ALAS1

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Herbert L. Bonkovsky - One of the best experts on this subject based on the ideXlab platform.

  • phase 3 trial of rnai therapeutic givosiran for acute intermittent porphyria
    The New England Journal of Medicine, 2020
    Co-Authors: Manisha Balwani, Herbert L. Bonkovsky, Eliane Sardh, David C Rees, Montgomery D Bissell, Paolo Ventura, Paula Aguilera Peiro, Ulrich Stolzel, Jerzy Windyga, Karl E. Anderson
    Abstract:

    Abstract Background Up-regulation of hepatic delta-aminolevulinic acid synthase 1 (ALAS1), with resultant accumulation of delta-aminolevulinic acid (ALA) and porphobilinogen, is central to the path...

  • phase 1 trial of an rna interference therapy for acute intermittent porphyria
    The New England Journal of Medicine, 2019
    Co-Authors: Eliane Sardh, Robert J Desnick, John D Phillips, Pauline Harper, Manisha Balwani, Penelope E Stein, David C Rees, Montgomery D Bissell, Charles J Parker, Herbert L. Bonkovsky
    Abstract:

    Abstract Background Induction of delta aminolevulinic acid synthase 1 (ALAS1) gene expression and accumulation of neurotoxic intermediates result in neurovisceral attacks and disease manifestations...

  • Differential regulation of human ALAS1 mRNA and protein levels by heme and cobalt protoporphyrin
    Molecular and Cellular Biochemistry, 2008
    Co-Authors: Jianyu Zheng, Ying Shan, Richard W. Lambrecht, Susan E. Donohue, Herbert L. Bonkovsky
    Abstract:

    5-Aminolevulinic acid synthase 1 (ALAS1) is the first and rate-controlling enzyme of heme biosynthesis. This study was to determine the effects of heme and selected nonheme metalloporphyrins on human ALAS1 gene expression in hepatocytes. We found that, upon heme and cobalt protoporphyrin (CoPP) treatments, ALAS1 mRNA levels were down-regulated significantly by ca. 50% or more. Measurement of mRNA in the presence of actinomycin D showed that these down-regulations were due to the decreases in mRNA half-lives. Furthermore, the levels of mitochondrial mature ALAS1 protein were down-regulated by 60–70%, but those of the cytosolic precursor protein were up-regulated by 2–5-fold. Measurement of protein in the presence of cycloheximide (CHX) suggests that elevation of the precursor form is due to the increase in protein half-lives. These results provide novel insights into the mechanisms of heme repressional effects on ALAS1 and provide a rationale for further investigation of CoPP as a therapeutic agent for acute porphyric syndromes.

  • Tissue-specific expression of ALA synthase-1 and heme oxygenase-1 and their expression in livers of rats chronically exposed to ethanol
    FEBS letters, 2008
    Co-Authors: Jianyu Zheng, Qing Tian, Weihong Hou, John A. Watts, Laura W. Schrum, Herbert L. Bonkovsky
    Abstract:

    5-Aminolevulinic acid synthase-1 (ALAS1) and heme oxygenase-1 (HO-1) are the rate-controlling enzymes for heme biosynthesis and degradation, respectively. Expression of these two genes showed tissue-specific expression pattern at both mRNA and protein levels in selected non-treated rat tissues. In the livers of rats receiving oral ethanol for 10 weeks, ALAS1 mRNA levels were increased by 65%, and the precursor and mature ALAS1 protein levels were increased by 1.8- and 2.3-fold, respectively, while no changes were observed in HO-1 mRNA and protein levels, compared with pair-fed controls. These results provide novel insights into the effects of chronic ethanol consumption on hepatic heme biosynthesis and porphyrias.

F. Annane - One of the best experts on this subject based on the ideXlab platform.

  • first principle investigation of alas and alp compounds and ordered ALAS1 xpx alloys
    Computational Materials Science, 2010
    Co-Authors: F. Annane, H. Meradji, S. Ghemid, El Haj F Hassan
    Abstract:

    Abstract We have investigated the structural and electronic properties of AlAs and AlP compounds and of ordered ALAS1−xPx alloys using the full potential linearized augmented plane wave plus local orbitals (FP-LAPW + lo) method based on density functional theory. The total energies and structural quantities of those compounds have been calculated for different approximations of exchange–correlation energy. The electronic quantities have been found to be in good agreement with the corresponding measured ones when the compounds were defined by the lattice constants of Perdew–Wang-generalized gradient approximation (PW-GGA) scheme. The PW-GGA approach was also applied on ordered ALAS1−xPx alloys to study the effect of composition on lattice constant, band gap, and refractive index of ALAS1−xPx ternary alloys. The calculated lattice constants scale linearly with composition (Vegard’s law). The microscopic origins of the gap bowing were explained by using the approach of Zunger and co-workers. In addition to (FP-LAPW + lo) method, the composition dependence of the refractive index was studied by Reedy and Nazeer model. The thermodynamic stability of these alloys was investigated by calculating the excess enthalpy of mixing ΔHm as well as the phase diagram.

