The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD-containing PAS domain. Here, we randomly mutagenized the region encoding residues 14-119 of the PAS domain and found 72 aerotaxis-defective mutants, 24 of which were gain-of-function, signal-on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS-FAD domain in NifL from Azotobacter vinlandii. Signal-on lesions clustered in the FAD binding pocket, the beta-scaffolding and in the N-cap loop. We suggest that the signal-on lesions mimic the 'signal-on' state of the PAS domain, and therefore may be markers for the signal-in and signal-out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the beta-scaffolding and the N-cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS beta-scaffold and residue Q248C in the HAMP AS-2 helix.

The Bacillus subtilis ResD-ResE two-component regulatory system activates genes involved in nitrate respiration in response to oxygen limitation or nitric oxide (NO). The sensor kinase ResE activates the response regulator ResD through phosphorylation, which then binds to the regulatory region of genes involved in anaerobiosis to activate their transcription. ResE is composed of an N-terminal signal input domain and a C-terminal catalytic domain. The N-terminal domain contains two transmembrane subdomains and a large extracytoplasmic loop. It also has a cytoplasmic PAS subdomain between the HAMP linker and C-terminal kinase domain. In an attempt to identify the signal-sensing subdomain of ResE, a series of deletions and amino acid substitutions were generated in the N-terminal domain. The results indicated that cytoplasmic ResE lacking the transmembrane segments and the extracytoplasmic loop retains the ability to sense oxygen limitation and NO, which leads to transcriptional activation of ResDE-dependent genes. This activity was eliminated by the deletion of the PAS subdomain, demonstrating that the PAS subdomain participates in signal reception. The study also raised the possibility that the extracytoplasmic region may serve as a second signal-sensing subdomain. This suggests that the extracytoplasmic region could contribute to amplification of ResE activity leading to the robust activation of genes required for anaerobic metabolism in B. subtilis.

Signal transduction systems comprising histidine kinases are suggested as new molecular targets of antibiotics. The important human fungal pathogen Candida albicans possesses three histidine kinases, one of which is the type III histidine kinase CaNik1, which activates the MAP kinase Hog1. We established a screening system for inhibitors of this class of histidine kinases by functional expression of the CaNIK1 gene in S. cerevisiae. This transformant was susceptible to fungicides to which the wild type strain was resistant, such as fludioxonil and ambruticin. Growth inhibition correlated with phosphorylation of Hog1 and was dependent on an intact Hog1 pathway. At the N-terminus the histidine kinase CaNik1 has four amino acid repeats of 92 amino acids each and one truncated repeat of 72 amino acids. Within these repeats we identified 9 HAMP domains with a paired structure. We constructed mutants in which one or two pairs of these domains were deleted. S. cerevisiae transformants expressing the full-length CaNIK1 showed the highest sensitivity to the fungicides, any truncation reduced the susceptibility of the transformants to the fungicides. This indicates that the HAMP domains are decisive for the mode of action of the antifungal compounds.

Macrophages are responsible for the control of inflammation and healing, and their malfunction results in cardiometabolic disorders. TGF-β is a pleiotropic growth factor with dual (protective and detrimental) roles in atherogenesis. We have previously shown that in human macrophages, TGF-β1 activates Smad2/3 signaling and induces a complex gene expression program. However, activated genes were not limited to known Smad2/3-dependent ones, which prompted us to study TGF-β1-induced signaling in macrophages in detail. Analysis of Id3 regulatory sequences revealed a novel enhancer, located between +4517 and 4662 bp, but the luciferase reporter assay demonstrated that this enhancer is not Smad2/3 dependent. Because Id3 expression is regulated by Smad1/5 in endothelial cells, we analyzed activation of Smad1/5 in macrophages. We demonstrate here for the first time, to our knowledge, that TGF-β1, but not BMPs, activates Smad1/5 in macrophages. We show that an ALK5/ALK1 heterodimer is responsible for the induction of Smad1/5 signaling by TGF-β1 in mature human macrophages. Activation of Smad1/5 by TGF-β1 induces not only Id3, but also HAMP and PLAUR, which contribute to atherosclerotic plaque vulnerability. We suggest that the balance between Smad1/5- and Smad2/3-dependent signaling defines the outcome of the effect of TGF-β on atherosclerosis where Smad1/5 is responsible for proatherogenic effects, whereas Smad2/3 regulate atheroprotective effects of TGF-β.

