RESUMEN
3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine metabolism. The disease is caused by mutations in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL). To date 26 different mutations have been described. A (betaalpha)(8) TIM barrel structure has been proposed for the protein, and almost all missense mutations identified so far localize in the beta sheets that define the inside cavity. We report an Italian patient who bears homozygously a novel HL mutation, c.608G > A (p. G203E) in beta sheet six. A structural model of the mutated protein suggests that glutamic acid 203 impedes catalysis by blocking the entrance to the inner cavity of the enzyme. Loss of functionality has been confirmed in expression studies in E. coli, which demonstrate that the G203E mutation completely abolishes enzyme activity. Beta sheet six and beta sheet two are the two protein regions that accumulate most missense mutations, indicating their importance in enzyme functionality. A model for the mechanism of enzyme function is proposed.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Meglutol/orina , Mutación Missense , Oxo-Ácido-Liasas/química , Oxo-Ácido-Liasas/genética , Secuencia de Aminoácidos , Animales , Niño , Análisis Mutacional de ADN , Humanos , Masculino , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Mitochondrial HMG-CoA synthase deficiency is an inherited metabolic disorder caused by a defect in the enzyme that regulates the formation of ketone bodies. Patients present with hypoketotic hypoglycaemia, encephalopathy and hepatomegaly, usually precipitated by an intercurrent infection or prolonged fasting. The diagnosis may easily be missed as previously reported results of routine metabolic investigations, urinary organic acids and plasma acylcarnitines may be nonspecific or normal, and a high index of suspicion is required to proceed to further confirmatory tests. We describe a further acute case in which the combination of urinary organic acids, low free carnitine and changes in the plasma acylcarnitine profile on carnitine supplementation were very suggestive of a defect in ketone synthesis. The diagnosis of mitochondrial HMG-CoA synthase deficiency was confirmed on genotyping, revealing two novel mutations: c.614G > A (R188H) and c.971T > C (M307T). A further sibling, in whom the diagnosis had not been made acutely, was also found to be affected. The possible effects of these mutations on enzyme activity are discussed.
Asunto(s)
Hidroximetilglutaril-CoA Sintasa/deficiencia , Errores Innatos del Metabolismo/diagnóstico , Enfermedades Mitocondriales/diagnóstico , Carnitina/análogos & derivados , Carnitina/sangre , Carnitina/farmacología , Análisis Mutacional de ADN , Genotipo , Heterocigoto , Humanos , Lactante , Masculino , MutaciónRESUMEN
3-Hydroxy-3-methylglutaric aciduria is a human autosomal recessive metabolic disorder that usually appears within the first year of life. The causes of this aciduria are lethal mutations in the gene encoding for 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL). HL is a mitochondrial matrix enzyme that catalyzes the last step of ketogenesis and leucine catabolism. This gene has been mapped to chromosome 1 at locus 1pter-p33 and its genomic organisation comprises 9 exons whose sizes vary between 64-678 bp. The human cDNA sequence was reported in 1993 with the first genetic study of two Acadian-French Canadian siblings. To date, 24 mutations in 36 patients have been described; most of them are single-base substitutions causing amino acid replacements and a variety of splicing defects. In the population studied two mutations appear predominant: g.122GA (8 patients and 15 alleles) frequent in Saudi Arabia, and g.109GT (6 patients and 12 alleles), prevalent in Spain. At least seven mutations are clustered in the second half of exon 2 affecting aminoacids E37, R41 and D42 and conforming a possible hot spot. The genotype-phenotype correlation is difficult to establish since the probands received different treatments, and the onset of an acute episode frequently depends on external factors such as fasting or acute illness.
