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Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in CLPX inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism. [ABSTRACT FROM AUTHOR]

Heme is an oxygen carrier and a cofactor of both industrial enzymes and food additives. The intracellular level of free heme is low, which limits the synthesis of heme proteins. Therefore, increasing heme synthesis allows an increased production of heme proteins. Using the genome-scale metabolic model (GEM) Yeast8 for the yeast Saccharomyces cerevisiae , we identified fluxes potentially important to heme synthesis. With this model, in silico simulations highlighted 84 gene targets for balancing biomass and increasing heme production. Of those identified, 76 genes were individually deleted or overexpressed in experiments. Empirically, 40 genes individually increased heme production (up to threefold). Heme was increased by modifying target genes, which not only included the genes involved in heme biosynthesis, but also those involved in glycolysis, pyruvate, Fe-S clusters, glycine, and succinyl-coenzyme A (CoA) metabolism. Next, we developed an algorithmic method for predicting an optimal combination of these genes by using the enzyme-constrained extension of the Yeast8 model, ecYeast8. The computationally identified combination for enhanced heme production was evaluated using the heme ligand-binding biosensor (Heme-LBB). The positive targets were combined using CRISPR-Cas9 in the yeast strain (IMX581- HEM15-HEM14-HEM3-Δshm1-HEM2-Δhmx1-FET4-Δgcv2-HEM1-Δgcv1-HEM13 ), which produces 70-fold-higher levels of intracellular heme.

Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Human ferrochelatase is a homodimeric, inner mitochondrial membrane-associated enzyme that possesses an essential [2Fe-2S] cluster. In this work, we report the crystal structure of human ferrochelatase with the substrate protoporphyrin IX bound as well as a higher resolution structure of the R115L variant without bound substrate. The data presented reveal that the porphyrin substrate is bound deep within an enclosed pocket. When compared with the location of N-methylmesoporphyrin in the Bacillus subtilis ferrochelatase, the porphyrin is rotated by ≈100° and is buried an additional 4.5 Å deeper within the active site. The propionate groups of the substrate do not protrude into solvent and are bound in a manner similar to what has been observed in uroporphyrinogen decarboxylase. Furthermore, in the substrate-bound form, the jaws of the active site mouth are closed so that the porphyrin substrate is completely engulfed in the pocket. These data provide insights that will aid in the determination of the mechanism for ferrochelatase. [ABSTRACT FROM AUTHOR]

Drug resistance in fungal pathogens has risen steadily over the past decades due to long-term azole therapy or triazole usage in agriculture. Modification of the drug target protein to prevent drug binding is a major recognized route to induce drug resistance. However, mechanisms for nondrug target-induced resistance remain only loosely defined. Here, we explore the molecular mechanisms of multidrug resistance resulted from an efficient adaptation strategy for survival in drug environments in the human pathogen Aspergillus fumigatus We show that mutants conferring multidrug resistance are linked with mitochondrial dysfunction induced by defects in heme A biosynthesis. Comparison of the gene expression profiles between the drug-resistant mutants and the parental wild-type strain shows that multidrug-resistant transporters, chitin synthases, and calcium-signaling-related genes are significantly up-regulated, while scavenging mitochondrial reactive oxygen species (ROS)-related genes are significantly down-regulated. The up-regulated-expression genes share consensus calcium-dependent serine threonine phosphatase-dependent response elements (the binding sites of calcium-signaling transcription factor CrzA). Accordingly, drug-resistant mutants show enhanced cytosolic Ca 2+ transients and persistent nuclear localization of CrzA. In comparison, calcium chelators significantly restore drug susceptibility and increase azole efficacy either in laboratory-derived or in clinic-isolated A. fumigatus strains. Thus, the mitochondrial dysfunction as a fitness cost can trigger calcium signaling and, therefore, globally up-regulate a series of embedding calcineurin-dependent-response-element genes, leading to antifungal resistance. These findings illuminate how fitness cost affects drug resistance and suggest that disruption of calcium signaling might be a promising therapeutic strategy to fight against nondrug target-induced drug resistance., Competing Interests: The authors declare no competing interest.

