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Ronald J.a. Wanders - One of the best experts on this subject based on the ideXlab platform.

  • Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient.
    Biochimica et biophysica acta, 2013
    Co-Authors: S Violante, Lodewijk Ijlst, Ronald J.a. Wanders, Janet Koster, Isabel Tavares De Almeida, F V Ventura, Heleen Te Brinke, Sander M Houten
    Abstract:

    Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine Translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.

  • Carnitine palmitoyltransferase 2 and carnitine/acylcarnitine Translocase are involved in the mitochondrial synthesis and export of acylcarnitines
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2013
    Co-Authors: S Violante, Lodewijk Ijlst, Ronald J.a. Wanders, Isabel Tavares De Almeida, F V Ventura, Heleen Te Brinke, Sander M Houten
    Abstract:

    Acylcarnitines are commonly used in the diagnosis of mitochondrial fatty acid β-oxidation disorders (mFAODs). It is generally assumed that this plasma acylcarnitine profile reflects the mitochondrial accumulation of acyl-CoAs. The identity of the enzymes and the mitochondrial and plasmalemmal transporters involved in the synthesis and export of these metabolites have remained undefined. We used lentiviral shRNA to knock down the expression of medium-chain acyl-CoA dehydrogenase (MCAD) in control and carnitine palmitoyltransferase 2 (CPT2)-, carnitine/acylcarnitine Translocase (CACT)-, and plasmalemmal carnitine transporter (OCTN2)-deficient human fibroblasts. These cell lines, including mock-transduced controls, were loaded with decanoic acid and carnitine, followed by the measurement of the acylcarnitine profile in the extracellular medium. In control fibroblasts, MCAD knockdown markedly increased the production of octanoylcarnitine (3-fold, P

  • Carnitine‐acylcarnitine Translocase deficiency: case report and review of the literature
    Acta paediatrica (Oslo Norway : 1992), 2007
    Co-Authors: M. E. Rubio-gozalbo, Ronald J.a. Wanders, P. Vos, P Ph Forget, S. B. Van Der Meer, Hans R. Waterham, Jaap A. Bakker
    Abstract:

    Carnitine-Acylcarnitine Translocase (CACT) deficiency is an inborn error of metabolism involving the mitochondrial beta-oxidation of long-chain fatty acids. The aim of this study was to report on a new case (neonatal phenotype) and review the literature data on 24 previously reported cases. Clinical data of the new case are described and compared with the previous reports. The patient with a novel mutation had clinical features and biochemical findings similar to those of the other reported patients. CACT is an entity in which clinical encephalopathy, hepatomegaly and arrythmias are common. Hyperammonaemia and elevation of creatine kinase seem to be constant findings as in other disorders of mitochondrial beta-oxidation of long-chain fatty acids. The mortality rate is very high.

  • dif-1 and colt, both implicated in early embryonic development, encode carnitine acylcarnitine Translocase
    Molecular genetics and metabolism, 2005
    Co-Authors: Nadia A. Oey, Lodewijk Ijlst, Carlo W.t. Van Roermund, Frits A. Wijburg, Ronald J.a. Wanders
    Abstract:

    It has always been assumed that during development the embryo and fetus depend only on glycolysis for energy generation and that they do not oxidize fatty acids. Recently, however, we found abundant expression and activity of fatty acid oxidation (FAO) enzymes in the human embryo and fetus. In a search for FAO gene expression during development we came across two embryonic differentiation genes: differentiation defective (dif-1) and congested-like trachea (colt) of Caenorhabditis elegans and Drosophila melanogaster, respectively. Earlier studies showed that expression of these two genes is essential during developmental stages with high energy requirements. Both dif-1 and colt encode proteins with sequence similarity to the mitochondrial carnitine acylcarnitine carrier (CACT), which suggests that the DIF-1 and COLT proteins might be functional orthologues of CACT. To investigate this, we expressed both dif-1 and colt in Saccharomyces cerevisiae. Our results show that DIF-1 and COLT can functionally complement a yeast CACT deletion strain and thus function as carnitine acylcarnitine transporters. This finding is well in line with the recent observation that embryos are capable of oxidizing fatty acids and furthermore implies that FAO is essential during early embryonic development when the energy demand is high.

