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Biomedical subjects

Jan A M Smeitink

Publications and source records attributed to Jan A M Smeitink.

At least 19 recordsLinked to original sources
PubMed2007

Identification of mitochondrial complex I assembly intermediates by tracing tagged NDUFS3 demonstrates the entry point of mitochondrial subunits.

Biogenesis of human mitochondrial complex I (CI) requires the coordinated assembly of 45 subunits derived from both the mitochondrial and nuclear genome. The presence of CI subcomplexes in CI-deficient cells suggests that assembly occurs in distinct steps. However, discriminating between products of assembly or instability is problematic. Using an inducible NDUFS3-green fluorescent protein (GFP) expression system in HEK293 cells, we here provide direct evidence for the stepwise assembly of CI. Upon induction, six distinct NDUFS3-GFP-containing subcomplexes gradually appeared on a blue native Western blot also observed in wild type HEK293 mitochondria. Their stability was demonstrated by differential solubilization and heat incubation, which additionally allowed their distinction from specific products of CI instability and breakdown. Inhibition of mitochondrial translation under conditions of steady state labeling resulted in an accumulation of two of the NDUFS3-GFP-containing subcomplexes (100 and 150 kDa) and concomitant disappearance of the fully assembled complex. Lifting inhibition reversed this effect, demonstrating that these two subcomplexes are true assembly intermediates. Composition analysis showed that this event was accompanied by the incorporation of at least one mitochondrial DNA-encoded subunit, thereby revealing the first entry point of these subunits.

Blotting, Western↗
PubMed2006

Simultaneous quantification of oxidative stress and cell spreading using 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein.

BACKGROUND: Mitochondrial dysfunction may lead to increased oxidative stress and consequent changes in cell spreading. Here, we describe and validate a novel method for simultaneous quantification of these two parameters. METHODS: Human skin fibroblasts were loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-H(2)DCF), and its oxidative conversion into CM-DCF was monitored as a function of time by video-rate confocal microscopy and real-time image averaging. Cell size was determined after binarization of the acquired images. RESULTS: At the lowest practical laser output, CM-DCF formation occurred with zero order kinetics, indicating that [CM-H(2)DCF] was not rate-limiting and that the rate of [CM-DCF] formation (V(CM-DCF)) was a function of the cellular oxidant level. Analysis of fibroblasts of a healthy control subject and a patient with a deficiency of NADH:ubiquinone oxidoreductase, the first complex of the oxidative phosphorylation system, revealed a significant increase in cellular oxidant level in the latter cells that was, however, not accompanied by a change in cell spreading. Conversely, chronic treatment with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a derivative of vitamin E, markedly decreased the oxidant level and cell spreading in both control and patient fibroblasts. CONCLUSIONS: We present a reliable method for simultaneous quantification of oxidant levels and cell spreading in living cells.

Antioxidants↗
PubMed2006

The mitochondrial 13513G > A mutation is most frequent in Leigh syndrome combined with reduced complex I activity, optic atrophy and/or Wolff-Parkinson-White.

The m.13513G > A transition in the mitochondrial gene encoding the ND5 subunit of respiratory chain complex I, can cause mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and has been reported to be a frequent cause of Leigh syndrome (LS). We determined the frequency of the mutation in a cohort of 123 patients with reduced complex I activity in muscle (n = 113) or fibroblast (n = 10) tissue. We describe a Pyrosequencing assay for rapid detection and quantification of the m.13513G > A mutation. Two patients with the mutation were identified; both had LS, optical atrophy and a Wolff-Parkinson-White Syndrome (WPWS)-like cardiac conduction defect. The clinical presentation of the m.13513G > A mutation is discussed. We conclude that the m.13513G > A mutation seems not as frequent as previously suggested and is most likely to be present in patients with Leigh (-like) syndrome combined with a complex I deficiency, optic atrophy and/ or WPWS. In addition, we confirmed that the adjacent m.13514A > G mutation is a rare cause of LS or MELAS since no cases with this transition were found.

Cohort Studies↗
PubMed2006

Mutated ND2 impairs mitochondrial complex I assembly and leads to Leigh syndrome.