It has been generally accepted that biosynthesis of protoheme (heme) uses a common set of core metabolic intermediates that includes protoporphyrin. Herein, we show that the Actinobacteria and Firmicutes (high-GC and low-GC Gram-positive bacteria) are unable to synthesize protoporphyrin. Instead, they oxidize coproporphyrinogen to coproporphyrin, insert ferrous iron to make Fe-coproporphyrin (coproheme), and then decarboxylate coproheme to generate protoheme. This pathway is specified by three genes named hemY, hemH, and hemQ. The analysis of 982 representative prokaryotic genomes is consistent with this pathway being the most ancient heme synthesis pathway in the Eubacteria. Our results identifying a previously unknown branch of tetrapyrrole synthesis support a significant shift from current models for the evolution of bacterial heme and chlorophyll synthesis. Because some organisms that possess this coproporphyrin-dependent branch are major causes of human disease, HemQ is a novel pharmacological target of significant therapeutic relevance, particularly given high rates of antimicrobial resistance among these pathogens.

Although cells express hundreds of metalloenzymes, the mechanisms by which apoenzymes receive their metal cofactors are largely unknown. Poly(rC)-binding proteins PCBP1 and PCBP2 are multifunctional adaptor proteins that bind iron and deliver it to ferritin for storage or to prolyl and asparagyl hydroxylases to metallate the mononuclear iron center. Here, we show that PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the translation factor eukaryotic initiation factor 5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo form of the enzyme in cells, particularly when cells were made mildly iron-deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNA-binding activity, a pattern suggesting the incomplete disassembly of the [4Fe-4S] cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic nonheme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.

DiGeorge syndrome critical region gene 8 (DGCR8) is the RNA-binding partner protein of the nuclease Drosha. DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and starfish DGCR8 bind heme when expressed in Escherichia coli and that Fe(III) heme activates apoDGCR8 in reconstituted pri-miRNA processing assays. However, the physiological relevance of heme in miRNA maturation has not been clear. Here, we present a live-cell pri-miRNA processing assay that produces robust signals and faithfully indicates DGCR8 and Drosha activities. We demonstrate that all known heme-binding-deficient DGCR8 mutants are defective in pri-miRNA processing in HeLa cells. DGCR8 contains a previously uncharacterized heme-binding motif, "IPCL," that is also required for its activity. Heme availability and biosynthesis in HeLa cells positively affect pri-miRNA processing and production of mature miRNA. These results establish an essential function for heme in pri-miRNA processing in mammalian cells. Our study suggests that abnormal heme biosynthesis and degradation may contribute to diseases via miRNA-mediated gene regulation networks.

Staphylococcus aureus is a significant infectious threat to global public health. Acquisition or synthesis of heme is required for S. aureus to capture energy through respiration, but an excess of this critical cofactor is toxic to bacteria. S. aureus employs the heme sensor system (HssRS) to overcome heme toxicity; however, the mechanism of heme sensing is not defined. Here, we describe the identification of a small molecule activator of HssRS that induces endogenous heme biosynthesis by perturbing central metabolism. This molecule is toxic to fermenting S. aureus, including clinically relevant small colony variants. The utility of targeting fermenting bacteria is exemplified by the fact that this compound prevents the emergence of antibiotic resistance, enhances phagocyte killing, and reduces S. aureus pathogenesis. Not only is this small molecule a powerful tool for studying bacterial heme biosynthesis and central metabolism; it also establishes targeting of fermentation as a viable antibacterial strategy.

Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae. Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.

Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.