  • First principle investigation of AlAs and AlP compounds and ordered ALAS1−xPx alloys
    Computational Materials Science, 2010
    Co-Authors: F. Annane, H. Meradji, S. Ghemid, F. El Haj Hassan
    Abstract:

    Abstract We have investigated the structural and electronic properties of AlAs and AlP compounds and of ordered ALAS1−xPx alloys using the full potential linearized augmented plane wave plus local orbitals (FP-LAPW + lo) method based on density functional theory. The total energies and structural quantities of those compounds have been calculated for different approximations of exchange–correlation energy. The electronic quantities have been found to be in good agreement with the corresponding measured ones when the compounds were defined by the lattice constants of Perdew–Wang-generalized gradient approximation (PW-GGA) scheme. The PW-GGA approach was also applied on ordered ALAS1−xPx alloys to study the effect of composition on lattice constant, band gap, and refractive index of ALAS1−xPx ternary alloys. The calculated lattice constants scale linearly with composition (Vegard’s law). The microscopic origins of the gap bowing were explained by using the approach of Zunger and co-workers. In addition to (FP-LAPW + lo) method, the composition dependence of the refractive index was studied by Reedy and Nazeer model. The thermodynamic stability of these alloys was investigated by calculating the excess enthalpy of mixing ΔHm as well as the phase diagram.

Robert J Desnick - One of the best experts on this subject based on the ideXlab platform.

  • 5-Aminolevulinate dehydratase porphyria: Update on hepatic 5-aminolevulinic acid synthase induction and long-term response to hemin.
    Molecular genetics and metabolism, 2020
    Co-Authors: Arian Pourmehdi Lahiji, Robert J Desnick, Karl E. Anderson, Amy Chan, Amy Simon, V. M. Sadagopa Ramanujam
    Abstract:

    Abstract Background 5-Aminolevulinic acid dehydratase (ALAD) porphyria (ADP) is an ultrarare autosomal recessive disease, with only eight documented cases, all of whom were males. Although classified as an acute hepatic porphyria (AHP), induction of the rate limiting hepatic enzyme 5-aminolevulinic acid synthase-1 (ALAS1) has not been demonstrated, and the marrow may also contribute excess 5-aminolevulinic acid (ALA). Two patients have died and reported follow up for the others is limited, so the natural history of this disease is poorly understood and treatment experience limited. Methods We report new molecular findings and update the clinical course and treatment of the sixth reported ADP patient, now 31 years old and the only known case in the Americas, and review published data regarding genotype-phenotype correlation and treatment. Results Circulating hepatic 5-aminolevulinic acid synthase-1 (ALAS1) mRNA was elevated in this case, as in other AHPs. Gain of function mutation of erythroid specific ALAS2 – an X-linked modifying gene in some other porphyrias – was not found. Seven reported ADP cases had compound heterozygous ALAD mutations resulting in very low residual ALAD activity and symptoms early in life or adolescence. One adult with a germline ALAD mutant allele developed ADP in association with a clonal myeloproliferative disorder, polycythemia vera. Conclusions Elevation in circulating hepatic ALAS1 and response to treatment with hemin indicate that the liver is an important source of excess ALA in ADP, although the marrow may also contribute. Intravenous hemin was effective in most reported cases for treatment and prevention of acute attacks of neurological symptoms.

  • phase 1 trial of an rna interference therapy for acute intermittent porphyria
    The New England Journal of Medicine, 2019
    Co-Authors: Eliane Sardh, Robert J Desnick, John D Phillips, Pauline Harper, Manisha Balwani, Penelope E Stein, David C Rees, Montgomery D Bissell, Charles J Parker, Herbert L. Bonkovsky
    Abstract:

    Abstract Background Induction of delta aminolevulinic acid synthase 1 (ALAS1) gene expression and accumulation of neurotoxic intermediates result in neurovisceral attacks and disease manifestations...

