Step In Heme Synthesis Research Articles

Preclinical Studies of the GlyT1 Inhibitor Bitopertin in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) results from the haploinsufficiency of 1 of 20 different ribosomal proteins. While DBA patients have hematopoietic stem/progenitor cell defects which broadly impact hematopoiesis, the dominant phenotype is ineffective erythropoiesis and severe anemia. In ~60% of patients, the anemia responds to corticosteroid therapy, leaving an unmet need for safe and effective treatment. In DBA, the synthesis of heme (a chemical chelate) proceeds at a near normal rate while the ribosomal haploinsufficiency leads to slowed translation and delayed globin protein synthesis, resulting in a heme-globin imbalance. DBA patient colony forming unit-erythroid (CFU-E)/proerythroblasts and murine Rpl11 haploinsufficient CFU-E/proerythroblasts show increased heme- and reactive oxygen species (ROS)-mediated cell damage, apoptosis, and ferroptosis. Single cell RNAseq analyses also suggest that excessive heme is a major factor in DBA erythroid marrow failure. When patient marrow cells are cultured in the presence of succinylacetone to reduce heme synthesis or hemopexin to facilitate heme export, erythroid output significantly increases. Succinylacetone competitively inhibits the 2 nd step in heme synthesis, while hemopexin enhances heme export 100-fold and scavenges the extracellular heme. Neither would be a pragmatic and safe therapy for DBA patients. In the first step of heme biosynthesis, glycine and succinyl-CoA combine to form aminolevulinic acid. Erythroid precursors are unable to synthesize enough glycine to support their robust heme synthesis and must import glycine via glycine transporter 1 (GlyT1) (PMID 28495915). Bitopertin is an investigational oral small-molecule inhibitor of GlyT1. In Phase 3 clinical studies of schizophrenia, it was found to be ineffective but exhibited a favorable safety profile (PMID 27816567). To determine whether bitopertin could rebalance heme and globin synthesis, reduce ineffective erythropoiesis, and improve red cell production in DBA, we cultured marrow cells from patients with DBA without and with 10 nM bitopertin (n=3). Following 7 days of culture, bitopertin increased the mean number of cells (21%) and the number of CD71+ erythroid precursors (31%) without impacting erythroid maturation. We next made human cord blood CD34+ deficient for RPS19 (44±2% reduction) with RPS19 shRNA transduction (or control luciferase shRNA) to model RPS19 haploinsufficient DBA. Treatment with bitopertin improved erythroid expansion of RPS19-deficient CD34+ cells 60% (P<0.01) but not control CD34+ cells. Additionally, to assess erythroid differentiation in vitro in the context of an erythroblastic island (EBI) niche, we cultured the CD34+ cell-derived erythroblasts with cord blood-derived macrophages. Erythroid expansion is 7.9±1.3-fold higher (P<0.02) in EBI cultures compared to cultures without macrophages. Treatment of the EBI cultures with bitopertin increased the expansion of RPS19-deficient CD34+ cell-derived erythroblasts 3.9±0.3-fold (P<0.002) but did not increase the expansion of control CD34+ cell-derived erythroblasts. As a final study, Rpl11 haploinsufficient mice were treated with bitopertin (10 to 150 ppm in chow, 2-30 mg/kg/day) for 8 weeks. Bitopertin treatment resulted in a dose-dependent improvement in anemia. After treatment with an optimal dose of bitopertin (100 ppm in chow, 20 mg/kg/day), there was an 8.8% increase in the hemoglobin values, and there was significant improvement in red cell numbers (7.5±1.1 M/µL to 8.9±1.3 M/µL, p=0.005), hematocrit (41.6±4.9% to 45.5±4.2%, p<0.05), and mean corpuscular volume (55.7±3.5 fL to 51.8±4.2 fL, p=0.01). Additionally, bitopertin treatment diminished the developmental block at the CFU-E/proerythroblast stage and reduced erythroblast ROS. The improved in vitro erythroid differentiation of DBA patient marrow and RPS19-deficient CD34+ cells and the improved anemia of RPL11 haploinsufficient mice suggest that bitopertin might mitigate anemia in DBA patients. These encouraging preclinical data supported the initiation of a Phase 1/2 clinical trial (NCT05828108: Young et al, abstract this meeting).

Maintenance of heme homeostasis in Staphylococcus aureus through post-translational regulation of glutamyl-tRNA reductase.