The mitochondrion is well known for its key role in energy transduction. However, it is less well appreciated that it is also a focal point of iron metabolism. Iron is needed not only for heme and iron sulfur cluster (ISC)-containing proteins involved in electron transport and oxidative phosphorylation, but also for a wide variety of cytoplasmic and nuclear functions, including DNA synthesis. The mitochondrial pathways involved in the generation of both heme and ISCs have been characterized to some extent. However, little is known concerning the regulation of iron uptake by the mitochondrion and how this is coordinated with iron metabolism in the cytosol and other organelles (e.g., lysosomes). In this article, we discuss the burgeoning field of mitochondrial iron metabolism and trafficking that has recently been stimulated by the discovery of proteins involved in mitochondrial iron storage (mitochondrial ferritin) and transport (mitoferrin-1 and -2). In addition, recent work examining mitochondrial diseases (e.g., Friedreich's ataxia) has established that communication exists between iron metabolism in the mitochondrion and the cytosol. This finding has revealed the ability of the mitochondrion to modulate whole-cell iron-processing to satisfy its own requirements for the crucial processes of heme and ISC synthesis. Knowledge of mitochondrial iron-processing pathways and the interaction between organelles and the cytosol could revolutionize the investigation of iron metabolism.

Cellular energy generation uses membrane-localized electron transfer chains for ATP synthesis. Formed ATP in turn is consumed for the biosynthesis of cellular building blocks. In contrast, heme cofactor biosynthesis was found driving ATP generation via electron transport after initial ATP consumption. The FMN enzyme protoporphyrinogen IX oxidase (HemG) of Escherichia coli abstracts six electrons from its substrate and transfers them via ubiquinone, cytochrome bo(3) (Cyo) and cytochrome bd (Cyd) oxidase to oxygen. Under anaerobic conditions electrons are transferred via menaquinone, fumarate (Frd) and nitrate reductase (Nar). Cyo, Cyd and Nar contribute to the proton motive force that drives ATP formation. Four electron transport chains from HemG via diverse quinones to Cyo, Cyd, Nar, and Frd were reconstituted in vitro from purified components. Characterization of E. coli mutants deficient in nar, frd, cyo, cyd provided in vivo evidence for a detailed model of heme biosynthesis coupled energy generation.

Plants depend on light signals to modulate many aspects of their development and optimize their photosynthetic capacity. Phytochromes (phys), a family of photoreceptors, initiate a signal transduction pathway that alters expression of a large number of genes to induce these responses. Recently, phyA and phyB were shown to bind members of a basic helix-loop-helix family of transcription factors called phy-interacting factors (PIFs). PIF1 negatively regulates chlorophyll biosynthesis and seed germination in the dark, and light-induced degradation of PIF1 relieves this negative regulation to promote photomorphogenesis. Here, we report that PIF1 regulates expression of a discrete set of genes in the dark, including protochlorophyllide oxidoreductase (POR), ferrochelatase (FeChII), and heme oxygenase (HO3), which are involved in controlling the chlorophyll biosynthetic pathway. Using ChIP and DNA gel-shift assays, we demonstrate that PIF1 directly binds to a G-box (CACGTG) DNA sequence element present in the PORC promoter. Moreover, in transient assays, PIF1 activates transcription of PORC in a G-box-dependent manner. These data strongly suggest that PIF1 directly and indirectly regulates key genes involved in chlorophyll biosynthesis to optimize the greening process in Arabidopsis.

Budding yeast undergo robust oscillations in oxygen consumption during continuous growth in a nutrient-limited environment. Using liquid chromatography-mass spectrometry and comprehensive 2D gas chromatography-mass spectrometry-based metabolite profiling methods, we have determined that the intracellular concentrations of many metabolites change periodically as a function of these metabolic cycles. These results reveal the logic of cellular metabolism during different phases of the life of a yeast cell. They may further indicate that oscillation in the abundance of key metabolites might help control the temporal regulation of cellular processes and the establishment of a cycle. Such oscillations in metabolic state might occur during the course of other biological cycles.