Iron is required for most forms of organisms, and it is the most essential element for the functions of many iron-containing proteins involved in oxygen transport, cellular respiration, DNA replication, and so on. Disorders of iron metabolism are associated with diverse diseases, including anemias (e.g., iron-deficiency anemia and anemia of chronic diseases) and iron overload diseases, such as hereditary hemochromatosis and β-thalassemia. Hepcidin (encoded by Hamp gene) is a peptide hormone synthesized by hepatocytes, and it plays an important role in regulating the systematic iron homeostasis. As the systemic iron regulator, hepcidin, not only controls dietary iron absorption and iron egress out of iron storage cells, but also induces iron redistribution in various organs. Deregulated hepcidin is often seen in a variety of iron-related diseases including anemias and iron overload disorders. In the case of iron overload disorders (e.g., hereditary hemochromatosis and β-thalassemia), hepatic hepcidin concentration is significantly reduced.Since hepcidin deregulation is responsible for iron disorder-associated diseases, the purpose of this review is to summarize the recent findings on therapeutics targeting hepcidin.Continuous efforts have been made to search for hepcidin mimics and chemical compounds that could be used to increase hepcidin level. Here, a literature search was conducted in PubMed, and research papers relevant to hepcidin regulation or hepcidin-centered therapeutic work were reviewed. On the basis of literature search, we recapitulated recent findings on therapeutic studies targeting hepcidin, including agonists and antagonists to modulate hepcidin expression or its downstream signaling. We also discussed the molecular mechanisms by which hepcidin level and iron metabolism are modulated.Elevating hepcidin concentration is an optimal strategy to ameliorate iron overload diseases, and also to relieve β-thalassemia phenotypes by improving ineffective erythropoiesis. Relative to the current conventional therapies, such as phlebotomy and blood transfusion, therapeutics targeting hepcidin would open a new avenue for treatment of iron-related diseases.

HAMP domains, found in many bacterial signal transduction proteins, generally transmit an intramolecular signal between an extracellular sensory domain and an intracellular signaling domain. Studies of HAMP domains in proteins where both the input and output signals occur intracellularly are limited to those of the Aer energy taxis receptor of Escherichia coli, which has both a HAMP domain and a sensory PAS domain. Campylobacter jejuni has an energy taxis system consisting of the domains of Aer divided between two proteins, CetA (HAMP domain containing) and CetB (PAS domain containing). In this study, we found that the CetA HAMP domain differs significantly from that of Aer in the predicted secondary structure. Using similarity searches, we identified 55 pairs of HAMP/PAS proteins encoded by adjacent genes in a diverse group of microorganisms. We propose that these HAMP/PAS pairs form a new family of bipartite energy taxis receptors. Within these proteins, we identified nine residues in the HAMP domain and proximal signaling domain that are highly conserved, at least three of which are required for CetA function. Additionally, we demonstrated that CetA contributes to the invasion of human epithelial cells by C. jejuni, while CetB does not. This finding supports the hypothesis that members of HAMP/PAS pairs possess the capacity to act independently of each other in cellular traits other than energy taxis.

