A promoter mutation in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causes X-linked sideroblastic anemia.
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X-linked sideroblastic anemia (XLSA) is caused by mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). XLSA was diagnosed in a 32-year-old woman with a mild phenotype and moderately late onset. Pyridoxine therapy had no effect in the proband, but in her affected son engendered a modest increase in hemoglobin concentration and a 4-fold reduction in ferritin iron. Molecular analysis identified a C to G transversion at nucleotide -206 from the transcription start site, as defined by primer extension, in the proximal promoter region of ALAS2. No other mutations were found in the promoter region, the flanking intronic sequences, the exons, or the 3' genomic region. The same mutation was found in her affected son but not in any other of her unaffected relatives. The mutation resulted in a 94% loss of activity relative to the wild-type sequence for a luciferase reporter construct containing the proximal 293 nucleotides (nt's) of the ALAS2 promoter when transfected into human erythroid K562 cells. Confirming the mutation's deleterious effect, the ALAS2 mRNA level in the proband's erythroid precursors was reduced 87%. The mutation occurred in or near 3 different putative transcription factor binding sites of unknown erythroid importance. The dramatic decreases in reporter activity and mRNA level suggest that the region of the mutation may bind a novel and important erythroid regulatory element. (+info)
5-Aminolevulinic acid synthase: mechanism, mutations and medicine.
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5-Aminolevulinic acid synthase (ALAS), the first enzyme of the heme biosynthesis pathway, catalyses the pyridoxal 5'-phosphate-dependent condensation between glycine and succinyl-CoA to yield 5-aminolevulinic acid (5-amino-4-oxopentanoate). A three-dimensional structural model of Rhodobacter spheroides ALAS has been constructed and used to identify amino acid residues at the active site that are likely to be important for the recognition of glycine, the only amino acid substrate. Several residues have been investigated by site-directed mutagenesis and enzyme variants have been generated that are able to use alanine, serine or threonine. A three-dimensional structure model of 5-aminolevulinic acid synthase from human erythrocytes (ALAS 2) has also been constructed and used to map a range of naturally occurring human mutants that give rise to X-linked sideroblastic anemia. A number of these anemias respond favourably to vitamin B(6) (pyridoxine) therapy, whereas others are either partially responsive or completely refractory. Detailed investigations with selected human mutants have highlighted the importance of arginine-517 that is implicated in glycine carboxyl group binding. (+info)
Occupational lead exposure in Finland. V. Shipyards and shipbreaking.
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Three Finnish shipyards and two shipbreaking enterprises were studied for lead exposure. Blood lead (Pb-B) concentration was measured for 568 workers in the shipyards and 13 workers in the shipbreaking enterprises. Erythrocyte delta-aminolevulinic acid dehydratase (ALAD) activity was determined for 139 workers in one shipyard and 7 workers in one shipbreaking enterprise. Hemoglobin level was determined for 545 workers in the three shipyards and 7 workers in one shipbreaking enterprise. Lead exposure in the shipyards was relatively low. No Pb-B value exceeded 70mug/100 ml. The most exposed occupations were welders, plumbers, painters, repairmen, and sheet metal workers. The mean Pb-B did not exceed 40 mug/100 ml in any of these groups. In both shipbreaking enterprises one Pb-B value exceeded 70 mug/100 ml, the mean Pb-B values of all the workers in the two enterprises being 51 and 46 mug/100 ml. respectively. ALAD values corresponded well with the respective Pb-B values. All the hemoglobin mean values were normal, and there were no statistically significant differences between the hemoglobin values of different groups. (+info)
Circular permutation of 5-aminolevulinate synthase: effect on folding, conformational stability, and structure.
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The first and regulatory step of heme biosynthesis in mammals begins with the pyridoxal 5'-phosphate-dependent condensation reaction catalyzed by 5-aminolevulinate synthase. The enzyme functions as a homodimer with the two active sites at the dimer interface. Previous studies demonstrated that circular permutation of 5-aminolevulinate synthase does not prevent folding of the polypeptide chain into a structure amenable to binding of the pyridoxal 5'-phosphate cofactor and assembly of the two subunits into a functional enzyme. However, while maintaining a wild type-like three-dimensional structure, active, circularly permuted 5-aminolevulinate synthase variants possess different topologies. To assess whether the aminolevulinate synthase overall structure can be reached through alternative or multiple folding pathways, we investigated the guanidine hydrochloride-induced unfolding, conformational stability, and structure of active, circularly permuted variants in relation to those of the wild type enzyme using fluorescence, circular dichroism, activity, and size exclusion chromatography. Aminolevulinate synthase and circularly permuted variants folded reversibly; the equilibrium unfolding/refolding profiles were biphasic and, in all but one case, protein concentration-independent, indicating a unimolecular process with the presence of at least one stable intermediate. The formation of this intermediate was preceded by the disruption of the dimeric interface or dissociation of the dimer without significant change in the secondary structural content of the subunits. In contrast to the similar stabilities associated with the dimeric interface, the energy for the unfolding of the intermediate as well as the overall conformational stabilities varied among aminolevulinate synthase and variants. The unfolding of one functional permuted variant was protein concentration-dependent and had a potentially different folding mechanism. We propose that the order of the ALAS secondary structure elements does not determine the ability of the polypeptide chain to fold but does affect its folding mechanism. (+info)
The regulation of haemoglobin synthesis in cultured chick blastoderms by steroids related to 5beta-androstane.
