Heme Deficiency Research Articles

15-Deoxyspergualin Primarily Targets the Trafficking of Apicoplast Proteins in Plasmodium falciparum

15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2alpha. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process.

The Eukaryotic Initiation Factor-2 Kinase Pathway Facilitates Differential GADD45a Expression in Response to Environmental Stress

Phosphorylation of eukaryotic initiation factor-2 (eIF2) regulates general and gene-specific translation in response to diverse environmental stresses. Central to gene expression induced by eIF2 phosphorylation is the preferential translation of ATF4, a basic zipper transcription activator. Phosphorylation of eIF2 and its attendant induction of ATF4 can lead to different patterns of gene expression depending on the environmental stress. This is of fundamental importance because eIF2 kinases can induce the expression of genes involved in survival as well as in apoptosis. In this report, we explore the molecular basis for why there can be differential expression of GADD45a, a stress-responsive protein that regulates genome stability, apoptosis, and immune responses. We find that whereas ATF4 is required for GADD45a transcription during many different environmental stresses, GADD45a protein accumulates only during a limited number of stress arrangements. The basis for this difference between measurable GADD45a mRNA and protein lies in the observation that GADD45a protein is labile. Those stress agents that enhance ATF4-directed GADD45a transcription and impede the turnover of GADD45a protein by blocking ubiquitin/proteasome-mediated degradation elevate GADD45a protein levels. By comparison, those stress arrangements that trigger ATF4 levels and GADD45a transcription, but do not perturb the proteasome pathway, only elevate GADD45a mRNA levels. This study highlights the molecular mechanisms by which environmental stresses can differentially control central regulatory proteins targeted by the eIF2 kinase pathway.

Biotin deficiency inhibits heme synthesis and impairs mitochondria in human lung fibroblasts.

Four of the 5 biotin-dependent carboxylases (BDC) are in the mitochondria. BDC replace intermediates in the Krebs [tricarboxylic acid (TCA)] cycle that are regularly removed for the synthesis of key metabolites such as heme or amino acids. Heme, unlike amino acids, is not recycled to regenerate these intermediates, is not utilized from the diet, and must be synthesized in situ. We studied whether biotin deficiency (BD) lowers heme synthesis and whether mitochondria would be disrupted. Biotin-deficient medium was prepared by using bovine serum stripped of biotin with charcoal/dextran or avidin. Biotin-deficient primary human lung fibroblasts (IMR90) lost their BDC and senesced before biotin-sufficient cells. BD caused heme deficiency; there was a decrease in heme content and heme synthesis, and biotin-deficient cells selectively lost mitochondrial complex IV, which contains heme-a. Loss of complex IV, which is part of the electron transport chain, triggered oxidant release and oxidative damage, hallmarks of heme deficiency. Restoring biotin to the biotin-deficient medium prevented the above changes. Old cells were more susceptible to biotin shortage than young cells. These findings highlight the biochemical connection among biotin, heme, and iron metabolism, and the mitochondria, due to the role of biotin in maintaining the biochemical integrity of the TCA cycle. The findings are discussed in relation to aging and birth defects in humans.

Heme as a Magnificent Molecule with Multiple Missions: Heme Determines Its Own Fate and Governs Cellular Homeostasis

