Expression of peptide transporter 1 has a positive correlation in protoporphyrin IX accumulation induced by 5-aminolevulinic acid with photodynamic detection of non-small cell lung cancer and metastatic brain tumor specimens originating from non-small cell lung cancer.

BACKGROUND: Recently, 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX fluorescence was reported to be a useful tool during total surgical resection of high-grade gliomas. However, the labeling efficacy of protoporphyrin IX fluorescence is lower in metastatic brain tumors compared to that in high-grade gliomas, and the mechanism underlying protoporphyrin IX fluorescence in metastatic brain tumors remains unclear. Lung cancer, particularly non-small cell lung cancer (NSCLC), is the most common origin for metastatic brain tumor. Therefore, we investigated the mechanism of protoporphyrin IX fluorescence in NSCLC and associated metastatic brain tumors. METHODS: Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to evaluate the protein and mRNA levels of five transporters and enzymes involved in the porphyrin biosynthesis pathway: peptide transporter 1 (PEPT1), hydroxymethylbilane synthase (HMBS), ferrochelatase (FECH), ATP-binding cassette 2 (ABCG2), and heme oxygenase 1 (HO-1). The correlation between protein, mRNA, and protoporphyrin IX levels in NSCLC cells were evaluated in vitro. Immunohistochemistry was used to determine proteins that played a key role in intraoperative protoporphyrin IX fluorescence in clinical samples from patients with NSCLC and pathologically confirmed metastatic brain tumors. RESULTS: A significant correlation between PEPT1 expression and protoporphyrin IX accumulation in vitro was identified by western blotting (P = 0.003) and qRT-PCR (P = 0.04). Immunohistochemistry results indicated that there was a significant difference in PEPT1 between the intraoperative protoporphyrin IX fluorescence-positive and protoporphyrin IX fluorescence-negative groups (P = 0.009). CONCLUSION: Expression of PEPT1 was found to be positively correlated with 5-ALA-induced protoporphyrin IX accumulation detected by photodynamic reaction in metastatic brain tumors originating from NSCLC.

Liaison between heme metabolism and bioenergetics pathways-a multimodal elucidation for early diagnosis of oral cancer.

Metabolic alterations of oral epithelial cells under oxidative stress are important signatures for early diagnosis of oral cancer. Amongst different metabolic alterations, non-invasive photo-diagnostic methods have been extensively used for determining cellular heme metabolism and accumulation of protoporphyrin IX (PpIX) under administration of suitable photosensitizer. In this study, we report these metabolic alterations by direct analysis of oral exfoliated cells obtained from individuals with prolonged smoking habit without the exogenous administration of any photosensitizer. The relative expression level of relevant biomolecules of study groups were compared with clinically diagnosed and histopathologically confirmed leukoplakia (OLPK) and oral squamous cell carcinoma (OSCC) patients. The energy imbalance and variation in 'redox ratio' were examined through spectroscopic studies which showed an increasing trend (p < 0.001) in smokers to OSCC groups in comparison to nonsmoker control. Gene expression of important intermediates of the heme metabolic pathway (viz. 5-aminolevulinate synthase 1 (ALAS1), Ferrochelatase (FECH), hemeoxygenase 1 (HO-1) and ATP binding cassette subfamily G member 2 (ABCG2)) which affect production of PpIX was assessed. Relative mRNA level of ALAS1 and HO1 was upregulated whereas mRNA level of other genes (viz. FECH and ABCG2) were found to be downregulated in smokers as well as in cancer groups. Outcome of different spectroscopic studies on exfoliated cells (viz. fluorescence, atomic absorption and Fourier transform infrared spectroscopy) corroborated with the expression of biomarkers related to cellular endogenous metabolism related to heme cycle. This study indicates significant alterations in endogenous metabolic products, and cellular functional groups in oral epithelial cells among the study groups. Our study reports a strong possibility of diagnosis of early cancer signatures amongst habitual smokers by direct and non-invasive assessment of metabolic status of oral epithelial cells without exogenous administration of photosensitizers. The knowledge accrued from the study may guide clinicians in precise detection of precancer trend in the susceptible population through a noninvasive rapid screening method.

Phytochrome B Mediates the Regulation of Chlorophyll Biosynthesis through Transcriptional Regulation of ChlH and GUN4 in Rice Seedlings.