  • Carnitine-Acylcarnitine Translocase deficiency, clinical, biochemical and genetic aspects.
    Molecular Aspects of Medicine, 2004
    Co-Authors: M. E. Rubio-gozalbo, Hans R. Waterham, Jaap A. Bakker, Ronald J.a. Wanders
    Abstract:

    The Carnitine-Acylcarnitine Translocase (CACT) is one of the components of the carnitine cycle. The carnitine cycle is necessary to shuttle long-chain fatty acids from the cytosol into the intramitochondrial space where mitochondrial beta-oxidation of fatty acids takes place. The oxidation of fatty acids yields acetyl-coenzyme A (CoA) units, which may either be degraded to CO(2) and H(2)O in the citric acid cycle to produce ATP or converted into ketone bodies which occurs in liver and kidneys. Metabolic consequences of a defective CACT are hypoketotic hypoglycaemia under fasting conditions, hyperammonemia, elevated creatine kinase and transaminases, dicarboxylic aciduria, very low free carnitine and an abnormal acylcarnitine profile with marked elevation of the long-chain acylcarnitines. Clinical signs and symptoms in CACT deficient patients, are a combination of energy depletion and endogenous toxicity. The predominantly affected organs are brain, heart and skeletal muscle, and liver, leading to neurological abnormalities, cardiomyopathy and arrythmias, skeletal muscle damage and liver dysfunction. Most patients become symptomatic in the neonatal period with a rapidly progressive deterioration and a high mortality rate. However, presentations at a later age with a milder phenotype have also been reported. The therapeutic approach is the same as in other long-chain fatty acid disorders and includes intravenous glucose (+/- insulin) administration to maximally inhibit lipolysis and subsequent fatty acid oxidation during the acute deterioration, along with other measures such as ammonia detoxification, depending on the clinical features. Long-term strategy consists of avoidance of fasting with frequent meals and a special diet with restriction of long-chain fatty acids. Due to the extremely low free carnitine concentrations, carnitine supplementation is often needed. Acylcarnitine profiling in plasma is the assay of choice for the diagnosis at a metabolite level. However, since the acylcarnitine profile observed in CACT-deficient patients is identical to that in CPT2-deficient patients, definitive identification of CACT-deficiency in a certain patient requires determination of the activity of CACT. Subsequently, mutational analysis of the CACT gene can be performed. So far, 9 different mutations have been identified in the CACT gene.

Cesare Indiveri - One of the best experts on this subject based on the ideXlab platform.

  • Mitochondrial carnitine/acylcarnitine Translocase: insights in structure/ function relationships. Basis for drug therapy and side effects prediction.
    Mini reviews in medicinal chemistry, 2015
    Co-Authors: Annamaria Tonazzi, Lara Console, Nicola Giangregorio, Cesare Indiveri
    Abstract:

    The mitochondrial carnitine/acylcarnitine Translocase has been identified, purified and reconstituted in liposomes in 1990. Since that time it has been object of studies aimed to characterize its function and to define the molecular determinants of the translocation pathway. Thanks to these tenacious studies the molecular map of the amino acids involved in the catalysis has been constructed and the roles of critical residues in the translocation pathway have been elucidated. This has been possible through the combination of transport assay in reconstituted liposomes, site-directed mutagenesis, chemical labeling and bioinformatics. Recently some molecules which modulate CACT activity have been identified, such as glutathione and hydrogen peroxide, constituting some of the few cases of control mechanisms of mitochondrial carriers. The vast knowledge on the carnitine/acylcarnitine Translocase is essential both as a progress in basic science and as instrument to foresee therapeutic or toxic effects of xenobiotics and drugs. Such studies have been already started pointing out the inhibitory action of drugs such as K + /H + -ATPase inhibitors (omeprazole) or antibiotics (β-lactams) on the carnitine/acylcarnitine Translocase, which can explain some of their adverse effects.

