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Robert J Desnick - One of the best experts on this subject based on the ideXlab platform.
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congenital erythropoietic porphyria and erythropoietic protoporphyria identification of 7 Uroporphyrinogen III Synthase and 20 ferrochelatase novel mutations
Molecular Genetics and Metabolism, 2019Co-Authors: Yedidyah Weiss, Makiko Yasuda, Manisha Balwani, Brenden Chen, Irina Nazarenko, Robert J DesnickAbstract:Abstract The erythropoietic porphyrias are inborn errors of heme biosynthesis with prominent cutaneous manifestations. They include autosomal recessive Congenital Erythropoietic Porphyria (CEP) due to loss-of-function (LOF) mutations in the Uroporphyrinogen III Synthase (UROS) gene, Erythropoietic Protoporphyria (EPP) due to LOF mutations in the ferrochelatase (FECH) gene, and X-Linked Protoporphyria (XLP) due to gain-of-function mutations in the terminal exon of the Aminolevulinic Acid Synthase 2 (ALAS2) gene. During the 11-year period from 01/01/2007 through 12/31/2017, the Mount Sinai Porphyrias Diagnostic Laboratory provided molecular diagnostic testing for one or more of these disorders in 628 individuals, including 413 unrelated individuals. Of these 628, 120 patients were tested for CEP, 483 for EPP, and 331 for XLP, for a total of 934 tests. For CEP, 24 of 78 (31%) unrelated individuals tested had UROS mutations, including seven novel mutations. For EPP, 239 of 362 (66%) unrelated individuals tested had pathogenic FECH mutations, including twenty novel mutations. The IVS3-48 T > C low-expression allele was present in 231 (97%) of 239 mutation-positive EPP probands with a pathogenic FECH mutation. In the remaining 3%, three patients with two different FECH mutations in trans were identified. For XLP, 24 of 250 (10%) unrelated individuals tested had ALAS2 exon 11 mutations. No novel ALAS2 mutations were identified. Among family members referred for testing, 33 of 42 (79%) CEP, 62 of 121 (51%) EPP, and 31 of 81 (38%) XLP family members had the respective family mutation. Mutation-positive CEP, EPP, and XLP patients who had been biochemically tested had marked elevations of the disease-appropriate porphyrin intermediates. These results expand the molecular heterogeneity of the erythropoietic porphyrias by adding a total of 27 novel mutations. The results document the usefulness of molecular testing to confirm the positive biochemical findings in these patients and to identify heterozygous family members.
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congenital erythropoietic porphyria a novel Uroporphyrinogen III Synthase branchpoint mutation reveals underlying wild type alternatively spliced transcripts
Blood, 2010Co-Authors: David F Bishop, Xiaoye Schneideryin, Sonia Clavero, Hanwook Yoo, Elisabeth I Minder, Robert J DesnickAbstract:Splicing mutations account for approximately 10% of lesions causing genetic diseases, but few branchpoint sequence (BPS) lesions have been reported. In 3 families with autosomal recessive congenital erythropoietic porphyria (CEP) resulting from Uroporphyrinogen III Synthase (URO-Synthase) deficiency, sequencing the promoter, all 10 exons and the intron/exon boundaries did not detect a mutation. Northern analyses of lymphoblast mRNAs from 2 patients and reverse-transcribed polymerase chain reaction (RT-PCR) of lymphoblast mRNAs from all 3 patients revealed multiple longer transcripts involving intron 9 and low levels of wild-type message. Sequencing intron 9 RT-PCR products and genomic DNA in each case revealed homozygosity for a novel BPS mutation (c.661-31T→G) and alternatively spliced transcripts containing 81, 246, 358, and 523 nucleotides from intron 9. RT-PCR revealed aberrant transcripts in both wild-type and CEP lymphoblasts, whereas BPS mutation reduced the wild-type transcript and enzyme activity in CEP lymphoblasts to approximately 10% and 15% of normal, respectively. Although the +81-nucleotide alternative transcript was in-frame, it only contributed approximately 0.2% of the lymphoblast URO-Synthase activity. Thus, the BPS mutation markedly reduced the wild-type transcript and enzyme activity, thereby causing the disease. This is the first BPS mutation in the last intron, presumably accounting for the observed 100% intron retention without exon skipping.
