ORCID Profile
0000-0002-4583-7790
Current Organisation
Queensland University of Technology
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Medical Biotechnology | Logistics and Supply Chain Management | Biomaterials | Regenerative Medicine (incl. Stem Cells and Tissue Engineering) |
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Publisher: Elsevier BV
Date: 07-2021
Publisher: Elsevier BV
Date: 2000
DOI: 10.1016/S0168-8278(00)80415-8
Abstract: Hereditary haemochromatosis (HHC) is a common inherited disorder of iron metabolism characterised by progressive iron loading of parenchymal cells of the liver, pancreas, heart and other organs ultimately leading to cirrhosis and organ failure. Despite HLA studies which localised the defective gene to the short arm of chromosome 6, the haemochromatosis gene remained elusive until 1996, when the gene was identified by a massive positional cloning effort. The haemochromatosis gene (HFE) encodes a novel nonclassical MHC class-1-like molecule. Two missense mutations have been identified in patients with HHC, a G to A at nucleotide 845, resulting in a substitution of tyrosine for cysteine at amino acid 282 (referred to as the C282Y mutation) and a C to G at nucleotide 187, resulting in a substitution of aspartate for histidine at amino acid 63 (H63D). An average of 85-90% of patients with typical clinical features of HHC are homozygous for the C282Y mutation. H63D is not associated with the same degree of iron loading as C282Y. Clinical expression is variable depending on environmental (dietary) iron, physiological and pathological blood loss and as yet unidentified modifying genetic factors. One recent Australian study indicates that only about 50% of homozygous subjects are fully expressing and symptomatic and that about 30% show no clinical or biochemical expression. Genetic tests for identifying mutations in the HFE gene provide precise means for diagnosis, family testing and population screening and have led to re-evaluation of the indications for liver biopsy in this disease. At the present time, however, the most practical and cost-effective method of screening is for phenotypic expression by transferrin saturation or unsaturated iron binding capacity measurement. In the future, population screening by genotype should be feasible once the relevant technical, legal and ethical issues are resolved.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 12-10-2009
DOI: 10.1002/HEP.23198
Abstract: Hepcidin is a central regulator of iron homeostasis. HFE and transferrin receptor 2 (TFR2) are mutated in adult-onset forms of hereditary hemochromatosis and regulate the expression of hepcidin in response to iron. Whether they act through the same or parallel pathways is unclear. To investigate this, we generated a mouse model with deletion of both Hfe and Tfr2 genes by crossing Hfe and Tfr2 null mice on a genetically identical background. Tissue and serum from wildtype, single-, and double-null mice were analyzed. Serum transferrin saturation and hepatic iron concentrations were determined. The expression of iron-related messenger RNA (mRNA) transcripts was analyzed by real-time polymerase chain reaction (PCR). Levels of the iron-related proteins Tfr1, Tfr2, ferritin, and prohepcidin, and the phosphorylation status of the cell signaling proteins extracellular signal-regulated kinase 1/2 (Erk1/2) and Smad1/5/8, were analyzed by immunoblotting. Double-null mice had more severe iron loading than mice lacking either Hfe or Tfr2 Tfr2 null mice had a greater iron burden than Hfe-null mice. Hepcidin expression relative to iron stores was reduced in the Hfe-null mice, with significantly lower values in the Tfr2-null mice. In the absence of both Hfe and Tfr2, hepcidin expression was reduced even further. A significant decrease in phospho-Erk1/2 in the livers of null mice and a reduction in phospho-Smad1/5/8 suggest that both the mitogen-activated protein kinase (MAPK) and bone morphogenetic protein / mothers against decapentaplegic homolog (BMP/SMAD) signaling pathways may be involved in Hfe- and Tfr2-mediated regulation of hepcidin. These studies demonstrate that iron overload due to deletion of Tfr2 is more severe than that due to Hfe, and that loss of both molecules results in pronounced iron overload. Analysis of Hfe/Tfr2 double-null mice suggests that Hfe and Tfr2 regulate hepcidin through parallel pathways involving Erk1/2 and Smad1/5/8.