In most free-living eukaryotes studied thus far, heme is synthesized from a series of intermediates through a well defined evolutionarily conserved pathway. We found that free-living worms, including the model genetic organism Caenorhabditis elegans, and parasitic helminths are unable to synthesize heme de novo, even though these animals contain hemoproteins that function in key biological processes. Radioisotope, fluorescence labeling, and heme analog studies suggest that C. elegans acquires heme from exogenous sources. Iron-deprived worms were unable to grow in the presence of adequate heme unless rescued by increasing heme levels in the growth medium. These data indicate that although worms use dietary heme for incorporation into hemoproteins, ingested heme is also used as an iron source when iron is limiting. Our results provide a biochemical basis for the dependence of worm growth and development on heme, and they suggest that pharmacologic targeting of heme transport pathways in worms could be an important control measure for helminthic infections.

The Bacillus subtilis mrgA gene encodes an abundant DNA-binding protein that protects cells against the lethal effects of H2O2. Transcription of mrgA is induced by H2O2 or by entry into stationary phase when manganese and iron levels are low. We have selected for strains derepressed for transcription of mrgA in the presence of Mn(II). The resulting cis-acting mutants define an operator site just upstream of the mrgA promoter. Similar sequences flank the promoters for the catalase gene, katA, and the heme biosynthesis operon, hemAXCDBL. Like mrgA, transcription of the katA and hem genes is repressed by Mn(II), which thereby potentiates the killing action of H2O2. We identified two classes of trans-acting mutants derepressed for mrgA transcription in the presence of Mn(II): some exhibit a coordinate derepression of MrgA, catalase, heme biosynthesis, and alkyl hydroperoxide reductase and are H2O2 resistant, while others have reduced catalase activity and are H2O2 sensitive. These data indicate that the peroxide stress response of B. subtilis is regulated by a repressor that senses both metal ion levels and H2O2.

The hemH gene of Escherichia coli encodes ferrochelatase (EC 4.99.1.1), the enzyme that catalyzes the last step in the production of heme, namely the synthesis of heme from protoporphyrin IX plus Fe2+. The behavioral responses to light were studied in E. coli carrying a hemH mutation. It was shown that the hemH mutant displayed a tumbling response upon illumination and a running response upon removal of the light. The most effect light to induce a tumbling response in the hemH mutant was blue light (396-450 nm). The chemotaxis machinery was needed for the light-induced tumbling response in the hemH mutant. The bacterial defect is an analog of the human inherited disease erythropoietic protoporphyria.

Treatment of murine erythroleukemia (MEL) cells with imidazole in the presence of dimethyl sulfoxide (Me2SO) has been shown to dissociate hemoglobin accumulation from commitment to terminal maturation. To explore the mechanism(s) of this effect, we studied iron transport and heme and hemoglobin synthesis in Me2SO-induced MEL cells that were then exposed to imidazole. Imidazole treatment (i) causes moderate inhibition of 125I-labeled transferrin binding to both control and Me2SO-treated MEL cells; (ii) markedly suppresses Me2SO-induced activation of iron uptake into MEL cells; (iii) markedly decreases the incorporation of iron into ferritin; and (iv) abolishes heme biosynthesis from [2-14C]glycine and hemoglobin accumulation in Me2SO-treated cells. Imidazole treatment does not inhibit other aspects of cellular maturation; cells treated with Me2SO in the presence or absence of imidazole exhibit similar changes in proliferative activity and protein synthesis and, as shown previously, in cell morphology. Inhibition of hemoglobin accumulation in MEL cells is reversible on withdrawal of imidazole but is not altered by exogenous hemin. These data indicate that commitment to terminal maturation is regulated independently from the systems for iron transport and heme biosynthesis during early phases of erythroid cell differentiation.