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human Uroporphyrinogen III Synthase nmr based mapping of the active site
Proteins, 2008Co-Authors: Luis F Cunha, Miklos Kuti, David F Bishop, Mihaly Mezei, Lei Zeng, Mingming Zhou, Robert J DesnickAbstract:Uroporphyrinogen III Synthase (URO-Synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to Uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-Synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-Synthase (HMB-Synthase) and/or Uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-Synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-Synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO Synthase that mapped the enzyme's active site. In silico docking of the URO-Synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-Synthase mixed with the homogeneous HMB-Synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex.
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Uroporphyrinogen III Synthase knock in mice have the human congenital erythropoietic porphyria phenotype including the characteristic light induced cutaneous lesions
American Journal of Human Genetics, 2006Co-Authors: David F Bishop, Amr A Shady, Annika Johansson, Robert G Phelps, Maria Celeste M Ramirez, Makiko Yasuda, Andres A Caro, Robert J DesnickAbstract:Congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error, results from the deficient but not absent activity of Uroporphyrinogen III Synthase (URO-Synthase), the fourth enzyme in the heme biosynthetic pathway. The major clinical manifestations include severe anemia, erythrodontia, and disfiguring cutaneous involvement due to the accumulation of phototoxic porphyrin I isomers. Murine models of CEP could facilitate studies of disease pathogenesis and the evaluation of therapeutic endeavors. However, URO-Synthase null mice were early embryonic lethals. Therefore, knock-in mice were generated with three missense mutations, C73R, V99A, and V99L, which had in vitro–expressed activities of 0.24%, 5.9%, and 14.8% of expressed wild-type activity, respectively. Homozygous mice for all three mutations were fetal lethals, except for mice homozygous for a spontaneous recombinant allele, V99AT/V99AT, a head-to-tail concatemer of three V99A targeting constructs. Although V99AT/V99AT and C73R/V99AT mice had ∼2% hepatic URO-Synthase activity and normal hepatic microsomal heme and hemoprotein levels, they had 20% and 13% of wild-type activity in erythrocytes, respectively, which indicates that sufficient erythroid URO-Synthase was present for fetal development and survival. Both murine genotypes showed marked porphyrin I isomer accumulation in erythrocytes, bone, tissues, and excreta and had fluorescent erythrodontia, hemolytic anemia with reticulocytosis and extramedullary erythropoiesis, and, notably, the characteristic light-induced cutaneous involvement. These mice provide insight into why CEP is an erythroid porphyria, and they should facilitate studies of the disease pathogenesis and therapeutic endeavors for CEP.
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congenital erythropoietic porphyria identification and expression of eight novel mutations in the Uroporphyrinogen III Synthase gene
British Journal of Haematology, 2002Co-Authors: Amr A Shady, Luis F Cunha, David F Bishop, Kenneth H. Astrin, Brandon R Colby, Robert J DesnickAbstract:Summary. Mutations in the Uroporphyrinogen III Synthase (URO-Synthase) gene cause congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of haem biosynthesis. Molecular analysis of the URO-Synthase gene in seven unrelated CEP patients revealed eight novel mutations. These included four missense mutations (A69T, E81D, G188W and I219S), a deletion (21delG), two insertions (398insG and 672ins28) and one complex mutation (627del6ins39), as well as three previously reported mutations, C73R, T228M, and −86CA. When the four novel missense mutations were expressed in Escherichia coli, only E81D expressed significant enzymatic activity (30% of expressed wild-type activity), which was thermolabile. In addition, reverse transcription polymerase chain reaction studies demonstrated that E81D, which altered the penultimate nucleotide in exon 4, impaired splicing and caused about 85% exon 4 skipping. The identification and expression of these mutations provided genotype–phenotype correlations and further evidence of the molecular heterogeneity underlying this erythropoietic porphyria.
Oscar Millet - One of the best experts on this subject based on the ideXlab platform.