Asunto(s)
Meglutol/orina , Mutación , Oxo-Ácido-Liasas/deficiencia , Oxo-Ácido-Liasas/genética , Empalme Alternativo , Cromosomas Humanos Par 1 , Análisis Mutacional de ADN , Exones , Genotipo , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/orina , Leucina/metabolismo , Mitocondrias/enzimología , Fenotipo , Polimorfismo GenéticoRESUMEN
Carnitine palmitoyltransferase I (CPT I) and carnitine octanoyltransferase (COT) catalyze the conversion of long- and medium-chain acyl-CoA to acylcarnitines in the presence of carnitine. We propose a common three-dimensional structural model for the catalytic domain of both, based on fold identification for 200 amino acids surrounding the active site through a threading approach. The model is based on the three-dimensional structure of the rat enoyl-CoA hydratase, established by x-ray diffraction analysis. The study shows that the structural model of 200 amino acids of the catalytic site is practically identical in CPT I and COT with identical distribution of 4 beta-sheets and 6 alpha-helices. Functional analysis of the model was done by site-directed mutagenesis. When the critical histidine residue 473 in CPT I (327 in COT), localized in the acyl-CoA pocket in the model, was mutated to alanine, the catalytic activity was abolished. Mutation of the conserved alanine residue to aspartic acid, A381D (in CPT I) and A238D (in COT), which are 92/89 amino acids far from the catalytic histidine, respectively (but very close to the acyl-CoA pocket in the structural model), decreased the activity by 86 and 80%, respectively. The K(m) for acyl-CoA increased 6-8-fold, whereas the K(m) for carnitine hardly changed. The inhibition of the mutant CPT I by malonyl-CoA was not altered. The structural model explains the loss of activity reported for the CPT I mutations R451A, W452A, D454G, W391A, del R395, P479L, and L484P, all of which occur in or near the modeled catalytic domain.
Asunto(s)
Carnitina Aciltransferasas/química , Carnitina O-Palmitoiltransferasa/química , Alanina/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Carnitina Aciltransferasas/genética , Carnitina O-Palmitoiltransferasa/genética , Catálisis , Dominio Catalítico , Relación Dosis-Respuesta a Droga , Histidina/química , Cinética , Malonil Coenzima A/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Mutagénesis Sitio-Dirigida , Mutación , Plásmidos/metabolismo , Conformación Proteica , Isoformas de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Deficiency of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGS) is a recessive disorder of ketogenesis that has been previously diagnosed in two children with hypoglycaemic hypoketotic coma during fasting periods. Here, we report the results of molecular investigations in a third patient affected by this disease. Sequencing of the entire coding region of the HMGCS2 gene revealed two missense mutations, G212R and R500H. Mendelian inheritance was confirmed by the analysis of parental samples and neither of the mutations was found on 200 control chromosomes. Functional relevance was confirmed by in vitro expression studies in cytosolic HMGS-deficient cells. Whereas wild-type cDNA of the HMGCS2 gene reverted the auxotrophy for mevalonate, the cDNAs of the mutants did not. The disease may be recognised by specific clinical and biochemical features but it is difficult to confirm enzymatically since the gene is expressed only in liver and testis. Molecular studies may facilitate or confirm future diagnoses in affected patients.