The purpose of this study was to assess the ability of hepatocytes to synthesize porphyrins and heme from delta-aminolevulinic acid derived from gamma, delta-dioxovalerate and alanine. An alternate pathway for delta-aminolevulinic acid synthesis, in contrast to the condensation of succinate and glycine by delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37] has been suggested. This has been supported by the isolation of gamma, delta-dioxovalerate transaminase from liver mitochondria (Varticovski, L., Kushner, J. P. & Burnham, B. F. (1980) J. Biol. Chem. 255, 3742-3747). Gamma, delta-dioxovalerate transaminase catalyzes the formation of delta-aminolevulinic acid by a transamination reaction involving gamma, delta-dioxovalerate and alanine. To assess the significance of this alternate route, we prepared suspensions of respiring rat hepatocytes, which were incubated with optimal concentrations of various additives and then analyzed spectrophotometrically for synthesized porphyrins. No porphyrin synthesis was detected in cell suspensions incubated with succinate(1 mM) and glycine (20 mM). Cell suspensions incubated with gamma, delta-dioxovalerate (0.5-1.0 mM) and alanine (20 mM) synthesized 0.19 nmol of porphyrin per 10(7) cells per 2 hr (SD, 0.057). Cell suspensions incubated with delta-aminolevulinic acid (0.1 mM) synthesized 1.26 nmol of porphyrin per 10(7) cells per 2 hr (range, 1.18-1.32). Incubations with chemically synthesized gamma, delta-dioxo[5-14C]valerate were followed by extraction and purification of porphyrin esters and heme. Liquid scintillation counting revealed radiolabel incorporation into both porphyrins and heme. These studies demonstrate significant tetrapyrrole synthesis by the gamma, delta-dioxovalerate transaminase reaction. That gamma, delta-dioxovalerate is an important precursor of heme in vivo must be considered.

The addition of appropriate doses of interferon (IF) to cultures of Friend erythroleukemic cells inhibits dimethyl sulfoxide (Me2SO)-stimulated erythroid differentiation. In this study, the synthesis of heme, hemoglobin, and globin mRNA in Me2SO-stimulated cultures, with or without IF added, was compared. Although the hemoglobin content in Me2SO+IF-treated cultures was reduced 6- to 9-fold compared to that of cultures treated with Me2SO alone, there was less than a 2-fold decrease in the amount of heme accumulated. Globin mRNA, although unchanged in size or base sequence, was reduced in content in the Me2SO+IF cultures. The level of reduction of globin mRNA was insufficient to account for the lack of globin synthesis. Thus, it appears that IF may operate on two levels--one involving the transcription of globin mRNA and the other involving its translation.

Although lead has no physiological function and is present in only negligible amounts in the blood of remote populations, it has become customary to accept the usual blood Pb level(s) (BPb) observed in industrialized society as "normal." Pb interferes with many biochemical systems, among them the heme biosynthetic pathway; this is reflected by an exponential increase in erythrocyte protoporphyrin concentration (EP) as PBb increases. The present study estimated the threshold PBb at which an increase of EP occurs in a population of urban children. In the 2,004 children studied, BPbs ranged from 2 to 98 micrograms/dl, with 1,852 having a BPb of less than or equal to 30 micrograms/dl, a value presently considered normal. Preliminary analysis suggested that an exponential increase in the concentration of EP occurred after a threshold BPb (apparently between 12 and 20 micrograms/dl) was reached. Precise definition of the threshold BPb for an increase of EP was next determined by two approaches: segmented line techniques and probit analysis. Whether the entire population was analyzed or only the subset of samples with "normal" BPb (less than or equal to 30 micrograms/dl), both methods yielded a threshold BPb of 15-18 micrograms/dl (average value, 16.5). These studies indicate that the heme synthetic pathway is affected by Pb at a level of exposure commonly observed in urban children, which is well below the limit that is presently too easily accepted as normal.