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natural and pharmacological chaperones against accelerated protein degradation Uroporphyrinogen III Synthase and congenital erythropoietic porphyria
2020Co-Authors: Ganeko Bernardoseisdedos, Emmanuel Richard, David Gil, Jeanmarc Blouin, Oscar MilletAbstract:Abstract Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disease derived from a deficient activity in the fourth enzyme of the heme biosynthetic pathway, Uroporphyrinogen III Synthase (UROIIIS). The impossibility of a correct heme production results in the accumulation of intermediate metabolites known as uroporhyrins, leading to pathological consequences such as skin photosensitivity and phototoxic cutaneous injuries. In this chapter, we discuss the importance of protein homeostasis in the heme pathway and its regulation, as well as how many of the CEP-causing mutations impair UROIIIS homeostasis, affecting thermodynamic stability but preserving normal or near-normal activity. As a proof of concept, we show how UROIIIS proteostasis can be restored by use of a proteasomal inhibitor in animal models. Finally, a new line of therapeutic intervention against CEP is presented, which involves the use of a pharmacological chaperone known as ciclopirox (CPX) to improve UROIIIS stability, reverting most of the CEP symptoms in mice models.
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tuning intracellular homeostasis of human Uroporphyrinogen III Synthase by enzyme engineering at a single hotspot of congenital erythropoietic porphyria
Human Molecular Genetics, 2014Co-Authors: Fredj Ben Bdira, Ana Lain, Esperanza Gonzalez, Juan M Falconperez, Paula Pluta, Arantza Sanzparra, Oscar MilletAbstract:Congenital erythropoietic porphyria (CEP) results from a deficiency in Uroporphyrinogen III Synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.
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intracellular rescue of the Uroporphyrinogen III Synthase activity in enzymes carrying the hotspot mutation c73r
Journal of Biological Chemistry, 2011Co-Authors: Arola Fortian, David Castano, Esperanza Gonzalez, Juan M Falconperez, Oscar MilletAbstract:A single mutation (C73R) in the enzyme Uroporphyrinogen III Synthase (UROIIIS) is responsible for more than one-third of all of the reported cases of the rare autosomal disease congenital erythropoietic porphyria (CEP). CEP patients carrying this hotspot mutation develop a severe phenotype of the disease, including reduced life expectancy. Here, we have investigated the molecular basis for the functional deficit in the mutant enzyme both in vitro and in cellular systems. We show that a Cys in position 73 is not essential for the catalytic activity of the enzyme but its mutation to Arg speeds up the process of irreversible unfolding and aggregation. In the mammalian cell milieu, the mutant protein levels decrease to below the detection limit, whereas wild type UROIIIS can be detected easily. The disparate response is not produced by differences at the level of transcription, and the results with cultured cells and in vitro are consistent with a model where the protein becomes very unstable upon mutation and triggers a degradation mechanism via the proteasome. Mutant protein levels can be restored upon cell treatment with the proteasome inhibitor MG132. The intracellularly recovered C73R-UROIIIS protein shows enzymatic activity, paving the way for a new line of therapeutic intervention in CEP patients.
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structural thermodynamic and mechanistical studies in Uroporphyrinogen III Synthase molecular basis of congenital erythropoietic porphyria
Advances in Protein Chemistry, 2011Co-Authors: Arola Fortian, Ana Lain, David Castano, Esperanza Gonzalez, Juan M Falconperez, Oscar MilletAbstract:Congenital erythropoietic porphyria (CEP) is a rare autosomal disease ultimately related to deleterious mutations in Uroporphyrinogen III Synthase (UROIIIS), the fourth enzyme of the biosynthetic route of the heme group. UROIIIS catalyzes the cyclization of the linear tetrapyrrol hydroxymethylbilane (HMB), inverting the configuration in one of the aromatic rings. In the absence of the enzyme (or when ill-functioning), HMB spontaneously degrades to the by-product Uroporphyrinogen I, which cannot lead to the heme group and accumulates in the body, producing some of the symptoms observed in CEP patients. In the present chapter, clinical, biochemical, and biophysical information has been compiled to provide an integrative view on the molecular basis of CEP. The high-resolution structure of UROIIIS sheds light on the enzyme reaction mechanism while thermodynamic analysis revealed that the protein is thermolabile. Pathogenic missense mutations are found throughout the primary sequence of the enzyme. All but one of these is rarely found in patients, whereas C73R is responsible for more than one-third of the reported cases. Most of the mutant proteins (C73R included) retain partial catalytic activity but the mutations often reduce the enzyme's stability. The stabilization of the protein in vivo is discussed in the context of a new line of intervention to complement existing treatments such as bone marrow transplantation and gene therapy.