Accurate regulation of chlorophyll synthesis is crucial for chloroplast formation during the greening process in angiosperms. In this study, we examined the role of phytochrome B (phyB) in the regulation of chlorophyll synthesis in rice seedlings (Oryza sativa L.) through the characterization of a pale-green phenotype observed in the phyB mutant grown under continuous red light (Rc) irradiation. Our results show that the Rc-induced chlorophyll accumulation can be divided into two components--a phyB-dependent and a phyB-independent component, and that the pale-green phenotype is caused by the absence of the phyB-dependent component. To elucidate the role of the missing component we established an Rc-induced greening experiment, the results of which revealed that several genes encoding proteins on the chlorophyll branch were repressed in the phyB mutant. Notable among them were ChlH and GUN4 genes, which encode subunit H and an activating factor of magnesium chelatase (Mg-chelatase), respectively, that were largely repressed in the mutant. Moreover, the kinetic profiles of chlorophyll precursors suggested that Mg-chelatase activity simultaneously decreased with the reduction in the transcript levels of ChlH and GUN4. These results suggest that phyB mediates the regulation of chlorophyll synthesis through transcriptional regulation of these two genes, whose products exert their action at the branching point of the chlorophyll biosynthesis pathway. Reduction of 5-aminolevulinic acid (5-ALA) synthesis could be detected in the mutant, but the kinetic profiles of chlorophyll precursors indicated that it was an event posterior to the reduction of the Mg-chelatase activity. It means that the repression of 5-ALA synthesis should not be a triggering event for the appearance of the pale-green phenotype. Instead, the repression of 5-ALA synthesis might be important for the subsequent stabilization of the pale-green phenotype for preventing excessive accumulation of hazardous chlorophyll precursors, which is an inevitable consequence of the reduction of Mg-chelatase activity.

The induction of two biosynthetic enzymes helps Escherichia coli sustain heme synthesis and activate catalase during hydrogen peroxide stress.

Hydrogen peroxide pervades many natural environments, including the phagosomes that mediate cell-based immunity. Transcriptomic analysis showed that during protracted low-grade H(2)O(2) stress, Escherichia coli responds by activating both the OxyR defensive regulon and the Fur iron-starvation response. OxyR induced synthesis of two members of the nine-step heme biosynthetic pathway: ferrochelatase (HemH) and an isozyme of coproporphyrinogen III oxidase (HemF). Mutations that blocked either adaptation caused the accumulation of porphyrin intermediates, inadequate activation of heme enzymes, low catalase activity, defective clearance of H(2)O(2) and a failure to grow. Genetic analysis indicated that HemH induction is needed to compensate for iron sequestration by the mini-ferritin Dps. Dps activity protects DNA and proteins by limiting Fenton chemistry, but it interferes with the ability of HemH to acquire the iron that it needs to complete heme synthesis. HemF is a manganoprotein that displaces HemN, an iron-sulfur enzyme whose synthesis and/or stability is apparently problematic during H(2)O(2) stress. Thus, the primary responses to H(2)O(2), including the sequestration of iron, require compensatory adjustments in the mechanisms of iron-cofactor synthesis. The results support the growing evidence that oxidative stress is primarily an iron pathology.

Salicylic acid induces mitochondrial injury by inhibiting ferrochelatase heme biosynthesis activity.

Salicylic acid is a classic nonsteroidal anti-inflammatory drug. Although salicylic acid also induces mitochondrial injury, the mechanism of its antimitochondrial activity is not well understood. In this study, by using a one-step affinity purification scheme with salicylic acid-immobilized beads, ferrochelatase (FECH), a homodimeric enzyme involved in heme biosynthesis in mitochondria, was identified as a new molecular target of salicylic acid. Moreover, the cocrystal structure of the FECH-salicylic acid complex was determined. Structural and biochemical studies showed that salicylic acid binds to the dimer interface of FECH in two possible orientations and inhibits its enzymatic activity. Mutational analysis confirmed that Trp301 and Leu311, hydrophobic amino acid residues located at the dimer interface, are directly involved in salicylic acid binding. On a gel filtration column, salicylic acid caused a shift in the elution profile of FECH, indicating that its conformational change is induced by salicylic acid binding. In cultured human cells, salicylic acid treatment or FECH knockdown inhibited heme synthesis, whereas salicylic acid did not exert its inhibitory effect in FECH knockdown cells. Concordantly, salicylic acid treatment or FECH knockdown inhibited heme synthesis in zebrafish embryos. Strikingly, the salicylic acid-induced effect in zebrafish was partially rescued by FECH overexpression. Taken together, these findings illustrate that FECH is responsible for salicylic acid-induced inhibition of heme synthesis, which may contribute to its antimitochondrial and anti-inflammatory function. This study establishes a novel aspect of the complex pharmacological effects of salicylic acid.