Intestinal iron absorption is extremely high in neonatal mammals but falls rapidly to adult levels following weaning. The aim of this study was to investigate the molecular basis of this elevated neonatal absorption using the rat as an experimental model. RNA was extracted from various sections of the intestine of 10-, 15-, 20-, 25-, and 300-day-old rats and the expression of the genes encoding DMT1 (Slc11a2), ferroportin (Slc40a1), Cybrd1 (Cybrd1), and hephaestin (heph) determined by ribonuclease protection assay. The hepatic expression of Hamp was studied at the same ages. Iron absorption was examined by following (59)Fe uptake in both whole animals and in isolated intestinal loops. Slc11a2, Slc40a1, and Cybrd1 mRNAs were highly expressed in all regions of the small intestine and colon studied in suckling rats. However, after weaning, when iron absorption declined significantly, strong expression was retained only in the duodenum. No change in hephaestin mRNA occurred in any part of the digestive tract. In the distal small intestine and colon, Slc40a1 expression most closely followed the change in absorption that occurred after weaning. Hamp expression was low during the neonatal period and increased to adult levels following weaning. Our results suggest that the distal small intestine and colon contribute significantly to the high intestinal iron absorption seen in neonatal animals and that this reflects increased expression of the iron transporters, particularly Slc40a1.

BACKGROUND

Hemojuvelin (Hjv) is a key component of the signaling cascade that regulates liver hepcidin (Hamp) expression. The purpose of this study was to determine Hjv protein levels in mice and rats subjected to iron overload and iron deficiency.

METHODS

C57BL/6 mice were injected with iron (200 mg/kg); iron deficiency was induced by feeding of an iron-deficient diet, or by repeated phlebotomies. Erythropoietin (EPO)-treated mice were administered recombinant EPO at 50 U/mouse. Wistar rats were injected with iron (1200 mg/kg), or fed an iron-deficient diet. Hjv protein was determined by immunoblotting, liver samples from Hjv-/- mice were used as negative controls. Mouse plasma Hjv content was determined by a commercial ELISA kit.

RESULTS

Liver crude membrane fraction from both mice and rats displayed a major Hjv-specific band at 35 kDa, and a weaker band of 20 kDa. In mice, the intensity of these bands was not changed following iron injection, repeated bleeding, low iron diet or EPO administration. No change in liver crude membrane Hjv protein was observed in iron-treated or iron-deficient rats. ELISA assay for mouse plasma Hjv did not show significant difference between Hjv+/+ and Hjv-/- mice. Liver Hamp mRNA, Bmp6 mRNA and Id1 mRNA displayed the expected response to iron overload and iron deficiency. EPO treatment decreased Id1 mRNA, suggesting possible participation of the bone morphogenetic protein pathway in EPO-mediated downregulation of Hamp mRNA.

CONCLUSIONS

Since no differences between Hjv protein levels were found following various experimental manipulations of body iron status, the results indicate that, in vivo, substantial changes in Hamp mRNA can occur without noticeable changes of membrane hemojuvelin content. Therefore, modulation of hemojuvelin protein content apparently does not represent the limiting step in the control of Hamp gene expression.

Recycled iron from reticuloendothelial macrophages to erythroid precursors is important to maintain the iron homeostasis. However, the molecular mechanisms underlying iron homeostasis in macrophages are poorly understood. In this study, male Sprague-Dawley rats were treated with recombinant human erythropoietin (rHuEpo, 500 IU/day, s.c.) for 3 days. At the fifth day, peritoneal exudate macrophages were harvested, and then (55)Fe uptake and release were measured by liquid scintillation counting method. The expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in peritoneal exudate macrophages was detected by RT-PCR and Western blot. In order to exclude the direct effect of rHuEpo on macrophages, the parallel experiments were performed with incubation normal peritoneal exudate macrophages with rHuEpo (2 IU/ml). Our results showed rHuEpo injection reduced the peritoneal exudate macrophages iron retention. The uptake of Fe(II) was decreased via the suppression of DMT1 (+IRE) expression and the release of Fe(II) was increased with increasing the expression of FPN1 in macrophages. Moreover, the expression of HAMP mRNA was four times lower in rHuEpo-treated liver of rats than control group (CG). HAMP mRNA expression was increased; the synthesis of DMT1 had no significant change, whereas the FPN1 was decreased in normal peritoneal exudate macrophages after treatment with rHuEpo in vitro. We conclude that hepcidin may play a major, causative role in the change of FPN1 synthesis and that decreased the iron retention in macrophages of rHuEpo-treated rats.