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1. After 24h of incubation, the blastoderm may be dissected from the early developing chick embryo and successfully maintained under conditions of organ culture in vitro. 2. Low concentrations of steroids related to 5beta-androstane stimulate the synthesis of foetal haemoglobins, types E and P, in a highly steroid- and tissue-specific manner. (+info)
Regulation of mitochondrial biogenesis: enzymatic changes in cytochrome-deficient yeast mutants requiring delta-aminolevulinic acid.
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Yeast cells almost completely deficient in all cytochromes were obtained by introducing two defective nuclear genes, cyd1 and cyc4, into the same haploid strain. The action of the two mutant genes is synergistic, since either gene acting singly results in only partial cytochrome deficiency. Normal synthesis of all cytochromes can be restored in the double mutant by adding delta-aminolevulinic acid to the growth medium. The optimum concentration of delta-aminolevulinate for restoration of cytochrome synthesis is about 40 muM; when higher concentrations are used, synthesis of cytochromes is partially suppressed, particularly that of cytochrome a.a3. Growth yield of the double mutant is stimulated by ergosterol and Tween 80, a source of unsaturated fatty acid. Methionine stimulates further. None of these nutrients is required for growth when sufficient delta-aminolevulinic acid is present in the growth medium. With respect to nutritional responses, the single-gene, cytochrome-deficient mutant, ole3, behaves like the double mutant. The frequency of the p-mutation in the double mutant grown in the absence of ergosterol, Tween 80, and delta-aminolevulinic acid is at least 15%. The frequency can be reduced to less than 1% by either delta-aminolevulinic acid or Tween 80. Ergosterol alone does not decrease the p- frequency. The ole3 mutant does not exhibit increased p-frequency under similar conditions of unsaturated fatty acid deficiency. (+info)
Bilirubin is highly effective in preventing in vivo delta-aminolevulinic acid-induced oxidative cell damage.
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Delta-aminolevulinic acid (ALA), precursor of heme, accumulates in a number of organs, particularly in liver of patients with acute porphyrias or lead intoxication. This study characterizes the involvement of bilirubin as an antioxidant in a chronic intoxication with ALA. Female Wistar rats were injected intraperitoneally a daily dose of 40 mg ALA/body wt., during 10 days. A marked increase in lipid peroxidation and a decrease in GSH content were observed 24 h after the last injection of ALA. The activities of liver antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase were also diminished. ALA synthase (ALA-S) and heme oxygenase-1 were induced. Both ALA dehydratase (ALA-D) and porphobilinogenase (PBG-ase) activities were inhibited. Administration of bilirubin (5 mmol/kg body wt.) 2 h before ALA treatment entirely prevented the effects of ALA. Co-administration of ALA and Sn-protoporphyrin IX (Sn-PPIX; 100 microg/body wt., i.p.), a potent inhibitor of heme oxygenase, completely abolished its induction and provoked a marked decrease in liver GSH levels as well as an increase in lipid peroxidation. These results add further support to the proposal assigning bilirubin a key protective role against oxidative damage here induced by ALA. (+info)
Nuclear receptors constitutive androstane receptor and pregnane X receptor activate a drug-responsive enhancer of the murine 5-aminolevulinic acid synthase gene.
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Nuclear receptors have been implicated in the transcriptional regulation of expression of a growing number of genes, including cytochromes P450 and 5-aminolevulinate synthase (ALAS1), the first and rate-limiting enzyme in the heme biosynthesis pathway. Although drugs that induce cytochromes P450 also induce ALAS1, the regulatory mechanisms governing these pathways have not been fully elucidated. We have identified a drug-responsive enhancer in the murine ALAS1 gene. This sequence mediates transcriptional activation by a wide range of compounds including typical cytochrome P450 pan-inducers phenobarbital and metyrapone, as well as specific activators of the pregnane X receptor and the constitutive androstane receptor. ALAS1 drug-responsive enhancer sequences were identified by transient transfection of reporter gene constructs in the drug-responsive leghorn male hepatoma cell line. Using the NUBIScan algorithm, DR4 nuclear receptor binding sites were identified within the elements and their roles in mediating transcriptional activation of ALAS1 were confirmed by site-directed mutagenesis. Electrophoretic mobility shift assays demonstrate clear interactions of mouse pregnane X receptor and constitutive androstane receptor on the ADRES. Transactivation assays in CV-1 cells implicate the nuclear receptors as major contributors to transcriptional activation of ALAS1. Moreover, in vivo studies in knock-out animals confirm the induction of ALAS1 is mediated at least in part by nuclear receptors. These studies are the first to explain drug induction via drug response elements for mammalian ALAS1. (+info)