  • Molecular expression, characterization and mechanism of ALAS2 gain-of-function mutants
    BMC, 2019
    Co-Authors: Vassili Tchaikovskii, Robert J Desnick, David F Bishop
    Abstract:

    Abstract Background X-linked protoporphyria (XLP) (MIM 300752) is an erythropoietic porphyria due to gain-of-function mutations in the last exon (Ducamp et al., Hum Mol Genet 22:1280-88, 2013) of the erythroid-specific aminolevulinate synthase gene (ALAS2). Five ALAS2 exon 11 variants identified by the NHBLI Exome sequencing project (p.R559H, p.E565D, p.R572C, p.S573F and p.Y586F) were expressed, purified and characterized in order to assess their possible contribution to XLP. To further characterize the XLP gain-of-function region, five novel ALAS2 truncation mutations (p.P561X, p.V562X, p.H563X, p.E569X and p.F575X) were also expressed and studied. Methods Site-directed mutagenesis was used to generate ALAS2 mutant clones and all were prokaryotically expressed, purified to near homogeneity and characterized by protein and enzyme kinetic assays. Standard deviations were calculated for 3 or more assay replicates. Results The five ALAS2 single nucleotide variants had from 1.3- to 1.9-fold increases in succinyl-CoA Vmax and 2- to 3-fold increases in thermostability suggesting that most could be gain-of-function modifiers of porphyria instead of causes. One SNP (p.R559H) had markedly low purification yield indicating enzyme instability as the likely cause for XLSA in an elderly patient with x-linked sideroblastic anemia. The five novel ALAS2 truncation mutations had increased Vmax values for both succinyl-CoA and glycine substrates (1.4 to 5.6-fold over wild-type), while the Kms for both substrates were only modestly changed. Of interest, the thermostabilities of the truncated ALAS2 mutants were significantly lower than wild-type, with an inverse relationship to Vmax fold-increase. Conclusions Patients with porphyrias should always be assessed for the presence of the ALAS2 gain-of-function modifier variants identified here. A key region of the ALAS2 carboxyterminal region is identified by the truncation mutations studied here and the correlation of increased thermolability with activity suggests that increased molecular flexibility/active site openness is the mechanism of enhanced function of mutations in this region providing further insights into the role of the carboxyl-terminal region of ALAS2 in the regulation of erythroid heme synthesis

  • interim data from a randomized placebo controlled phase 1 study of aln as1 an investigational rnai therapeutic for the treatment of acute hepatic porphyria
    Blood, 2016
    Co-Authors: Eliane Sardh, Robert J Desnick, Karl E. Anderson, Pauline Harper, Manisha Balwani, Charles J Parker, Nabil Altawil, Joseph R Bloomer, Montgomery D Bissel, John D Phillips
    Abstract:

    Acute hepatic porphyria (AHP) is a family of rare metabolic disorders including acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP), caused by a deficiency in one of the eight enzymes required for heme biosynthesis in the liver. When ALA synthetase (ALAS1), the first and rate limiting step in the pathway, is induced by triggers such as exposure to certain drugs or fasting, the neurotoxic heme intermediates aminolevulinic acid (ALA) and porphobilinogen (PBG) can accumulate upstream of the deficient enzyme leading to acute and potentially life threatening neurovisceral attacks in AHP patients. RNA interference is a naturally occurring cellular mechanism mediated by small interfering RNA (siRNA) that allows for the inhibition of protein synthesis through the cleavage and degradation of a specific mRNA. ALN-AS1 is an investigational RNAi therapeutic that targets ALAS1 in order to decrease ALA and PBG levels and subsequent porphyria attacks. We are currently conducting a phase 1, multinational, randomized, placebo-controlled, study in 3 parts; Part A single ascending dose (SAD), Part B multiple ascending dose (MAD) and Part C multiple dose (MD) study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics (i.e. changes in ALA, PBG and circulating ALAS1 mRNA levels) of subcutaneously administered ALN-AS1 in AIP patients. Part C also has exploratory analyses of clinical activity, including the impact of ALN-AS1 on porphyria attacks, acute healthcare visits, and heme treatment (ClinicalTrials.gov Identifier: NCT02452372). We previously presented interim SAD data demonstrating that ALN-AS1 was generally well tolerated with no serious adverse events (SAEs) or clinically significant laboratory abnormalities related to study drug, and no discontinuations due to AEs. Circulating ALAS1 mRNA levels had a mean (SEM) maximal reduction of 44% ± 8% relative to baseline (p ≤ 0.01 compared to placebo), with concomitant mean (SEM) maximal reductions in ALA and PBG of 77% ± 7% and 73% ± 6%, respectively (p= 0.03 and 0.06 compared to placebo, respectively) at the 0.35 mg/kg dose. In addition, changes in circulating ALAS1 mRNA were highly correlated with changes in urinary ALA and PBG (R2=0.82, p Disclosures Sardh:Alnylam Pharmaceuticals: Consultancy. Harper:Alnylam Pharmaceuticals: Consultancy. Balwani:ICGG Gaucher Registry (Sanofi Genzyme sponsored): Membership on an entity9s Board of Directors or advisory committees; Sanofi Genzyme: Honoraria, Speakers Bureau; Alnylam Pharmaceuticals: Consultancy. Anderson:Alnylam Pharmaceuticals: Consultancy. Bloomer:Alnylam Pharamceuticals: Consultancy. Bissel:Alnylam Pharmaceuticals: Consultancy. Desnick:Alnylam Pharmaceuticals: Consultancy. Bonkovsky:Alnylam Pharamceuticals: Consultancy. Penz:Alnylam Pharmaceuticals: Employment, Equity Ownership. Chan:Alnylam Pharmaceuticals: Employment, Equity Ownership. Soh:Alnylam Pharmaceuticals: Employment, Equity Ownership. Querbes:Alnylam Pharmaceuticals: Employment, Equity Ownership. Simon:Alnylam Pharmaceuticals: Employment, Equity Ownership. Rees:Alnylam Pharmaceuticals: Consultancy.