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Uroporphyrinogen III Synthase mutations related to congenital erythropoietic porphyria identify a key helix for protein stability
Biochemistry, 2009Co-Authors: Arola Fortian, Ana Lain, David Castano, Gabriel Ortega, Miquel Pons, Oscar MilletAbstract:In the present study we have investigated deleterious mutants in the Uroporphyrinogen III Synthase (UROIIIS) that are related to the congenital erythropoietic porphyria (CEP). The 25 missense mutan...
Hubert De Verneuil - One of the best experts on this subject based on the ideXlab platform.
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Congenital erythropoietic porphyria: mutation update and correlations between genotype and phenotype.
Cellular and molecular biology (Noisy-le-Grand France), 2009Co-Authors: Cécile Ged, François Moreau-gaudry, Emmanuel Richard, Elodie Robert-richard, Hubert De VerneuilAbstract:High quality genotype/phenotype analysis is a difficult issue in rare genetic diseases such as congenital erythropoietic porphyria (CEP) or Gunther's disease, a heme biosynthesis defect due to Uroporphyrinogen III Synthase deficiency. The historical background and the main phenotypic features of the disease are depicted together with an update of published mutants and genotype/phenotype correlations. General rules concerning the prediction of disease severity are drawn as a guide for patient management and therapeutic choices. The phenotypic heterogeneity of the disease is presented in relation with a likely influence of modifying factors, either genetic or acquired.
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lentivirus mediated gene transfer of Uroporphyrinogen III Synthase fully corrects the porphyric phenotype in human cells
Journal of Molecular Medicine, 2003Co-Authors: Fabien Geronimi, Cécile Ged, Emmanuel Richard, Isabelle Lamrissigarcia, Magalie Lalanne, Isabelle Redonnetvernhet, F Moreaugaudry, Hubert De VerneuilAbstract:Congenital erythropoietic porphyria (CEP) is an inherited disease due to a deficiency in the Uroporphyrinogen III Synthase, the fourth enzyme of the heme biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor, with death often occurring early in adult life. For severe transfusion-dependent cases, when allogeneic cell transplantation cannot be performed, the autografting of genetically modified primitive/stem cells may be the only alternative. In vitro gene transfer experiments have documented the feasibility of gene therapy via hematopoietic cells to treat this disease. In the present study lentiviral transduction of porphyric cell lines and primary CD34+ cells with the therapeutic human Uroporphyrinogen III Synthase (UROS) cDNA resulted in both enzymatic and metabolic correction, as demonstrated by the increase in UROS activity and the suppression of porphyrin accumulation in transduced cells. Very high gene transfer efficiency (up to 90%) was achieved in both cell lines and CD34+ cells without any selection. Expression of the transgene remained stable over long-term liquid culture. Furthermore, gene expression was maintained during in vitro erythroid differentiation of CD34+ cells. Therefore the use of lentiviral vectors is promising for the future treatment of CEP patients by gene therapy.