We show that HEM1 (encoding 5-aminolevulinate synthase) expression, while constitutive under all steady-state growth conditions tested, is activated by the HAP2-HAP3 global activation system that controls expression of apocytochromes. This finding creates a paradox because apocytochrome activation by HAP2-HAP3 is highly regulated, subject to induction by heme, and subject to further derepression by a shift from glucose medium to one containing a nonfermentable carbon source. We clarify this issue by showing that HEM1 is subject to two additional layers of control that mask regulatory changes. First is a second activation system acting at a site close to the HAP2-HAP3 target sequence that keeps HEM1 turned on under conditions of heme deficiency. Second is a regulated negative control site downstream of the upstream activation site that counteracts derepression in medium containing a nonfermentable carbon source. Thus, transcription of the constitutive gene is actually a composite of opposing regulatory sites. This complex regulatory arrangement may exist to allow HEM1 to be coordinated transiently with apocytochromes for transition to respiratory growth. Conversely, it may reflect the alteration of HEM1 from a regulated to a constitutive gene over evolution.

Erythrodifferentiation and hemoglobin synthesis in dimethyl sulfoxide-stimulated Friend erythroleukemia cells were inhibited by hydrocortisone (HC) and four other steroids: dexamethasone, deoxycorticosterone, corticosterone, and aldosterone. The effect was specific, because no significant cytotoxicity occurred with any of these compounds at the concentrations that were inhibitory. The mechanism of action of HC was studied in detail. In the absence of dimethyl sulfoxide, it had no effect on hemoglobin levels; but, in the presence of this inducer, the synthesis of heme and globin were each inhibited by approximately 90%. There was no alteration in the synthesis of any major protein other than globin, as determined by gel electrophoresis of cell lysates. The activities of two enzymes in the heme biosynthetic pathway, delta-aminolevulinate dehydratase and uroporphyrinogen-I synthase, were inhibited by 80% and 70%, respectively. Globin mRNA induction was reduced by approximately 90%. This demonstrated that the HC inhibition of globin synthesis occurred at a pretranslational step. The dimethyl sulfoxide-induced single-stranded breaks in DNA, which have been suggested to play a role in Friend leukemia cell differentiation, were reduced in number but not eliminated. HC reduced the dimethyl sulfoxide-stimulation of virus release into the medium by approximately 50%. HC treatment in the absence of dimethyl sulfoxide doubled the production of virus.

In Bradyrhizobium japonicum/soybean symbiosis, the leghemoglobin (legume hemoglobin) apoprotein is a plant product, but the origin of the heme prosthetic group is not known. B. japonicum strain LO505 is a transposon Tn5-induced cytochrome-deficient mutant; it excreted the oxidized heme precursor coproporphyrin III into the growth medium. Mutant strain LO505 was specifically deficient in protoporphyrinogen oxidase (protoporphyrinogen-IX:oxygen oxidoreductase, EC 1.3.3.4) activity, and thus it could not catalyze the penultimate step in heme biosynthesis. Soybean root nodules formed from this mutant did not contain leghemoglobin, but the apoprotein was synthesized nevertheless. Data show that bacterial heme synthesis is required for leghemoglobin expression, but the heme moiety is not essential for apoleghemoglobin synthesis by the plant. Soybean leghemoglobin, therefore, is a product of both the plant and bacterial symbionts.

Previous studies demonstrated that 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a tumor promoter, is a potent inhibitor of inducer-mediated differentiation of murine erythroleukemia cells. Inhibition of cell differentiation was associated with inhibition of cell growth. The present studies, employing a cell line adapted for growth in TPA, demonstrate that inhibition of differentiation is not dependent upon inhibition of cell growth or a change in the cell division cycle; neither is inhibition of differentiation accompanied by detectable effect on cell uptake of [3H]hexamethylene bisacetamide, the inducer used in these studies. TPA causes an inhibition of expression of all hexamethylene bisacetamide-inducible erythroid characteristics measured, including commitment to terminal cell division, accumulation of globin mRNA, and synthesis of globins, spectrin, heme synthetic enzymes (delta-aminolevulinic acid dehydratase and uroporphyrinogen-I synthase) and heme. A hypothetical model for the inhibitory action of tumor promoters on terminal cell differentiation is discussed.