Co-expression of ferrochelatase allows for complete heme incorporation into recombinant proteins produced in E. coli.

Over-expression of heme binding proteins in Escherichia coli often results in sub-optimal heme incorporation and the amount of heme-bound protein produced usually varies with the protein of interest. Complete heme incorporation is important for biochemical characterization, spectroscopy, structural studies, and for the production of homogeneous commercial proteins with high activity. We have determined that recombinant proteins expressed in E. coli often contain less than a full complement of heme because they rather are partially incorporated with free-base porphyrin. Porphyrin-incorporated proteins have similar spectral characteristics as the desired heme-loaded targets, and thus are difficult to detect, even in purified samples. We present a straightforward and inexpensive solution to this problem that involves the co-expression of native ferrochelatase with the protein of interest. The method is shown to be effective for proteins that contain either Cys- or His-ligated hemes.

The role of nitric oxide in delta-aminolevulinic acid (ALA)-induced photosensitivity of cancerous cells.

Application of delta-aminolevulinic acid (ALA) results in the endogenous accumulation of protoporphyrin IX and is a useful approach in the photodynamic therapy (PDT) of cancers. To investigate the role of nitric oxide (NO) in the specific accumulation of protoporphyrin and ALA-induced PDT of cancerous cells, we transfected inducible-nitric oxide synthase (NOS2) cDNA into human embryonic kidney (HEK) 293T cells and examined the ALA-induced photo-damage as well as the accumulation of porphyrin in the cells. When the NOS2-expressing HEK293T cells were treated with ALA and then exposed to visible light, they became more sensitive to the light with accumulating porphyrins, as compared with the ALA-treated control cells. An increase in the generation of NO in transfected cells led to the accumulation of protoporphyrin with a concomitant decrease of ferrochelatase, the final step enzyme of heme biosynthesis. When mouse macrophage-like RAW264.7 cells were cultured with lipopolysaccharide and interferon-gamma, the expression of NOS2 was induced. The addition of ALA to these cells led to the accumulation of protoporphyrin and cell death upon exposure to light. The treatment of cells with an NOS inhibitor, NG-monomethyl-L-arginine acetate, resulted in the inhibition of protoporphyrin accumulation and cell death. The levels of mitochondrial ferrochelatase and rotenone-sensitive NADH dehydrogenase in the NOS2-induced cells decreased. These results indicated that the generation of NO augments the ALA-induced accumulation of protoporphyrin IX and subsequent photo-damage in cancerous cells by decreasing the levels of mitochondrial iron-containing enzymes. Based on the fact that the production of NO in cancerous cells is elevated, NO in the cells is responsible for susceptibility with ALA-induced PDT.

Involvement of ABC7 in the biosynthesis of heme in erythroid cells: interaction of ABC7 with ferrochelatase.