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immunological enzymatic and biochemical studies of Uroporphyrinogen III Synthase deficiency in 20 patients with congenital erythropoietic porphyria
FEBS Journal, 1998Co-Authors: Anne Georga Freesemann, Hubert De Verneuil, M. Bensidhoum, U Gross, M DossAbstract:Congenital erythropoietic porphyria (CEP), a rare autosomal recessive inborn error of heme biosynthesis, results from reduced activity of Uroporphyrinogen III Synthase (URO-III-S) leading to an excessive production and accumulation of porphyrins. Various clinical and biochemical observations point to a relationship between degree of disease expression and metabolic disturbance. We investigated 20 patients with early onset of clinical symptoms of CEP and, additionally, the relatives of six patients. CEP was confirmed by porphyrinemia and porphyrinuria with dominance of uroporphyrin and its isomer I. The investigation of the immunological nature of the defective URO-III-S gene from unrelated patients with unknown mutations was possible thanks to an antibody against the human enzyme. URO-III-S concentration in erythrocytes was determined by ELISA. No signal was achieved when assaying nonimmune serum by ELISA, whereas there was a positive reaction with the serum after immunisation. Furthermore, specificity of immune sera is demonstrated by immunoprecipitation of URO-III-S activity which caused a 33 % reduction of enzyme activity. Normal levels of immunoreactive enzyme protein 100 ± 10 % of control (x¯ ± SD, n = 12) with a reduced specific activity 15 ± 8.5 % (x¯ ± SD, n = 12) were found in erythrocytes from all patients, with the exception of a girl with a remarkably high enzyme concentration of 149 % of controls and a very low specific activity of 4 %. In consequence, all patients had cross-reacting immunological material (CRIM)-positive mutations. CRIM-ratios varied between 3.2 and 24.5. The CRIM-positive nature of the gene defect indicated that the mutations altered the activity of URO-III-S. The different CRIM ratios implied the presence of various mutations, which is further evidence for the heterogeneity in the genetic defect found in CEP. URO-III-S activity was determined in erythrocyte lysates by a coupled enzyme assay. Erythrocyte URO-III-S activities of patients were reduced to 4−33 % of the normal value (1.72 ± 0.14 pkat/mg protein). An increase of urinary coproporphyrin isomer I (40−61 %, norm = 17−31 %) and a halved URO-III-S activity can serve as a biochemical test for asymptomatic heterozygous gene carriers of CEP.
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Immunological, enzymatic and biochemical studies of Uroporphyrinogen III‐Synthase deficiency in 20 patients with congenital erythropoietic porphyria
European journal of biochemistry, 1998Co-Authors: Anne Georga Freesemann, Hubert De Verneuil, M. Bensidhoum, U Gross, Manfred O. DossAbstract:Congenital erythropoietic porphyria (CEP), a rare autosomal recessive inborn error of heme biosynthesis, results from reduced activity of Uroporphyrinogen III Synthase (URO-III-S) leading to an excessive production and accumulation of porphyrins. Various clinical and biochemical observations point to a relationship between degree of disease expression and metabolic disturbance. We investigated 20 patients with early onset of clinical symptoms of CEP and, additionally, the relatives of six patients. CEP was confirmed by porphyrinemia and porphyrinuria with dominance of uroporphyrin and its isomer I. The investigation of the immunological nature of the defective URO-III-S gene from unrelated patients with unknown mutations was possible thanks to an antibody against the human enzyme. URO-III-S concentration in erythrocytes was determined by ELISA. No signal was achieved when assaying nonimmune serum by ELISA, whereas there was a positive reaction with the serum after immunisation. Furthermore, specificity of immune sera is demonstrated by immunoprecipitation of URO-III-S activity which caused a 33 % reduction of enzyme activity. Normal levels of immunoreactive enzyme protein 100 ± 10 % of control (x¯ ± SD, n = 12) with a reduced specific activity 15 ± 8.5 % (x¯ ± SD, n = 12) were found in erythrocytes from all patients, with the exception of a girl with a remarkably high enzyme concentration of 149 % of controls and a very low specific activity of 4 %. In consequence, all patients had cross-reacting immunological material (CRIM)-positive mutations. CRIM-ratios varied between 3.2 and 24.5. The CRIM-positive nature of the gene defect indicated that the mutations altered the activity of URO-III-S. The different CRIM ratios implied the presence of various mutations, which is further evidence for the heterogeneity in the genetic defect found in CEP. URO-III-S activity was determined in erythrocyte lysates by a coupled enzyme assay. Erythrocyte URO-III-S activities of patients were reduced to 4−33 % of the normal value (1.72 ± 0.14 pkat/mg protein). An increase of urinary coproporphyrin isomer I (40−61 %, norm = 17−31 %) and a halved URO-III-S activity can serve as a biochemical test for asymptomatic heterozygous gene carriers of CEP.