HAMP domains are sensory transduction modules that connect input and output domains in diverse signaling proteins from archaea, bacteria, and lower eukaryotes. Here, we employed in vivo disulfide cross-linking to explore the structure of the HAMP domain in the Escherichia coli aerotaxis receptor Aer. Using an Aer HAMP model based on the structure of Archaeoglobus fulgidus Af1503-HAMP, the closest residue pairs at the interface of the HAMP AS-1 and AS-2' helices were determined and then replaced with cysteines and cross-linked in vivo. Except for a unique discontinuity in AS-2, the data suggest that the Aer HAMP domain forms a parallel four-helix bundle that is similar to the structure of Af1503. The HAMP discontinuity was associated with a segment of AS-2 that was recently shown to interact with the Aer-PAS sensing domain. The four-helix HAMP bundle and its discontinuity were maintained in both the kinase-on and kinase-off states of Aer, although differences in the rates of disulfide formation also indicated the existence of different HAMP conformations in the kinase-on and kinase-off states. In particular, the kinase-on state was accompanied by significantly increased disulfide formation rates at the distal end of the HAMP four-helix bundle. This indicates that HAMP signaling may be associated with a tilting of the AS-1 and AS-2' helices, which may be the signal that is transmitted to the kinase control region of Aer.

Congenital portosystemic shunts are developmental anomalies of the splanchnic vascular system that cause portal blood to bypass the liver. Large-breed dogs are predisposed for intrahepatic portosystemic shunts (IHPSS) and small-breed dogs for extrahepatic portosystemic shunts (EHPSS). While the phenotype resulting from portal bypass of the liver of the two types of shunt is identical, the genotype and molecular pathways involved are probably different. The aim of this study was to gain insight into the pathways involved in the different types of portosystemic shunting. Microarray analysis of mRNA expression in liver tissue from dogs with EHPSS and IHPSS revealed that the expression of 26 genes was altered in either IHPSS or EHPSS samples compared with that in liver samples from control dogs. Quantitative real-time PCR of these genes in 14 IHPSS, 17 EHPSS, and 8 control liver samples revealed a significant differential expression of ACBP, CCBL1, GPC3, HAMP, PALLD, VCAM1, and WEE1. Immunohistochemistry and Western blotting confirmed an increased expression of VCAM1 in IHPSS but its absence in EHPSS, an increased WEE1 expression in IHPSS but not in EHPSS, and a decreased expression of CCBL1 in both shunt types. Regarding their physiologic functions, these findings may indicate a causative role for VCAM1 in EHPSS [corrected] and WEE1 for IHPSS. CCBL1 could be an interesting candidate to study not yet elucidated aspects in the pathophysiology of hepatic encephalopathy.

Because genes that are highly expressed in the cochlea after noise stress may have crucial regulatory roles in hearing, the identification of these genes may be useful for restoring normal auditory function. This study assessed altered gene expression at 1h following the cessation of noise exposure by using microarrays and real-time polymerase chain reaction (qPCR) in rats. In addition, the auditory threshold shifts and morphological changes of hair cells were observed. This study indicated that applied noise induced outer hair cell loss and a 40-50 dB hearing loss. Totally 239 altered genes were involved in the immune system process, response to stress, or response to stimulus. The expression of five up-regulated genes (Reg3b, Lcn2, Serpina3n, Nob1 and Hamp) was confirmed by qPCR. Future experiments will focus on several of these new candidate genes and may provide insight into the underlying auditory pathophysiology.

HAMP domains, ∼55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function.