Asunto(s)
Hidroximetilglutaril-CoA Sintasa/deficiencia , Hidroximetilglutaril-CoA Sintasa/genética , Mitocondrias/enzimología , Animales , Secuencia de Bases , Células CHO , Cricetinae , Análisis Mutacional de ADN , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Expresión Génica , Genes Recesivos , Humanos , Técnicas In Vitro , Lactante , Masculino , Mutagénesis Sitio-Dirigida , Mutación MissenseRESUMEN
Mitochondrial metabolism plays a pivotal role in the pancreatic beta cell by generating signals that couple glucose sensing to insulin secretion. We have demonstrated previously that mitochondrially derived glutamate participates directly in the stimulation of insulin exocytosis. The aim of the present study was to impose altered cellular glutamate levels by overexpression of glutamate decarboxylase (GAD) to repress elevation of cytosolic glutamate. INS-1E cells infected with a recombinant adenovirus vector encoding GAD65 showed efficient overexpression of the GAD protein with a parallel increase in enzyme activity. In control cells glutamate levels were slightly increased by 7.5 mm glucose (1.4-fold) compared with the effect at 15 mm (2.3-fold) versus basal 2.5 mm glucose. Upon GAD overexpression, glutamate concentrations were no longer elevated by 15 mm glucose as compared with controls (-40%). Insulin secretion was stimulated in control cells by glucose at 7.5 mm (2.5-fold) and more efficiently at 15 mm (5.2-fold). INS-1E cells overexpressing GAD exhibited impaired insulin secretion on stimulation with 15 mm glucose (-37%). The secretory response to 30 mm KCl, used to raise cytosolic Ca(2+) levels, was unaffected. Similar results were obtained in perifused rat pancreatic islets following adenovirus transduction. This GAD65-mediated glutamate decarboxylation correlating with impaired glucose-induced insulin secretion is compatible with a role for glutamate as a glucose-derived factor participating in insulin exocytosis.
Asunto(s)
Glucosa/metabolismo , Glutamato Descarboxilasa/metabolismo , Ácido Glutámico/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Isoenzimas/metabolismo , Adenosina Trifosfato/metabolismo , Adenoviridae/genética , Animales , Calcio/metabolismo , Células Cultivadas , Citosol/metabolismo , Exocitosis , Glutamatos/metabolismo , Humanos , Immunoblotting , Secreción de Insulina , Islotes Pancreáticos/citología , Potenciales de la Membrana , Microscopía Fluorescente , Mitocondrias/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Factores de Tiempo , Transducción Genética , Células Tumorales CultivadasRESUMEN
In man, activated N-, K- and H-ras oncogenes have been found in around 30% of the solid tumours tested. An exon known as IDX, which has been described previously and is located between exon 3 and exon 4A of the c-H-ras pre-mRNA, allows an alternative splicing process that results in the synthesis of the mRNA of a putative protein named p19. It has been suggested that this alternative pathway is less tumorigenic than that which results in the activation of p21. We have used the mammalian trans-splicing mechanism as a tool with which to modulate this particular pre-mRNA processing to produce mRNA similar to that of mature p19 RNA. The E4A exon of the activated H-ras gene was found to be a good target for external trans-splicing. We reprogrammed the rat carnitine octanoyltransferase exon 2 to specifically invade the terminal region of H-ras. Assays performed with this reprogrammed trans-exon showed that the trans-splicing product was obtained in competition with cis-splicing of the D intron of the H-ras gene, and was associated with concomitant down-modulation of D intron cis-splicing. We also found that the exon 4A of the human c-H-ras gene underwent successive trans-splicing rounds with an external exon.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes ras , Trans-Empalme , Animales , Elementos de Facilitación Genéticos , Exones , Células HeLa , Humanos , Intrones , RatasRESUMEN
Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which may lead to the synthesis of two proteins. Generation of the three COT transcripts in rat does not depend on sex, development, fat feeding, the inclusion of the peroxisome proliferator diethylhexyl phthalate in the diet or hyperinsulinemia. In addition, trans-splicing was not detected in COT of other mammals, such as human, pig, cow and mouse, or in Cos7 cells from monkey. Rat COT exon 2 contains two purine-rich sequences. Mutation of the rat COT exon 2 upstream box does not affect the trans-splicing in vitro between two truncated constructs containing exon 2 and its adjacent intron boundaries. In contrast, mutation of the downstream box from the rat sequence (GAAGAAG) to a random sequence or the sequence observed in the other mammals (AAAAAAA) decreased trans-splicing in vitro. In contrast, mutation of the AAAAAAA box of human COT exon 2 to GAAGAAG increases trans-splicing. Heterologous reactions between COT exon 2 from rat and human do not produce trans-splicing. HeLa cells transfected with minigenes of rat COT sequences produced cis- and trans-spliced bands. Mutation of the GAAGAAG box to AAAAAAA abolished trans-splicing and decreased cis-splicing in vivo. We conclude that GAAGAAG is an exonic splicing enhancer that could induce natural trans-splicing in rat COT.