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novel point mutation in the Uroporphyrinogen III Synthase gene causes congenital erythropoietic porphyria of a japanese family
American Journal of Medical Genetics, 1997Co-Authors: Noboru Takamura, Hubert De Verneuil, I. Hombrados, Ken Tanigawa, Hiroyuki Namba, Yuji Nagayama, Shunichi YamashitaAbstract:The molecular basis of the Uroporphyrinogen III Synthase (UROIIIS) deficiency was investigated in a member of a Japanese family. This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Gunther's disease. The patient was homozygous for a novel missense mutation: a G to T transition of nucleotide 7 that predicted a valine to phenylalanine substitution at residue 3 (V3F). The parents were heterozygous for the same mutation. The loss of UROIIIS activity was verified by an in vitro assay system. The corresponding mutated protein was expressed in Escherichia coli and no residual activity was observed. Further studies are needed to determine whether the mutations of the UROIIIS gene (UROS) have a specific profile in Japan compared to European or American countries.
Alan R. Battersby - One of the best experts on this subject based on the ideXlab platform.
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biosynthesis of porphyrins and related macrocycles part 44 synthetic and stereochemical studies on the proposed spiro intermediate for biosynthesis of the natural porphyrins
Journal of The Chemical Society-perkin Transactions 1, 1996Co-Authors: Mark A Cassidy, Nigel Crockett, Finian J Leeper, Alan R. BattersbyAbstract:A route is devised for synthesis of both enantiomers of the spiro lactam 4. The enzyme Uroporphyrinogen III Synthase (cosynthetase), which converts hydroxymethylbilane 1 into Uroporphyrinogen III 3, is competitively inhibited more than twenty times more strongly by one enantiomer of 4 than by the other. This finding adds further strong support to the view that cosynthetase acts by generating the spiro pyrrolenine 2 as an intermediate.
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expression purification and characterisation of the product from the bacillus subtilis hemd gene Uroporphyrinogen III Synthase
FEBS Journal, 1995Co-Authors: Patrick N J Stamford, Alfredo Capretta, Alan R. BattersbyAbstract:Uroporphyrinogen III Synthase, the product of the hemD gene, is the enzyme responsible for the cyclisation of the linear tetrapyrrole, hydroxymethylbilane. The hemD gene isolated from Bacillus subtilis was manipulated by PCR to enable direct cloning behind a synthetic ribosome-binding site downstream of tandem bacteriophage λPR and PL promoters in a pCE30-derived vector. Following thermal induction of transcription, the resulting plasmid (pPS21) directed the synthesis of Uroporphyrinogen III Synthase. The protein produced was soluble and was readily purified. Pure Uroporphyrinogen III Synthase is monomeric with an isoelectric point of 4.1 and an optimum pH for activity of 8.3. Its specific activity by assay using synthetic hydroxymethylbilane as substrate is 565 units mg−1 and the Km for this substrate is 330 ± 30 nM. The N -terminal sequence of the enzyme is Met-Glu-Asn-Asp-Phe-Pro-Leu, in agreement with the gene-derived sequence. Studies based on amino acid modifications suggest that arginine, lysine and probably histidine residues are essential for the activity of Uroporphyrinogen III Synthase. Significantly, this Synthase from B. subtilis is substantially more thermostable than the enzymes from previously studied sources.
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stereochemical studies on the proposed spiro intermediate for the biosynthesis of the natural porphyrins determination by a novel x ray method of the absolute configuration of the spirolactam which inhibits cosynthetase
Journal of The Chemical Society Chemical Communications, 1995Co-Authors: Alan C Spivey, Alfredo Capretta, Christopher S Frampton, Finian J Leeper, Alan R. BattersbyAbstract:A novel X-ray analysis, combined with correlations by circular dichroism, has been used to establish the (R)-configuration at the stereocentre in that enantiomer of the spirolactam 4 which strongly inhibits Uroporphyrinogen III Synthase.