A mitochondrial half-type ATP-binding cassette (ABC) protein, ABC7, plays a role in iron homeostasis in mitochondria, and defects in human ABC7 were shown to be responsible for the inherited disease X-linked sideroblastic anemia/ataxia. We examined the role of ABC7 in the biosynthesis of heme in erythroid cells where hemoglobin is a major product of iron-containing compounds. RNA blots showed that the amount of ABC7 mRNA in dimethylsulfoxide (Me(2)SO)-treated mouse erythroleukemia (MEL) cells increased markedly in parallel with the induction of the mRNA expression of ferrochelatase, the last enzyme in the pathway to synthesize heme. The transfection of the antisense oligonucleotide to mouse ABC7 mRNA into Me(2)SO-treated MEL cells led to a decrease of heme production, as compared with sense oligonucleotide-transfected cells. ABC7 protein was shown to be colocalized with ferrochelatase in mitochondria, as assessed by immunostaining. Furthermore, in vitro and in vivo pull-down assays revealed that ABC7 protein is interacted with the carboxy-terminal region containing the iron-sulfur cluster of ferrochelatase. The transient expression of ABC7 in mouse embryo liver BNL-CL2 cells resulted in an increase in the activity and level of ferrochelatase and thioredoxin, a cytosolic protein containing iron-sulfur. These increases were also observed in MEL cells stably expressing ABC7. When ABC7 transfectants were treated with Me(2)SO, an increase in cellular heme concomitant with a marked induction of the expression of ferrochelatase was observed. The extent of these increases was 3-fold greater than in control cells. The results indicated that ABC7 positively regulates not only the expression of extramitochondrial thioredoxin but also that of an intramitochondrial iron-sulfur-containing protein, ferrochelatase. Then, the expression of ABC7 contributes to the production of heme during the differentiation of erythroid cells.

Porphyrinogenic xenobiotic-induced N-alkylprotoporphyrin IX formation: a bioassay utilizing chick embryo hepatic ferrochelatase.

INTRODUCTION: The porphyrinogenicity of some xenobiotics results from mechanism-based inactivation of selected cytochrome P450 (CYP) enzymes accompanied by conversion of prosthetic heme groups to N-alkylprotoporphyrins (N-alkylPPs), some of which inhibit ferrochelatase (FC). Problems have arisen in extrapolating xenobiotic porphyrinogenicity observed in test animals to humans, due in part to differences among CYP enzymes. Our goal was to develop a bioassay to detect N-alkylPPs formed following interaction of porphyrinogenic xenobiotics with rat liver microsomal CYP. METHODS: Seventeen-day-old chick embryo livers were homogenized, and the mitochondrial fraction was isolated. The FC activity of this fraction was determined by means of the pyridine hemochromogen method. Inhibition of FC was used to detect N-alkylPP formation following interaction of porphyrinogenic xenobiotics with rat liver microsomes. RESULTS: The 17-day-old chick embryo hepatic mitochondrial preparation served as a stable source of FC activity, which was linear with respect to time and protein concentration. FC activity was higher than previously reported in a homogenate of 17-day-old chick embryo hepatocytes in culture and in an aqueous extract of 17-day-old chick embryo mitochondria. The EC(50) of N-methylprotoporphyrin IX in the chick embryo liver mitochondrial preparation was similar to that in the homogenate of chick embryo liver cell culture. The FC bioassay could detect N-alkylPPs formed following the interaction of porphyrinogenic xenobiotics with rat liver microsomes containing 2.4-9.0 nmol of CYP. DISCUSSION: In future studies investigating N-alkylPP formation following interaction of xenobiotics with CYP enzymes, we recommend using a combination of a fluorescence technique and the chick embryo hepatic mitochondrial FC assay. This would provide information both on the formation of N-alkylPPs and distinguish between those N-alkylPPs that produced porphyrin accumulation via FC inhibition and those that do not.

Identification of [2Fe-2S] clusters in microbial ferrochelatases.

The terminal enzyme of heme biosynthesis, ferrochelatase (EC 4.99.1.1), catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. Prior to the present work, [2Fe-2S] clusters have been identified and characterized in animal ferrochelatases but not in plant or prokaryotic ferrochelatases. Herein we present evidence that ferrochelatases from the bacteria Caulobacter crescentus and Mycobacterium tuberculosis possess [2Fe-2S] clusters. The enzyme from C. crescentus is a homodimeric, membrane-associated protein while the enzyme from M. tuberculosis is monomeric and soluble. The clusters of the C. crescentus and M. tuberculosis ferrochelatases are ligated by four cysteines but possess ligand spacings that are unlike those of any previously characterized [2Fe-2S] cluster-containing protein, including the ferrochelatase of the yeast Schizosaccharomyces pombe. Thus, the microbial ferrochelatases represent a new group of [2Fe-2S] cluster-containing proteins.

The penetrance of dominant erythropoietic protoporphyria is modulated by expression of wildtype FECH.

