The Experts below are selected from a list of 999 Experts worldwide ranked by ideXlab platform
Gloria C. Ferreira - One of the best experts on this subject based on the ideXlab platform.
-
Anti-Correlation between the Dynamics of the Active Site Loop and C-Terminal Tail in Relation to the Homodimer Asymmetry of the Mouse Erythroid 5-Aminolevulinate Synthase
MDPI AG, 2018Co-Authors: Dominique Catena, Gloria C. Ferreira, Min J. Kong, Vladimir N. UverskyAbstract:Biosynthesis of heme represents a complex process that involves multiple stages controlled by different enzymes. The first of these proteins is a pyridoxal 5′-phosphate (PLP)-dependent homodimeric enzyme, 5-Aminolevulinate Synthase (ALAS), that catalyzes the rate-limiting step in heme biosynthesis, the condensation of glycine with succinyl-CoA. Genetic mutations in human erythroid-specific ALAS (ALAS2) are associated with two inherited blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA is caused by diminished ALAS2 activity leading to decreased ALA and heme syntheses and ultimately ineffective erythropoiesis, whereas XLPP results from “gain-of-function” ALAS2 mutations and consequent overproduction of protoporphyrin IX and increase in Zn2+-protoporphyrin levels. All XLPP-linked mutations affect the intrinsically disordered C-terminal tail of ALAS2. Our earlier molecular dynamics (MD) simulation-based analysis showed that the activity of ALAS2 could be regulated by the conformational flexibility of the active site loop whose structural features and dynamics could be changed due to mutations. We also revealed that the dynamic behavior of the two protomers of the ALAS2 dimer differed. However, how the structural dynamics of ALAS2 active site loop and C-terminal tail dynamics are related to each other and contribute to the homodimer asymmetry remained unanswered questions. In this study, we used bioinformatics and computational biology tools to evaluate the role(s) of the C-terminal tail dynamics in the structure and conformational dynamics of the murine ALAS2 homodimer active site loop. To assess the structural correlation between these two regions, we analyzed their structural displacements and determined their degree of correlation. Here, we report that the dynamics of ALAS2 active site loop is anti-correlated with the dynamics of the C-terminal tail and that this anti-correlation can represent a molecular basis for the functional and dynamic asymmetry of the ALAS2 homodimer
-
Molecular dynamics analysis of the structural and dynamic properties of the functionally enhanced hepta-variant of mouse 5-Aminolevulinate Synthase.
Journal of biomolecular structure & dynamics, 2017Co-Authors: Shelly Deforte, Gloria C. Ferreira, Bosko M. Stojanovski, Vladimir N. UverskyAbstract:Heme biosynthesis, a complex, multistage, and tightly controlled process, starts with 5-Aminolevulinate (ALA) production, which, in metazoa and certain bacteria, is a reaction catalyzed by 5-Aminolevulinate Synthase (ALAS), a pyridoxal 5′-phosphate (PLP)-dependent enzyme. Functional aberrations in ALAS are associated with several human diseases. ALAS can adopt open and closed conformations, with segmental rearrangements of a C-terminal, 16-amino acid loop and an α-helix regulating accessibility to the ALAS active site. Of the murine erythroid ALAS (mALAS2) forms previously engineered to assess the role of the flexible C-terminal loop versus mALAS2 function one stood out due to its impressive gain in catalytic power. To elucidate how the simultaneously introduced seven mutations of this activity-enhanced variant affected structural and dynamic properties of mALAS2, we conducted extensive molecular dynamics simulation analysis of the dimeric forms of wild-type mALAS2, hepta-variant and Rhodobacter capsulatu...
-
The unfolding pathways of the native and molten globule states of 5-Aminolevulinate Synthase.
