition of the NADPH oxidase using gp91ds-tat prevented the Ang II-induced increase in DCF fluorescence, without affecting cells loaded with H2-calcein. Diphenylene iodonium (DPI), which inhibits all flavin-dependent enzymes, including those in the respiratory chain, had little effect on the basal but prevented the Ang II-induced oxidation of H2-DCF. In contrast, DPI inhibited H2-calcein oxidation in non-stimulated cells by almost 50%. Blockade of respiratory chain complex I inhibited H2-calcein oxidation, whereas inhibitors of complex III were without effect. Calcein accumulated in the mitochondria, whereas DCF was localized in the cytoplasm. In submitochondrial particles, H2-calcein, but not H2-DCF inhibited complex I activity. These observations indicate that H2-DCF is an indicator for intracellular ROS, whereas the oxidation of H2-calcein most likely occurs as a consequence of direct electron transfer to mitochondrial complex I.

Angiotensin II↗

PubMed2003

Proton pumping by NADH:ubiquinone oxidoreductase. A redox driven conformational change mechanism?

The modular evolutionary origin of NADH:ubiquinone oxidoreductase (complex I) provides useful insights into its functional organization. Iron-sulfur cluster N2 and the PSST and 49 kDa subunits were identified as key players in ubiquinone reduction and proton pumping. Structural studies indicate that this 'catalytic core' region of complex I is clearly separated from the membrane. Complex I from Escherichia coli and Klebsiella pneumoniae was shown to pump sodium ions rather than protons. These new insights into structure and function of complex I strongly suggest that proton or sodium pumping in complex I is achieved by conformational energy transfer rather than by a directly linked redox pump.

Amino Acid Sequence↗

PubMed2002

Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids.

NADH:ubiquinone oxidoreductase (complex I) is the largest multiprotein complex of the mitochondrial respiratory chain. His-tagged complex I purified from the strictly aerobic yeast Yarrowia lipolytica exhibited electron transfer rates from NADH to n-decylubiquinone of less than 2% when compared to turnover numbers calculated for native mitochondrial membranes from this organism. Reactivation was observed upon addition of asolectin, purified phospholipids and different phospholipid mixtures. Maximal activities of 6-7 micromol NADH min(-1) mg(-1) were observed following incubation with a mixture of 76% phosphatidylcholine, 19% phosphatidylethanolamine and 5% cardiolipin. For full reactivation, 400-500 phospholipid molecules per complex I were needed. This demonstrated that the inactivation of complex I from Y. lipolytica by general delipidation could be fully reversed simply by returning the phospholipids that had been removed during the purification procedure. Thus, our homogeneous and highly pure complex I preparation had retained its full catalytic potential and no specific, functionally essential component had been lost. As the purified enzyme was also found to contain only substoichiometric amounts of ubiquinone-9 (0.2-0.4 mol/mol), a functional requirement of this endogeneous ubiquinone could also be excluded.

Electron Transport Complex I↗

PubMed2002

Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I.

The obligate aerobic yeast Yarrowia lipolytica is introduced as a powerful new model for the structural and functional analysis of mitochondrial complex I. A brief introduction into the biology and the genetics of this nonconventional yeast is given and the relevant genetic tools that have been developed in recent years are summarized. The respiratory chain of Y. lipolytica contains complexes I-IV, one "alternative" NADH-dehydrogenase (NDH2) and a non-heme alternative oxidase (AOX). Because the NADH binding site of NDH2 faces the mitochondrial intermembrane space rather than the matrix, complex I is an essential enzyme in Y. lipolytica. Nevertheless, complex I deletion strains could be generated by attaching the targeting sequence of a matrix protein, thereby redirecting NDH2 to the matrix side. Deletion strains for several complex I subunits have been constructed that can be complemented by shuttle plasmids carrying the deleted gene. Attachment of a hexa-histidine tag to the NUGM (30 kDa) subunit allows fast and efficient purification of complex I from Y. lipolytica by affinity-chromatography. The purified complex has lost most of its NADH:ubiquinone oxidoreductase activity, but is almost fully reactivated by adding 400-500 molecules of phosphatidylcholine per complex I. The established set of genetic tools has proven useful for the site-directed mutagenesis of individual subunits of Y. lipolytica complex I. Characterization of a number of mutations already allowed for the identification of several functionally important amino acids, demonstrating the usefulness of this approach.

Electron Spin Resonance Spectroscopy↗

PubMed2002

Concanamycin A, the specific inhibitor of V-ATPases, binds to the V(o) subunit c.

Vacuolar-type ATPase (V-ATPase) purified from the midgut of the tobacco hornworm Manduca sexta is inhibited 50% by 10 nm of the plecomacrolide concanamycin A, the specific inhibitor of V-ATPases. To determine the binding site(s) of that antibiotic in the enzyme complex, labeling with the semisynthetic 9-O-[p-(trifluoroethyldiazirinyl)-benzoyl]-21,23-dideoxy-23-[(125)I]iodo-concanolide A (J-concanolide A) was performed, which still inhibits the V-ATPase 50% at a concentration of 15-20 microm. Upon treatment with UV light, a highly reactive carbene is generated from this concanamycin derivative, resulting in the formation of a covalent bond to the enzyme. In addition, the radioactive tracer (125)I makes the detection of the labeled subunit(s) feasible. Treatment of the V(1)/V(o) holoenzyme, the V(o) complex, and the V-ATPase containing goblet cell apical membranes with concanolide resulted in the labeling of only the proteolipid, subunit c, of the proton translocating V(o) complex. Binding of J-concanolide A to subunit c was prevented in a concentration-dependent manner by concanamycin A, indicating that labeling was specific. Binding was also prevented by the plecomacrolides bafilomycin A(1) and B(1), respectively, but not by the benzolactone enamide salicylihalamide, a member of a novel class of V-ATPase inhibitors.

Amino Acid Sequence↗