Staphylococcus aureus is an important human pathogen responsible for a variety of infections including skin and soft tissue infections, endocarditis, and sepsis. The combination of increasing antibiotic resistance in this pathogen and the lack of an efficacious vaccine underscores the importance of understanding how S. aureus maintains metabolic homeostasis in a variety of environments, particularly during infection. Within the host, S. aureus must regulate cellular levels of the cofactor heme to support enzymatic activities without encountering heme toxicity. Glutamyl tRNA reductase (GtrR), the enzyme catalyzing the first committed step in heme synthesis, is an important regulatory node of heme synthesis in Bacteria, Archaea, and Plantae. In many organisms, heme status negatively regulates the abundance of GtrR, controlling flux through the heme synthesis pathway. We identified two residues within GtrR, H32 and R214, that are important for GtrR-heme binding. However, in strains expressing either GtrRH32A or GtrRR214A, heme homeostasis was not perturbed, suggesting an alternative mechanism of heme synthesis regulation occurs in S. aureus. In this regard, we report that heme synthesis is regulated through phosphorylation and dephosphorylation of GtrR by the serine/threonine kinase Stk1 and the phosphatase Stp1, respectively. Taken together, these results suggest that the mechanisms governing staphylococcal heme synthesis integrate both the availability of heme and the growth status of the cell. IMPORTANCE Staphylococcus aureus represents a significant threat to human health. Heme is an iron-containing enzymatic cofactor that can be toxic at elevated levels. During infection, S. aureus must control heme levels to replicate and survive within the hostile host environment. We identified residues within a heme biosynthetic enzyme that are critical for heme binding in vitro; however, abrogation of heme binding is not sufficient to perturb heme homeostasis within S. aureus. This marks a divergence from previously reported mechanisms of heme-dependent regulation of the highly conserved enzyme glutamyl tRNA reductase (GtrR). Additionally, we link cell growth arrest to the modulation of heme levels through the post-translational regulation of GtrR by the kinase Stk1 and the phosphatase Stp1.

FC2 stabilizes POR and suppresses ALA formation in the tetrapyrrole biosynthesis pathway.

During photoperiodic growth, the light-dependent nature of chlorophyll synthesis in angiosperms necessitates robust control of the production of 5-aminolevulinic acid (ALA), the rate-limiting step in the initial stage of tetrapyrrole biosynthesis (TBS). We are interested in dissecting the post-translational control of this process, which suppresses ALA synthesis for chlorophyll synthesis in dark-grown plants. Using biochemical approaches for analysis of Arabidopsis wild-type (WT) and mutant lines as well as complementation lines, we show that the heme-synthesizing ferrochelatase 2 (FC2) interacts with protochlorophyllide oxidoreductase and the regulator FLU which both promote the feedback-controlled suppression of ALA synthesis by inactivation of glutamyl-tRNA reductase, thus preventing excessive accumulation of potentially deleterious tetrapyrrole intermediates. Thereby, FC2 stabilizes POR by physical interaction. When the interaction between FC2 and POR is perturbed, suppression of ALA synthesis is attenuated and photoreactive protochlorophyllide accumulates. FC2 is anchored in the thylakoid membrane via its membrane-spanning CAB (chlorophyll-a-binding) domain. FC2 is one of the two isoforms of ferrochelatase catalyzing the last step of heme synthesis. Although FC2 belongs to the heme-synthesizing branch of TBS, its interaction with POR potentiates the effects of the GluTR-inactivation complex on the chlorophyll-synthesizing branch and ensures reciprocal control of chlorophyll and heme synthesis.

The immunometabolite itaconate inhibits heme synthesis and remodels cellular metabolism in erythroid precursors

AbstractAs part of the inflammatory response by macrophages, Irg1 is induced, resulting in millimolar quantities of itaconate being produced. This immunometabolite remodels the macrophage metabolome and acts as an antimicrobial agent when excreted. Itaconate is not synthesized within the erythron but instead may be acquired from central macrophages within the erythroid island. Previously, we reported that itaconate inhibits hemoglobinization of developing erythroid cells. Herein we show that this action is accomplished by inhibition of tetrapyrrole synthesis. In differentiating erythroid precursors, cellular heme and protoporphyrin IX synthesis are reduced by itaconate at an early step in the pathway. In addition, itaconate causes global alterations in cellular metabolite pools, resulting in elevated levels of succinate, 2-hydroxyglutarate, pyruvate, glyoxylate, and intermediates of glycolytic shunts. Itaconate taken up by the developing erythron can be converted to itaconyl–coenzyme A (CoA) by the enzyme succinyl-CoA:glutarate-CoA transferase. Propionyl-CoA, propionyl-carnitine, methylmalonic acid, heptadecanoic acid, and nonanoic acid, as well as the aliphatic amino acids threonine, valine, methionine, and isoleucine, are increased, likely due to the impact of endogenous itaconyl-CoA synthesis. We further show that itaconyl-CoA is a competitive inhibitor of the erythroid-specific 5-aminolevulinate synthase (ALAS2), the first and rate-limiting step in heme synthesis. These findings strongly support our hypothesis that the inhibition of heme synthesis observed in chronic inflammation is mediated not only by iron limitation but also by limitation of tetrapyrrole synthesis at the point of ALAS2 catalysis by itaconate. Thus, we propose that macrophage-derived itaconate promotes anemia during an inflammatory response in the erythroid compartment.