Biochemical and biophysical research communications, 2016Co-Authors: Bosko M. Stojanovski, Vladimir N. Uversky, Leonid Breydo, Gloria C. FerreiraAbstract:In this communication, we report the equilibrium and kinetic properties of the unfolding pathways of the native (pH 7.5) and alkaline molten globule (pH 10.5) states of the pyridoxal 5'-phosphate (PLP)-dependent enzyme 5-Aminolevulinate Synthase (ALAS). The stability of the molten globule state is adversely affected by thermal- and guanidine hydrochloride (GuHCl)-induced denaturation, and the equilibrium unfolding pathways, irrespective of pH, cannot be described with simple two-state models. Rapid kinetic measurements, in the presence of denaturing GuHCl concentrations, reveal that at pH 10.5, the rate of ALAS denaturation is 3 times faster than at pH 7.5. From pH jump experiments, comparable rates for the denaturation of the tertiary structure and PLP-microenvironment were discerned, indicating that the catalytic active site geometry strongly depends on the stable tertiary structural organization. Lastly, we demonstrate that partially folded ALAS tends to self-associate into higher oligomeric species at moderate GuHCl concentrations.
-
Expression of Murine 5-Aminolevulinate Synthase Variants Causes Protoporphyrin IX Accumulation and Light-Induced Mammalian
2016Co-Authors: Cell Death, Erica J. Fratz, Gregory A. Hunter, Gloria C. FerreiraAbstract:5-Aminolevulinate Synthase (ALAS; EC 2.3.1.37) catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2) is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX), the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21–22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. The delivery of stable and highl
-
Murine erythroid 5-Aminolevulinate Synthase: Truncation of a disordered N-terminal extension is not detrimental for catalysis.
Biochimica et biophysica acta, 2016Co-Authors: Bosko M. Stojanovski, Vladimir N. Uversky, Leonid Breydo, Gloria C. FerreiraAbstract:5-Aminolevulinate Synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme, catalyzes the initial step of heme biosynthesis in non-plant eukaryotes. The precursor form of the enzyme is translated in the cytosol, and upon mitochondrial import, the N-terminal targeting presequence is proteolytically cleaved to generate mature ALAS. In bone marrow-derived erythroid cells, a mitochondrial- and site-specific endoprotease of yet unknown primary structure, produces a protein shorter than mature erythroid ALAS (ALAS2) found in peripheral blood erythroid cells. This truncated ALAS2 lacks the presequence and the N-terminal sequence (corresponding to ~7 KDa molecular mass) present in ALAS2 from peripheral blood erythroid cells. How the truncation affects the structural topology and catalytic properties of ALAS2 is presently not known. To address this question, we created a recombinant, truncated, murine ALAS2 (ΔmALAS2) devoid of the cleavable N-terminal region and examined its catalytic and biophysical properties. The N-terminal truncation of mALAS2 did not significantly affect the organization of the secondary structure, but a subtle reduction in the rigidity of the tertiary structure was noted. Furthermore, thermal denaturation studies revealed a decrease of 4.3°C in the Tm value of ΔmALAS2, implicating lower thermal stability. While the kcat of ΔmALAS2 is slightly increased over that of the wild-type enzyme, the slowest step in the ΔmALAS2-catalyzed reaction remains dominated by ALA release. Importantly, intrinsic disorder algorithms imply that the N-terminal region of mALAS2 is highly disordered, and thus susceptible to proteolysis. We propose that the N-terminal truncation offers a cell-specific ALAS2 regulatory mechanism without hindering heme synthesis.
Brian K. May - One of the best experts on this subject based on the ideXlab platform.
-
The major splice variant of human 5-Aminolevulinate Synthase-2 contributes significantly to erythroid heme biosynthesis.