  • rnai mediated silencing of hepatic ALAS1 effectively prevents and treats the induced acute attacks in acute intermittent porphyria mice
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Makiko Yasuda, John D Phillips, Brenden Chen, Lin Gan, Senkottuvelan Kadirvel, Maria I New, Abigail Liebow, Kevin Fitzgerald, William Querbes, Robert J Desnick
    Abstract:

    The acute hepatic porphyrias are inherited disorders of heme biosynthesis characterized by life-threatening acute neurovisceral attacks. Factors that induce the expression of hepatic 5-aminolevulinic acid synthase 1 (ALAS1) result in the accumulation of the neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), which recent studies indicate are primarily responsible for the acute attacks. Current treatment of these attacks involves i.v. administration of hemin, but a faster-acting, more effective, and safer therapy is needed. Here, we describe preclinical studies of liver-directed small interfering RNAs (siRNAs) targeting ALAS1 (ALAS1-siRNAs) in a mouse model of acute intermittent porphyria, the most common acute hepatic porphyria. A single i.v. dose of ALAS1-siRNA prevented the phenobarbital-induced biochemical acute attacks for approximately 2 wk. Injection of ALAS1-siRNA during an induced acute attack significantly decreased plasma ALA and PBG levels within 8 h, more rapidly and effectively than a single hemin infusion. ALAS1-siRNA was well tolerated and a therapeutic dose did not cause hepatic heme deficiency. These studies provide proof-of-concept for the clinical development of RNA interference therapy for the prevention and treatment of the acute attacks of the acute hepatic porphyrias.

David F Bishop - One of the best experts on this subject based on the ideXlab platform.

  • Molecular expression, characterization and mechanism of ALAS2 gain-of-function mutants
    BMC, 2019
    Co-Authors: Vassili Tchaikovskii, Robert J Desnick, David F Bishop
    Abstract:

    Abstract Background X-linked protoporphyria (XLP) (MIM 300752) is an erythropoietic porphyria due to gain-of-function mutations in the last exon (Ducamp et al., Hum Mol Genet 22:1280-88, 2013) of the erythroid-specific aminolevulinate synthase gene (ALAS2). Five ALAS2 exon 11 variants identified by the NHBLI Exome sequencing project (p.R559H, p.E565D, p.R572C, p.S573F and p.Y586F) were expressed, purified and characterized in order to assess their possible contribution to XLP. To further characterize the XLP gain-of-function region, five novel ALAS2 truncation mutations (p.P561X, p.V562X, p.H563X, p.E569X and p.F575X) were also expressed and studied. Methods Site-directed mutagenesis was used to generate ALAS2 mutant clones and all were prokaryotically expressed, purified to near homogeneity and characterized by protein and enzyme kinetic assays. Standard deviations were calculated for 3 or more assay replicates. Results The five ALAS2 single nucleotide variants had from 1.3- to 1.9-fold increases in succinyl-CoA Vmax and 2- to 3-fold increases in thermostability suggesting that most could be gain-of-function modifiers of porphyria instead of causes. One SNP (p.R559H) had markedly low purification yield indicating enzyme instability as the likely cause for XLSA in an elderly patient with x-linked sideroblastic anemia. The five novel ALAS2 truncation mutations had increased Vmax values for both succinyl-CoA and glycine substrates (1.4 to 5.6-fold over wild-type), while the Kms for both substrates were only modestly changed. Of interest, the thermostabilities of the truncated ALAS2 mutants were significantly lower than wild-type, with an inverse relationship to Vmax fold-increase. Conclusions Patients with porphyrias should always be assessed for the presence of the ALAS2 gain-of-function modifier variants identified here. A key region of the ALAS2 carboxyterminal region is identified by the truncation mutations studied here and the correlation of increased thermolability with activity suggests that increased molecular flexibility/active site openness is the mechanism of enhanced function of mutations in this region providing further insights into the role of the carboxyl-terminal region of ALAS2 in the regulation of erythroid heme synthesis