We describe a novel mitochondrial ND2 mutation (T4681C) in a patient presenting with Leigh Syndrome. Biochemical analyses revealed a low isolated complex I activity in patient's fibroblasts, blood and skeletal muscle. Mutant transmitochondrial cybrid clones retained the specific complex I defect, demonstrating the mitochondrial genetic origin of the disease. The mutation leads to a L71P substitution at an evolutionary conserved amino acid stretch. By two-dimensional blue native electrophoresis (2D-BN-SDS-PAGE), decreased complex I levels were observed together with an accumulation of specific assembly intermediates, suggesting that the mutation disturbs the complex I assembly pathway.

Amino Acid Sequence↗
PubMed2006

Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs.

The 13 polypeptides encoded in mitochondrial DNA (mtDNA) are synthesized in the mitochondrial matrix on a dedicated protein-translation apparatus that resembles that found in prokaryotes. Here, we have investigated the genetic basis for a mitochondrial protein-synthesis defect associated with a combined oxidative phosphorylation enzyme deficiency in two patients, one of whom presented with encephalomyopathy and the other with hypertrophic cardiomyopathy. Sequencing of candidate genes revealed the same homozygous mutation (C997T) in both patients in TSFM, a gene coding for the mitochondrial translation elongation factor EFTs. EFTs functions as a guanine nucleotide exchange factor for EFTu, another translation elongation factor that brings aminoacylated transfer RNAs to the ribosomal A site as a ternary complex with guanosine triphosphate. The mutation predicts an Arg333Trp substitution at an evolutionarily conserved site in a subdomain of EFTs that interacts with EFTu. Molecular modeling showed that the substitution disrupts local subdomain structure and the dimerization interface. The steady-state levels of EFTs and EFTu in patient fibroblasts were reduced by 75% and 60%, respectively, and the amounts of assembled complexes I, IV, and V were reduced by 35%-91% compared with the amounts in controls. These phenotypes and the translation defect were rescued by retroviral expression of either EFTs or EFTu. These data clearly establish mutant EFTs as the cause of disease in these patients. The fact that the same mutation is associated with distinct clinical phenotypes suggests the presence of genetic modifiers of the mitochondrial translation apparatus.

Amino Acid Sequence↗
PubMed2006

Multiple oxidative phosphorylation deficiencies in severe childhood multi-system disorders due to polymerase gamma (POLG1) mutations.

Failure to thrive, feeding difficulties, variable forms of infantile epilepsy or psychomotor developmental delay and hypotonia were the most frequent clinical disease presentations in eight children with combined oxidative phosphorylation enzyme complex deficiencies carrying mutations in the polymerase gamma (POLG1) gene. Five out of eight patients developed severe liver dysfunction during the course of the disease. Three of these patients fulfilled the disease criteria for Alpers syndrome. Most children showed deficiencies of respiratory chain enzyme complexes I and III, in combination with complex II, complex IV and/or PDHc in muscle, whereas in fibroblasts normal enzyme activities were measured. All children carried homozygous or compound heterozygous mutations in the POLG1 gene, including two novel mutations in association with mtDNA depletion. Conclusion We suggest performing POLG1 mutation analysis in children with combined oxidative phosphorylation deficiencies in muscle, even if the clinical picture is not Alpers syndrome.

DNA Mutational Analysis↗
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

Mitochondrial complex I: structure, function and pathology.

Oxidative phosphorylation (OXPHOS) has a prominent role in energy metabolism of the cell. Being under bigenomic control, correct biogenesis and functioning of the OXPHOS system is dependent on the finely tuned interaction between the nuclear and the mitochondrial genome. This suggests that disturbances of the system can be caused by numerous genetic defects and can result in a variety of metabolic and biochemical alterations. Consequently, OXPHOS deficiencies manifest as a broad clinical spectrum. Complex I, the biggest and most complicated enzyme complex of the OXPHOS system, has been subjected to thorough investigation in recent years. Significant progress has been made in the field of structure, composition, assembly, and pathology. Important gains in the understanding of the Goliath of the OXPHOS system are: exposing the electron transfer mechanism and solving the crystal structure of the peripheral arm, characterization of almost all subunits and some of their functions, and creating models to elucidate the assembly process with concomitant identification of assembly chaperones. Unravelling the intricate mechanisms underlying the functioning of this membrane-bound enzyme complex in health and disease will pave the way for developing adequate diagnostic procedures and advanced therapeutic treatment strategies.