The international journal of biochemistry & cell biology, 2004Co-Authors: Timothy C. Cox, Sylvia S. Bottomley, Christopher S Matthews, Timothy J. Sadlon, Quenten Schwarz, Phillip D Wise, Liza L Cox, Brian K. MayAbstract:Abstract The initial step of the heme biosynthetic pathway in erythroid cells is catalyzed by an erythroid-specific isoform of 5-Aminolevulinate Synthase-2 (ALAS2). Previously, an alternatively spliced mRNA isoform of ALAS2 was identified although the functional significance of the encoded protein was unknown. We sought to characterize the contribution of this ALAS2 isoform to overall erythroid heme biosynthesis. Here, we report the identification of three novel ALAS2 mRNA splice isoforms in addition to the previously described isoform lacking exon 4-derived sequence. Quantitation of these mRNAs using ribonuclease protection experiments revealed that the isoform without exon 4-derived sequence represents ∼35–45% of total ALAS2 mRNA while the newly identified transcripts together represent ∼15%. Despite the significant amounts of these three new transcripts, their features indicate that they are unlikely to substantially contribute to overall mitochondrial ALAS2 activity. In contrast, in vitro studies show that the major splice variant (lacking exon 4-encoded sequence) produces a functional enzyme, albeit with slightly reduced activity and with affinity for the ATP-specific, beta subunit of succinyl CoA Synthase, comparable to that of mature ALAS2. It was also established that the first 49 amino acids of the ALAS2 pre-protein are necessary and sufficient for translocation across the mitochondrial inner membrane and that this process is not affected by the absence of exon 4-encoded sequence. We conclude that the major splice isoform of ALAS2 is functional in vivo and could significantly contribute to erythroid heme biosynthesis and hemoglobin formation.
-
Regulation of erythroid 5-Aminolevulinate Synthase expression during erythropoiesis.
The international journal of biochemistry & cell biology, 1999Co-Authors: Timothy J. Sadlon, Tania Dell’oso, Kathy H. Surinya, Brian K. MayAbstract:Erythroid tissue is the major site of heme production in the body. The synthesis of heme and globin chains is coordinated at both the transcriptional and post-transcriptional levels to ensure that virtually no free heme or globin protein accumulates. The key rate-controlling enzyme of the heme biosynthetic pathway is 5-Aminolevulinate Synthase (ALAS) and an erythroid-specific isoform (ALAS2) is up-regulated during erythropoiesis. Differentiation of embryonic stem cells with a disrupted ALAS2 gene has established that expression of this gene is critical for erythropoiesis and cannot be compensated by expression of the ubiquitous isoform of the enzyme (ALAS1). Interestingly, heme appears to be important for expression of globin and other late erythroid genes and for erythroid cell differentiation although the mechanism of this effect is not clear. Transcriptional control elements that regulate the human gene for ALAS2 have been identified both in the promoter and in intronic enhancer regions. Subsequent translation of the ALAS2 mRNA is dependent on an adequate iron supply. The mechanism by which transcription of the gene for ALAS2 is increased by erythropoietin late in erythropoiesis remains an interesting issue. Erythropoietin action may result in altered levels of critical erythroid transcription factors or modulate the phosphorylation/acetylation status of these factors. Defects in the coding region of the gene for ALAS2 underlie the disease state X-linked sideroblastic anemia. In this review, we focus on the regulation and function of erythroid-specific 5-Aminolevulinate Synthase during erythropoiesis and its role in the X-linked sideroblastic anemia.