Increased Heme Synthesis in Ovarian Cancer and Triple Negative Breast Cancer Cells Leads to Downregulation of Glycolysis, Mitochondrial Respiration, and Cell Migration.

Cancer cells undergo a complex rearrangement of metabolic pathways involved in biosynthetic processes to support tumor initiation and progression. Targeting glycolysis and TCA cycle that provide precursors for biosynthetic pathways emerged as a potential therapeutic strategy in some cancers. Heme has a potential to regulate both glycolysis and TCA cycle. Heme serves as a prosthetic group in proteins and enzymes that sense, transport, or use oxygen, such as hemoglobin, myoglobin, cytochrome complexes, catalases, and cyclooxygenases. Heme is also required for ATP production by the mitochondrial electron transport chain (ETC) and oxidative phosphorylation (OXPHOS). The first and rate‐limiting step in heme synthesis is production of 5‐aminolevulinic acid (ALA). In this study, we show that promoting heme synthesis by ALA supplementation in ovarian cancer and triple negative breast cancer cells leads to downregulation of glycolysis and lactate production, mitochondrial respiration, as well as cell migration. The mechanism responsible for reduced glycolysis and cell migration involves destabilization of heme‐regulated transcription factor Bach1.Support or Funding InformationSupported by NIH GM120710

A Dominant Mutation in Mitochondrial Unfoldase CLPX Results in Erythropoietic Protoporphyria

▪Red cells synthesize large quantities of heme during terminal differentiation. Central to erythropoiesis is heme synthesis, which requires tight coordination between mitochondrial iron import and synthesis of protoporphyrin IX (PPIX).Most individuals with erythropoietic porphyria carry loss of function mutations in FECH, or gain of function mutations in ALAS2, resulting in protoporphyrin IX accumulation. We performed whole exome sequencing to identify novel mutations in an individual exhibiting symptoms in whom FECH and ALAS2 mutations were absent (Fig. A, asterisk). We identified a novel CLPX point mutation in this individual (III.2), her father (II.4) and her paternal uncle (II.2), who also exhibited increased porphyrin levels relative to healthy individuals. The individual's mother was healthy and had a wild-type CLPX genotype (Fig. A). CLPX encodes a mitochondrial protein unfoldase that partially unfolds ALA synthase (ALAS) to allow efficient incorporation of its cofactor, pyridoxal phosphate (Kardon et al. 2015 Cell). This greatly stimulates the synthesis of d-aminolevulinic acid (ALA), the first step in heme biosynthesis.To determine if the CLPX mutation was causative for porphyria, we expressed mutant CLPX in HEK293T embryonic kidney cells and Friend mouse erythroleukemia (MEL) cells. Mutant CLPX expression (MUT) caused a significant increase in ALAS1 (non-erythroid isoform) (Fig. B) and ALAS2 (erythroid isoform) (Fig. C) activity relative to control and wild-type CLPX expressing samples (WT). This increase in ALAS enzymatic activity translated to an increase in PPIX levels (Fig. D, E), consistent with the porphyria phenotype observed in the individuals in this study.We observed that MUT-expressing samples had increased levels of ALAS1 and ALAS2. To determine if mutant CLPX altered ALAS protein stability, we transfected WT or MUT CLPX into HEK293T and MEL cells. Cells were treated with cycloheximide to block translation. We quantitated degradation rate of ALAS by western blot analysis of cell lysates obtained at several time points after cycloheximide treatment (chase). Expression of MUT CLPX stabilized both ALAS1 and ALAS2, accounting for the increase in ALAS protein levels, activity and downstream production of PPIX.The control of ALAS enzymatic activity and protein stability by CLPX unveils a novel cause of protoporphyria and insights revealing the ways in which mitochondrial physiology and heme synthesis are interdependent. Our results reveal an important regulatory node where the mitochondrial protein quality control machinery intersects with a key step in heme synthesis and provides an important genetic tool for understanding the pathology of porphyrias. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The mutual control of iron and erythropoiesis.