Animals↗
PubMed2006

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency.

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic Ca2+ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca2+ indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial Ca2+ concentration ([Ca2+]M) and, consequently, the Ca2+-stimulated increase in [ATP]M, thus allowing studies of how the increase in [ATP]M and the cytosolic Ca2+ removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [Ca2+]M and [ATP]M by maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic Ca2+ indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca2+ removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca2+ removal during stimulation with a Ca2+-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic Ca2+ removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca2+ pumps. This work reveals the usefulness of rhodamine-based Ca2+ indicators in examining the role of intramitochondrial Ca2+ in mitochondrial (patho) physiology.

Adenosine Triphosphate↗
PubMed2006

Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology.

BACKGROUND: Diagnosis of mitochondrial disorders usually requires a muscle biopsy to examine mitochondrial function. We describe our diagnostic procedure and results for 29 patients with mitochondrial disorders. METHODS: Muscle biopsies were from 43 healthy individuals and 29 patients with defects in one of the oxidative phosphorylation (OXPHOS) complexes, the pyruvate dehydrogenase complex (PDHc), or the adenine nucleotide translocator (ANT). Homogenized muscle samples were used to determine the oxidation rates of radiolabeled pyruvate, malate, and succinate in the absence or presence of various acetyl Co-A donors and acceptors, as well as specific inhibitors of tricarboxylic acid cycle or OXPHOS enzymes. We determined the rate of ATP production from oxidation of pyruvate. RESULTS: Each defect in the energy-generating system produced a specific combination of substrate oxidation impairments. PDHc deficiencies decreased substrate oxidation reactions containing pyruvate. Defects in complexes I, III, and IV decreased oxidation of pyruvate plus malate, with normal to mildly diminished oxidation of pyruvate plus carnitine. In complex V defects, pyruvate oxidation improved by addition of carbonyl cyanide 3-chlorophenyl hydrazone, whereas other oxidation rates were decreased. In most patients, ATP production was decreased. CONCLUSION: The proposed method can be successfully applied to the diagnosis of defects in PDHc, OXPHOS complexes, and ANT.

Adenosine Triphosphate↗
PubMed2006

Simultaneous quantitative measurement and automated analysis of mitochondrial morphology, mass, potential, and motility in living human skin fibroblasts.

BACKGROUND: Understanding the interdependence of mitochondrial and cellular functioning in health and disease requires detailed knowledge about the coupling between mitochondrial structure, motility, and function. Currently, no rapid approach is available for simultaneous quantification of these parameters in single living cells. METHODS: Human skin fibroblasts were pulse-loaded with the mitochondria-selective fluorescent cation rhodamine 123. Next, mitochondria were visualized using video-rate (30 Hz) confocal microscopy and real-time image averaging. To highlight the mitochondria, the acquired images were binarized using a novel image processing strategy. RESULTS: Our approach enabled rapid and simultaneous quantification of mitochondrial morphology, mass, potential, and motility. It was found that acute inhibition of mitochondrial complex I (NADH:ubiquinone oxidoreductase) by means of rotenone transiently reduced mitochondrial branching, area, and potential. In contrast, mitochondrial motility was permanently reduced. CONCLUSIONS: We present and validate a novel approach for rapid, unbiased, and simultaneous quantification of multiple mitochondrial parameters in living cells. Because this method is automated, large numbers of cells can be analyzed in a short period of time.

Cells, Cultured↗
PubMed2006

Sequence analysis of the structural nuclear encoded subunits and assembly genes of cytochrome c oxidase in a cohort of 10 isolated complex IV-deficient patients revealed five mutations.