-
Phenobarbital-Induced Activation ofCYP2H1and 5-Aminolevulinate Synthase Genes in Chick Embryo Hepatocytes Is Blocked by an Inhibitor of Protein Phosphorylation
Archives of biochemistry and biophysics, 1996Co-Authors: Satish C. Dogra, Brian K. MayAbstract:Abstract The phenobarbital-induced activation of cytochrome P4502H1 ( CYP2H1 ) and 5-Aminolevulinate Synthase (ALAS-1) genes in chick embryo hepatocytes occurs at the level of gene transcription, but the molecular mechanism underlying this induction is not understood in detail. In the present study, we report that the protein kinase inhibitor 2-aminopurine markedly inhibits the phenobarbital-induced activation of CYP2H1 and ALAS-1 genes as measured by Northern blot analysis, but does not alter the basal expression of these genes in the absence of drug. Transient expression studies confirmed these findings. The construct pCATBg4.8 contains a 4.8-kb drug-responsive domain of the CYP2H1 gene fused to the enhancerless SV40 promoter and the drug-induced expression of this construct in chick embryo hepatocytes was inhibited by 2-aminopurine. Another construct pCAT, with the first 547 bp of 5′ flanking region of the CYP2H1 gene, is not responsive to drug and basal expression of this construct was not altered by the addition of 2-aminopurine. The evidence presented here demonstrates that the inhibitory action of 2-aminopurine on drug-induction is not due to a toxic effect on the cells. The induction of the CYP2H1 gene by phenobarbital was not altered by treating cells with the specific inhibitors for protein kinase C (GF 109203X and Ro 31-8220) or prolonged exposure to 12-0-tetradecanoyl- phorbol 13-acetate (TPA) or treatment with the specific inhibitors for tyrosine kinase (genistein and tyrphostin A25). Overall, the data indicate that a 2-aminopurine-sensitive protein kinase activity is required for the phenobarbital-induction mechanism but this is unlikely to be a protein kinase C or tyrosine kinase. It can be postulated that phosphorylation of a drug receptor protein may be an important step in the drug-induction process.
-
molecular defects of erythroid 5 aminolevulinate Synthase in x linked sideroblastic anemia
Journal of Bioenergetics and Biomembranes, 1995Co-Authors: Sylvia S. Bottomley, Philip D Cotter, Timothy C. Cox, Brian K. May, David F BishopAbstract: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.
-
x linked pyridoxine responsive sideroblastic anemia due to a thr388 to ser substitution in erythroid 5 aminolevulinate Synthase
The New England Journal of Medicine, 1994Co-Authors: Timothy C. Cox, Michael J. Bawden, Sylvia S. Bottomley, James S Wiley, Christopher S Matthews, Brian K. MayAbstract:Background X-linked sideroblastic anemia is usually associated with reduced 5-Aminolevulinate Synthase activity in erythroid cells, and some cases are responsive to treatment with pyridoxine, the p...
Gregory A. Hunter - One of the best experts on this subject based on the ideXlab platform.
-
Expression of Murine 5-Aminolevulinate Synthase Variants Causes Protoporphyrin IX Accumulation and Light-Induced Mammalian
2016Co-Authors: Cell Death, Erica J. Fratz, Gregory A. Hunter, Gloria C. FerreiraAbstract:5-Aminolevulinate Synthase (ALAS; EC 2.3.1.37) catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2) is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX), the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21–22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. The delivery of stable and highl
-
Human Erythroid 5-Aminolevulinate Synthase Mutations Associated with X-Linked Protoporphyria Disrupt the Conformational Equilibrium and Enhance Product Release.
Biochemistry, 2015Co-Authors: Erica J. Fratz, Gregory A. Hunter, Vladimir N. Uversky, Leonid Breydo, Jerome Clayton, Sarah Ducamp, Jean-charles Deybach, Laurent Gouya, Hervé Puy, Gloria C. FerreiraAbstract:Regulation of 5-Aminolevulinate Synthase (ALAS) is at the origin of balanced heme production in mammals. Mutations in the C-terminal region of human erythroid-specific ALAS (hALAS2) are associated with X-linked protoporphyria (XLPP), a disease characterized by extreme photosensitivity, with elevated blood concentrations of free protoporphyrin IX and zinc protoporphyrin. To investigate the molecular basis for this disease, recombinant hALAS2 and variants of the enzyme harboring the gain-of-function XLPP mutations were constructed, purified, and analyzed kinetically, spectroscopically, and thermodynamically. Enhanced activities of the XLPP variants resulted from increases in the rate at which the product 5-Aminolevulinate (ALA) was released from the enzyme. Circular dichroism spectroscopy revealed that the XLPP mutations altered the microenvironment of the pyridoxal 5′-phosphate cofactor, which underwent further and specific alterations upon succinyl-CoA binding. Transient kinetic analyses of the variant-ca...