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Uroporphyrinogen III Synthase: Studies on its mechanism of action, molecular biology and biochemistry
Tetrahedron, 1991Co-Authors: Nigel Crockett, Peter R. Alefounder, Alan R. Battersby, Chris AbellAbstract:Abstract A review is given of studies on the mechanism of action of Uroporphyrinogen III Synthase (cosynthetase, EC 4.2.1.75) based on synthesis of an inhibitory spiro-lactam. HemD, the Escherichia coli gene coding for Uroporphyrinogen III Synthase has been cloned and overexpressed at levels sixteen fold higher than in wild type E. coli. Evidence for hemD being part of an operon is reviewed. Uroporphyrinogen III Synthase, which has been purified approx. 6000 fold, shows Mr 28000 under denaturing conditions and has a pH optimum of 8.0 ± 0.2. The results from chemical modification of the enzyme point to the presence of arginine and lysine residues at or close to the active site.
Kenneth H. Astrin - One of the best experts on this subject based on the ideXlab platform.
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congenital erythropoietic porphyria identification and expression of eight novel mutations in the Uroporphyrinogen III Synthase gene
British Journal of Haematology, 2002Co-Authors: Amr A Shady, Luis F Cunha, David F Bishop, Kenneth H. Astrin, Brandon R Colby, Robert J DesnickAbstract:Summary. Mutations in the Uroporphyrinogen III Synthase (URO-Synthase) gene cause congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of haem biosynthesis. Molecular analysis of the URO-Synthase gene in seven unrelated CEP patients revealed eight novel mutations. These included four missense mutations (A69T, E81D, G188W and I219S), a deletion (21delG), two insertions (398insG and 672ins28) and one complex mutation (627del6ins39), as well as three previously reported mutations, C73R, T228M, and −86CA. When the four novel missense mutations were expressed in Escherichia coli, only E81D expressed significant enzymatic activity (30% of expressed wild-type activity), which was thermolabile. In addition, reverse transcription polymerase chain reaction studies demonstrated that E81D, which altered the penultimate nucleotide in exon 4, impaired splicing and caused about 85% exon 4 skipping. The identification and expression of these mutations provided genotype–phenotype correlations and further evidence of the molecular heterogeneity underlying this erythropoietic porphyria.
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Uroporphyrinogen III Synthase erythroid promoter mutations in adjacent gata1 and cp2 elements cause congenital erythropoietic porphyria
Journal of Clinical Investigation, 2001Co-Authors: Constanza Solis, Kenneth H. Astrin, David F Bishop, Gerardo I. Aizencang, Robert J DesnickAbstract:Congenital erythropoietic porphyria, an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of Uroporphyrinogen III Synthase. Extensive mutation analyses of 40 unrelated patients only identified approximately 90% of mutant alleles. Sequencing the recently discovered erythroid-specific promoter in six patients with a single undefined allele identified four novel mutations clustered in a 20-bp region: (a) a -70T to C transition in a putative GATA-1 consensus binding element, (b) a -76G to A transition, (c) a -86C to A transversion in three unrelated patients, and (d) a -90C to A transversion in a putative CP2 binding motif. Also, a -224T to C polymorphism was present in approximately 4% of 200 unrelated Caucasian alleles. We inserted these mutant sequences into luciferase reporter constructs. When transfected into K562 erythroid cells, these constructs yielded 3 +/- 1, 54 +/- 3, 43 +/- 6, and 8 +/- 1%, respectively, of the reporter activity conferred by the wild-type promoter. Electrophoretic mobility shift assays indicated that the -70C mutation altered GATA1 binding, whereas the adjacent -76A mutation did not. Similarly, the -90C mutation altered CP2 binding, whereas the -86A mutation did not. Thus, these four pathogenic erythroid promoter mutations impaired erythroid-specific transcription, caused CEP, and identified functionally important GATA1 and CP2 transcriptional binding elements for erythroid-specific heme biosynthesis.
