Biomedical subjects

Lambert P van den Heuvel

Publications and source records attributed to Lambert P van den Heuvel.

At least 19 recordsLinked to original sources

PubMed2006

Proteomic inventory of "anchorless" proteins on the colon adenocarcinoma cell surface.

Surface proteins play important pathophysiological roles in health and disease, and accumulating proteomics-based studies suggest that several "non-membrane" proteins are sorted to the cell surface by unconventional mechanisms. Importantly, these proteins may comprise attractive therapeutic targets and novel disease markers for colon cancer. To perform a proteomics-based inventory of these so-called "anchorless" surface proteins, intact colon adenocarcinoma SW480 cells were labeled with membrane-impermeable biotin after which only soluble biotinylated proteins were isolated and identified by nanoLC-MS/MS. Computer-assisted analysis predicted that only 9 of the 97 identified surface-exposed proteins have predicted secretory signal peptides, whereas 2 other proteins have a putative transmembrane segment. Of the 9 proteins with putative signal peptides, 1 was predicted to be retained at the cell surface by a GPI-anchor, whereas 5 other proteins contained an ER-retention motif (KDEL) that should prevent them from being sorted to the cell surface. The remaining 86 soluble "surface" proteins lack known export signals and the possibility that these proteins are candidate substrates of non-classical transporters or exported by unconventional mechanisms is discussed. Alternatively, the large number of "intracellular" and ER-resident proteins may imply that biotinylation approaches are not only specific for surface proteins, but also biased against a certain subset of non-surface proteins. This underscores the importance of post-proteomic verification of proteomics-based inventories on surface-exposed proteins, which eventually should reveal to which extent non-classical export and retention mechanisms contribute to the sorting of "anchorless" proteins to the surface of colon tumor cells.

Adenocarcinoma↗

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

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

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

Protein complexes in the archaeon Methanothermobacter thermautotrophicus analyzed by blue native/SDS-PAGE and mass spectrometry.

Methanothermobacter thermautotrophicus is a thermophilic archaeon that produces methane as the end product of its primary metabolism. The biochemistry of methane formation has been extensively studied and is catalyzed by individual enzymes and proteins that are organized in protein complexes. Although much is known of the protein complexes involved in methanogenesis, only limited information is available on the associations of proteins involved in other cell processes of M. thermautotrophicus. To visualize and identify interacting and individual proteins of M. thermautotrophicus on a proteome-wide scale, protein preparations were separated using blue native electrophoresis followed by SDS-PAGE. A total of 361 proteins, corresponding to almost 20% of the predicted proteome, was identified using peptide mass fingerprinting after MALDI-TOF MS. All previously characterized complexes involved in energy generation could be visualized. Furthermore the expression and association of the heterodisulfide reductase and methylviologen-reducing hydrogenase complexes depended on culture conditions. Also homomeric supercomplexes of the ATP synthase stalk subcomplex and the N5-methyl-5,6,7,8-tetrahydromethanopterin:coenzyme M methyltransferase complex were separated. Chemical cross-linking experiments confirmed that the multimerization of both complexes was not experimentally induced. A considerable number of previously uncharacterized protein complexes were reproducibly visualized. These included an exosome-like complex consisting of four exosome core subunits, which associated with a tRNA-intron endonuclease, thereby expanding the constituency of archaeal exosomes. The results presented show the presence of novel complexes and demonstrate the added value of including blue native gel electrophoresis followed by SDS-PAGE in discovering protein complexes that are involved in catabolic, anabolic, and general cell processes.

Archaeal Proteins↗

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↗

PubMed2004

Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency.

Although most components of the mitochondrial translation apparatus are encoded by nuclear genes, all known molecular defects associated with impaired mitochondrial translation are due to mutations in mitochondrial DNA. We investigated two siblings with a severe defect in mitochondrial translation, reduced levels of oxidative phosphorylation complexes containing mitochondrial DNA (mtDNA)-encoded subunits, and progressive hepatoencephalopathy. We mapped the defective gene to a region on chromosome 3q containing elongation factor G1 (EFG1), which encodes a mitochondrial translation factor. Sequencing of EFG1 revealed a mutation affecting a conserved residue of the guanosine triphosphate (GTP)-binding domain. These results define a new class of gene defects underlying disorders of oxidative phosphorylation.

Chromosomes, Human, Pair 3↗

PubMed2004

Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis.

Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity.

Adenosine Triphosphate↗

PubMed2004

Inhibition of mitochondrial Na+-Ca2+ exchange restores agonist-induced ATP production and Ca2+ handling in human complex I deficiency.

Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca2+ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca2+ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca2+ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca2+ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.

Adenosine Triphosphate↗

PubMed2004

Differences in assembly or stability of complex I and other mitochondrial OXPHOS complexes in inherited complex I deficiency.