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by a partial deficiency of ferrochelatase (FECH, EC 4.99.1.1). EPP is transmitted as an autosomal dominant disorder with an incomplete penetrance. Using haplotype segregation analysis, we have identified an intronic single nucleotide polymorphism (SNP), IVS3-48T/C, that modulates the use of a constitutive aberrant acceptor splice site. The aberrantly spliced mRNA is degraded by a nonsense-mediated decay mechanism (NMD), producing a decreased steady-state level of mRNA and the additional FECH enzyme deficiency necessary for EPP phenotypic expression.

Two types of ferrochelatase in photosynthetic and nonphotosynthetic tissues of cucumber: their difference in phylogeny, gene expression, and localization.

Ferrochelatase catalyzes the insertion of Fe(2+) into protoporphyrin IX to generate protoheme. In higher plants, there is evidence for two isoforms of this enzyme that fulfill different roles. Here, we describe the isolation of a second ferrochelatase cDNA from cucumber (CsFeC2) that was less similar to a previously isolated isoform (CsFeC1) than it was to some ferrochelatases from other higher plants. In in vitro import experiments, the two cucumber isoforms showed characteristics similar to their respective ferrochelatase counterparts of Arabidopsis thaliana. The C-terminal region of CsFeC2 but not CsFeC1 contained a conserved motif found in light-harvesting chlorophyll proteins, and CsFeC2 belonged to a phylogenetic group of plant ferrochelatases containing this conserved motif. We demonstrate that CsFeC2 was localized predominantly in thylakoid membranes as an intrinsic protein, and forming complexes probably with the C-terminal conserved motif, but a minor portion was also detected in envelope membranes. CsFeC2 mRNA was detected in all tissues and was light-responsive in cotyledons, whereas CsFeC1 mRNA was detected in nonphotosynthetic tissues and was not light-responsive. Interestingly, tissue-, light-, and cycloheximide-dependent expressions of the two isoforms of ferrochelatase were similar to those of two glutamyl-tRNA reductase isoforms involved in the early step of tetrapyrrole biosynthesis, suggesting the existence of distinctly controlled tetrapyrrole biosynthetic pathways in photosynthetic and nonphotosynthetic tissues.

Two course illuminating scheme improves aminolevulinic acid photodynamic therapy in cell cultures.

Photodynamic therapy with the pro-drug 5-aminolaevulinic acid (ALA-PDT) is being used for the treatment of Barrett's oesophagus. We postulated that a first early course of ALA-PDT would increase protoporphyrin IX (PPIX) accumulation and thus the efficacy of a second course of ALA-PDT, by manipulating ferrochelatase (FC) and porphobilinogen deaminase (PBG-d) activity. Human EBV-transformed lymphoblastoid cells were used as a model of human tumour cells for the ability to form haem is present in all cells. After a single course of illumination (633 nm, 100 mW/cm2) the FC activity decreased significantly whereas the PBG-d activity did not change. During continued incubation with ALA following the first illumination, cells accumulated up to four times more PPIX than non-illuminated controls [220% +/- 30% versus (vs) 55% +/- 5%; p<0.001]. Two illuminations resulted in more cell death than one illumination (97% +/- 1% vs 80% +/- 2%; p<0.001). Since a second course of ALA-PDT within 3 hr after the first course resulted in a four fold increase in PPIX accumulation and significantly more cell death, we propose that a two course ALA-PDT scheme might improve the efficacy of this treatment for Barrett's oesophagus.

Impaired expression of the plastidic ferrochelatase by antisense RNA synthesis leads to a necrotic phenotype of transformed tobacco plants.

Protoporphyrin IX is the last common intermediate of tetrapyrrole biosynthesis. The chelation of a Mg2+ ion by magnesium chelatase and of a ferrous ion by ferrochelatase directs protoporphyrin IX towards the formation of chlorophyll and heme, respectively. A full length cDNA clone encoding a ferrochelatase was identified from a Nicotiana tabacum cDNA library. The encoded protein consists of 497 amino acid residues with a molecular weight of 55.4 kDa. In vitro import of the protein into chloroplasts and its location in stroma and thylakoids confirm its close relationship to the previously described Arabidopsis thaliana plastid-located ferrochelatase (FeChII). A 1700-bp tobacco FeCh cDNA sequence was expressed in Nicotiana tabacum cv. Samsun NN under the control of the CaMV 35S promoter in antisense orientation allowing investigation into the consequences of selective reduction of the plastidic ferrochelatase activity for protoporphyrin IX channeling in chloroplasts and for interactions between plastidic and mitochondrial heme synthesis. Leaves of several transformants showed a reduced chlorophyll content and, during development, a light intensity-dependent formation of necrotic leaf lesions. In comparison with wild-type plants the total ferrochelatase activity was decreased in transgenic lines leading to an accumulation of photosensitizing protoporphyrin IX. Ferrochelatase activity was reduced only in plastids but not in mitochondria of transgenic plants. By means of the specifically diminished ferrochelatase activity consequences of the selective inhibition of protoheme formation for the intracellular supply of heme can be investigated in the future.