Growth delay is common in children with chronic kidney disease (CKD), often associated with poor quality of life. The role of anemia in uremic growth delay is poorly understood. Here we describe an induction of uremic growth retardation by a 0.2% adenine diet in wild-type (WT) and hepcidin gene (Hamp) knockout (KO) mice, compared with their respective littermates fed a regular diet. Experiments were started at weaning (3 wk). After 8 wk, blood was collected and mice were euthanized. Adenine-fed WT mice developed CKD (blood urea nitrogen 82.8 ± 11.6 mg/dl and creatinine 0.57 ± 0.07 mg/dl) and were 2.1 cm shorter compared with WT controls. WT adenine-fed mice were anemic and had low serum iron, elevated Hamp, and elevated IL6 and TNF-α. WT adenine-fed mice had advanced mineral bone disease (serum phosphorus 16.9 ± 3.1 mg/dl and FGF23 204.0 ± 115.0 ng/ml) with loss of cortical and trabecular bone volume seen on microcomputed tomography. Hamp disruption rescued the anemia phenotype resulting in improved growth rate in mice with CKD, thus providing direct experimental evidence of the relationship between Hamp pathway and growth impairment in CKD. Hamp disruption ameliorated CKD-induced growth hormone-insulin-like growth factor 1 axis derangements and growth plate alterations. Disruption of Hamp did not mitigate the development of uremia, inflammation, and mineral and bone disease in this model. Taken together, these results indicate that an adenine diet can be successfully used to study growth in mice with CKD. Hepcidin appears to be related to pathways of growth retardation in CKD suggesting that investigation of hepcidin-lowering therapies in juvenile CKD is warranted.

BACKGROUND

Iron overload syndromes include a wide spectrum of genetic and acquired conditions. Recent studies suggest suppressed hepcidin synthesis in the liver to be the molecular basis of hemochromatosis. However, a liver with acquired iron overload synthesizes an adequate amount of hepcidin. Thus, hepcidin could function as a biochemical marker for differential diagnosis of iron overload syndromes.

METHODS

We measured serum iron parameters and hepcidin-25 levels followed by sequencing HFE, HJV, HAMP, TFR2, and SLC40A1 genes in 13 Japanese patients with iron overload syndromes. In addition, we performed direct measurement of serum hepcidin-25 levels using liquid chromatography-tandem mass spectrometry in 3 Japanese patients with aceruloplasminemia and 4 Italians with HFE hemochromatosis.

RESULTS

One patient with HJV hemochromatosis, 2 with TFR2 hemochromatosis, and 3 with ferroportin disease were found among the 13 Japanese patients. The remaining 7 Japanese patients showed no evidence for genetic basis of iron overload syndrome. As far as the serum hepcidin-25 was concerned, seven patients with hemochromatosis and 3 with aceruloplasminemia showed markedly decreased serum hepcidin-25 levels. In contrast, 3 patients with ferroportin disease and 7 with secondary iron overload syndromes showed serum hepcidin levels parallel to their hyperferritinemia. Patients with iron overload syndromes were divided into 2 phenotypes presenting as low and high hepcidinemia. These were then associated with their genotypes.

CONCLUSIONS

Determining serum hepcidin-25 levels may aid differential diagnosis of iron overload syndromes prior to genetic analysis.

The 50-residue HAMP domain converts input signal into output response in a variety of transmembrane signal transduction proteins, including methyl-accepting chemotaxis proteins and histidine kinases. HAMP domains are present in many other contexts as well. Despite focused study over the past decade, the question remains: How does this small domain play such a large role for so many different proteins? Analysis of structural models for the Afl1503 and Aer2 HAMP domains has generated hypotheses in which the HAMP domain assumes either of two discrete forms that generate opposing signal output. In contrast, genetic analysis of the HAMP domain from the Tsr methyl-accepting chemotaxis protein resulted in a distinct hypothesis, the biphasic dynamic bundle. In this hypothesis, signalling involves differential packing stabilities of the HAMP domain four-helix bundle, marked by at least three distinct states. Here I summarize and compare these hypotheses in the context of a deletion analysis that further explores the biphasic dynamic bundle hypothesis.