  • Mitochondrial Carnitine/Acylcarnitine Translocase: Insights in Structure/Function Relationship.
    Mini reviews in medicinal chemistry, 2015
    Co-Authors: Annamaria Tonazzi, Lara Console, Nicola Giangregorio, Cesare Indiveri
    Abstract:

    The mitochondrial carnitine/acylcarnitine Translocase has been identified, purified and reconstituted in liposomes in 1990. Since that time it has been object of studies aimed to characterize its function and to define the molecular determinants of the translocation pathway. Thanks to these tenacious studies the molecular map of the amino acids involved in the catalysis has been constructed and the roles of critical residues in the translocation pathway have been elucidated. This has been possible through the combination of transport assay in reconstituted liposomes, site-directed mutagenesis, chemical labeling and bioinformatics. Recently some molecules which modulate CACT activity have been identified, such as glutathione and hydrogen peroxide, constituting some of the few cases of control mechanisms of mitochondrial carriers. The vast knowledge on the carnitine/acylcarnitine Translocase is essential both as a progress in basic science and as instrument to foresee therapeutic or toxic effects of xenobiotics and drugs. Such studies have been already started pointing out the inhibitory action of drugs such as KM+/H+-ATPase inhibitors (omeprazole) or antibiotics (β-lactams) on the carnitine/acylcarnitine Translocase, which can explain some of their adverse effects.

  • Carnitine/acylcarnitine Translocase and carnitine palmitoyltransferase 2 form a complex in the inner mitochondrial membrane.
    Molecular and cellular biochemistry, 2014
    Co-Authors: Lara Console, Nicola Giangregorio, Cesare Indiveri, Annamaria Tonazzi
    Abstract:

    Carnitine/acylcarnitine Translocase and carnitine palmitoyltransferase 2 are members of the carnitine system, which are responsible of the regulation of the mitochondrial CoA/acyl-CoA ratio and of supplying substrates for the ß-oxidation to mitochondria. This study, using cross-Linking reagent, Blue native electrophoresis and immunoprecipitation followed by detection with immunoblotting, shows conclusive evidence about the interaction between carnitine palmitoyltransferase 2 and carnitine/acylcarnitine Translocase supporting the channeling of acylcarnitines and carnitine at level of the inner mitochondrial membrane.

  • Localization of Mitochondrial Carnitine/Acylcarnitine Translocase in Sensory Neurons from Rat Dorsal Root Ganglia
    Neurochemical research, 2013
    Co-Authors: Annamaria Tonazzi, Cristina Mantovani, Matilde Colella, Giorgio Terenghi, Cesare Indiveri
    Abstract:

    The carnitine/acylcarnitine transporter is a transport system whose function is essential for the mitochondrial β-oxidation of fatty acids. Here, the presence of carnitine/acylcarnitine carrier (CACT) in nervous tissue and its sub-cellular localization in dorsal root ganglia (DRG) neurons have been investigated. Western blot analysis using a polyclonal anti-CACT antibody produced in our laboratory revealed the presence of CACT in all the nervous tissue extracts analyzed. Confocal microscopy experiments performed on fixed and permeabilized DRG neurons co-stained with the anti-CACT antibody and the mitochondrial marker MitoTracker Red clearly showed a mitochondrial localization for the carnitine/acylcarnitine transporter. The transport activity of CACT from DRG extracts reconstituted into liposomes was about 50 % in respect to liver extracts. The experimental data here reported represent the first direct evidence of the expression of the carnitine/acylcarnitine transporter in sensory neurons, thus supporting the existence of the β-oxidation pathway in these cells.

  • Functional characterization of residues within the carnitine/acylcarnitine Translocase RX2PANAAXF distinct motif.
    Molecular membrane biology, 2008
    Co-Authors: J. Ramón De Lucas, Nicola Giangregorio, Annamaria Tonazzi, Cesare Indiveri, Vito Iacobazzi, Patricia Perez, Ferdinando Palmieri
    Abstract:

    The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane α-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using mole...