Publisher: American Medical Association (AMA)
Date: 13-02-2006
DOI: 10.1001/ARCHINTE.166.3.294
Abstract: Hemochromatosis in white subjects is mostly due to homozygosity for the common C282Y substitution in HFE. Although clinical symptoms are preventable by early detection of the genetic predisposition and prophylactic treatment, population screening is not currently advocated because of the discrepancy between the common mutation prevalence and apparently lower frequency of clinical disease. This study compared screening for hemochromatosis in subjects with or without a family history. We assessed disease expression by clinical evaluation and liver biopsy in 672 essentially asymptomatic C282Y homozygous subjects identified by either family screening or health checks. We also observed a subgroup of untreated homozygotes with normal serum ferritin levels for up to 24 years. Prevalence of hepatic iron overload and fibrosis were comparable between the 2 groups. Disease-related conditions were more common in male subjects identified by health checks, but they were older. Hepatic iron overload (grades 2-4) was present in 56% and 34.5% of male and female subjects, respectively hepatic fibrosis (stages 2-4) in 18.4% and 5.4% and cirrhosis in 5.6% and 1.9%. Hepatic fibrosis and cirrhosis correlated significantly with the hepatic iron concentration, and except in cases of cirrhosis, there was a 7.5-fold reduction in the mean fibrosis score after phlebotomy. All subjects with cirrhosis were asymptomatic. Screening for hemochromatosis in apparently healthy subjects homozygous for the C282Y mutation with or without a family history reveals comparable levels of hepatic iron overload and disease. Significant hepatic fibrosis is frequently found in asymptomatic subjects with hemochromatosis and, except when cirrhosis is present, is reversed by iron removal.
Publisher: Oxford University Press (OUP)
Date: 28-02-2014
Abstract: We investigated the relationship between microbial translocation, immune activation, and liver disease in human immunodeficiency virus (HIV)/hepatitis B virus (HBV) coinfection. Lipopolysaccharide (LPS), soluble CD14, CXCL10, and CCL-2 levels were elevated in patients with HIV/HBV coinfection. Levels of LPS, soluble CD14, and CCL-2 declined following receipt of HBV-active combination antiretroviral therapy (cART), but the CXCL10 level remained elevated. No markers were associated with liver disease severity on liver biopsy (n = 96), but CXCL10, interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor α, and interferon γ (IFN-γ) were all associated with elevated liver enzyme levels during receipt of HBV-active cART. Stimulation of hepatocyte cell lines in vitro with IFN-γ and LPS induced a profound synergistic increase in the production of CXCL10. LPS may contribute to liver disease via stimulating persistent production of CXCL10.
Publisher: Elsevier
Date: 2021
Publisher: Elsevier BV
Date: 09-2010
Publisher: Wiley
Date: 18-05-2004
Publisher: SAGE Publications
Date: 22-05-2016
Abstract: We have investigated the effects of a multispecies probiotic preparation containing a combination of probiotic bacterial genera that included Bifidobacteria, Lactobacilli and a Streptococcus in a mouse model of high-fat diet or obesity-induced liver steatosis. Three groups of C57B1/6J mice were fed either a standard chow or a high-fat diet for 20 weeks, while a third group was fed a high-fat diet for 10 weeks and then concomitantly administered probiotics for a further 10 weeks. Serum, liver and large bowel s les were collected for analysis. The expression of the tight-junction proteins ZO-1 and ZO-2 was reduced ( p 0.05) in high-fat diet-fed mice compared to chow-fed mice. Probiotic supplementation helped to maintain tight ZO-1 and ZO-2 expression compared with the high-fat diet group ( p 0.05), but did not restore ZO-1 or ZO-2 expression compared with chow-fed mice. Mice fed a high-fat diet ± probiotics had significant steatosis development compared with chow-fed mice ( p 0.05) steatosis was less severe in the probiotics group compared with the high-fat diet group. Hepatic triglyceride concentration was higher in mice fed a high-fat diet ± probiotics compared with the chow group ( p 0.05), and was lower in the probiotics group compared with the high-fat diet group ( p 0.05). Compared with chow-fed mice, serum glucose, cholesterol concentration and the activity of alanine transaminase were higher ( p 0.05), whereas serum triglyceride concentration was lower ( p 0.05) in mice fed a high-fat diet ± probiotics. Supplementation with a multispecies probiotic formulation helped to maintain tight-junction proteins ZO-1 and ZO-2, and reduced hepatic triglyceride concentration compared with a high-fat diet alone.