In this study, the effects of iron depletion and repletion on biochemical and molecular indices of iron status were investigated in growing male Wistar rats. We hypothesized that iron from Moringa leaves could overcome the effects of iron deficiency and modulate the expression of iron-responsive genes better than conventional iron supplements. Iron deficiency was induced by feeding rats an iron-deficient diet for 10 weeks, whereas control rats were maintained on an iron-sufficient diet (35.0-mg Fe/kg diet). After the depletion period, animals were repleted with different source of iron, in combination with ascorbic acid. Iron deficiency caused a significant (P < .05) decrease in serum iron and ferritin levels by 57% and 40%, respectively, as compared with nondepleted control animals. Significant changes in the expression (0.5- to100-fold) of liver hepcidin (HAMP), transferrin, transferrin receptor-2, hemochromatosis type 2, ferroportin 1, ceruloplasmin, and ferritin-H were recorded in iron-depleted and iron-repleted rats, as compared with nondepleted rats (P < .05). Dietary iron from Moringa leaf was found to be superior compared with ferric citrate in overcoming the effects of iron deficiency in rats. These results suggest that changes in the relative expression of liver hepcidin messenger RNA can be used as a sensitive molecular marker for iron deficiency.

Transfusion-dependent myelodysplastic (MDS) patients are prone to iron overload. We evaluated 43 transfused MDS patients with T2* magnetic resonance imaging scans. 81% had liver and 16·8% cardiac iron overload. Liver R2* (1000/T2*), but not cardiac R2*, was correlated with number of units transfused (r=0·72, P<0·0001) and ferritin (r=0·53, P<0·0001). The area under the curve of a time-ferritin plot was found to be much greater in patients with cardiac iron loading (median 53·7x10(5) Megaunits vs. 12·2x10(5) Megaunits, P=0·002). HFE, HFE2, HAMP or SLC40A1 genotypes were not predictors of iron overload in these patients.

Plants perceive injury and herbivore attack via the recognition of damage-associated molecular patterns (DAMPs) and herbivore-associated molecular patterns (HAMPs). Although HAMPs in particular are cues that can indicate the presence of a specific enemy, the application of pure DAMPs or HAMPs frequently activates general downstream responses: membrane depolarization, Ca(2+) influxes, oxidative stress, MAPKinase activation and octadecanoid signaling at the molecular level, and the expression of digestion inhibitors, cell wall modifications and other general defenses at the phenotypic level. We discuss the relative benefits of perceiving the non-self versus the damaged-self and of specific versus non-specific responses and suggest that the perception of a complex mixture of DAMPs and HAMPs triggers fine-tuned plant responses. DAMPs such as extracellular ATP (eATP), cell wall fragments, signaling peptides, herbivore-induced volatile organic compounds (HI-VOCs) and eDNA hold the key for a more complete understanding of how plants perceive that and by whom they are attacked.

Archaeal photoreceptors, together with their cognate transducer proteins, mediate phototaxis by regulating cell motility through two-component signal transduction pathways. This sensory pathway is closely related to the bacterial chemotactic system, which has been studied in detail during the past 40 years. Structural and functional studies applying site-directed spin labelling and electron paramagnetic resonance spectroscopy on the sensory rhodopsin II/transducer (NpSRII/NpHtrII) complex of Natronomonas pharaonis have yielded insights into the structure, the mechanisms of signal perception, the signal transduction across the membrane and provided information about the subsequent information transfer within the transducer protein towards the components of the intracellular signalling pathway. Here, we provide an overview about the findings of the last decade, which, combined with the wealth of data from research on the Escherichia coli chemotaxis system, served to understand the basic principles microorganisms use to adapt to their environment. We document the time course of a signal being perceived at the membrane, transferred across the membrane and, for the first time, how this signal modulates the dynamic properties of a HAMP domain, a ubiquitous signal transduction module found in various protein classes.