Michèle Brivet - One of the best experts on this subject based on the ideXlab platform.

  • A novel SLC25A20 splicing mutation in patients of different ethnic origin with neonatally lethal Carnitine-Acylcarnitine Translocase (CACT) deficiency
    Molecular genetics and metabolism, 2006
    Co-Authors: Stanley H. Korman, James Pitt, Avihu Boneh, Imad Dweikat, Mokhtar Zater, Vardiella Meiner, Alisa Gutman, Michèle Brivet
    Abstract:

    Carnitine-Acylcarnitine Translocase (CACT) deficiency is a rare disorder of fatty acid oxidation associated with high mortality. Two female newborns of different ethnic origin (the first Anglo-Celtic and the second Palestinian Arab) both died after sudden collapse on day 2 of life. Both had elevated bloodspot long-chain acylcarnitines consistent with either CACT or carnitine palmitoyltransferase II (CPT2) deficiency; the latter was excluded by demonstrating normal CPT2 activity in fibroblasts. Direct sequencing of all SLC25A20 (CACT) gene exons and exon-intron boundaries revealed that Patient 1 was compound heterozygous for a novel c.609-3c>g (IVS6-3c>g) mutation on the paternal allele and a previously described c.326delG mutation on the maternal allele. Patient 2 was homozygous for the same, novel c.609-3c>g mutation. Previously reported SLC25A20 mutations have been almost exclusively confined to a single family or ethnic group. Analysis of fibroblast cDNA by RT-PCR, agarose gel electrophoresis and sequencing of extracted bands showed that both mutations produce aberrant splicing. c.609-3C>G results in exon 7 skipping leading to a frameshift with premature termination seven amino acids downstream. c.326delG was confirmed to produce skipping of exons 3 or 3 plus 4. CACT activity in both patients' fibroblasts was near-zero. For both families, prenatal diagnosis of an unaffected fetus was performed by mutation analysis on CVS tissue in a subsequent pregnancy. Due to the urgency of prenatal diagnosis in the second family, molecular diagnosis was performed prior to demonstration of CACT enzyme deficiency, illustrating that mutation analysis is a rapid and reliable approach to first-line diagnosis of CACT deficiency.

  • Mutational spectrum and DNA-based prenatal diagnosis in Carnitine-Acylcarnitine Translocase deficiency.
    Molecular genetics and metabolism, 2003
    Co-Authors: Catherine Costa, A. Slama, A. Boutron, J. M. Costa, C. Vequaud, Alain Legrand, Michèle Brivet
    Abstract:

    Abstract Carnitine-Acylcarnitine Translocase (CAC) deficiency is a rare autosomal recessive disorder of long-chain fatty acid oxidation with a severe outcome. We report mutation analysis in a cohort of 12 patients. Twelve mutations were identified of which 9 have not been reported so far (G28C, D32N, R178Q, P230R, D231H, 179delG, 802delG, 69–70insTGTGC, and 609−1g > a). Altogether, including our results, 22 mutations of the CAC gene have been published to date in 23 patients demonstrating the allelic heterogeneity of CAC deficiency. DNA-based prenatal diagnosis was performed for the first time in pregnancies at risk for CAC deficiency. Two fetuses were affected and one pregnancy was terminated by family decision. Two other fetuses had normal genotype and five others were heterozygotes. All the offspring of these seven pregnancies are alive and apparently healthy.

  • Carnitine/acylcarnitine Translocase deficiency (neonatal phenotype): successful prenatal and postmortem diagnosis associated with a novel mutation in a single family.
    Molecular genetics and metabolism, 2001
    Co-Authors: Bing-zhi Yang, Diane S. Roe, Michèle Brivet, Jason M. Mallory, G.d. Strobel, Kerri M. Jones, Jia-huan Ding, Charles R. Roe
    Abstract:

    Abstract The neonatal phenotype of Carnitine-Acylcarnitine Translocase (CACT) deficiency is one of the most severe and usually lethal mitochondrial fat oxidation disorders characterized by hypoketotic hypoglycemia, hyperammonemia, cardiac abnormalities, and early death. In this study, the proband was the daughter of consanguineous Hispanic parents. At 36 h of life, she had bradycardia and died at 4 days of age without a specific diagnosis. In a subsequent pregnancy, prenatal counseling and amniocentesis were provided. Incubation of the amniocytes from this pregnancy and fibroblasts (from the dead proband) with [16-2H3]palmitic acid and analysis by tandem mass spectrometry revealed an increasedconcentration of [16-2H3]palmitoylcarnitine, suggesting the diagnoses of either CACT or carnitine palmitoyltransferase II (CPT-II) deficiency. CACT enzyme activity was absent in both cell lines. Molecular investigation of cDNA from the dead proband and her affected sibling revealed aberrant CACT cDNA species, including exon 3 skipping, both exon 3 and 4 skipping, and a 13-bp insertion at cDNA position 388. Investigation of these cell lines for mutations affecting CACT RNA processing by analysis of CACT gene sequences, including intron and exon boundaries, revealed a single nucleotide G deletion at the donor site in intron 3 which resulted in exon skipping and a 13-bp insertion. The proband and her affected sibling were homozygous for this deletion.

  • Evidence for a short-chain Carnitine-Acylcarnitine Translocase in mitochondria specifically related to the metabolism of branched-chain amino acids.
    Molecular genetics and metabolism, 2000
    Co-Authors: Diane S. Roe, Charles R. Roe, Michèle Brivet, Lawrence Sweetman
    Abstract:

    Carnitine-Acylcarnitine Translocase (CATR) deficiency is a severe defect in fatty acid oxidation which presents early in life most frequently with hypoglycemia, hyperammonemia, and severe cardiac abnormalities. CATR exchanges acylcarnitines of various chain lengths for free carnitine across the mitochondrial membrane. In vitro studies in intact fibroblasts from patients with documented deficiency of CATR were probed with stable-isotope-labeled precursors and the resulting acylcarnitines were analyzed by tandem mass spectrometry. After a 72-h incubation with l-[(2)H(3)]carnitine the Translocase-deficient cells produced acylcarnitines in which the deuterium was incorporated into short-chain acylcarnitines, C2-C5. Experiments with simultaneous incubation of l-[(2)H(3)]carnitine and l-[(13)C(6)]isoleucine produced [(13)C(5)]2-methylbutyryl-[(2)H(3)]carnitine and [(13)C(3)]propionyl-[(2)H(3)]carnitine indicating exchange of labeled acylcarnitine from inside the mitochondrial matrix with labeled free carnitine. These studies support the possible existence of a "branched-chain" Carnitine-Acylcarnitine translocator in mitochondria.

  • Neonatal hyperammonemia caused by a defect of Carnitine-Acylcarnitine Translocase☆☆☆★
    The Journal of pediatrics, 1995
    Co-Authors: H. Ogier De Baulny, Abdelhamid Slama, Guy Touati, Douglass M. Turnbull, Morteza Pourfarzam, Michèle Brivet
    Abstract:

    Carnitine-Acylcarnitine Translocase deficiency is a newly recognized inborn error of metabolism that involves transport of long-chain fatty acids into mitochondria, which in turn impairs mitochondrial beta-oxidation, and ketogenesis. We report a new familial example; the affected twins had neonatal distress, hyperammonemia, and transient intracardiac conduction defects. Clinical and biochemical analysis of both our patients and the two previously reported patients revealed that this inherited defect could be manifested during the neonatal period without any of the signs classically associated with fatty oxidation defects. In contrast, all four patients had sustained and "isolated" hyperammonemia, which could be misinterpreted as being caused by urea cycle defects. We conclude that Carnitine-Acylcarnitine Translocase deficiency is a potential differential diagnosis in neonates with unexplained neonatal hyperammonemia. Cardiac and muscle involvement may represent further early pivotal symptoms.

Vito Iacobazzi - One of the best experts on this subject based on the ideXlab platform.