Publisher: Wiley
Date: 06-06-2013
DOI: 10.1111/ACER.12155
Abstract: Combined iron overload and alcohol may promote synergistic chronic liver injury and toxicity. The role of specific dietary fats in influencing the development of co-toxic alcoholic liver disease needs further evaluation and is investigated in this study. Wild-type (WT) and the iron-loaded Hfe-null (Hfe(-/-) ) mice were fed chow (CC), a AIN-93G standard control (SC), or a corn oil-modified, AIN-93G-based (CO) diet with or without the addition of 20% ethanol (EtOH) in the drinking water for 8 weeks and assessed for liver injury. WT mice on CC, SC, and CO diets had no liver injury, although mild steatosis developed in the SC and CO groups. The addition of EtOH resulted in mild steatohepatitis in WT mice fed SC but not those on a CO diet. EtOH administration in Hfe(-/-) animals on the CC and SC diets caused marked oxidative stress, inflammatory activity, and subsinusoidal and portal-portal tract linkage fibrosis with significant up-regulation of genes involved in cellular stress signaling and fibrogenic pathways. These effects were abrogated in the CO-fed mice, despite elevated serum EtOH levels and hepatic iron concentrations, reduced hepatic glutathione and mitochondrial superoxide dismutase activities. Feeding with the CO diet led to increased hepatic glutathione peroxidase and catalase activities and attenuated alcohol-induced hepatic steatosis in the Hfe(-/-) animals. Iron and EtOH feeding markedly reduced p-STAT3 and p-AMPK protein levels, but this effect was significantly attenuated when a CO diet was consumed. A CO-based diet is protective against combined EtOH- and iron-induced liver toxicity, likely via attenuation of hepatic steatosis and oxidative stress and may have a role in the prevention of fibrosis development in chronic liver disease.
Publisher: Elsevier BV
Date: 04-1999
Abstract: Transport of proteins along the exocytotic pathway is primarily achieved by vesicular intermediates. Two proteins, Golgi SNAREs of 27 kDa, GS27, and of 28 kDa, GS28 (HGMW-approved nomenclature GOSR2 and GOSR1, respectively), are important trafficking membrane proteins between the endoplasmic reticulum and the Golgi and between Golgi subcompartments. Here, we present the human GS27 and GS28 cDNA sequences. They encode predicted proteins of 212 and 250 amino acids, respectively. Chromosomal mapping analyses reveal that human GS27 is located on chromosome 17q21 and GS28 on approximately 17q11. The chromosomal location of GS27 near a locus implicated in familial essential hypertension and its known function in trafficking indicate that it is a potential candidate gene for this disease.
Publisher: Wiley
Date: 29-07-2017
DOI: 10.1002/AJH.24844
Publisher: Wiley
Date: 17-11-2014
DOI: 10.1111/BJH.13225
Abstract: Effective erythropoiesis requires an appropriate supply of iron and mechanisms regulating iron homeostasis and erythropoiesis are intrinsically linked. Iron dysregulation, typified by iron-deficiency anaemia and iron overload, is common in many clinical conditions and impacts the health of up to 30% of the world's population. The proteins transmembrane protease, serine 6 (TMPRSS6 also termed matriptase-2), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis, by regulating expression of the iron regulatory hormone hepcidin. We have performed a systematic analysis of mice deficient in these three proteins and show that TMPRSS6 predominates over HFE and TFR2 in hepcidin regulation. The phenotype of mice lacking TMPRSS6 and TFR2 is characterized by severe anaemia and extramedullary haematopoiesis in the spleen. Stress erythropoiesis in these mice results in increased expression of the newly identified erythroid iron regulator erythroferrone, which does not appear to overcome the hepcidin overproduction mediated by loss of TMPRSS6. Extended analysis reveals that TFR2 plays an important role in erythroid cells, where it is involved in terminal erythroblast differentiation and the regulation of erythropoietin. In conclusion, we have identified an essential role for TFR2 in erythropoiesis that may provide new targets for the treatment of anaemia.
Publisher: Portland Press Ltd.