-
Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-Aminolevulinate Synthase.
Biochimica et biophysica acta, 2014Co-Authors: Bosko M. Stojanovski, Vladimir N. Uversky, Gregory A. Hunter, Leonid Breydo, Gloria C. FerreiraAbstract:Abstract 5-Aminolevulinate Synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the kcat value is significantly decreased. Finally, the binding of 5-Aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.
-
Expression of Murine 5-Aminolevulinate Synthase Variants Causes Protoporphyrin IX Accumulation and Light-Induced Mammalian Cell Death
PloS one, 2014Co-Authors: Erica J. Fratz, Gregory A. Hunter, Gloria C. FerreiraAbstract:5-Aminolevulinate Synthase (ALAS; EC 2.3.1.37) catalyzes the first committed step of heme biosynthesis in animals. The erythroid-specific ALAS isozyme (ALAS2) is negatively regulated by heme at the level of mitochondrial import and, in its mature form, certain mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX (PPIX), the precursor for heme. Importantly, generation of PPIX is a crucial component in the widely used photodynamic therapies (PDT) of cancer and other dysplasias. ALAS2 variants that cause high levels of PPIX accumulation provide a new means of targeted, and potentially enhanced, photosensitization. In order to assess the prospective utility of ALAS2 variants in PPIX production for PDT, K562 human erythroleukemia cells and HeLa human cervical carcinoma cells were transfected with expression plasmids for ALAS2 variants with greater enzymatic activity than the wild-type enzyme. The levels of accumulated PPIX in ALAS2-expressing cells were analyzed using flow cytometry with fluorescence detection. Further, cells expressing ALAS2 variants were subjected to white light treatments (21–22 kLux) for 10 minutes after which cell viability was determined. Transfection of HeLa cells with expression plasmids for murine ALAS2 variants, specifically for those with mutated mitochondrial presequences and a mutation in the active site loop, caused significant cellular accumulation of PPIX, particularly in the membrane. Light treatments revealed that ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid (ALA) treatment producing a similar amount of PPIX. The delivery of stable and highly active ALAS2 variants has the potential to expand and improve upon current PDT regimes.
-
Functional Asymmetry for the Active Sites of Linked 5-Aminolevulinate Synthase and 8-Amino-7-Oxononanoate Synthase
Archives of Biochemistry and Biophysics, 2011Co-Authors: Tracy D. Turbeville, Junshun Zhang, Gregory A. Hunter, W. Christopher Adams, Gloria C. FerreiraAbstract:5-Aminolevulinate Synthase (ALAS) and 8-amino-7-oxononanoate Synthase (AONS) are homodimeric members of the α-oxoamine Synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. Previously, linking two ALAS subunits into a single polypeptide chain dimer yielded an enzyme (ALAS/ALAS) with a significantly greater turnover number than that of wild-type ALAS. To examine the contribution of each active site to the enzymatic activity of ALAS/ALAS, the catalytic lysine, which also covalently binds the PLP cofactor, was substituted with alanine in one of the active sites. Albeit the chemical rate for the pre-steady-state burst of ALA formation was identical in both active sites of ALAS/ALAS, the k(cat) values of the variants differed significantly (4.4±0.2 vs. 21.6±0.7 min(-1)) depending on which of the two active sites harbored the mutation. We propose that the functional asymmetry for the active sites of ALAS/ALAS stems from linking the enzyme subunits and the introduced intermolecular strain alters the protein conformational flexibility and rates of product release. Moreover, active site functional asymmetry extends to chimeric ALAS/AONS proteins, which while having a different oligomeric state, exhibit different rates of product release from the two ALAS and two AONS active sites due to the created intermolecular strain.
David F Bishop - One of the best experts on this subject based on the ideXlab platform.