Iron is essential for hemoglobin synthesis during terminal erythropoiesis. To supply adequate iron the carrier transferrin is required together with transferrin receptor endosomal cycle and normal mitochondrial iron utilization. Iron and iron protein deficiencies result in different types of anemia. Iron-deficiency anemia is the commonest anemia worldwide due to increased requirements, malnutrition, chronic blood losses and malabsorption. Mutations of transferrin, transferrin receptor cycle proteins, enzymes of the first step of heme synthesis and iron sulfur cluster biogenesis lead to rare anemias, usually accompanied by iron overload. Hepcidin plays an indirect role in erythropoiesis by controlling plasma iron. Inappropriately high hepcidin levels characterize the rare genetic iron-refractory iron-deficiency anemia (IRIDA) and the common anemia of chronic disease. Iron modulates both effective and ineffective erythropoiesis: iron restriction reduces heme and alpha-globin synthesis that may be of benefit in thalassemia. This review relies on the analysis of the most recent literature and personal data. Erythropoiesis controls iron homeostasis, by releasing erythroferrone that inhibits hepcidin transcription to increase iron acquisition in iron deficiency, hypoxia and EPO treatment. Erythroferrone, produced by EPO-stimulated erythropoiesis, inhibits hepcidin only when the activity of BMP/SMAD pathway is low, suggesting that EPO somehow modulates the latter signaling. Erythroblasts sense circulating iron through the second transferrin receptor (TFR2) that, in animal models, modulates the sensitivity of the erythroid cells to EPO. The advanced knowledge of the regulation of systemic iron homeostasis and erythropoiesis-mediated hepcidin regulation is leading to the development of targeted therapies for anemias and iron disorders.

Truncated Human ATP-Specific Succinyl-CoA Synthetase

Two isoforms of the heterodimeric enzyme succinyl-CoA synthetase (SCS) exist in the mitochondria of humans. One is specific for ATP, while the other is specific for GTP. Both catalyze the reversible reaction: succinate + CoA + NTP ⇌ succinyl-CoA + NDP + Pi, where N denotes adenosine or guanosine. SCS is best known as an enzyme of the citric acid cycle where the reaction generates NTP. In the reverse direction, SCS replenishes succinyl-CoA required for the catabolism of ketone bodies and for heme synthesis. Nucleotide-specific forms are thought to be required for SCS to serve its different metabolic roles. The nucleotide specificity lies in the β-subunit [1], and the β-subunit of human ATP-specific SCS has been shown to interact with the C-terminus of erythroid-specific aminolevulinic acid synthase (ALAS2) [2]. ALAS2 catalyzes the committed step in heme synthesis: succinyl-CoA + Gly ⇌ 5-aminolevulinate + CoA + CO2. An interaction between SCS and ALAS2 makes biological sense, since this could provide channeling of succinyl-CoA from SCS to ALAS2. We hypothesize that the interaction is with the carboxy-terminus of the β-subunit of ATP-specific SCS because sequence comparisons show that the β-subunit of ATP-specific SCS has a carboxy-terminal extension when compared to other SCSs' β-subunits. To test this hypothesis, we added a carboxy-terminal His8-tag to the α-subunit of human ATP-specific SCS and mutated the codon for Thr 396β to a stop codon. This truncated version of human ATP-specific SCS has been produced in E. coli and purified. As well as testing to see if truncated human ATP-specific SCS interacts with ALAS2, we are using the truncated version in crystallization trials. Crystals of full-length human ATP-specific SCS diffract to only 3.2 Å and our goal is to obtain better-diffracting crystals of the complex of ATP with truncated human ATP-specific SCS.