Heme is a prosthetic group of various types of proteins, such as hemoglobin, myoglobin, cytochrome c, cytochrome p450, catalase and peroxidase. In addition, heme is involved in a variety of biological events by modulating the function or the state of hemoproteins. For example, protein synthesis is inhibited in erythroid cells under heme deficiency, as the consequence of the activation of heme-regulated inhibitor (HRI). Iron concentration in the cell is sensed and regulated by the heme-mediated oxidization and subsequent degradation of iron regulatory protein 2 (IRP2). Heme also binds to certain types of potassium channels, thereby inhibiting transmembrane K(+) currents. Importantly, heme determines its own fate; namely, heme regulates its synthesis and degradation through the feedback mechanisms, by which intracellular heme level is precisely maintained. Heme reduces heme synthesis by suppressing the expression of non-specific 5-aminolevulinate synthase (ALAS1) and stimulates heme breakdown by inducing heme oxygenase (HO)-1 expression. ALAS1 and HO-1 are the rate limiting enzymes in heme biosynthesis and catabolism, respectively. Accordingly, under the heme-rich condition, heme binds to cysteine-proline (CP) motifs of ALAS1 and those of transcriptional repressor Bach1, thereby leading to repression of mitochondrial transport of ALAS1 and induction of HO-1 transcription, respectively. Moreover, chemosensing functions of HO-2 containing CP motifs, another isozyme of HO, have been unveiled recently. In this review article, we summarize and update the pleiotropic effects of heme on various biological events and the regulatory network of heme biosynthesis and catabolism.

HRI Protects Erythroid Precursors in Iron Deficiency and in β-Thalassemia by Maintaining GATA-1 and Fog-1 Expressions.

HRI is the heme-regulated eIF2a kinase that controls the translation of mRNAs in erythroid precursors by phosphorylating the a-subunit of eukaryotic translational initiation factor 2 (eIF2a). HRI is essential for translational regulation of a and b globins and the survival of erythroid precursors in iron and heme deficiency. Furthermore, HRI modifies the severity of β-thalassemic mice that are devoid of the b-globin major gene. To gain insights into the molecular mechanisms by which HRI exert its protection during stress erythropoiesis, micrroarray gene profilings of erythroid precursors were carried out under the conditions of iron deficiency and HRI deficiency. HRI +/+ (Wt) and −/ − (Ko) mice were maintained in an iron deficient diet for 2 months and during gestation to induce iron deficiency in developing embryos. Under these conditions, both Wt and Ko fetal liver cells at E14.5 exhibited impaired erythroid differentiation, increased proliferation and elevated apoptosis, hallmarks of ineffective erythropoiesis. All these parameters were aggravated by HRI deficiency. Furthermore, examinations of embryonic blood smears and fetal liver cytospins revealed the presence of inclusions in reticulocytes of Ko-Fe mice (HRI−/− in iron deficiency). These phenotypes of Ko-Fe in fetal definitive erythropoiesis were the same as those observed earlier in adult erythropoiesis. Hence, mRNAs from E14.5 fetal liver nucleated erythroid precursors were used for mouse expression arrays. In Wt-Fe, expression of 213 genes was altered more than 2-fold. The number of genes altered in Ko-Fe increased dramatically by 15-fold to 3,135. In contrast, the number of the genes changed in Ko+Fe was relatively small (85), consistent with the mild phenotype in Ko+Fe mice. Therefore, HRI is crucial in maintaining proper gene expression in iron deficient erythroid precursors. Large numbers of genes altered in Ko-Fe were involved in cell death, protein synthesis, cancer and the cell cycle, consistent with the phenotypes of elevated apoptosis and increased proliferation in Ko-Fe erythroid precursors. In addition, there were more known erythroid genes (29) and iron/heme homoeostasis genes (12) that were changed in Ko-Fe as compared to Wt-Fe. Most of these genes were targets of GATA-1. When gene profiling of Ko-Fe erythroid precursors was compared with that of G1E-Er4 cells upon induction of GATA-1 by estradiol, there were 892 overlapping genes affected in both cases. GATA-1 mRNA and protein levels in fetal liver cells were reduced in iron deficiency and in b-thalassemia, and were decreased further in HRI deficiencies. Fog-1 mRNA level was not significantly altered in iron deficiency or b-thlassemia alone, but were reduced significantly upon combined deficiencies with HRI. These results demonstrate for the first time the impairment of GATA-1 and Fog-1 expressions in iron deficiency and in b-thalassemia with deletion of the b-globin gene. Furthermore, in iron deficiency and b-thalassemia HRI is necessary to maintain both expressions of GATA-1 and Fog-1, which are essential for definitive erythropoiesis.