Regulation of the expression of human ferrochelatase by intracellular iron levels.

Mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of a ferrous ion into protoporphyrin and contains a labile [2Fe-2S] cluster center at the C-terminus. To clarify the roles of the iron-sulfur cluster in the expression of mammalian ferrochelatase, enzyme activity in human erythroleukemia K562 cells under iron-depleted conditions was examined. Treatment of cells with an iron chelator, desferrioxamine, resulted in a decrease in enzyme activity, in a dose- and time-dependent manner. Heme content decreased during desferrioxamine treatment of the cells. Addition of ferric ion-nitrilotriacetate [Fe (III)NTA] to desferrioxamine-containing cultures led to restoration of the reduction in the enzyme activity. While RNA blots showed that the amount of ferrochelatase mRNA remained unchanged during these treatments, the amount of ferrochelatase decreased with a concomitant decrease in enzyme activity. When full-length human ferrochelatase was expressed in Cos7 cells, the activity was found mainly in the mitochondria and was decreased markedly by treatment with desferrioxamine. The activity in Cos7 cells expressing human ferrochelatase in cytoplasm decreased with desferrioxamine, but to a lesser extent. When Escherichia coli ferrochelatase, which lacks the iron-sulfur cluster, was expressed in Cos7 cells, the activity did not change following any treatment. Conversely, the addition of Fe (III)NTA to the culture of K562 and Cos7 cells led to an increase in ferrochelatase activity. These results indicate that the expression of mammalian ferrochelatase is regulated by intracellular iron levels, via the iron-sulfur cluster center at the C-terminus, and this contributes to the regulation of the biosynthesis of heme at the terminal step.

The role of HNF-1alpha in controlling hepatic catalase activity.

Mice deficient in hepatocyte nuclear factor 1alpha (HNF-1alpha) develop Laron dwarfism and non-insulin-dependent diabetes mellitus (Lee et al., 1998). Oxidative stress was present in the diabetic HNF-1alpha-null mice. To understand the mechanism underlying the oxidative stress in HNF-1alpha-null mice, we examined whether HNF-1alpha deficiency affects the integrity of the cellular defense system against oxidative stress. The glutathione level and activities of superoxide dismutase and glutathione reductase in liver and other tissues examined were not affected by HNF-1alpha deficiency. However, activities of cytosolic glutathione peroxidase and catalase, two enzymes responsible for detoxification of hydrogen peroxide within cells, were reduced specifically in liver of HNF-1alpha-null mice. The mRNA and protein levels of hepatic catalase in HNF-1alpha-null mice did not differ from those in normal mice. The loss of hepatic catalase activity in HNF-1alpha-null mice is probably caused by an insufficient heme pool in liver cells, because the mRNA level of ferrochelatase, the enzyme that catalyzes the last step of heme biosynthesis, was significantly reduced in liver, and the daily hemin treatment restored partial catalase activity in liver of HNF-1alpha-null mice. Furthermore, our results of cell transfection and luciferase reporter assay indicated that the mouse ferrochelatase promoter could be trans-activated directly by HNF-1alpha.

Long-term cure of the photosensitivity of murine erythropoietic protoporphyria by preselective gene therapy.

Definitive cure of an animal model of a human disease by gene transfer into hematopoietic stem cells has not yet been accomplished in the absence of spontaneous in vivo selection for transduced cells. Erythropoietic protoporphyria is a genetic disease in which ferrochelatase is defective. Protoporphyrin accumulates in erythrocytes, leaks into the plasma and results in severe skin photosensitivity. Using a mouse model of erythropoietic protoporphyria, we demonstrate here that ex vivo preselection of hematopoietic stem cells transduced with a polycistronic retrovirus expressing both human ferrochelatase and green fluorescent protein results in complete and long-term correction of skin photosensitivity in all transplanted mice.