Abstract 161: ABCB10 Plays a Role in Heme Synthesis and Mitochondrial δ-Aminolevulinic Acid Export

Introduction: Heme is essential for cardiovascular health, as it is a component of myoglobin, cytochrome P450, electron transport chain complexes, and antioxidant enzymes. The eight steps of heme synthesis are well characterized; however, it is not known how the synthetic intermediates are transported between mitochondria and cytosol. We hypothesized that the mitochondrial ATP-binding cassette protein-B10 (ABCB10) plays a role in heme synthesis by mediating the export of δ-aminolevulinic acid (ALA) out of the mitochondria. Results: Lentiviral shRNA-based knockdown of ABCB10 in H9c2 cardiac myoblasts significantly reduced cytosolic and mitochondrial heme levels and the activities of heme-containing proteins (catalase, peroxidase and complex IV), while their mRNA levels remained unchanged. The observed heme deficiency was not due to a defect in heme synthesis, iron incorporation, or enhanced heme degradation, as ABCB10 shRNA had no effect on the expression of enzymes mediating these steps. We then tested whether ABCB10 regulates mitochondrial export of ALA. Addition of exogenous ALA completely reversed the reduction in heme levels and normalized the activities of heme containing enzymes in cells with ABCB10 knockdown. However, overexpression of ALA synthase 1 (ALAS1), an enzyme catalyzing the production of ALA in the mitochondria, resulted in increased heme levels at baseline, but failed to restore heme content in ABCB10 shRNA-treated cells. To examine the role of ABCB10 in cardiac disease, we measured ABCB10 expression in the hearts of mice subjected to myocardial infarction and in explanted human hearts with ischemic cardiomyopathy. ABCB10 protein was significantly elevated in mouse and human hearts, and adenoviral overexpression of ABCB10 in H9c2 cardiac myoblasts significantly attenuated the production of reactive oxygen species (ROS), suggesting the protective role for ABCB10 in heart disease. Conclusions: Our studies place ABCB10 between the mitochondrial synthesis of ALA by ALAS1 and the cytosolic conversion of ALA into heme, suggesting that ABCB10 plays a role ALA export out of the mitochondria. Furthermore, its overexpression exerts protective effects against oxidant induced cell death in cardiac cells.

Nitrogen Monoxide Inhibits Heme Synthesis In Reticulocytes

AbstractAbstract 4252Anemia of chronic disease (ACD) is a condition that often manifests in patients with chronic immune activation due to chronic infections, autoimmune disorders, cancer and other diseases. The pathogenesis of ACD is complex and involves inefficient erythropoietin production, immune-mediated inhibition of erythropoiesis, and retention of iron in hemoglobin-processing macrophages. During their development, erythroid cells are closely associated with macrophages. In inflammatory conditions, activated macrophages generate large quantities of the gaseous molecule, nitric oxide (NO), which has numerous effects on iron metabolism. In this study, we explored the possibility that NO affects iron metabolism in erythroid cells. We treated reticulocytes with the NO donors, sodium nitroprusside (SNP) and S-Nitroso-N-acetyl-D,L-penicillamine (SNAP). We show that NO inhibits 59Fe incorporation from 59Fe-transferrin into reticulocytes and their heme. Significantly, 5-aminolevulinic acid (ALA, the product of ALA synthase, which catalyzes the first step of heme synthesis) reversed the SNP-mediated decrease in 59Fe incorporation into heme but not the cellular 59Fe uptake. In addition, SNP treatment led to an increase in eIF2α phosphorylation (which is known to occur in heme-deficient cells) and decreased globin translation. Importantly, the addition of ALA to SNP-treated reticulocytes prevented the effect of SNP on eIF2α phosphorylation and reversed globin synthesis inhibition. This indicates that in SNP treated reticulocytes, the phosphorylation of eIF2α and inhibition of globin synthesis occur indirectly, via NO's effect on erythroid-specific ALA synthase (ALA-S2). These results led us to conclude that NO has two distinct effects on reticulocytes, namely: a decrease in ALA-S2 activity and a decrease in transferrin-mediated iron uptake. The profound impact of NO on heme synthesis, iron uptake and globin translation in reticulocytes raises the possibility that NO production by macrophages could also contribute to ACD. Disclosures:No relevant conflicts of interest to declare.

Erythropoiesis and Iron Sulfur Cluster Biogenesis

Erythropoiesis in animals is a synchronized process of erythroid cell differentiation that depends on successful acquisition of iron. Heme synthesis depends on iron through its dependence on iron sulfur (Fe-S) cluster biogenesis. Here, we review the relationship between Fe-S biogenesis and heme synthesis in erythropoiesis, with emphasis on the proteins, GLRX5, ABCB7, ISCA, and C1orf69. These Fe-S biosynthesis proteins are highly expressed in erythroid tissues, and deficiency of each of these proteins has been shown to cause anemia in zebrafish model. GLRX5 is involved in the production and ABCB7 in the export of an unknown factor that may function as a gauge of mitochondrial iron status, which may indirectly modulate activity of iron regulatory proteins (IRPs). ALAS2, the enzyme catalyzing the first step in heme synthesis, is translationally controlled by IRPs. GLRX5 may also provide Fe-S cofactor for ferrochelatase, the last enzyme in heme synthesis. ISCA and C1orf69 are thought to assemble Fe-S clusters for mitochondrial aconitase and for lipoate synthase, the enzyme producing lipoate for pyruvate dehydrogenase complex (PDC). PDC and aconitase are involved in the production of succinyl-CoA, a substrate for heme biosynthesis. Thus, many steps of heme synthesis depend on Fe-S cluster assembly.