The mitochondrial oxidative phosphorylation system is composed of five multiprotein complexes. The fourth complex of this system, cytochrome c oxidase (complex IV), consists of 13 subunits: 3 encoded by mitochondrial DNA and 10 encoded by the nuclear genome. Patients with an isolated complex IV deficiency frequently harbor mutations in nuclear genes encoding for proteins necessary for the assembly of the complex. Strikingly, until now, no mutations have been detected in the nuclear encoded structural subunits of complex IV in these patients. We report the results of a mutational analysis study in patients with isolated complex IV deficiency screened for mutations in all structural genes as well as assembly genes known to cause complex IV deficiency. Four patients carried mutations in the complex IV assembly gene SURF1. One patient harbored a mutation in the COX10 gene involved in heme A synthesis. Mutations in the 10 nuclear encoded structural genes were not present.

Alkyl and Aryl Transferases↗
PubMed2005

Decreased agonist-stimulated mitochondrial ATP production caused by a pathological reduction in endoplasmic reticulum calcium content in human complex I deficiency.

Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.

Adenosine Triphosphate↗
PubMed2005

Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency.

Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency.

Cell Cycle↗
PubMed2005

Inhibition of complex I of the electron transport chain causes O2-. -mediated mitochondrial outgrowth.

Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial O(2)(-)* production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial O(2)(-)* production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIPY(581/591). Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by O(2)(-)* to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a twofold increase in the rate of O(2)(-)* production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial O(2)(-)* production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this O(2)(-)* to prevent alterations in mitochondrial morphology.

Cells, Cultured↗
PubMed2005

Human mitochondrial complex I assembly is mediated by NDUFAF1.

Complex I (NADH:ubiquinone oxidoreductase) is the largest multiprotein enzyme of the oxidative phosphorylation system. Its assembly in human cells is poorly understood and no proteins assisting this process have yet been described. A good candidate is NDUFAF1, the human homologue of Neurospora crassa complex I chaperone CIA30. Here, we demonstrate that NDUFAF1 is a mitochondrial protein that is involved in the complex I assembly process. Modulating the intramitochondrial amount of NDUFAF1 by knocking down its expression using RNA interference leads to a reduced amount and activity of complex I. NDUFAF1 is associated to two complexes of 600 and 700 kDa in size of which the relative distribution is altered in two complex I deficient patients. Analysis of NDUFAF1 expression in a conditional complex I assembly system shows that the 700 kDa complex may represent a key step in the complex I assembly process. Based on these data, we propose that NDUFAF1 is an important protein for the assembly/stability of complex I.

Cell Fractionation↗
PubMed2004

Optimized spectrophotometric assay for the completely activated pyruvate dehydrogenase complex in fibroblasts.

BACKGROUND: Analysis of the pyruvate dehydrogenase complex (PDHc) activity in human skin fibroblasts is hampered by low enzyme activity in the cells. The most commonly used radiochemical method detects the formation of (14)CO(2), an endproduct of the E1 component of PDHc, from [1-(14)C]pyruvate. METHODS: We report a spectrophotometric method for the analysis of PDHc activity in fibroblasts based on detection of NADH formation via a p-iodonitrotetrazolium violet (INT)-coupled system. We investigated in detail the specific requirements of this assay, such as cofactor requirements and the effects of suggested stimulatory compounds and different cell disruption procedures. The reliability of the optimized assay was studied by investigation of patients previously diagnosed with PDHc deficiency and by comparison with results from the radiochemical method. RESULTS: Mean (SD) total PDHc activities were 136 (31) and 58 (21) mU/U of citrate synthase in fibroblast homogenates from 10 healthy volunteers and 7 PDHc-deficient patients, respectively, by the spectrophotometric assay. Similar results were obtained in a mitochondrial fraction. Dithiothreitol (DTT) increased the nonspecific inhibitor-insensitive rate with less pronounced effect on the specific rate of PDHc activity. Administration of DTT increased PDHc activity to 193 (3)% of control activity (without DTT), but decreased the inhibitor-sensitive rate from 99 (0.3)% (without DTT) to 69 (2)% (with 0.3 mmol/L DTT). CONCLUSION: The simple, optimized spectrophotometric assay for PDHc analysis allows reliable investigation of the enzyme complex in human skin fibroblasts.

Cells, Cultured↗