Activity and cellular location in Saccharomyces cerevisiae of chimeric mouse/yeast and Bacillus subtilis/yeast ferrochelatases.

We have constructed a series of chimeric yeast/mouse and yeast/Bacillus subtilis ferrochelatase genes in order to investigate domains of the ferrochelatase that are important for activity and/or association with the membrane. These genes were expressed in a Saccharomyces cerevisiae mutant in which the endogenous ferrochelatase gene (HEM15) had been deleted, and the phenotypes of the transformants were characterized. Exchanging the approximately 40-amino-acid C-terminus between the yeast and mouse ferrochelatases caused a total loss of activity and the hybrid proteins were unstable when overproduced in Escherichia coli. The water-soluble ferrochelatase of B. subtilis did not complement the yeast mutant, although a large amount of active protein accumulated in the cytosol. Addition of the N-terminal leader sequence of yeast ferrochelatase to the B. subtilis enzyme targeted the fusion protein to mitochondria, but both the precursor and the mature forms of the enzyme were inactive in vivo and had residual activity when measured in vitro. An internal approximately 45-amino-acid segment located at the N-terminus of yeast ferrochelatase was identified, which, when replaced with the corresponding 30-amino-acid segment of the B. subtilis enzyme, caused the yeast enzyme to be located in the mitochondrial matrix as a soluble protein. The fusion protein was inactive in vivo and had residual activity in vitro. We speculate that this segment, which shows the greatest variability between species, is responsible for the association of the enzyme with the membrane.

[Expression of transferrin receptor and ferrochelatase mRNAs in MEL cells].

OBJECTIVE: To investigate the changes of transferrin receptor (TfR) mRNA and ferrochelatase mRNA levels under different iron status of DMSO induced or non-induced MEL cells. METHODS: MEL cells were induced with 1.5% DMSO. TfR mRNA and ferrochelatase mRNA were assayed by Northern blot after adding transferrin, deferrioxamine, anti-TfR monoclonal antibody (McAb) or Epo, respectively. RESULTS AND CONCLUSION: 1. TfR mRNA level increased with cell proliferation in non-induced MEL cells, while in the induced MEL cells, although the proliferating capacity was losing with cell maturation, TfR mRNA level increased with hemoglobin synthesis and its amplitude was higher than that in non-induced cells. 2. In both non-induced and induced MEL cells, TfR mRNA levels increased when intracellular iron level decreased; and vice versa. 3. Stimulation of erythropoiesis by Epo increased the expression of TfR mRNA, indicating that its expression in erythroid cells was regulated by the level of intracellular iron and the synthesis of heme. 4. The expression of ferrochelatase mRNA had no change in non-induced MEL cells, but increased significantly in induced MEL cells. 5. The level of intracellular iron in non-induced and induced MEL cells had less effects on the expression of ferrochelatase mRNA.

A novel stop codon mutation (X417L) of the ferrochelatase gene in bovine protoporphyria, a natural animal model of the human disease.

Protoporphyria (PP) is caused by a deficiency of ferrochelatase (FC) activity, which catalyzes the final step in the heme biosynthesis pathway. Bovine are the only species other than man with naturally occurring PP. For expression of the PP phenotype, two copies of the mutated gene are necessary in bovine, whereas one copy is sufficient in humans. We report the first potential disease-causing mutation in the bovine FC gene. The coding region of FC was sequenced from the liver tissue of protoporphyric and normal bovine. A transversion was identified at nucleotide position 1250 which changed the stop codon to leucine (TGA-->TTA) in the protoporphyric FC sequence. As a consequence, the mutant protein is predicted to have an additional 27 amino acids. To screen other bovine for the G-->T transversion, cDNAs from liver tissue of clinically and biochemically normal, and from heterozygous and homozygous affected animals were used for allele-specific polymerase chain reaction. Three normal animals had only the G allele, five affected animals had only the T allele, and three heterozygous animals had both the G and T alleles. These results support our hypothesis that this mutation causes PP in bovine.