  • familial skewed x chromosome inactivation as a predisposing factor for late onset x linked sideroblastic anemia in carrier females
    Blood, 2000
    Co-Authors: Mario Cazzola, Alison May, Gaetano Bergamaschi, Paola Cerani, Vittorio Rosti, David F Bishop
    Abstract:

    X-linked sideroblastic anemia (XLSA) is caused by mutations in the erythroid-specific 5-aminolevulinic acid synthase ( ALAS2 ) gene. An elderly woman who presented with an acquired sideroblastic anemia is studied. Molecular analysis revealed that she was heterozygous for a missense mutation in the ALAS2 gene, but she expressed only the mutated gene in reticulocytes. Her 2 daughters and a granddaughter were heterozygous for this mutation, had normal hemoglobin levels, and expressed the normal ALAS2 gene in reticulocytes. A grandson with a previous diagnosis of thalassemia intermedia was found to be hemizygous for the ALAS2 mutation. Treatment with pyridoxine completely corrected the anemia both in the proband and her grandson. All women who were analyzed in this family showed skewed X-chromosome inactivation in leukocytes, which indicated a hereditary condition associated with unbalanced lyonization. Because the preferentially active X chromosome carried the mutant ALAS2 allele, acquired skewing in the elderly likely worsened the genetic condition and abolished the normal ALAS2 allele expression in the proband.

  • molecular defects of erythroid 5 aminolevulinate synthase in x linked sideroblastic anemia
    Journal of Bioenergetics and Biomembranes, 1995
    Co-Authors: Sylvia S. Bottomley, Timothy C. Cox, Brian K. May, Philip D Cotter, David F Bishop
    Abstract:

    The erythroid-specific isozyme of 5-aminolevulinate synthase (ALAS2), the first and ratelimiting enzyme of heme biosynthesis, is expressed concomitantly with the differentiation and maturation of the erythroid cell in order to accommodate generation of the large amounts of heme required for hemoglobin production. During the past few years the ALAS2 gene and its transcript have been characterized and the amino acid sequence of the enzyme deduced. The human genetic disorder X-linked sideroblastic anemia, previously postulated to be caused by defects of ALAS, has now been analyzed at the molecular and tissue-specific level. A heterogeneous group of point mutations in the catalytic domain of the ALAS2 enzyme has been found to cause the disorder. Impaired activity of recombinant mutant ALAS2 enzymes has also been demonstrated. Characterization of molecular defects in individuals with X-linked sideroblastic anemia has provided improved diagnosis for at-risk family members.

  • Assignment of the human housekeeping δ-aminolevulinate synthase gene (ALAS1) to chromosome band 3p21.1 by PCR analysis of somatic cell hybrids
    Cytogenetics and cell genetics, 1995
    Co-Authors: Philip D Cotter, Harry A. Drabkin, T.a. Varkony, David I. Smith, David F Bishop
    Abstract:

    The precise map position of the human housekeeping δ-aminolevulinate synthase (ALAS1) gene has been localized to chromosome band 3p21.1. PCR analysis of somatic cell and radiation hybrids localized AL

  • Assignment of human erythroid δ-aminolevulinate synthase (ALAS2) to a distal subregion of band Xp11.21 by PCR analysis of somatic cell hybrids containing X;Autosome translocations
    Genomics, 1992
    Co-Authors: Philip D Cotter, Huntington F. Willard, Jerome L. Gorski, David F Bishop
    Abstract:

    The erythroid-specific (ALAS2) and housekeeping (ALAS1) genes encoding delta-aminolevulinate synthase have recently been mapped to chromosomes Xp21.1----q21 and 3p21, respectively. The erythroid-specific gene is a candidate for mutations resulting in X-linked sideroblastic anemia. Analysis of DNA from hybrid clones containing translocations in the region Xp11.21----Xq21.3 permitted the finer localization of the ALAS2 gene with respect to other loci and breakpoints within this region. These studies localized the ALAS2 gene to the distal subregion of Xp11.21 in Interval 5 indicating the following gene order: Xpter-OATL2-[L62-3A, Xp11.21; A62-1A-4b, Xp11.21]-(ALAS2, DXS323)-[B13-3, Xp11.21; C9-5, Xp11.21]-(DXS14, DXS429)-DXS422-(DXZ1, Xcen). Thus, the reported linkage of acquired sideroblastic anemia and sideroblastic anemia with ataxia to Xq13 presumably results from genes other than ALAS2.

Urs A. Meyer - One of the best experts on this subject based on the ideXlab platform.

  • Identification of the xenosensors regulating human 5-aminolevulinate synthase.
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Michael Podvinec, Christoph Handschin, Renate Looser, Urs A. Meyer
    Abstract:

    Heme is an essential component of numerous hemoproteins with functions including oxygen transport, energy metabolism, and drug biotransformation. In nonerythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the rate-limiting enzyme in heme biosynthesis. Upon exposure to drugs that induce cytochromes P450 and other drug-metabolizing enzymes, ALAS1 is transcriptionally up-regulated, increasing the rate of heme biosynthesis to provide heme for cytochrome P450 hemoproteins. We used a combined in silico-in vitro approach to identify sequences in the ALAS1 gene that mediate direct transcriptional response to xenobiotic challenge. We have characterized two enhancer elements, located 20 and 16 kb upstream of the transcriptional start site. Both elements respond to prototypic inducer drugs and interact with the human pregnane X receptor NR1I2 and the human constitutive androstane receptor NR1I3. Our results suggest that the fundamental mechanism of drug induction is the same for cytochromes P450 and ALAS1. Transcriptional activation of the ALAS1 gene is the first step in the coordinated up-regulation of apoprotein and heme synthesis in response to exogenous and endogenous signals controlling heme levels. Understanding the direct effects of drugs on heme synthesis is of clinical interest, particularly in patients with hepatic porphyrias.

  • Nuclear receptors constitutive androstane receptor and pregnane X receptor activate a drug-responsive enhancer of the murine 5-aminolevulinic acid synthase gene.
    The Journal of biological chemistry, 2003
    Co-Authors: David J. Fraser, Adrian Zumsteg, Urs A. Meyer
    Abstract:

    Abstract 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.

  • Drugs Mediate the Transcriptional Activation of the 5-Aminolevulinic Acid Synthase (ALAS1) Gene via the Chicken Xenobiotic-sensing Nuclear Receptor (CXR)
    The Journal of biological chemistry, 2002
    Co-Authors: David J. Fraser, Michael Podvinec, Michel R. Kaufmann, Urs A. Meyer
    Abstract:

    Heme is an essential component in oxygen transport and metabolism in living systems. In non-erythropoietic cells, 5-aminolevulinate synthase (ALAS1) is the first and rate-limiting enzyme in the heme biosynthesis pathway. ALAS1 expression and heme levels are increased in vivo by drugs and other chemical inducers of cytochrome P450 hemoproteins through mechanisms that are poorly understood. In the present studies, a chicken genomic cosmid library was employed to isolate a major portion of the ALAS1 gene. Two drug-responsive enhancer sequences, 176 and 167 base pairs in length, were identified in the 5'-flanking region of the gene in reporter gene assays in the hepatoma cell line LMH. The relative potency of inducers to activate these enhancers corresponds to induction of ALAS1 mRNA levels in LMH cells. Analysis of putative transcription factor binding sites within the enhancers revealed DR5 and DR4 type recognition sequences for nuclear receptors. Drug activation of the enhancer elements was reduced at least 60% after mutagenesis of individual nuclear receptor binding sites and was virtually eliminated following alteration of both recognition sites within the respective elements. Electrophoretic mobility shift assays and transactivation studies demonstrate direct interactions between the nuclear receptor binding sites and the recently described chicken xenobiotic-sensing receptor, (CXR) implicating drug activation mechanisms for ALAS1 similar to those found in inducible cytochrome(s) P450. This is the first report describing direct transcriptional activation of ALAS1 by drugs via drug-responsive enhancer sequences.