Frataxin knockdown causes loss of cytoplasmic iron–sulfur cluster functions, redox alterations and induction of heme transcripts

Frataxin protein deficiency causes the neurodegenerative disease Friedreich ataxia. We used inducible siRNA to order the consequences of frataxin deficiency that we and others have previously observed. The earliest consequence of frataxin deficiency was a defect in cytoplasmic iron–sulfur proteins. In the second phase, protein oxidative damage increased, and CuZnSOD was induced, as was the unfolded protein response (UPR), long before any decline in mitochondrial aconitase activity. In the third phase, mitochondrial aconitase activity declined. And in the fourth phase, coincident with the decrease in heme-containing cytochrome c protein, a transcriptional induction of the heme-dependent transcripts ALAS1 and MAOA occurred. These observations suggest that the earliest consequences of frataxin deficiency occur in ISC proteins of the cytoplasm, resulting in oxidative damage and stress and activation of the unfolded protein response which has been associated with neurological disease, and that later consequences involve mitochondrial iron–sulfur cluster deficiency, heme deficiency, and then increased heme biosynthesis.

Modulation of inhibition of ferrochelatase by N-methylprotoporphyrin

Protoporhyrin IX ferrochelatase catalyses the terminal step of the haem-biosynthetic pathway by inserting ferrous iron into protoporphyrin IX. NMPP (N-methylprotoporphyrin), a transition-state analogue and potent inhibitor of ferrochelatase, is commonly used to induce haem deficiency in mammalian cell cultures. To create ferrochelatase variants with different extents of tolerance towards NMPP and to understand further the mechanism of ferrochelatase inhibition by NMPP, we isolated variants with increased NMPP resistance, bearing mutations in an active-site loop (murine ferrochelatase residues 248-257), which was previously shown to mediate a protein conformational change triggered by porphyrin binding. The kinetic mechanisms of inhibition of two variants, in which Pro255 was replaced with either arginine (P255R) or glycine (P255G), were investigated and compared with that of wild-type ferrochelatase. While the binding affinity of the P255X variants for NMPP decreased by one order of magnitude in relation to that of wild-type enzyme, the inhibition constant increased by approximately two orders of magnitude (K(i)(app) values of 1 microM and 2.3 microM for P255R and P255G respectively, as against 3 nM for wild-type ferrochelatase). Nonetheless, the drastically reduced inhibition of the variants by NMPP was not paralleled with a decrease in specificity constant (kcat/K(m, protoporhyrin IX)) and/or catalytic activity (kcat). Further, although NMPP binding to either wild-type ferrochelatase or P255R occurred via a similar two-step kinetic mechanism, the forward and reverse rate constants associated with the second and rate-limiting step were comparable for the two enzymes. Collectively, these results suggest that Pro255 has a crucial role in maintaining an appropriate protein conformation and modulating the selectivity and/or regiospecificity of ferrochelatase.

P3-366: The complexation of amyloid-β with heme forms a peroxidase: Mechanism of neurotoxicity in Alzheimer’s disease

The molecular mechanism by which Amyloid–β (Aβ) peptide causes the neurodegeneration in Alzheimer's disease (AD) is not known. We previously proposed that excessive Aβ binds to regulatory heme, triggering functional heme deficiency (HD), causing the key cytopathologies of AD including mitochondrial dysfunction, loss of complex IV, iron accumulation, and oxidative stress (Atamna et al. 2004, PNAS). In the current study we searched for additional evidence that Ab binding to regulatory heme is the mechanism by which Aβ causes HD. The experiments included techniques of tissue culture, biochemistry, radiobiology, and HPLC. We found that heme binds to Aβ forming an Aβ–heme complex. We also found that Aβ induces heme synthesis and iron uptake in human neuroblastoma cells, which provides strong support that Aβ triggers HD. Interestingly, we also found that the Aβ–heme complex is a peroxidase, which catalyzes the oxidation of serotonin and DOPA by H2O2. Curcumin, which lowers oxidative damage in the brain in a mouse model for AD, inhibits the Aβ–heme peroxidase. The binding of Aβ to heme supports a unifying mechanism by which elevated levels of Aβ induces HD, causes oxidative damage to macromolecules, and depletes specific neurotransmitters. The relevance of the depletion of regulatory heme by excessive Aβ and Aβ–heme peroxidase to mitochondrial dysfunction, oxidative stress, neurotoxicity and other cytopathologies of AD will be discussed.