NADH-ubiquinone oxidoreductase (complex I) deficiency is amongst the most encountered defects of the mitochondrial oxidative phosphorylation (OXPHOS) system and is associated with a wide variety of clinical signs and symptoms. Mutations in complex I nuclear structural genes are the most common cause of isolated complex I enzyme deficiencies. The cell biological consequences of such mutations are poorly understood. In this paper we have used blue native electrophoresis in order to study how different nuclear mutations affect the integrity of mitochondrial OXPHOS complexes in fibroblasts from 15 complex I-deficient patients. Our results show an important decrease in the levels of intact complex I in patients harboring mutations in nuclear-encoded complex I subunits, indicating that complex I assembly and/or stability is compromised. Different patterns of low molecular weight subcomplexes are present in these patients, suggesting that the formation of the peripheral arm is affected at an early assembly stage. Mutations in complex I genes can also affect the stability of other mitochondrial complexes, with a specific decrease of fully-assembled complex III in patients with mutations in NDUFS2 and NDUFS4. We have extended this analysis to patients with an isolated complex I deficiency in which no mutations in structural subunits have been found. In this group, we can discriminate between complex I assembly and catalytic defects attending to the fact whether there is a correlation between assembly/activity levels or not. This will help us to point more selectively to candidate genes for pathogenic mutations that could lead to an isolated complex I defect.

Blotting, Western↗

PubMed2004

Cytochrome c oxidase biogenesis in a patient with a mutation in COX10 gene.

We report a cytochrome c oxidase (COX)-deficient patient, clinically affected with Leigh-like disease, with a homozygous mutation in the COX10 start codon. Two-dimensional gel electrophoresis showed a decrease of fully assembled COX without the accumulation of partially assembled COX subcomplexes. Western blot analysis with antibodies directed to COX subunits I, II, and IV showed a decrease of these subunits in this patient compared with control. Overexpression of the COX10 protein in the patient's fibroblasts proved that the detected mutation was indeed the disease cause.

Alkyl and Aryl Transferases↗

PubMed2004

Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

BACKGROUND: The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. METHODS: Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. RESULTS: As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. CONCLUSION: The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

Animals↗

PubMed2004

Genetic defects in the oxidative phosphorylation (OXPHOS) system.

The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual genetic control. Due to this bigenomic control, the inheritance of OXPHOS system defects is either maternal, in the case of mitochondrial DNA mutations, autosomal or X-linked, in the case of nuclear gene defects. In this review, our current genetic understanding of OXPHOS system enzyme deficiencies will be summarized, and future directions that the field might take to unravel so-far genetically unresolved OXPHOS system enzyme deficiencies will be described, with special emphasis on complex I biogenesis.

DNA, Mitochondrial↗

PubMed2003

Pathological mutations of the human NDUFS4 gene of the 18-kDa (AQDQ) subunit of complex I affect the expression of the protein and the assembly and function of the complex.

Presented is a study of the impact on the structure and function of human complex I of three different homozygous mutations in the NDUFS4 gene coding for the 18-kDa subunit of respiratory complex I, inherited by autosomal recessive mode in three children affected by a fatal neurological Leigh-like syndrome. The mutations consisted, respectively, of a AAGTC duplication at position 466-470 of the coding sequence, a single base deletion at position 289/290, and a G44A nonsense mutation in the first exon of the gene. All three mutations were found to be associated with a defect of the assembly of a functional complex in the inner mitochondrial membrane. In all the mutations, in addition to destruction of the carboxyl-terminal segment of the 18-kDa subunit, the amino-terminal segment of the protein was also missing. In the mutation that was expected to produce a truncated subunit, the disappearance of the protein was associated with an almost complete disappearance of the NDUFS4 transcript. These observations show the essential role of the NDUFS4 gene in the structure and function of complex I and give insight into the pathogenic mechanism of NDUFS4 gene mutations in a severe defect of complex I.

Amino Acid Sequence↗

PubMed2003

Impaired complex I assembly in a Leigh syndrome patient with a novel missense mutation in the ND6 gene.

We describe a novel mutation in the ND6 gene (T14487C) in a patient with Leigh syndrome. Biochemical analyses indicated a low complex I activity in the patient's fibroblasts but normal values in muscle and liver. Cybrid clones showed a specific complex I defect that correlates with the mutant heteroplasmy levels. Additionally, we demonstrate an altered mobility and a decrease in the levels of fully assembled complex I in the patient's fibroblasts and cybrids, suggesting that the mutation has a profound effect on complex I assembly and/or stability.

Cells, Cultured↗

PubMed2003

Real-time analysis of tyrosine hydroxylase gene expression: a sensitive and semiquantitative marker for minimal residual disease detection of neuroblastoma.

PURPOSE: The purpose of this study was to establish a sensitive and semiquantitative method for the detection of minimal residual disease of neuroblastoma, the most common solid tumor in childhood. EXPERIMENTAL DESIGN: Analysis was performed on a molecular level by reverse transcription-PCR using a new, real-time detection method. We measured two genes simultaneously, tyrosine hydroxylase (TH) as the target gene and glyceraldehyde-3-phosphate dehydrogenase as a reference gene, in blood and bone marrow samples at diagnosis and after follow-up from six patients with neuroblastoma, one patient with ganglioneuroma, and one patient with ganglioneuroblastoma. RESULTS: The sensitivity of the assay was 1:10(6) peripheral WBCs. Four patients with stage IV neuroblastoma and one patient with stage III neuroblastoma were scored positive. The other stage III patient and the other two patients with ganglioneuroma and ganglioneuroblastoma followed by acute lymphoblastic leukemia, respectively, were scored negative. Control bone marrow aspirates were also negative. The TH assay is more sensitive than immunohistochemical detection, and the results of the TH assay corresponded with the results of MYCN amplification. CONCLUSIONS: The described TH assay is specific, sensitive, and semiquantitative and can be used for the detection of neuroblastoma cell involvement in bone marrow and blood at diagnosis and during therapy. Furthermore, the TH assay is a possible prognostic marker for neuroblastoma.

Base Sequence↗