-
Molecular Medicine Loss-of-Function Ferrochelatase and Gain-of-Function Erythroid 5-Aminolevulinate Synthase Mutations Causing Erythropoietic Protoporphyria and X-Linked Protoporphyria in North American Patients Reveal Novel Mutations and a High Prev
2013Co-Authors: Manisha Balwani, David F Bishop, Harry A. Dailey, Dana Doheny, Irina Nazarenko, Makiko Yasuda, Karl E. Anderson, Montgomery D. Bissell, Joseph Bloomer, Herbert LAbstract:www.molmed.org 1 Molecular Medicine Erythropoietic Protoporphyria (EPP) and X-Linked Protoporphyria (XLP) are inborn errors of UNCORRECTED PROOF heme biosynthesis with the same phenotype, but resulting from autosomal recessive “loss-offunction” mutations in the ferrochelatase (FECH) gene and “gain-of-function ” mutations in the X-linked erythroid-specific 5-Aminolevulinate Synthase (ALAS2) gene, respectively. The “EPPphenotype” is characterized by acute, painful, cutaneous photosensitivity and elevate
-
a promoter mutation in the erythroid specific 5 aminolevulinate Synthase alas2 gene causes x linked sideroblastic anemia
Blood, 2003Co-Authors: Soumeya Bekri, Philip D Cotter, A I Alsabah, Alison May, Xiaojun Guo, Gillian S Masters, David F BishopAbstract: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.
-
four new mutations in the erythroid specific 5 aminolevulinate Synthase alas2 gene causing x linked sideroblastic anemia increased pyridoxine responsiveness after removal of iron overload by phlebotomy and coinheritance of hereditary hemochromatosis
Blood, 1999Co-Authors: Philip D Cotter, A I Alsabah, Edward J Fitzsimons, Mario Cazzola, Liping Li, David F BishopAbstract:X-linked sideroblastic anemia (XLSA) in four unrelated male probands was caused by missense mutations in the erythroid-specific 5-Aminolevulinate Synthase gene ( ALAS2 ). All were new mutations: T647C, C1283T, G1395A, and C1406T predicting amino acid substitutions Y199H, R411C, R448Q, and R452C. All probands were clinically pyridoxine-responsive. The mutation Y199H was shown to be the first de novo XLSA mutation and occurred in a gamete of the proband’s maternal grandfather. There was a significantly higher frequency of coinheritance of the hereditary hemochromatosis (HH) HFE mutant allele C282Y in 18 unrelated XLSA hemizygotes than found in the normal population, indicating a role for coinheritance of HFE alleles in the expression of this disorder. One proband (Y199H) with severe and early iron loading coinherited HH as a C282Y homozygote. The clinical and hematologic histories of two XLSA probands suggest that iron overload suppresses pyridoxine responsiveness. Notably, reversal of the iron overload in the Y199H proband by phlebotomy resulted in higher hemoglobin concentrations during pyridoxine supplementation. The proband with the R452C mutation was symptom-free on occasional phlebotomy and daily pyridoxine. These studies indicate the value of combined phlebotomy and pyridoxine supplementation in the management of XLSA probands in order to prevent a downward spiral of iron toxicity and refractory anemia.
-
molecular defects of erythroid 5 aminolevulinate Synthase in x linked sideroblastic anemia
Journal of Bioenergetics and Biomembranes, 1995Co-Authors: Sylvia S. Bottomley, Philip D Cotter, Timothy C. Cox, Brian K. May, David F BishopAbstract: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.
Timothy C. Cox - One of the best experts on this subject based on the ideXlab platform.
-
The major splice variant of human 5-Aminolevulinate Synthase-2 contributes significantly to erythroid heme biosynthesis.