Asunto(s)
Empalme Alternativo , Carnitina Aciltransferasas/genética , Elementos de Facilitación Genéticos/genética , Exones/genética , Animales , Secuencia de Bases , Northern Blotting , Células COS , Bovinos , ADN Complementario/química , ADN Complementario/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Células HeLa , Humanos , Intestinos/enzimología , Hígado/enzimología , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , PorcinosRESUMEN
Carnitine octanoyltransferase (COT) produces three different transcripts in rat through cis- and trans-splicing reactions, which can lead to the synthesis of two proteins. The occurrence of the three COT transcripts in rat has been found in all tissues examined and does not depend on sex, fat feeding, peroxisome proliferators or hyperinsulinaemia. Rat COT exon 2 contains a putative exonic splicing enhancer (ESE) sequence. Mutation of this ESE (GAAGAAG) to AAAAAAA decreased trans-splicing in vitro, from which it is deduced that this ESE sequence is partly responsible for the formation of the three transcripts. The protein encoded by cis-spliced mRNA of rat COT is inhibited by malonyl-CoA and etomoxir. cDNA species encoding full-length wild-type COT and one double mutant COT were expressed in Saccharomyces cerevisiae. The recombinant enzymes showed full activity towards both substrates, carnitine and decanoyl-CoA. The activity of the doubly mutated H131A/H340A enzyme was similar to that of the rat peroxisomal enzyme but was completely insensitive to malonyl-CoA and etomoxir. These results indicate that the histidine residues His-131 and His-340 are the sites responsible for the interaction of these two inhibitors, which inhibit COT by interacting with the same sites.
Asunto(s)
Carnitina Aciltransferasas/genética , Trans-Empalme/genética , Acilcoenzima A/metabolismo , Acilcoenzima A/farmacología , Animales , Secuencia de Bases , Carnitina Aciltransferasas/antagonistas & inhibidores , Carnitina Aciltransferasas/biosíntesis , Carnitina Aciltransferasas/metabolismo , Inhibidores Enzimáticos/farmacología , Compuestos Epoxi/farmacología , Exones/genética , Femenino , Masculino , Malonil Coenzima A/metabolismo , Malonil Coenzima A/farmacología , Mutación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
The outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) catalyzes the initial and regulatory step in the beta-oxidation of fatty acids. The genes for the two isoforms of CPTI-liver (L-CPTI) and muscle (M-CPTI) have been cloned and expressed, and the genes encode for enzymes with very different kinetic properties and sensitivity to malonyl-CoA inhibition. Pig L-CPTI encodes for a 772 amino acid protein that shares 86 and 62% identity, respectively, with rat L- and M-CPTI. When expressed in Pichia pastoris, the pig L-CPTI enzyme shows kinetic characteristics (carnitine, K(m) = 126 microM; palmitoyl-CoA, K(m) = 35 microM) similar to human or rat L-CPTI. However, the pig enzyme, unlike the rat liver enzyme, shows a much higher sensitivity to malonyl-CoA inhibition (IC(50) = 141 nM) that is characteristic of human or rat M-CPTI enzymes. Therefore, pig L-CPTI behaves like a natural chimera of the L- and M-CPTI isotypes, which makes it a useful model to study the structure--function relationships of the CPTI enzymes.