  • Functional characterization of residues within the carnitine/acylcarnitine Translocase RX2PANAAXF distinct motif.
    Molecular membrane biology, 2008
    Co-Authors: J. Ramón De Lucas, Nicola Giangregorio, Annamaria Tonazzi, Cesare Indiveri, Vito Iacobazzi, Patricia Perez, Ferdinando Palmieri
    Abstract:

    The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane α-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using mole...

  • Response to therapy in carnitine/acylcarnitine Translocase (CACT) deficiency due to a novel missense mutation.
    American journal of medical genetics. Part A, 2004
    Co-Authors: Vito Iacobazzi, Ferdinando Palmieri, Marzia Pasquali, Rani Singh, Dietrich Matern, Piero Rinaldo, Cristina Amat Di San Filippo, Nicola Longo
    Abstract:

    Deficiency of carnitine/acylcarnitine Translocase (CACT) is an autosomal recessive disorder of the carnitine cycle resulting in the inability to transfer fatty acids across the inner mitochondrial membrane. Only a limited number of affected patients have been reported and the effect of therapy on this condition is still not well defined. Here, we report a new patient with this disorder and follow the response to therapy. Our patient was the product of a consanguineous marriage. He presented shortly after birth with cardiac myopathy and arrhythmia coupled with severe non-ketotic hypoglycemia. Initial metabolic studies indicated severe non-ketotic C6-C10 dicarboxylic aciduria, plasma carnitine deficiency, and a characteristic elevation of plasma C:16:0, C18:1, and C18:2 acylcarnitine species. Enzyme assay confirmed deficiency of CACT activity. Molecular studies indicated that this child was homozygous, and both parents heterozygous, for a single bp change converting glutamine 238 to arginine (Q238R). Therapy with a formula providing most of the fat via medium chain triglycerides (MCT) and carnitine supplementation reduced the concentration of long-chain acylcarnitines and reversed cardiac symptoms and the hypoglycemia. These results suggest that carnitine and MCT may be effective in treating this defect of long-chain fatty acid oxidation.

  • Molecular and functional analysis of SLC25A20 mutations causing Carnitine-Acylcarnitine Translocase deficiency.
    Human mutation, 2004
    Co-Authors: Vito Iacobazzi, Rossella Parini, Federica Invernizzi, Barbara Garavaglia, Silvia Baratta, Roser Pons, Wendy K. Chung, Carlo Dionisi-vici, Antonia Ribes, Maria Dolores Huertas
    Abstract:

    The enzyme Carnitine-Acylcarnitine Translocase (CACT) is involved in the transport of long-chain fatty acids into mitochondria. CACT deficiency is a life-threatening, recessively inherited disorder of lipid beta-oxidation which manifests in early infancy with hypoketotic hypoglycemia, cardiomyopathy, liver failure, and muscle weakness. We report here the clinical, biochemical, and molecular features of six CACT-deficient patients from Italy, Spain, and North America who exhibited significant clinical heterogeneity. In five patients (Patients 1, 2, 4, 5, and 6) the disease manifested in the neonatal period, while the remaining patient (Patient 3), the younger sibling of an infant who had died with clinical suspicion of fatty acid oxidation defect, has been treated since birth and was clinically asymptomatic at 4.5 years of age. Patients 1 and 4 were deceased within 6 months from the onset of this study, while the remaining four are still alive at 8, 4.5, 3.5, and 2 years, respectively. Sequence analysis of the CACT gene (SLC25A20) disclosed five novel mutations and three previously reported mutations. Three patients were homozygous for the identified mutations. Two of the novel mutations (c.718+1G>C and c.843+4_843+50del) altered the donor splice site of introns 7 and 8, respectively. The 47-nt deletion in intron 8 caused both skipping of exon 8 only and skipping of exons 6-8. Four mutations [[c.159dupT;c.163delA] ([p.Gly54Trp;p.Thr55Ala]) c.397C>T (p.Arg133Trp), c.691G>C (p.Asp231His), and c.842C>T (p.Ala281Val)] resulted in amino acid substitutions affecting evolutionarily conserved regions of the protein. Interestingly, one of these exonic mutations (p.Ala281Val) was associated with a splicing defect also characterized by skipping of exons 6-8. The deleterious effect of the p.Arg133Trp substitution was demonstrated by measuring CACT activity upon expression of the normal and the mutant protein in E. coli and functional reconstitution into liposomes. Combined analysis of clinical, biochemical, and molecular data failed to indicate a correlation between the phenotype and the genotype.