The international journal of biochemistry & cell biology, 2004Co-Authors: Timothy C. Cox, Sylvia S. Bottomley, Christopher S Matthews, Timothy J. Sadlon, Quenten Schwarz, Phillip D Wise, Liza L Cox, Brian K. MayAbstract:Abstract The initial step of the heme biosynthetic pathway in erythroid cells is catalyzed by an erythroid-specific isoform of 5-Aminolevulinate Synthase-2 (ALAS2). Previously, an alternatively spliced mRNA isoform of ALAS2 was identified although the functional significance of the encoded protein was unknown. We sought to characterize the contribution of this ALAS2 isoform to overall erythroid heme biosynthesis. Here, we report the identification of three novel ALAS2 mRNA splice isoforms in addition to the previously described isoform lacking exon 4-derived sequence. Quantitation of these mRNAs using ribonuclease protection experiments revealed that the isoform without exon 4-derived sequence represents ∼35–45% of total ALAS2 mRNA while the newly identified transcripts together represent ∼15%. Despite the significant amounts of these three new transcripts, their features indicate that they are unlikely to substantially contribute to overall mitochondrial ALAS2 activity. In contrast, in vitro studies show that the major splice variant (lacking exon 4-encoded sequence) produces a functional enzyme, albeit with slightly reduced activity and with affinity for the ATP-specific, beta subunit of succinyl CoA Synthase, comparable to that of mature ALAS2. It was also established that the first 49 amino acids of the ALAS2 pre-protein are necessary and sufficient for translocation across the mitochondrial inner membrane and that this process is not affected by the absence of exon 4-encoded sequence. We conclude that the major splice isoform of ALAS2 is functional in vivo and could significantly contribute to erythroid heme biosynthesis and hemoglobin formation.
-
molecular defects of erythroid 5 aminolevulinate Synthase in x linked sideroblastic anemia
Journal of Bioenergetics and Biomembranes, 1995Co-Authors: Sylvia S. Bottomley, Philip D Cotter, Timothy C. Cox, Brian K. May, David F BishopAbstract: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.
-
x linked pyridoxine responsive sideroblastic anemia due to a thr388 to ser substitution in erythroid 5 aminolevulinate Synthase
The New England Journal of Medicine, 1994Co-Authors: Timothy C. Cox, Michael J. Bawden, Sylvia S. Bottomley, James S Wiley, Christopher S Matthews, Brian K. MayAbstract:Background X-linked sideroblastic anemia is usually associated with reduced 5-Aminolevulinate Synthase activity in erythroid cells, and some cases are responsive to treatment with pyridoxine, the p...
-
Human erythroid 5-Aminolevulinate Synthase. Gene structure and species-specific differences in alternative RNA splicing.
The Journal of biological chemistry, 1992Co-Authors: J G Conboy, Timothy C. Cox, Michael J. Bawden, S S Bottomley, Brian K. MayAbstract:Abstract Erythroid 5-Aminolevulinate Synthase (ALAS) is expressed exclusively in differentiating erythroid cells as the principal isoform of the enzyme to catalyze the first step of the heme biosynthetic pathway. The human gene encoding this isozyme was isolated from a cosmid library, and its structure was characterized with restriction mapping followed by sequencing of fragments. The gene is 22 kilobases long and has 11 exons. Exon 2 encodes the N-terminal signal sequence required for mitochondrial import, exons 3 and 4 encode a variable portion of the N-terminal end, and exons 5-11 the highly conserved C-terminal portion of the mature protein, respectively. Enzymatic amplification of human reticulocyte RNA using PCR techniques revealed two erythroid ALAS mRNA transcripts predicted to encode both the prototypical 64-kDa isoform as well as a novel smaller isoform with a deletion of 37 amino acids near the N terminus. The two mRNA isoforms are generated by alternative splicing of exon 4 and are expressed in fetal erythroid cells as well as at all stages of erythroid development tested, so that there is no evidence of differentiation-specific regulation of exon 4 splicing. However, striking species-specific differences were observed in that alternative splicing of exon 4 was found in man but not dog or mouse; also, the previously described alternative splicing within exon 3 in mouse was not observed in man. This transcript heterogeneity suggests the existence of erythroid ALAS protein isoforms with potentially distinct functional or regulatory roles. The occurrence of species-specific splicing in the least conserved region of the enzyme may reflect another mechanism of gene evolution in eukaryotes.