Heme biosynthetic activity in the symbiotic association involving crithidial flagellates and intracellular bacteroids was studied by enzymic, nutritional, and isotope incorporation experiments. Component organisms and their complexes in this association were analyzed separately to determine the underlying cause of the hemin requirement of hemoflagellates and the role of symbiotes in sparing this requirement of two crithidial species. Nutritional study of symbiote-free flagellates showed that their growth requires at least 0.1 mug/ml of hemin, which can be substituted by protoporphyrin IX, but not by the porphyrin precursors, delta-amino-levulinic acid or porphobilinogen. These flagellates, in the presence of protoporphyrin IX, incorporated 59Fe into heme, indicating that they possess ferrochelatase (EC 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, which catalyzes the insertion of iron into protoporphyrin IX. In symbiote-containing flagellates serially cultured in a defined medium free of tetrapyrrole compounds, heme and porphyrins can be detected by a fluorophotometric method, indicative of heme biosynthesis. Study of [14C]glycine incorporation into heme showed that the rate is much higher in symbiote-containing flagellates than in those without symbiotes. Microassay of uroporphyrinogen I synthase [EC 4.3.1.8; porphobilinogen ammonia-lyase (polymerizing)] revealed that the specific activity is high in symbiote-containing flagellates and higher still in isolated symbiotes, but essentially negligible in symbiote-free organisms. It is concluded that the bacterial symbiotes augment a very limited heme biosynthetic capacity of host flagellates by supplying uroporphyrinogen I synthase and perhaps other enzymes preceding ferrochelatase in the heme biosynthetic chain.

Mouse bone marrow cells infected in vitro with the anemia strain of Friend leukemia virus from large clusters (bursts) of erythroblasts after 5 days in culture in methylcellulose medium. Two types of erythroblast populations can be isolated from bursts of infected cells by manipulation of the culture conditions. One type of erythroblast, which is obtained when erythropoietin (EP) is added to the culture, has proliferated and undergoes differentiation to become an erythrocyte. The second type of erythroblast, which is obtained when no EP is added to the culture, is the product of extensive proliferation, but it fails to undergo the terminal stages of erythroblast differentiation. Comparisons of these two types of erythroblasts demonstrate that specific EP effects include changes in the nucleus, cytoplasm, and membrane of the treated cells. Those events of erythroid differentiation shown to be directed by EP were extrusion of the nucleus from the erythroblast, induction of uroporphyrinogen I synthetase activity, increased iron incorporation into protoporphyrin, synthesis of alpha- and beta-globin polypeptides due largely to increased mRNA production, and synthesis and incorporation of spectrin into the cell membrane. In this system, EP promotes these effects without observable stimulation of progenitor proliferation in addition to that caused by the virus alone. Thus, the role of EP in terminal erythrocyte differentiation is not simply that of an erythroid-specific mitogen.

Friend erythroleukemia cells of line Fw are noninducible for hemoglobin synthesis by dimethyl sulfoxide, butyric acid, and other agents. However, these agents were found to induce hemoglobin synthesis if the cells were also treated with exogenous hemin. Butyric acid (or, to a lesser extent, dimethyl sulfoxide) by itself induced accumulation of the erythroid-specific membrane protein spectrin. The basis of the control of globin gene expression by hemin was investigated. Hemin does not control globin and total protein synthesis via the action of the hemin-controlled repressor. Rather, hemin, alone or in combination with dimethyl sulfoxide, induces accumulation of globin mRNA and nuclear globin RNA sequences. Direct measurements of heme synthesis indicate that Fw cells may be significantly deficient in heme metabolism.