Asunto(s)
Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina/análogos & derivados , Carnitina/metabolismo , Inhibidores Enzimáticos/metabolismo , Malonil Coenzima A/metabolismo , Mitocondrias Hepáticas/enzimología , Mitocondrias Musculares/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carnitina O-Palmitoiltransferasa/biosíntesis , Carnitina O-Palmitoiltransferasa/genética , Clonación Molecular , ADN Complementario/aislamiento & purificación , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Pichia/genética , Ratas , Alineación de Secuencia , PorcinosRESUMEN
The unusually low hepatic ketogenic capacity of piglets has been correlated with lack of expression of the mitochondrial HMG-CoA synthase gene. However, we have shown that starvation of 2-week-old piglets increased the mRNA levels of mitochondrial HMG-CoA synthase to a level similar to that observed in starved rats (S. H. Adams, C. S. Alho, G. Asins, F. G. Hegardt, and P. F. Marrero, 1997, Biochem. J. 324, 65-73). We now report that antibodies against pig mitochondrial HMG-CoA synthase detected the pig enzyme in mitochondria of 2-week-old starved piglets and that the pig mitochondrial HMG-CoA synthase cDNA encodes an active enzyme in the eukaryotic cell line Mev-1, with catalytic behavior similar to that of the rat enzyme when expressed in the same system. We also show that low activity of pig mitochondrial HMG-CoA synthase correlates with low expression of the pig enzyme. The discrepancy in mitochondrial HMG-CoA synthase gene expression between the high levels of mRNA and low levels of enzyme was not associated with differences in transcript maturation, which suggests that an attenuated translation of the pig mRNA is responsible for the diminished ketogenic capacity of pig mitochondria.
Asunto(s)
Compuestos de Boro/farmacología , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Hígado/enzimología , Mitocondrias/enzimología , Biosíntesis de Proteínas/genética , Inanición/enzimología , Animales , Anticuerpos/inmunología , Western Blotting , Compuestos de Boro/química , Células CHO , Catálisis , Coenzima A Ligasas/inmunología , Cricetinae , Dosificación de Gen , Hígado/metabolismo , Mitocondrias/metabolismo , Ensayos de Protección de Nucleasas , Procesamiento Postranscripcional del ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Ribonucleasa H/metabolismo , Inanición/genética , PorcinosRESUMEN
Carnitine octanoyltransferase (COT), which facilitates the transport of shortened fatty acyl-CoAs from peroxisomes to mitochondria, is expressed in the intestinal mucosa of suckling rats; its mRNA levels increase rapidly after birth, remain steady until day 15, and decrease until weaning, when basal, adult values are established, which remain unchanged thereafter. The process seems to be controlled at the transcriptional level since the developmental pattern of mRNA coincides with that of pre-mRNA values. Dam's milk may influence the intestinal expression of COT, since mRNA levels at birth are low and increase after the first lactation. Moreover, mRNA levels decrease in rats weaned on day 18 or 21. COT is also expressed in the liver of suckling rats. Hepatic COT mRNA is maximal at day 3, remains constant until day 9, and decreases thereafter; this pattern is also similar to that of pre-mRNA values. The profile of expression of COT in intestine and liver strongly resembles that of mitochondrial 3-hydroxy 3-methylglutaryl-coenzyme A synthase and carnitine palmitoyltransferase I, suggesting that analogous transcription factors modulate ketogenesis and mitochondrial and peroxisomal fatty acid oxidation.