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Uroporphyrinogen III Synthase AN ALTERNATIVE PROMOTER CONTROLS ERYTHROID-SPECIFIC EXPRESSION IN THE MURINE GENE
The Journal of biological chemistry, 2000Co-Authors: Gerardo I. Aizencang, Kenneth H. Astrin, David F Bishop, Douglas Forrest, Robert J DesnickAbstract:Uroporphyrinogen III Synthase (URO-Synthase, EC 4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria. To investigate the erythroid-specific expression of murine URO-Synthase, the cDNA and approximately 24-kilobase genomic sequences were isolated and characterized. Three alternative transcripts were identified containing different 5'-untranslated regions (5'-UTRs), but identical coding exons 2B through 10. Transcripts with 5'-UTR exon 1A alone or fused to exon 1B were ubiquitously expressed (housekeeping), whereas transcripts with 5'-UTR exon 2A were only present in erythroid cells (erythroid-specific). Analysis of the TATA-less housekeeping promoter upstream of exon 1A revealed binding sites for ubiquitously expressed transcription factors Sp1, NF1, AP1, Oct1, and NRF2. The TATA-less erythroid-specific promoter upstream of exon 2A had nine putative GATA1 erythroid enhancer binding sites. Luciferase promoter/reporter constructs transfected into NIH 3T3 and mouse erythroleukemia cells indicated that the housekeeping promoter was active in both cell lines, while the erythroid promoter was active only in erythroid cells. Site-specific mutagenesis of the first GATA1 binding site markedly reduced luciferase activity in K562 cells (
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Uroporphyrinogen III Synthase an alternative promoter controls erythroid specific expression in the murine gene
Journal of Biological Chemistry, 2000Co-Authors: Gerardo I. Aizencang, Kenneth H. Astrin, David F Bishop, Douglas Forrest, Robert J DesnickAbstract:Uroporphyrinogen III Synthase (URO-Synthase, EC 4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria. To investigate the erythroid-specific expression of murine URO-Synthase, the cDNA and approximately 24-kilobase genomic sequences were isolated and characterized. Three alternative transcripts were identified containing different 5'-untranslated regions (5'-UTRs), but identical coding exons 2B through 10. Transcripts with 5'-UTR exon 1A alone or fused to exon 1B were ubiquitously expressed (housekeeping), whereas transcripts with 5'-UTR exon 2A were only present in erythroid cells (erythroid-specific). Analysis of the TATA-less housekeeping promoter upstream of exon 1A revealed binding sites for ubiquitously expressed transcription factors Sp1, NF1, AP1, Oct1, and NRF2. The TATA-less erythroid-specific promoter upstream of exon 2A had nine putative GATA1 erythroid enhancer binding sites. Luciferase promoter/reporter constructs transfected into NIH 3T3 and mouse erythroleukemia cells indicated that the housekeeping promoter was active in both cell lines, while the erythroid promoter was active only in erythroid cells. Site-specific mutagenesis of the first GATA1 binding site markedly reduced luciferase activity in K562 cells (<5% of wild type). Thus, housekeeping and erythroid-specific transcripts are expressed from alternative promoters of a single mouse URO-Synthase gene.
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molecular basis of congenital erythropoietic porphyria mutations in the human Uroporphyrinogen III Synthase gene
Human Mutation, 1996Co-Authors: Kenneth H. Astrin, Robert J DesnickAbstract:Congenital erythropoietic porphyria (CEP) is an autosomal recessive inborn error of metabolism that results from the markedly deficient activity of the fourth enzyme in the heme biosynthetic pathway, Uroporphyrinogen III Synthase (URO-Synthase). To date, 17 mutations have been described including 11 missense, one nonsense, two mRNA splicing defects, one deletion and two coding region insertions. Most mutations have been identified in one or a few unrelated families with the exception of C73R and L4F which occurred in 29.6% and 9.3% of the 54 mutant alleles studied, respectively. Interestingly, analysis of the mutant alleles identified only 83% of the causative mutations, suggesting that about 20% of the mutations causing CEP lie elsewhere in the gene. Of note, mutation V82F, resulting from a G to T transversion of the last nucleotide of exon 4, caused both a missense mutation and an aberrantly spliced RNA transcript. Prokaryotic expression of the mutant URO-Synthase alleles identified those with significant residual activity, thereby permitting genotype/phenotype predictions for this clinically heterogeneous disease.