In somatic cell hybrids formed by the fusion of mouse erythroleukemic cells with mouse primary bone marrow cells, retention of the X chromosome contributed by the bone marrow parent is correlated with inhibition of hemoglobin accumulation in response to dimethyl sulfoxide. The inhibition of hemoglobin accumulation is not due to the absence of globin mRNA. Dimethyl sulfoxide-treated hybrid cells accumulate polyribosomal globin mRNA to levels comparable to those of the parental erythroleukemic cells under the same conditions. Heme, or its precursor delta-aminolevulinc acid, can overcome the effects of the bone marrow X chromosome and induce hemoglobin accumulation in the dimethyl sulfoxide-treated hybrid cells. The data suggest that the X chromosome contributed by the bone marrow cells inhibits hemoglobin production by inhibiting inducible heme biosynthesis, most probably at the step catalyzed by delta-aminolevulinic acid synthetase (EC 2.3.1.37).

The activities of the enzymes of heme biosynthesis (except protoporphyrin oxidase) have been followed during the induction of Friend cells in culture. All the enzyme activities increased after induction with dimethyl sulfoxide. The activities of the intermediate enzymes were much higher than those of delta-aminolevulinate synthase [succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37], the initial enzyme, or ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the final enzyme of the pathway. Ferrochelatase activity was not detectable in the uninduced cell. delta-Aminolevulinate synthase activity increased during the first 24 hr of induction; porphobilinogen deaminase activity began to increase after 48 hr and ferrochelatase activity, after 72 hr. However, the induction of heme synthesis followed the same time course as that of ferrochelatase activity, not that of delta-aminolevulinate synthase activity. The cellular growth medium was found to contain traces of protoporphyrins. Thus, ferrochelatase is shown to be rate limiting for heme synthesis during early stages of Friend cell induction. A Friend cell variant (Fw), which is not inducible except in the presence of exogenous hemin, was also studied. All the enzymes of heme synthesis except ferrochelatase were inducible by butyric acid. Ferrochelatase was not inducible by butyric acid or hemin plus butyric acid. These cells also excrete protoporphyrin, The failure to induce ferrochelatase activity is believed to be the cause of, not a consequence of, the noninducibility of this cell line.

Hepatic conversion of porphobilinogen to porphyrins was less than 50% of control levels in human subjects with the genetic disease, intermittent acute porphyria. This relative block in heme biosynthesis may be relevant to a concomitant 6- to 10-fold elevation in delta-aminolevulinic acid synthetase activity, since this first and rate-controlling enzyme in the biosynthetic pathway is subject to negative feedback regulation by the end product, heme. A micro-radio-chemical assay of delta-aminolevulinic acid synthetase, and some of its applications, are described.

A cloned line of murine proerythroblastoid cells (T-3-Cl-2), transformed by Friend leukemia virus, undergoes changes associated with erythroid differentiation when treated with dimethylsulfoxide in culture. This line, which does not undergo spontaneous differentiation, develops specific erythrocyte-membrane antigen and accumulates detectable amounts of heme within four days of dimethylsulfoxide treatment. In the present study, we have followed the phenotypic expression of the globin genes by measuring globin mRNA in differentiating cells. Our hybridization probe for this purpose is [(3)H]DNA, which is complementary to purified globin mRNA, synthesized by viral RNA-directed DNA polymerase. This probe is sufficiently sensitive to detect less than 1 ng of globin mRNA. Using it, we find little or no hybridizable globin mRNA in either uninduced cells or in treated control lymphoid cells. In contrast, globin mRNA can be detected in T-3-Cl-2 cell 2 days after induction by dimethylsulfoxide; it reaches a maximum concentration four days after induction. At this time, cells that stain positively for heme appear. The hybridizable cytoplasmic RNA induced in these cells has the sedimentation properties of 9S globin mRNA. Considering the stable character of globin mRNA, our results are most readily explained in terms of a transcriptional activation of the globin genes.

Cultures initiated with isolated goat-kidney glomeruli produce erythropoietin over periods of 7 months. Production seems dependent upon a minimum interval of about 30 days between changes of medium. Experiments distinguishing between erythropoietin, renal activator, and serum substrate support the conclusion that the factor released to the medium of long-term glomerular cultures is erythropoietin itself. The system offers promising opportunities for studying regulation of erythropoietin production, and possibly could be developed as a source of significant quantities of the hormone.