Shigeru Sassa

Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1

Heme controls expression of genes involved in the synthesis of globins and heme. The mammalian transcription factor Bach1 functions as a repressor of the Maf recognition element (MARE) by forming antagonizing hetero‐oligomers with the small Maf family proteins. We show here that heme binds specifically to Bach1 and regulates its DNA‐binding activity. Deletion studies demonstrated that a heme‐binding region of Bach1 is confined within its C‐terminal region that possesses four dipeptide cysteine–proline (CP) motifs. Mutations in all of the CP motifs of Bach1 abolished its interaction with heme. The DNA‐binding activity of Bach1 as a MafK hetero‐oligomer was markedly inhibited by heme in gel mobility shift assays. The repressor activity of Bach1 was lost upon addition of hemin in transfected cells. These results suggest that increased levels of heme inactivate the repressor Bach1, resulting in induction of a host of genes with MAREs.

Identification of a human heme exporter that is essential for erythropoiesis

FLVCR, a member of the major facilitator superfamily of transporter proteins, is the cell surface receptor for feline leukemia virus, subgroup C. Retroviral interference with FLVCR display results in a loss of erythroid progenitors (colony-forming units-erythroid, CFU-E) and severe anemia in cats. In this report, we demonstrate that human FLVCR exports cytoplasmic heme and hypothesize that human FLVCR is required on developing erythroid cells to protect them from heme toxicity. Inhibition of FLVCR in K562 cells decreases heme export, impairs their erythroid maturation and leads to apoptosis. FLVCR is upregulated on CFU-E, indicating that heme export is important in primary cells at this stage. Studies of FLVCR expression in cell lines suggest this exporter also impacts heme trafficking in intestine and liver. To our knowledge, this is the first description of a mammalian heme transporter.

Positional cloning of the zebrafish sauternes gene: a model for congenital sideroblastic anaemia.

Many human anaemias are caused by defects in haemoglobin synthesis. The zebrafish mutant sauternes (sau) has a microcytic, hypochromic anaemia, suggesting that haemoglobin production is perturbed. During embryogenesis, sau mutants have delayed erythroid maturation and abnormal globin gene expression. Using positional cloning techniques, we show that sau encodes the erythroid-specific isoform of δ-aminolevulinate synthase (ALAS2; also known as ALAS-E), the enzyme required for the first step in haem biosynthesis. As mutations in ALAS2 cause congenital sideroblastic anaemia (CSA) in humans, sau represents the first animal model of this disease.

Heme Oxygenase-1: A Novel Therapeutic Target in Oxidative Tissue Injuries

Oxidative stresses such as oxidant stimuli, inflammation, exposure to xenobiotics, or ionizing irradiation provoke cellular protective responses, principally involving transcriptional activation of genes encoding proteins which participate in the defense against oxidative tissue injuries. Excess of free heme, which is released from hemeproteins under such conditions, may constitute a major threat because it can catalyze the formation of reactive oxygen species (ROS). Exposure of mammalian cells to oxidative stimuli induces heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, as well as a 33-kDa heat shock protein. In various model systems, HO-1 induction confers protection on tissues from further injuries, while the abrogation of its induction accelerates cellular injuries. In this article, we review recent advances in the regulatory mechanism of ho-1 gene expression and the role of HO-1 in various models of experimental oxidative tissue injuries, and its potential therapeutic implications.

Studies in lead poisoning: II. Correlation between the ratio of activated to inactivated δ-aminolevulinic acid dehydratase of whole blood and the blood lead level☆

Abstract 1. 1. A method is described, using aliquots of 5 μl heparinized blood, to determine the activity of δ-aminolevulinic acid dehydratase in the aliquot incubated with δ-aminolevulinic acid, and in the aliquot incubated with δ-aminolevulinic acid + dithiothreitol. 2. 2. The log of the activity of δ-aminolevulinic acid dehydratase in the absence of dithiothreitol is inversely correlated with the blood lead level with a correlation coefficient of 0.72. 3. 3. The ratio: dithiothreitol-activated enzyme/nonactivated enzyme, vs the blood lead level gives a linear regression line with the higher correlation coefficient of 0.8. 4. 4. Dithiothreitol (20 m M ) fully activates the enzyme in lead-poisoned blood to the levels of activated normal blood. Thus lead 0.09 mg 100 ml blood does not appear to inhibit the formation, or affect the destruction of δ-aminolevulinic acid dehydratase. 5. 5. Kinetic analysis indicates that lead changes the affinity of the dehydratase for δ-aminolevulinic acid (i.e., K m ) slightly, but markedly lowers the V max , i.e., the inhibition is primarily of the noncompetitive type.