Endoplasmic Reticulum Stress Induction of Insulin-like Growth Factor-binding Protein-1 Involves ATF4

Endoplasmic reticulum (ER) stress is sensed by cells in different physiopathological conditions in which there is an accumulation of unfolded proteins in the ER. A coordinated adaptive program called the unfolded protein response is triggered and includes translation inhibition, transcriptional activation of a set of genes encoding mostly intracellular proteins, and ultimately apoptosis. Here we show that insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), a secreted protein that modulates IGF bioavailability and has other IGF-independent effects, is potently induced during ER stress in human hepatocytes. Various ER stress-inducing agents were able to increase IGFBP-1 mRNA levels, as well as cellular and secreted IGFBP-1 protein up to 20-fold. A distal regulatory region of the human IGFBP-1 gene (-6682/-6384) containing an activating transcription factor 4 (ATF4) composite site was required for promoter activation upon ER stress. Mutation of the ATF4 composite site led to the loss of IGFBP-1 regulation. Electrophoretic mobility shift assay revealed an ER stress-inducible complex that was displaced by an ATF4 antibody. Knockdown of ATF4 expression using two specific small interfering RNAs impaired up-regulation of IGFBP-1 mRNA, which highlights the relevance of ATF4 in endogenous IGFBP-1 gene induction. In addition to intracellular proteins involved in secretory and metabolic pathways, we conclude that ER stress induces the synthesis of secreted proteins. Increased secretion of IGFBP-1 during hepatic ER stress may thus constitute a signal to modulate cell growth and metabolism and induce a systemic adaptive response.

Antiviral effect of the mammalian translation initiation factor 2α kinase GCN2 against RNA viruses

In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

Amyloid-β peptide binds with heme to form a peroxidase: Relationship to the cytopathologies of Alzheimer’s disease

Amyloid-beta peptide (Abeta) is the toxic agent in Alzheimer's disease (AD), although the mechanism causing the neurodegeneration is not known. We previously proposed a mechanism in which excessive Abeta binds to regulatory heme, triggering functional heme deficiency (HD), causing the key cytopathologies of AD. We demonstrated that HD triggers the release of oxidants (e.g., H(2)O(2)) from mitochondria due to the loss of complex IV, which contains heme-a. Now we add more evidence that Abeta binding to regulatory heme in vivo is the mechanism by which Abeta causes HD. Heme binds to Abeta, thus preventing Abeta aggregation by forming an Abeta-heme complex in a cell-free system. We suggest that this complex depletes regulatory heme, which would explain the increase in heme synthesis and iron uptake we observe in human neuroblastoma cells. The Abeta-heme complex is shown to be a peroxidase, which catalyzes the oxidation of serotonin and 3,4-dihydroxyphenylalanine by H(2)O(2). Curcumin, which lowers oxidative damage in the brain in a mouse model for AD, inhibits this peroxidase. The binding of Abeta to heme supports a unifying mechanism by which excessive Abeta induces HD, causes oxidative damage to macromolecules, and depletes specific neurotransmitters. The relevance of the binding of regulatory heme with excessive Abeta for mitochondrial dysfunction and neurotoxicity and other cytopathologies of AD is discussed.