Elucidation of the molecular pathways of iron transport through cells and its control is leading to an understanding of genetic iron loading conditions. The general phenotype of haemochromatosis is iron accumulation in liver parenchymal cells, a raised serum transferrin saturation and ferritin concentration. Four types have been identified: type 1 is the common form and is an autosomal recessive disorder of low penetrance strongly associated with mutations in the HFE gene on chromosome 6(p21.3); type 2 (juvenile haemochromatosis) is autosomal recessive, of high penetrance with causative mutations identified in the HFE2 gene on chromosome 1 (q21) and the HAMP gene on chromosome 19 (q13); type 3 is also autosomal recessive with mutations in the TfR2 gene on chromosome 3 (7q22); type 4 is an autosomal dominant condition with heterozygous mutations in the ferroportin 1 gene. In type 4, iron accumulates in both parenchymal and reticuloendothelial cells and the transferrin saturation may be normal. There are also inherited neurodegenerative conditions associated with iron accumulation. The current research challenges include understanding the central role of the HAMP gene (hepcidin) in controlling iron absorption and the reasons for the variable penetrance in HFE type 1.

NT-26 is a chemolithoautotrophic arsenite oxidizer. Understanding the mechanisms of arsenite signalling, tolerance and oxidation by NT-26 will have significant implications for its use in bioremediation and arsenite sensing. We have identified the histidine kinase (AroS) and the cognate response regulator (AroR) involved in the arsenite-dependent transcriptional regulation of the arsenite oxidase aroBA operon. AroS contains a single periplasmic sensory domain that is linked through transmembrane helices to the HAMP domain that transmits the signal to the kinase core of the protein. AroR belongs to a family of AAA+ transcription regulators that interact with DNA through a helix-turn-helix domain. The presence of the AAA+ domain as well as the RNA polymerase σ(54) -interaction sequence motif suggests that this protein regulates transcription through interaction with RNA polymerase in a σ(54) -dependent fashion. The kinase core of AroS and the receiver domain of AroR were heterologously expressed and purified and their autophosphorylation and transphosphorylation activities were confirmed. Using site-directed mutagenesis, we have identified the phosphorylation sites on both proteins. Mutational analysis in NT-26 confirmed that both proteins are essential for arsenite oxidation and the AroS mutant affected growth with arsenite, also implicating it in the regulation of arsenite tolerance. Lastly, arsenite sensing does not appear to involve thiol chemistry.

OBJECTIVE

Hereditary haemochromatosis is a disease that affects iron metabolism and leads to iron overload. Homozygosity for the H63D mutation is associated with increased transferrin saturation (TS) and ferritin levels. Our objective was to find out if the homozygosity of H63D mutation was the primary cause of iron overload.

METHODS

We studied 45 H63D homozygotes (31 males and 14 females) with biochemical iron overload and/or clinical features of haemochromatosis. The simultaneous detection of 18 known HFE, TFR2 and FPN1 mutations and sequencing of the HAMP gene were performed to rule out the possible existence of genetic modifier factors related with iron overload.

RESULTS

Values of biochemical iron overload, measured as percentage TS and serum ferritin concentration (SF), in our H63D homozygotes were significantly higher in patients than in controls: TS 55 +/- 15% vs. 35 +/- 15% and SF 764 (645-883) microg/L vs. 115 (108-123) microg/L for patients and controls, respectively. These H63D homozygotes presented extreme hyperferritinaemia and no additional mutations in HFE, TFR2, FPN1 and HAMP genes were detected.

CONCLUSIONS

The lack of additional mutations in our H63D homozygotes suggests that this genotype could be the primary cause of iron overload in these patients. Despite our results, we cannot entirely discount the possibility that one or more genetic modifier factor exists, simply because we were unable to find it, although there was a precedent in the HFE gene. Genetic modifier factors have been described for C282Y mutations in the HFE gene, but at the present time they have never been reported in H63D homozygotes.