  • Functional analysis of mutant human carnitine acylcarnitine Translocases in yeast
    Biochemical and biophysical research communications, 2001
    Co-Authors: Lodewijk Ijlst, Vito Iacobazzi, Ferdinando Palmieri, C. W. T. Van Roermund, W. Oostheim, Jos P.n. Ruiter, J.c. Williams, R. J. A. Wanders
    Abstract:

    Long chain fatty acids are translocated as carnitine esters across the mitochondrial inner membrane by carnitine acylcarnitine Translocase (CACT). We report functional studies on the mutant CACT proteins from a severe and a mild patient with CACT deficiency. CACT activities in fibroblasts of both patients were markedly deficient with some residual activity ( A (G81R) in the severe and a c.955insC mutation (C-terminal extension of 21 amino acids (CACT(+21aa)) in the milder patient. The effect of both mutations on the protein was studied in a sensitive expression system based on the ability of human CACT to functionally complement a CACT-deletion strain of yeast. Expression in this strain revealed significant residual activity for CACT(+21aa), while the CACT(G81R) was inactive.

  • The structure and organization of the human carnitine/acylcarnitine Translocase (CACT1) gene2
    Biochemical and biophysical research communications, 1998
    Co-Authors: Vito Iacobazzi, Ronald J.a. Wanders, Charles A. Stanley, Maria Antonia Naglieri, Ferdinando Palmieri
    Abstract:

    Abstract The carnitine/acylcarnitine Translocase (CACT) transports acylcarnitines into mitochondria in exchange for free carnitine and it is, therefore, essential for the fatty acid β-oxidation pathway. We have determined the exon–intron structure of the human CACT gene, which is responsible for a genetic disorder of fatty acid oxidation called CACT deficiency. The gene spans about 16.5 kb and consists of nine exons with the translation start site in exon 1. All the splice acceptor and donor sites conform to the AG/GT rules. All the introns except one are located at the level of the sequences coding for the extramembranous loops of CACT. We have designed a series of intronic oligonucleotide primers for amplifying each of the CACT exons together with their flanking intronic sequences, in segments well suited to detect mutations that would affect splicing of mRNA as well as the coding sequence itself.

Annamaria Tonazzi - One of the best experts on this subject based on the ideXlab platform.

  • Mitochondrial carnitine/acylcarnitine Translocase: insights in structure/ function relationships. Basis for drug therapy and side effects prediction.
    Mini reviews in medicinal chemistry, 2015
    Co-Authors: Annamaria Tonazzi, Lara Console, Nicola Giangregorio, Cesare Indiveri
    Abstract:

    The mitochondrial carnitine/acylcarnitine Translocase has been identified, purified and reconstituted in liposomes in 1990. Since that time it has been object of studies aimed to characterize its function and to define the molecular determinants of the translocation pathway. Thanks to these tenacious studies the molecular map of the amino acids involved in the catalysis has been constructed and the roles of critical residues in the translocation pathway have been elucidated. This has been possible through the combination of transport assay in reconstituted liposomes, site-directed mutagenesis, chemical labeling and bioinformatics. Recently some molecules which modulate CACT activity have been identified, such as glutathione and hydrogen peroxide, constituting some of the few cases of control mechanisms of mitochondrial carriers. The vast knowledge on the carnitine/acylcarnitine Translocase is essential both as a progress in basic science and as instrument to foresee therapeutic or toxic effects of xenobiotics and drugs. Such studies have been already started pointing out the inhibitory action of drugs such as K + /H + -ATPase inhibitors (omeprazole) or antibiotics (β-lactams) on the carnitine/acylcarnitine Translocase, which can explain some of their adverse effects.