Date: 07-2021
DOI: 10.1042/BSR20210720
Abstract: The flavonol rutin has been shown to possess antioxidant and iron chelating properties in vitro and in vivo. These dual properties are beneficial as therapeutic options to reduce iron accumulation and the generation of reactive oxygen species (ROS) resultant from excess free iron. The effect of rutin on iron metabolism has been limited to studies performed in wildtype mice either injected or fed high-iron diets. The effect of rutin on iron overload caused by genetic dysregulation of iron homoeostasis has not yet been investigated. In the present study we examined the effect of rutin treatment on tissue iron loading in a genetic mouse model of iron overload, which mirrors the iron loading associated with Type 3 hereditary haemochromatosis patients who have a defect in Transferrin Receptor 2 (TFR2). Male TFR2 knockout (KO) mice were administered rutin via oral gavage for 21 continuous days. Following treatment, iron levels in serum, liver, duodenum and spleen were assessed. In addition, hepatic ferritin protein levels were determined by Western blotting, and expression of iron homoeostasis genes by quantitative real-time PCR. Rutin treatment resulted in a significant reduction in hepatic ferritin protein expression and serum transferrin saturation. In addition, trends towards decreased iron levels in the liver and serum, and increased serum unsaturated iron binding capacity were observed. This is the first study to explore the utility of rutin as a potential iron chelator and therapeutic in an animal model of genetic iron overload.
Publisher: American Society of Hematology
Date: 05-07-2018
DOI: 10.1182/BLOOD-2018-02-830562
Abstract: This comprehensive comparison of the genetic subtypes of hemochromatosis reveals more severe iron overload and disease in non-HFE forms. Arthropathy is more common in HFE-related hemochromatosis, suggesting that joint disease may not be associated with iron.
Publisher: Springer Science and Business Media LLC
Date: 03-06-2008
DOI: 10.1007/S00109-008-0346-Y
Abstract: Hepcidin, a small cationic liver derived peptide, is a master regulator of body iron homeostasis. Cytokines and iron availability have so far been identified as regulators of hepcidin expression. Herein, we investigated the functional role of Kupffer cells for hepcidin expression because of their vicinity to the hepatocytes and their importance for iron recycling via erythrophagocytosis. We investigated C57Bl6 mice and littermates, in which Kupffer cells were eliminated in vivo upon intravenous injection of liposome-encapsulated clodronate. Primary cultures of hepatocytes and Kupffer cells were used to study direct regulatory effects ex vivo. The in vivo depletion of Kupffer cells resulted in a significant increase in liver hepcidin expression, which was paralleled by a significant reduction in serum iron levels. The same pattern of regulation by Kupffer cell depletion was observed upon injection of bacterial lipopolysaccharide into mice and in primary (Hfe -/-) and in secondary iron-overloaded mice. Accordingly, the messenger ribonucleic acid (mRNA) concentrations of the hepcidin iron-sensing molecule hemojuvelin were not significantly changed upon Kupffer cell depletion. When primary hepatocytes were cocultivated with Kupffer cells or stimulated with a Kupffer cell-conditioned medium ex vivo, a significant reduction in hepatocyte hepcidin mRNA expression was observed. Our data suggest that Kupffer cells control body iron homeostasis by exerting negative regulatory signals toward hepcidin expression, which may be primarily referred to the secretion of yet unidentified hepcidin-suppressing molecules by Kupffer cells.
Publisher: BMJ
Date: 05-03-2014
DOI: 10.1136/GUTJNL-2013-305317
Abstract: Hypoxia affects body iron homeostasis however, the underlying mechanisms are incompletely understood. Using a standardised hypoxia chamber, 23 healthy volunteers were subjected to hypoxic conditions, equivalent to an altitude of 5600 m, for 6 h. Subsequent experiments were performed in C57BL/6 mice, CREB-H knockout mice, primary hepatocytes and HepG2 cells. Exposure of subjects to hypoxia resulted in a significant decrease of serum levels of the master regulator of iron homeostasis hepcidin and elevated concentrations of platelet derived growth factor (PDGF)-BB. Using correlation analysis, we identified PDGF-BB to be associated with hypoxia mediated hepcidin repression in humans. We then exposed mice to hypoxia using a standardised chamber and observed downregulation of hepatic hepcidin mRNA expression that was paralleled by elevated serum PDGF-BB protein concentrations and higher serum iron levels as compared with mice housed under normoxic conditions. PDGF-BB treatment in vitro and in vivo resulted in suppression of both steady state and BMP6 inducible hepcidin expression. Mechanistically, PDGF-BB inhibits hepcidin transcription by downregulating the protein expression of the transcription factors CREB and CREB-H, and pharmacological blockade or genetic ablation of these pathways abrogated the effects of PDGF-BB toward hepcidin expression. Hypoxia decreases hepatic hepcidin expression by a novel regulatory pathway exerted via PDGF-BB, leading to increased availability of circulating iron that can be used for erythropoiesis.