Heme Deficiency in Alzheimer’s Disease: A Possible Connection to Porphyria

Mechanisms that cause Alzheimer's disease (AD), an invariably fatal neurodegenerative disease, are unknown. Important recent data indicate that neuronal heme deficiency may contribute to AD pathogenesis. If true, factors that contribute to the intracellular heme deficiency could potentially alter the course of AD. The porphyrias are metabolic disorders characterized by enzyme deficiencies in the heme biosynthetic pathway. We hypothesize that AD may differ significantly in individuals possessing the genetic trait for an acute hepatic porphyria. We elaborate on this hypothesis and briefly review the characteristics of the acute hepatic porphyrias that may be relevant to AD. We note the proximity of genes encoding enzymes of the heme biosynthesis pathway to genetic loci linked to sporadic, late-onset AD. In addition, we suggest that identification of individuals carrying the genetic trait for acute porphyria may provide a unique resource for investigating AD pathogenesis and inform treatment and management decisions.

Heme Deficiency Is Associated with Senescence and Causes Suppression ofN-Methyl-d-aspartate Receptor Subunits Expression in Primary Cortical Neurons

Heme is a crucial component of many pharmacological and toxicological processes, and studies have suggested that heme deficiency may play a role in cellular ageing. A model of ageing neurons was established using prolonged cultures of BALB/c mouse primary cortical neurons. Aged neurons displayed a senescent phenotype and a marked up-regulation of cathepsin-L expression. Down-regulation of the candidate neuron-specific genes for N-methyl-D-aspartate (NMDA) receptor subunits (NMDAzeta1 and -epsilon2) and neurofilament light peptide (NF-L) were found to be characteristic of the aging process as reported in vivo (Brain Res 907:71-83, 2001; Brain Res Mol Brain Res 99:40-45, 2002). In contrast, the genes for the controlling enzymes of heme synthesis and degradation (5-aminolevulinate synthase 1 and heme oxygenase 1, respectively) were up-regulated, implying depletion of a regulatory heme pool. Inhibition of heme synthesis (by 70-80%) at different enzymic steps by succinyl acetone and N-methylprotoporphyrin IX resulted in the earlier lowered expression of NMDAzeta1 and -epsilon2 and NF-L. Exogenous hemin added to heme-depleted cells rescued the expression of these neuron-specific genes. Culture of cortical neurons from BALB/c Fech(m1Pas) mutant mice demonstrating depressed heme synthesis showed premature senescence and reduced expression of NMDAzeta1 and -epsilon2 receptor subunits and NF-L compared with wild-type cells. Our findings suggest that reduced availability of heme in neurons associated with senescence may have significant effects on synaptic function.

Frataxin deficiency alters heme pathway transcripts and decreases mitochondrial heme metabolites in mammalian cells

Deficiency of the frataxin mRNA alters the transcriptome, triggering neuro- and cardiodegeneration in Friedreich's ataxia. We microarrayed murine frataxin-deficient heart tissue, liver tissue and cardiocytes and observed a transcript down-regulation to up-regulation ratio of nearly 2:1 with a mitochondrial localization of transcriptional changes. Combining all mouse and human microarray data for frataxin-deficient cells and tissues, the most consistently decreased transcripts were mitochondrial coproporphyrinogen oxidase (CPOX) of the heme pathway and mature T-cell proliferation 1, a homolog of yeast COX23, which is thought to function as a mitochondrial metallochaperone. Quantitative RT-PCR studies confirmed the significant down-regulation of Isu1, CPOX and ferrochelatase at 10 weeks in mouse hearts. We observed that mutant cells were resistant to aminolevulinate-dependent toxicity, as expected if the heme pathway was inhibited. Consistent with this, we observed increased cellular protoporphyrin IX levels, reduced mitochondrial heme a and heme c levels and reduced activity of cytochrome oxidase, suggesting a defect between protoporphyrin IX and heme a. Fe-chelatase activities were similar in mutants and controls, whereas Zn-chelatase activities were slightly elevated in mutants, supporting the idea of an altered metal-specificity of ferrochelatase. These results suggest that frataxin deficiency causes defects late in the heme pathway. As ataxic symptoms occur in other diseases of heme deficiency, the heme defect we observe in frataxin-deficient cells could be primary to the pathophysiological process.