  • Mitochondrial Carnitine/Acylcarnitine Translocase: Insights in Structure/Function Relationship.
    Mini reviews in medicinal chemistry, 2015
    Co-Authors: Annamaria Tonazzi, Lara Console, Nicola Giangregorio, Cesare Indiveri
    Abstract:

    The mitochondrial carnitine/acylcarnitine Translocase has been identified, purified and reconstituted in liposomes in 1990. Since that time it has been object of studies aimed to characterize its function and to define the molecular determinants of the translocation pathway. Thanks to these tenacious studies the molecular map of the amino acids involved in the catalysis has been constructed and the roles of critical residues in the translocation pathway have been elucidated. This has been possible through the combination of transport assay in reconstituted liposomes, site-directed mutagenesis, chemical labeling and bioinformatics. Recently some molecules which modulate CACT activity have been identified, such as glutathione and hydrogen peroxide, constituting some of the few cases of control mechanisms of mitochondrial carriers. The vast knowledge on the carnitine/acylcarnitine Translocase is essential both as a progress in basic science and as instrument to foresee therapeutic or toxic effects of xenobiotics and drugs. Such studies have been already started pointing out the inhibitory action of drugs such as KM+/H+-ATPase inhibitors (omeprazole) or antibiotics (β-lactams) on the carnitine/acylcarnitine Translocase, which can explain some of their adverse effects.

  • Carnitine/acylcarnitine Translocase and carnitine palmitoyltransferase 2 form a complex in the inner mitochondrial membrane.
    Molecular and cellular biochemistry, 2014
    Co-Authors: Lara Console, Nicola Giangregorio, Cesare Indiveri, Annamaria Tonazzi
    Abstract:

    Carnitine/acylcarnitine Translocase and carnitine palmitoyltransferase 2 are members of the carnitine system, which are responsible of the regulation of the mitochondrial CoA/acyl-CoA ratio and of supplying substrates for the ß-oxidation to mitochondria. This study, using cross-Linking reagent, Blue native electrophoresis and immunoprecipitation followed by detection with immunoblotting, shows conclusive evidence about the interaction between carnitine palmitoyltransferase 2 and carnitine/acylcarnitine Translocase supporting the channeling of acylcarnitines and carnitine at level of the inner mitochondrial membrane.

  • Localization of Mitochondrial Carnitine/Acylcarnitine Translocase in Sensory Neurons from Rat Dorsal Root Ganglia
    Neurochemical research, 2013
    Co-Authors: Annamaria Tonazzi, Cristina Mantovani, Matilde Colella, Giorgio Terenghi, Cesare Indiveri
    Abstract:

    The carnitine/acylcarnitine transporter is a transport system whose function is essential for the mitochondrial β-oxidation of fatty acids. Here, the presence of carnitine/acylcarnitine carrier (CACT) in nervous tissue and its sub-cellular localization in dorsal root ganglia (DRG) neurons have been investigated. Western blot analysis using a polyclonal anti-CACT antibody produced in our laboratory revealed the presence of CACT in all the nervous tissue extracts analyzed. Confocal microscopy experiments performed on fixed and permeabilized DRG neurons co-stained with the anti-CACT antibody and the mitochondrial marker MitoTracker Red clearly showed a mitochondrial localization for the carnitine/acylcarnitine transporter. The transport activity of CACT from DRG extracts reconstituted into liposomes was about 50 % in respect to liver extracts. The experimental data here reported represent the first direct evidence of the expression of the carnitine/acylcarnitine transporter in sensory neurons, thus supporting the existence of the β-oxidation pathway in these cells.

  • Functional characterization of residues within the carnitine/acylcarnitine Translocase RX2PANAAXF distinct motif.
    Molecular membrane biology, 2008
    Co-Authors: J. Ramón De Lucas, Nicola Giangregorio, Annamaria Tonazzi, Cesare Indiveri, Vito Iacobazzi, Patricia Perez, Ferdinando Palmieri
    Abstract:

    The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane α-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using mole...