Publisher: Elsevier BV
Date: 2018
Publisher: Elsevier BV
Date: 04-2009
Publisher: Wiley
Date: 03-1995
Publisher: Elsevier BV
Date: 08-1997
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 22-09-2008
DOI: 10.1002/HEP.22650
Abstract: Diagnosing the presence of cirrhosis is crucial for the management of patients with C282Y hereditary hemochromatosis (HH). HH patients with serum ferritin >1,000 microg/L are at risk of cirrhosis however, the majority of these patients do not have cirrhosis. Noninvasive markers of hepatic fibrosis may assist in determining which patients with a serum ferritin >1,000 microg/L have cirrhosis and require liver biopsy. This study evaluated the utility of current diagnostic algorithms for detecting cirrhosis, including serum ferritin concentration, platelet counts, and aspartate aminotransferase (AST) levels, in combination with serum markers of fibrosis, hyaluronic acid and collagen type IV (CLIV), in predicting cirrhosis in HH patients. Stage of fibrosis, serum hyaluronic acid and CLIV levels, were measured in 56 patients with HH. No patient with a serum ferritin 1,000 microg/L were cirrhotic. A combination of platelet count ( 1,000 microg/L did not detect 30% of cirrhotic subjects. Serum hyaluronic acid was increased in HH compared with controls (42.0 +/- 9.8 ng/mL versus 19.3 +/- 1.8 ng/mL P = 0.02). A hyaluronic acid concentration >46.5 ng/mL was 100% sensitive and 100% specific in identifying patients with cirrhosis. In patients with serum ferritin >1,000 microg/L, hyaluronic acid levels were significantly elevated in patients with cirrhosis versus those without cirrhosis (137 +/- 34.4 ng/mL versus 18.6 +/- 1.5 ng/mL, respectively P = 0.006). CLIV >113 ng/mL was 100% sensitive but only 56% specific for cirrhosis (area under the curve = 0.78 P = 0.01). In HH, the measurement of hyaluronic acid in patients with serum ferritin >1,000 microg/L is a noninvasive, accurate, and cost-effective method for the diagnosis of cirrhosis. (HEPATOLOGY 2009 :418-425.).
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 18-12-2015
DOI: 10.1002/HEP.28260
Publisher: Elsevier BV
Date: 04-2004
Publisher: The Endocrine Society
Date: 07-2009
DOI: 10.1210/JC.2009-0048
Abstract: The serum protein transthyretin (TTR) plays an important role in the transport of thyroid hormone and retinol, which are critical for normal development of the human fetus. TTR is not only synthesized and secreted into the circulation by the liver and other tissues but is also synthesized by placental trophoblasts, which separate the maternal and fetal circulations. Whether it is secreted or taken up by these cells and whether it carries thyroid hormone is unknown. Our objective was to study placental handling of TTR and determine whether TTR participates in placental thyroid hormone transport. We investigated the capacity of human placenta and choriocarcinoma cell lines to secrete and internalize TTR and its ligands by Western blotting, immunofluorescence, and uptake of radiolabeled TTR. Human placental explants and TTR expressing JEG-3 cells secrete TTR. JEG-3 cells grown in bicameral chambers secrete TTR, predominantly from the apical surface. Human placental explants and JEG-3 cells internalize Alexa Fluor488-labeled TTR and (125)I-TTR. Furthermore, binding to thyroid hormones (T(4), T(3)) increases (125)I-TTR uptake by enhancing tetramer formation. Cross-linking experiments confirm internalization of the TTR-(125)I-T(4) complex. Our results suggest that human placenta and choriocarcinoma cells secrete transthyretin, which binds extracellular T(4), and that T(4) binding results in increased internalization of TTR-T(4) complex. TTR production by trophoblasts may represent a mechanism to allow transfer of maternal thyroid hormone to the fetal circulation that could have important implications for fetal development.