Studies in lead poisoning: I. Microanalysis of erythrocyte protoporphyrin levels by spectrofluorometry in the detection of chronic lead intoxication in the subclinical range☆

Abstract This study describes the application of a rapid, simple, extremely sensitive new assay for blood protoporphyrin to the detection of lead poisoning in the subclinical range. This protoporphyrin method has proved to be especially useful for the detection of chronic lead intoxication in children because of its precision and the fact that only 2 μl of blood are required for the analysis. Blood protoporphyrin was found to reflect better the level of bone marrow lead existing 2–3 months prior to sampling rather than the circulating blood lead level. The correlation coefficient between log blood protoporphyrin and blood lead was found to be 0.72. Over 95% of the population with a blood lead level of 0.06 mg/100 ml had a protoporphyrin concentration of ≧ 140 μg/100 ml RBC. When a group of children was selected whose blood lead was in equilibrium with bone marrow lead, the correlation coefficient was 0.91. Several micromethods for the detection of lead poisoning are discussed; using a combination of this new protoporphyrin assay together with either the determination of blood lead, the presence of increased eythrocytic osmotic resistance or the measurement of δ-aminolevulinic acid dehydratase, permits a distinction to be made between acute and chronic lead poisoning and/or iron deficiency anemia.

Drug metabolism by the human hepatoma cell, Hep G2

The human liver-derived cell line, Hep G2, has aryl hydrocarbon hydroxylase and 7-ethoxycoumarin o-de-ethylase activities. Partial purification of cytochrome P-450 from Hep G2 cells provided spectral evidence of this hemeprotein in the purified fraction. These results suggest that Hep G2 cells will be useful for the study of cytochrome P-450 and the regulation of mixed function oxidase activities in liver cells of human origin.

Modern diagnosis and management of the porphyrias

Recent advances in the molecular understanding of the porphyrias now offer specific diagnosis and precise definition of the types of genetic mutations involved in the disease. Molecular diagnostic testing is powerful and very useful in kindred evaluation and genetic counselling when a disease‐responsible mutation has been identified in the family. It is also the only way to properly screen asymptomatic gene carriers, facilitating correct treatment and appropriate genetic counselling of family members at risk. However, it should be noted that DNA‐based testing is for the diagnosis of the gene carrier status, but not for the diagnosis of clinical syndrome or severity of the disease, e.g. an acute attack. For the diagnosis of clinically expressed porphyrias, a logical stepwise approach including the analysis of porphyrins and their precursors should not be underestimated, as it is still very useful, and is often the best from the cost‐effective point of view.

Hereditary Tyrosinemia and the Heme Biosynthetic Pathway. PROFOUND INHIBITION OF δ-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY BY SUCCINYLACETONE

Succinylacetone (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumarylacetoacetate hydrolase. It is known that patients with this hereditary disease excrete excessive amounts of delta-aminolevulinic acid (ALA) in urine and that certain patients have an accompanying clinical syndrome resembling that of acute intermittent porphyria (AIP). In order to elucidate the relation of succinylacetone to the heme biosynthetic pathway, we have examined the effects of this metabolite on the cellular heme content of cultured avian hepatocytes and on the activity of purified ALA dehydratase from normal human erythrocytes and from mouse and bovine liver. Our data indicate that succinylacetone is an extremely potent competitive inhibitor of ALA dehydratase in human as well as in animal tissues. By using purified preparations of the enzyme from human erythrocytes and mouse and bovine liver, an inhibitor constant ranging from 2 x 10(-7) M to 3 x 10(-7) M was obtained. In cultured hepatocytes, succinylacetone also inhibited ALA dehydratase activity, decreased the cellular content of heme and cytochrome P-450, and greatly potentiated the induction response of ALA synthase to drugs such as phenobarbital, chemicals such as allylisopropylacetamide and 3,5-dicarbethoxy-1,4-dihydrocollidine, and natural steroids such as etiocholanolone. Four patients with hereditary tyrosinemia have been studied and all were found to have greatly depressed levels of erythrocyte ALA dehydratase activity and elevated concentrations of this inhibitor in urine. These findings indicate that tyrosinemia is a disorder of special pharmacogenetic interest because succinylacetone, an abnormal product of the tyrosine metabolic pathway, resulting from the primary gene defect of the disease, profoundly inhibits heme biosynthesis in normal cells through a blockade at the ALA dehydratase level, leading to clinical and metabolic consequences that mimic another genetic disease, AIP.

Heme deficiency in erythroid lineage causes differentiation arrest and cytoplasmic iron overload

Erythroid 5‐aminolevulinate synthase (ALAS‐E) catalyzes the first step of heme biosynthesis in erythroid cells. Mutation of human ALAS‐E causes the disorder X‐linked sideroblastic anemia. To examine the roles of heme during hematopoiesis, we disrupted the mouse ALAS‐E gene. ALAS‐E‐null embryos showed no hemoglobinized cells and died by embryonic day 11.5, indicating that ALAS‐E is the principal isozyme contributing to erythroid heme biosynthesis. In the ALAS‐E‐null mutant embryos, erythroid differentiation was arrested, and an abnormal hematopoietic cell fraction emerged that accumulated a large amount of iron diffusely in the cytoplasm. In contrast, we found typical ring sideroblasts that accumulated iron mostly in mitochondria in adult mice chimeric for ALAS‐E‐null mutant cells, indicating that the mode of iron accumulation caused by the lack of ALAS‐E is different in primitive and definitive erythroid cells. These results demonstrate that ALAS‐E, and hence heme supply, is necessary for differentiation and iron metabolism of erythroid cells.