Deficiency of MnSOD in Hematopoietic Stem Cells Causes a Sideroblastic Anemia-Like Phenotype.

Abstract 1991Poster Board I-1013 Introduction:Sideroblastic anemia is caused by an inappropriate incorporation of iron into hemoglobin, and as such a phenotype demonstrating ringed sideroblasts, improper iron deposition, and malignancy is observed. The only known hereditary cause of this disease is a mutation in delta-aminolevulinate synthase 2 (ALAS2), which is a mitochondrially located enzyme that catalyzes the first step in heme synthesis, while the causes of the acquired or idiopathic forms remain unknown. With the knowledge that the free radical superoxide (O2•-) is able to displace iron from heme-containing proteins, it is hypothesized that the loss of the mitochondrial anti-oxidant enzyme manganese superoxide dismutase (MnSOD), encoded by the SOD2 gene, may cause disruption of proper heme synthesis and as such may contribute to a sideroblastic anemia-like phenotype. Methods: Due to the early lethality of the constitutive MnSOD knock-out mouse, a floxed (flanked by loxP) MnSOD mouse was created. Homozygous hematopoietic stem cell specific SOD2 knock-outs were generated by breeding the floxed MnSOD mice with mice expressing Cre-recombinase under the control of the hematopoietic stem cell specific promoter Vav. Results: Mice harboring the hematopoietic knock-out of MnSOD are born viable, and are able to live to maturity. Complete blood count (CBC) analysis of knock-out mice demonstrated a decrease in red blood cells (RBC), thymic white blood cells, and splenic white blood cells. The knock-outs additionally showed a decrease in hemoglobin to 5 g/dL compared to 11 g/dL in controls, a reduction in hematocrit to 32% versus 43.5% in controls, and a reticulocyte count of 31.6% compared to only 7.9% in controls. CBC analysis also revealed many RBC morphology flags including hypochromasia, ansiocytosis, and hematocrit variance. These findings were visualized in blood smears (see below). Finally, electron paramagnetic resonance spectrometry of plasma from the knock-out mice showed a ∼2-fold decrease in transferrin levels compared to controls. Conclusions: The loss of MnSOD in the hematopoietic stem cell population of a mouse creates a viable organism, but with an apparent sideroblastic anemia phenotype. Additionally, the white blood cells within these organisms also may be affected due to the decrease in total cellularity, and thus could contribute to immunodeficiency and/or malignancy later in the animal's life. Overall, this phenotype is very comparable to those observed in human patients, and as such MnSOD may play an undiscovered role in acquired or idiopathic sideroblastic anemia. Future work will focus on further characterization of the animals, examination of human blood samples for MnSOD mutations, and the use of SOD mimetics to potentially reverse the observed phenotype. This work was supported by NIH grants CA73612 and CA115438 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Trafficking of heme and porphyrins in metazoa.

Trafficking of heme and porphyrins in metazoa.

Ethnicity affects the distribution of δ-aminolevulinic acid dehydratase (ALAD) genetic variants

Background δ-Aminolevulinic acid dehydratase (ALAD) catalyzes the second step of heme synthesis. The ALAD gene shows a polymorphism (G-to-C transversion at position 177) leading to 2 alleles (ALAD1 and ALAD2) and 3 phenotypes (ALAD 1-1, ALAD 1-2 and ALAD 2-2). This polymorphism has been shown to affect lead toxicity and the risk of meningioma. In addition, there is little evidence showing interethnic differences in the distribution this polymorphism, especially in heterogeneous populations such as the present-day Brazilian population. We examined the distribution of genetic variants of the G177C ALAD polymorphism in black and white Brazilians. Methods We studied 115 subjects self-reported as black and 119 subjects as white (total N = 234; 135 men and 99 women; age range: 18–60 years). Genomic DNA was extracted from venous blood and the genotypes for the ALAD polymorphism were determined by PCR followed by RFLP digestion and gel electrophoresis. Results We found a notable interethnic disparity in the distribution of G177C ALAD genotypes and alleles. The ALAD2 allele was more common in whites (12%) than in blacks (4%) ( P < 0.05). Correspondingly, the heterozygote (ALAD 1-2) or homozygote variant (ALAD 2-2) genotypes for this polymorphism were more common in whites than in blacks ( P < 0.05). Conclusions The significant interethnic differences in the distribution of G177C ALAD variants found in the Brazilian population is consistent with differences previously reported in other countries. These findings may help us understand the interethnic disparities in susceptibility to lead toxicity and brain tumors.