Interaction of RNA-binding Proteins HuR and AUF1 with the Human ATF3 mRNA 3′-Untranslated Region Regulates Its Amino Acid Limitation-induced Stabilization

ATF3 expression is induced in cells exposed to a variety of stress conditions, including nutrient limitation. Here we demonstrated that the mechanism by which the ATF3 mRNA content is increased following amino acid limitation of human HepG2 hepatoma cells is mRNA stabilization. Analysis of ATF3 mRNA turnover revealed that the half-life was increased from about 1 h in control cells to greater than 8 h in the histidine-deprived state, demonstrating mRNA stabilization in response to nutrient deprivation. Treatment of HepG2 cells with thapsigargin, which causes endoplasmic reticulum stress, also increased the half-life of ATF3 mRNA. HuR is an RNA-binding protein that regulates both the stability and cytoplasmic/nuclear localization of mRNA species containing AU-rich elements. Another RNA-binding protein, AUF1, regulates target mRNA molecules by enhancing their decay. Amino acid limitation caused a slightly elevated mRNA level for HuR and AUF1 mRNA. The nuclear HuR protein content was unchanged, and AUF1 protein increased slightly after amino acid limitation, whereas the cytoplasmic levels of both HuR and AUF1 protein increased. Immunoprecipitation of HuR-RNA complexes followed by reverse transcriptase-PCR analysis showed that HuR interacted with ATF3 mRNA in vivo and that this interaction increased following amino acid limitation. In contrast, the interaction of AUF1 with the ATF3 mRNA is decreased in histidine-deprived cells relative to control cells. Suppression of HuR expression by RNA interference partially blocked the accumulation of ATF3 mRNA following amino acid deprivation. The results demonstrated that coordinated regulation of mRNA stability by HuR and AUF1 proteins contributes to the observed increase in ATF3 expression following amino acid limitation.

Ferric Cycle Activity and Alzheimer Disease

Elevated plasma homocysteine is an independent risk factor for the development of Alzheimer disease, however, the precise mechanisms underlying this are unclear. In this article, we expound on a novel hypothesis depicting the involvement of homocysteine in a vicious circle involving iron dysregulation and oxidative stress designated as the ferric cycle (Dwyer et al., 2004). Moreover, we suspect that the development of a critical heme deficiency in vulnerable neurons is an additional consequence of ferric cycle activity. Oxidative stress and heme deficiency are consistent with many pathological changes found in Alzheimer disease including mitochondrial abnormalities and impaired energy metabolism, cell cycle and cell signaling abnormalities, neuritic pathology, and other features of the disease involving alterations in iron homeostasis such as the abnormal expression of heme oxygenase-1 and iron response protein 2. Based on the ferric cycle concept, we have developed a model of Alzheimer disease development and progression, which offers an explanation for why sporadic Alzheimer disease is different than normal aging and why familial Alzheimer disease and sporadic Alzheimer disease could have different etiologies but a common end-stage.

Heme-regulated eIF2α kinase modifies the phenotypic severity of murine models of erythropoietic protoporphyria and β-thalassemia

Heme-regulated eIF2alpha kinase (HRI) controls protein synthesis by phosphorylating the alpha-subunit of eukaryotic translational initiation factor 2 (eIF2alpha). In heme deficiency, HRI is essential for translational regulation of alpha- and beta-globins and for the survival of erythroid progenitors. HRI is also activated by a number of cytoplasmic stresses other than heme deficiency, including oxidative stress and heat shock. However, to date, HRI has not been implicated in the pathogenesis of any known human disease or mouse phenotype. Here we report the essential role of HRI in 2 mouse models of human rbc disorders, namely erythropoietic protoporphyria (EPP) and beta-thalassemia. In both cases, lack of HRI adversely modifies the phenotype: HRI deficiency exacerbates EPP and renders beta-thalassemia embryonically lethal. This study establishes the protective function of HRI in inherited rbc diseases in mice and suggests that HRI may be a significant modifier of many rbc disorders in humans. Our findings also demonstrate that translational regulation could play a critical role in the clinical manifestation of rbc diseases.