Asunto(s)
Carnitina Aciltransferasas/genética , Regulación Enzimológica de la Expresión Génica , Intestinos/enzimología , Hígado/enzimología , Animales , Animales Recién Nacidos , Animales Lactantes , Secuencia de Bases , Carnitina Aciltransferasas/metabolismo , Exones/genética , Perfilación de la Expresión Génica , Mucosa Intestinal/citología , Mucosa Intestinal/enzimología , Mucosa Intestinal/metabolismo , Intestinos/citología , Intrones/genética , Hígado/citología , Hígado/metabolismo , Datos de Secuencia Molecular , Músculo Liso/enzimología , Músculo Liso/metabolismo , Peroxisomas/enzimología , Peroxisomas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transcripción Genética/genética , DesteteRESUMEN
Mitochondrial and cytosolic 3-hydroxy-3-methylglutaryl CoA synthase (m-HMS and c-HMS) genes show high identity at the nucleotide and amino acid level, but no homology has been found in the promoter area. The main regulator for c-HMS is SREBP. The best known transcription factor that regulates m-HMS is PPAR alpha. Three types of PPAR, alpha, gamma and delta have been described in vertebrates. Here we found that they display distinct ligand response profiles in the m-HMS promoter. In some conditions PPAR gamma is a significant activator of m-HMS. Thus, the m-HMS gene is transiently expressed during the clonal expansion phase of 3T3-L1 differentiation. We found that C/EBP delta and PPAR gamma activate the m-HMS promoter in 3T3-L1 cells synergistically. This synergistic effect was only observed in the whole promoter (-1148 to +28). A small construct (-116 to +28) which contains the PPRE did not respond to C/EBP delta and/or PPAR gamma. This suggests that a putative C/EBP site lie somewhere between -1148 and -116 bp. We also show that C/EBP delta was more efficient that C/EBP alpha and C/EBP beta to activate the m-HMS promoter. The time course of c-HMS mRNA expression during 3T3-L1 differentiation was different, with a significant increase at terminal adipogenesis. We found that the transcription factor C/EBP alpha did not activate the c-HMS promoter. The differential pattern of expression shown by these two genes, which have a common ancestor, exemplifies refinements of transcriptional control during evolution.
Asunto(s)
Adipocitos/citología , Adipocitos/enzimología , Regulación Enzimológica de la Expresión Génica , Hidroximetilglutaril-CoA Sintasa/genética , Mitocondrias/enzimología , Animales , Northern Blotting , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular , Línea Celular , Citosol/enzimología , Perfilación de la Expresión Génica , Humanos , Hidroximetilglutaril-CoA Sintasa/biosíntesis , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Blattella germanica has two cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase genes, HMG-CoA synthase-1 and -2. HMG-CoA synthase-1 gene shows several features of processed genes (retroposons): it contains no introns but has a short direct-repeat sequence (ATTATTATT) at both ends. An atypical feature is the presence at both ends of the gene of short inverse repeats flanked by direct repeats. There is neither a TATA box nor a CAAT box in the 5' region. Comparative analysis with other species suggests that the HMG-CoA synthase-1 gene derives from HMG-CoA synthase-2. Cultured embryonic B. germanica UM-BGE-1 cells express HMG-CoA synthase-1 but not HMG-CoA synthase-2, suggesting that the intron-less gene is functional. In addition, it can complement MEV-1 cell line, which is auxotrophic for mevalonate. We show that compactin and mevalonate do not significantly affect the mRNA levels of HMG-CoA synthase-1 in UM-BGE-1 cells. Compactin induces a 6.7-fold increase in HMG-CoA reductase activity, which is restored to normal levels by mevalonate. HMG-CoA synthase activity is not modified by either of these effectors, suggesting that the mevalonate pathway in this insect cell line is regulated by post-transcriptional mechanisms affecting HMG-CoA reductase but not HMG-CoA synthase.
Asunto(s)
Cucarachas/genética , Genes de Insecto , Proteínas de Insectos/genética , Lovastatina/análogos & derivados , Retroelementos , Animales , Secuencia de Bases , Cucarachas/citología , Cucarachas/enzimología , Regulación Enzimológica de la Expresión Génica , Hidroximetilglutaril-CoA Reductasas/biosíntesis , Hidroximetilglutaril-CoA Reductasas/genética , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hidroximetilglutaril-CoA Sintasa/biosíntesis , Hidroximetilglutaril-CoA Sintasa/genética , Intrones , Lovastatina/farmacología , Ácido Mevalónico/metabolismo , Datos de Secuencia Molecular , Especificidad de la EspecieRESUMEN
Rat peroxisomal carnitine octanoyltransferase (COT), which facilitates the transport of medium-chain fatty acids through the peroxisomal membrane, is irreversibly inhibited by the hypoglycaemia-inducing drug etomoxir. To identify the molecular basis of this inhibition, cDNAs encoding full-length wild-type COT, two different variant point mutants and one variant double mutant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae, an organism devoid of endogenous COT activity. The recombinant mutated enzymes showed activity towards both carnitine and decanoyl-CoA in the same range as the wild type. Whereas the wild-type version expressed in yeast was inhibited by etomoxir in an identical manner to COT from rat liver peroxisomes, the activity of the enzyme containing the double mutation H131A/H340A was completely insensitive to etomoxir. Individual point mutations H131A and H340A also drastically reduced sensitivity to etomoxir. Taken together, these results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by etomoxir and that the full inhibitory properties of the drug will be shown only if both histidines are intact at the same time. Our data demonstrate that both etomoxir and malonyl-CoA inhibit COT by interacting with the same sites.