δ-Aminolevulinic Acid Dehydratase Polymorphism and Risk of Brain Tumors in Adults

The enzyme δ -aminolevulinic acid dehydratase (ALAD), which catalyzes the second step of heme synthesis, can be inhibited by several chemicals, including lead, a potential risk factor for brain tumors, particularly meningioma. In this study we examined whether the ALAD G177C polymorphism in the gene coding for ALAD is associated with risk of intracranial tumors of the brain and nervous system. We use data from a case–control study with 782 incident brain tumor cases and 799 controls frequency matched on hospital, age, sex, race/ethnicity, and residential proximity to the hospital. Blood samples were drawn and DNA subsequently sent for genotyping for 73% of subjects. ALAD genotype was determined for 94% of these samples (355 glioma, 151 meningioma, 67 acoustic neuroma, and 505 controls). Having one or more copy of the ALAD2 allele was associated with increased risk for meningioma [odds ratio (OR) = 1.6; 95% confidence interval (CI), 1.0–2.6], with the association appearing stronger in males (OR = 3.5; 95% CI, 1.3–9.2) than in females (OR = 1.2; 95% CI, 0.7–2.2). No increased risk associated with the ALAD2 variant was observed for glioma or acoustic neuroma. These findings suggest that the ALAD2 allele may increase genetic susceptibility to meningioma.

A Mechanism of Oxygen Sensing in Yeast: MULTIPLE OXYGEN-RESPONSIVE STEPS IN THE HEME BIOSYNTHETIC PATHWAY AFFECT Hap1 ACTIVITY

Heme plays central roles in oxygen sensing and utilization in many living organisms. In yeast, heme mediates the effect of oxygen on the expression of many genes involved in using or detoxifying oxygen. However, a direct link between intracellular heme level and oxygen concentration has not been vigorously established. In this report, we have examined the relationships among oxygen levels, heme levels, Hap1 activity, and HAP1 expression. We found that Hap1 activity is controlled in vivo by heme and not by its precursors and that heme activates Hap1 even in anoxic cells. We also found that Hap1 activity exhibits the same oxygen dose-response curves as Hap1-dependent aerobic genes and that these dose-response curves have a sharp break at approximately 1 microM O2. The results show that the intracellular signaling heme level, reflected as Hap1 activity, is closely correlated with oxygen concentration. Furthermore, we found that bypass of all heme synthetic steps but ferrochelatase by deuteroporphyrin IX does not circumvent the need for oxygen in Hap1 full activation by heme, suggesting that the last step of heme synthesis, catalyzed by ferrochelatase, is also subjected to oxygen control. Our results show that multiple heme synthetic steps can sense oxygen concentration and provide significant insights into the mechanism of oxygen sensing in yeast.

Delta-aminolevulinic acid dehydratase genotype and lead toxicity: a HuGE review.

The ALAD gene (chromosome 9q34) codes for delta-aminolevulinic acid dehydratase (ALAD) (E.C. 4.2.1.24). ALAD catalyzes the second step of heme synthesis and is polymorphic. The ALAD G177C polymorphism yields two codominant alleles, ALAD-1 and ALAD-2, and it has been implicated in susceptibility to lead toxicity. Genotype frequencies vary by geography and race. The rarer ALAD-2 allele has been associated with high blood lead levels and has been thought to increase the risk of lead toxicity by generating a protein that binds lead more tightly than the ALAD-1 protein. Other evidence suggests that ALAD-2 may confer resistance to the harmful effects of lead by sequestering lead, making it unavailable for pathophysiologic participation. Recent studies have shown that individuals who are homozygous for the ALAD-1 allele have higher cortical bone lead levels; this implies that they may have a greater body lead burden and may be at higher risk of the long-term effects of lead. Individuals exposed to lead in occupational settings have been the most frequent subjects of study. Genotype selection bias may limit inferences from these studies. No firm evidence exists for an association between ALAD genotype and susceptibility to lead toxicity at background exposure levels; therefore, population testing for the ALAD polymorphism is not justified.