Acute intermittent porphyria

Acute intermittent porphyria (AIP) is characterised by neurovisceral crises the most common clinical presentation of which is abdominal pain. It is an autosomal dominant condition with incomplete penetrance and is potentially life-threatening. The key point in management is to suspect and confirm the diagnosis as early as possible in order to treat the attack and to avoid inappropriate treatments which may exacerbate the crisis. In this chapter we briefly outline the haem biosynthetic pathway and how deficiencies in individual enzymes give rise to the different porphyrias. We then describe the clinical features and diagnosis of AIP, followed by a discussion of pathogenesis, highlighting advances in the molecular biology of AIP and introducing the debate as to whether neurovisceral crises might result from porphyrin precursor neurotoxicity or from haem deficiency. Finally we discuss management, including family screening, avoidance of triggering factors, analgesia, maintenance of a high calorie intake, and administration of haem derivatives.

Heme deficiency suppresses the expression of key neuronal genes and causes neuronal cell death

Defective heme synthesis may cause acute porphyrias, which are associated with a wide array of neurological disturbances involving both the central and peripheral nervous systems. Thus, the understanding of the roles of heme in neuronal cell function may provide insights into the molecular events underlying the pathogenesis of neuropathies associated with defective heme synthesis. In this report, we use rat pheochromocytoma (PC12) clonal cells as a model system for studying the role of heme in neuronal cell survival. We examined the effects of inhibition of heme synthesis on signaling pathways and gene expression in nerve growth factor (NGF)-induced PC12 cells. We found that succinyl acetone-induced heme deficiency selectively caused apoptosis in NGF-induced PC12 cells. Further, we found that in succinyl acetone-treated, NGF-induced cells, the pro-survival Ras-ERK1/2 signaling pathway was inactivated and the pro-apoptotic JNK signaling pathway was activated. In these cells, the activation of caspase and the cleavage of nuclear poly (ADP-ribose) polymerase (PARP) were also evident. Importantly, microarray gene expression analysis showed that more than 20 key neuronal genes that were induced by NGF were suppressed by succinyl acetone. These genes include those encoding survival motor neuron protein, synaptic vesicle protein SVOP, and neural cell adhesion molecule NCAM. These results indicate that heme is important for neuronal cell signaling and the proper functioning of neuronal cells.

Cytosolic persistence of mouse brain CYP1A1 in chronic heme deficiency

Previous work has demonstrated that the function of extrahepatic cytochrome P450 CYP1A1 is dependent on the availability of heme. CYP1A1 is involved in the activation of polyaromatic hydrocarbons. In the present study we used a transgenic mouse model with chronic impairment of heme synthesis - female porphobilinogen deaminase-deficient (PBGD-/-) mice - to investigate the effects of limited heme in untreated and beta-naphthoflavone (beta-NF)-treated animals on the function of CYP1A1 in brain. The heme content of PBGD-/- mice was diminished in the liver and brain compared to wild types. In the liver, partial heme deficiency led to less potent induction of CYP1A1 mRNA after beta-NF treatment. In the brain, CYP1A1 protein was detected not only at the endoplasmic reticulum (ER), but also in the cytosol of PBGD-/- mice. Furthermore, 7-deethylation of ethoxyresorufin, an indicator of CYP1A1 metabolic activity, could be restored by heme in cytosol of PBGD-/- mouse brain. Independent of the genotype, we found only one cyp1a1 gene product, indicating that the cytosolic appearance of CYP1A1 most likely did not originate from mutant alleles. We conclude that heme deficiency in the brain leads to incomplete heme saturation of CYP1A1, which causes its improper incorporation into the ER membrane and persistence in the cytosol. It is suggested that diseases caused by relative heme deficiency, such as hepatic porphyrias, may lead to impaired hemoprotein function in brain.