Asunto(s)
Carnitina Aciltransferasas/antagonistas & inhibidores , Compuestos Epoxi/farmacología , Histidina/metabolismo , Hígado/enzimología , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Carnitina/metabolismo , Carnitina Aciltransferasas/genética , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Histidina/química , Humanos , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Peroxisomas/enzimología , Plásmidos/metabolismo , Mutación Puntual , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Steroidogenic factor 1 (SF-1) is an orphan member of the nuclear receptor family expressed in steroidogenic tissues, where it has an essential role in the regulation of the steroid hormone biosynthesis, adrenal and gonadal development and endocrine responses fundamental for reproduction. Here we show that SF-1 regulates the transcription of cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene, which is essential for the endogenous synthesis of cholesterol. We have identified an element located 365 bp upstream of the gene for cytosolic HMG-CoA synthase; SF-1 binds as a monomer to this element and confers SF-1 responsiveness to homologous and heterologous promoters. It has been shown that in tissues with a high demand for cholesterol to be used in steroid synthesis, there is a lack of correlation between the cholesterol levels and the activity of the limiting enzymes of the mevalonate pathway. In accord with those results, we observed that cholesterol synthesis from acetate and either cytosolic HMG-CoA mRNA expression or transcriptional activity were not changed in response to 25-hydroxycholesterol in the SF-1-expressing steroidogenic Leydig tumour MA-10 cells. Moreover, the overexpression of SF-1 in non-steroidogenic CV-1 cells renders them less sensitive to the regulatory effects of cholesterol. This observation led to the hypothesis that in steroidogenic tissues the expression of SF-1 permits high levels of endogenous synthesis of cholesterol irrespective of the intracellular levels of this metabolite.
Asunto(s)
Colesterol/biosíntesis , Proteínas de Unión al ADN/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Cricetinae , Citosol/enzimología , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Fushi Tarazu , Proteínas de Homeodominio , Hidroxicolesteroles/farmacología , Hidroximetilglutaril-CoA Sintasa/genética , Receptores Citoplasmáticos y Nucleares , Factor Esteroidogénico 1 , Factores de Transcripción/metabolismo , Transcripción Genética/fisiologíaRESUMEN
The effects of acute treatment with fluvastatin, a hypocholesteremic drug, on the mRNA levels of several regulatory enzymes of cholesterogenesis and of the LDL receptor were determined in rat liver. Fluvastatin increased the hepatic mRNA levels for HMG-CoA reductase up to 12-fold in 5 weeks of treatment at a daily dose of 6. 3 mg/kg. The effect was less marked in cytosolic HMG-CoA synthase, farnesyl-PP synthase, squalene synthetase, and LDL receptor. SREBP-2 mRNA levels were also increased, but SREBP-1 were not. De novo synthesis of cholesterol in several cultured cells was reduced by increasing concentrations of fluvastatin, and the IC(50) values of fluvastatin in HepG2, CV-1, and CHO cells were respectively 0.01, 0. 05, and 0.1 microM. When CHO cells stably transfected with a chimeric gene composed of the promoter of cytosolic HMG-CoA synthase and the CAT gene as a reporter were incubated with fluvastatin, the CAT gene was overexpressed, an effect which was similar to the cotransfection with the processed form of SREBP-1a. Both ALLN and fluvastatin increased the transcriptional activity of cytosolic HMG-CoA synthase. Mutation in either SRE or NF-Y boxes abolished the increase in transcriptional rate caused by fluvastatin in the promoter of cytosolic HMG-CoA synthase. These results indicate that the increase in transcriptional activity in the HMG-CoA synthase gene attributable to fluvastatin is a consequence of the activation of the proteolytic cleavage of SREBPs by reduced levels of intracellular cholesterol.