Inhibition of hemoglobin expression by heterologous production of nitric oxide synthase in the K562 erythroleukemic cell line

Recent studies have indicated that nitric oxide may affect iron metabolism through disruption of the iron-sulfur complex of iron regulatory protein-1, a translational regulator. In the present study, we report that heterologous expression of murine macrophage nitric oxide synthase (NOS-2) in the human erythroleukemic K562 cell line results in constitutive production of nitric oxide associated with inhibition of hemoglobin expression. K562 cells were transfected with an episomally-maintained, hygromycin-selectable expression vector bearing the coding region of NOS-2. Constitutive NOS expression was detected by Western blotting of cell lysates and by the accumulation of nitrite in the culture media. Although NOS-transfected cells grew more slowly than control cells, they were able to maintain constitutive expression of NOS and production of nitric oxide for more than 1 month following transfection. The hemoglobin content of NOS-transfected K562 cells was less than one-fifth that of control cells, but increased markedly if NOS inhibitor was included in the culture media. The nitric oxide-mediated inhibition of hemoglobin expression was reversed by supplementing the culture media with 20 mumol/L hemin or 0.5 mmol/L 5- amino-levulinate, indicating that nitric oxide did not directly inhibit hemoglobin synthesis, but likely acted on a step in heme synthesis. mRNA levels for globin and erythroid aminolevulinic acid synthase (eALAS) were the same in both NOS-transfected and control cells. Our observations indicate that hemoglobin expression is inhibited by nitric oxide in NOS-transfected K562 cells by posttranscriptional repression of eALAS, the first enzyme of the heme biosynthetic pathway. The most likely mechanism is a nitric oxide-mediated translational repression of eALAS, as was recently demonstrated for ferritin synthesis. These observations further illustrate the potential for endogenously produced nitric oxide to regulate cellular posttranscriptional events. In particular, our observations may be relevant to the role of nitric oxide in anemia and lowered blood hemoglobin concentrations that are associated with chronic infections, such as tuberculosis or parasitic disease.

Inhibition of Hemoglobin Expression by Heterologous Production of Nitric Oxide Synthase in the K562 Erythroleukemic Cell Line

Recent studies have indicated that nitric oxide may affect iron metabolism through disruption of the iron-sulfur complex of iron regulatory protein-1, a translational regulator. In the present study, we report that heterologous expression of murine macrophage nitric oxide synthase (NOS-2) in the human erythroleukemic K562 cell line results in constitutive production of nitric oxide associated with inhibition of hemoglobin expression. K562 cells were transfected with an episomally-maintained, hygromycin-selectable expression vector bearing the coding region of NOS-2. Constitutive NOS expression was detected by Western blotting of cell lysates and by the accumulation of nitrite in the culture media. Although NOS-transfected cells grew more slowly than control cells, they were able to maintain constitutive expression of NOS and production of nitric oxide for more than 1 month following transfection. The hemoglobin content of NOS-transfected K562 cells was less than one-fifth that of control cells, but increased markedly if NOS inhibitor was included in the culture media. The nitric oxide-mediated inhibition of hemoglobin expression was reversed by supplementing the culture media with 20 mumol/L hemin or 0.5 mmol/L 5-amino-levulinate, indicating that nitric oxide did not directly inhibit hemoglobin synthesis, but likely acted on a step in heme synthesis. mRNA levels for globin and erythroid aminolevulinic acid synthase (eALAS) were the same in both NOS-transfected and control cells. Our observations indicate that hemoglobin expression is inhibited by nitric oxide in NOS-transfected K562 cells by posttranscriptional repression of eALAS, the first enzyme of the heme biosynthetic pathway. The most likely mechanism is a nitric oxide-mediated translational repression of eALAS, as was recently demonstrated for ferritin synthesis. These observations further illustrate the potential for endogenously produced nitric oxide to regulate cellular posttranscriptional events. In particular, our observations may be relevant to the role of nitric oxide in anemia and lowered blood hemoglobin concentrations that are associated with chronic infections, such as tuberculosis or parasitic disease.

Nucleotide sequence of the Rhodobacter capsulatus hemH gene

The last step in heme synthesis is the insertion of iron into the ring of protoporphyrin IX. The enzyme which catalyzes this reaction, ferrochelatase (FC), is encoded by the hemH gene. A clone containing this gene from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium, has been sequenced. A single open reading frame was found which could encode a protein of 351 amino acids. This putative protein is very similar to other FC and contains the FC signature sequence