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT , Proteínas de Unión al ADN/metabolismo , Ácidos Grasos Monoinsaturados/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Hidroximetilglutaril-CoA Sintasa/genética , Indoles/farmacología , Proteínas Nucleares/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Células CHO , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Cricetinae , Citosol/enzimología , Cartilla de ADN , Fluvastatina , Humanos , Hígado/enzimología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/efectos de los fármacos , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Activación Transcripcional/efectos de los fármacos , Transfección , Células Tumorales CultivadasRESUMEN
Carnitine octanoyltransferase (COT), an enzyme that facilitates the transport of medium chain fatty acids through peroxisomal membranes, is inhibited by malonyl-CoA. cDNAs encoding full-length wild-type COT and one double mutant variant from rat peroxisomal COT were expressed in Saccharomyces cerevisiae. Both expressed forms were expressed similarly in quantitative terms and exhibited full enzyme activity. The wild-type-expressed COT was inhibited by malonyl-CoA like the liver enzyme. The activity of the enzyme encoded by the double mutant H131A/H340A was completely insensitive to malonyl-CoA in the range assayed (2-200 microM). These results indicate that the two histidine residues, H131 and H340, are the sites responsible for inhibition by malonyl-CoA. Another mutant variant, H327A, abolishes the enzyme activity, from which it is concluded that it plays an important role in catalysis.
Asunto(s)
Carnitina Aciltransferasas/antagonistas & inhibidores , Carnitina Aciltransferasas/química , Hígado/enzimología , Malonil Coenzima A/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carnitina Aciltransferasas/genética , Dominio Catalítico/genética , Cartilla de ADN/genética , ADN Complementario/genética , Expresión Génica , Histidina/química , Humanos , Técnicas In Vitro , Cinética , Mutagénesis Sitio-Dirigida , Peroxisomas/enzimología , Mutación Puntual , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
cAMP increases transcription of the mitochondrial (mit.) gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase, which encodes an enzyme that has been proposed as a control site of ketogenesis. The incubation of Caco-2 cells with cAMP increased mit.HMG-CoA synthase mRNA levels 4-fold within 24 h. We have identified an active cAMP-response element (CRE) located 546 bp upstream of the mit. HMG-CoA synthase promoter that is necessary for the induction of expression by dibutyryl cAMP. Co-transfections of constructs, containing the CRE element of the mit.HMG-CoA synthase promoter fused to the gene for chloramphenicol acetyltransferase, with protein kinase A and a dominant-negative mutant of cAMP-response-element-binding protein (CREB) show that the response to cAMP is mediated by the transcription factor CREB. The CRE element confers responsiveness of protein kinase A to a heterologous promoter in transfection assays in Caco-2 cells. Gel-retardation assays revealed that the mit.HMG-CoA synthase CRE binds to recombinant CREB. The shifted band obtained with the putative mit. HMG-CoA synthase CRE sequence and nuclear proteins from Caco-2 cells competed with CRE sequences of other genes such as somatostatin and phosphoenolpyruvate carboxykinase. We conclude that the regulation of the expression of the gene for mit.HMG-CoA synthase in Caco-2 cells by cAMP is mediated by a CRE sequence in the promoter.