Publisher: Elsevier BV
Date: 11-2015
DOI: 10.1016/J.JHEP.2015.06.027
Abstract: The development of targeted next-generation sequencing (NGS) applications now promises to be a clinically viable option for the diagnosis of rare disorders. This approach is proving to have significant utility where standardized testing has failed to identify the underlying molecular basis of disease. We have developed a unique targeted NGS panel for the systematic sequence-based analysis of atypical iron disorders. We report the analysis of 39 genes associated with iron regulation in eight cases of atypical iron dysregulation, in which five cases we identified the definitive causative mutation, and a possible causative mutation in a sixth. We further provide a molecular and cellular characterization study of one of these mutations (TFR2, p.I529N) in a familial case as proof of principle. Cellular analysis of the mutant protein indicates that this amino acid substitution affects the localization of the protein, which results in its retention in the endoplasmic reticulum and thus failure to function at the cell surface. Our unique NGS panel presents a rapid and cost-efficient approach to identify the underlying genetic cause in cases of atypical iron homeostasis disorders.
Publisher: Oxford University Press (OUP)
Date: 17-08-2021
Abstract: Hereditary hemochromatosis (HH) is a genetic disease, leading to iron accumulation and possible organ damage. Patients are usually homozygous for p. Cys282Tyr in the homeostatic iron regulator gene but may have mutations in other genes involved in the regulation of iron. Next-generation sequencing is increasingly being utilized for the diagnosis of patients, leading to the discovery of novel genetic variants. The clinical significance of these variants is often unknown. Determining the pathogenicity of such variants of unknown significance is important for diagnostics and genetic counseling. Predictions can be made using in silico computational tools and population data, but additional evidence is required for a conclusive pathogenicity classification. Genetic disease models, such as in vitro models using cellular overexpression, induced pluripotent stem cells or organoids, and in vivo models using mice or zebrafish all have their own challenges and opportunities when used to model HH and other iron disorders. Recent developments in gene-editing technologies are transforming the field of genetic disease modeling. In summary, this review addresses methods and developments regarding the discovery and classification of genetic variants, from in silico tools to in vitro and in vivo models, and presents them in the context of HH. It also explores recent gene-editing developments and how they can be applied to the discussed models of genetic disease.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-06-2015
DOI: 10.1002/HEP.27851
Publisher: Springer Science and Business Media LLC
Date: 25-04-2018
Publisher: Elsevier BV
Date: 10-2011
Publisher: Elsevier BV
Date: 11-2029
Publisher: Elsevier BV
Date: 05-2007
DOI: 10.1016/J.JHEP.2007.01.033
Abstract: Hereditary iron overload is associated with mutations in a number of genes involved in the regulation of iron metabolism. In this study we examined the molecular basis of iron overload in an in idual from New Zealand and characterised the molecular and cellular defect. We analysed the ferroportin gene and a control population was screened using allele-specific PCR and denaturation analysis. Molecular characterisation was performed by immunofluorescence microscopy analysis of transfected cells. We analysed the ferritin levels of cells expressing wild-type and mutant ferroportin to define the nature of the molecular defect on iron transport. We identified a novel nucleotide substitution (c. 1014T>G) in the ferroportin gene leading to the S338R mutation. This mutation is not a common polymorphism. Cellular analysis of the mutant protein indicates that this amino acid change does not affect the localisation of the protein or its ability to transport iron. The S338R mutation results in a mutated ferroportin associated with iron overload and is predicted insensitive to regulation by the iron regulatory hormone hepcidin.
Publisher: Portland Press Ltd.
Date: 05-2020
DOI: 10.1042/BSR20191499
Abstract: Mutations in the only known iron exporter ferroportin (FPN) in humans are associated with the autosomal dominantly inherited iron overload disorder ferroportin disease or type IV hereditary hemochromatosis (HH). While our knowledge of the central role of FPN in iron homeostasis has grown in the last 20 years, there exist some questions surrounding the structure and membrane topology of FPN with conflicting data on whether this receptor acts as a monomer or a multimer. To investigate and determine if FPN dimerization occurs in cells, we used novel tools including a variety of different FPN constructs expressing different tagged versions of the protein, a novel antibody that only detects cell surface FPN and proximity ligation assays. The results of the present study suggest that both the carboxy- and amino-termini of the FPN protein are intracellular. We also show that exogenously transfected FPN forms dimers these dimers can be formed between the wild-type and mutant FPN proteins. This is the first study to examine the intracellular dimerization of FPN protein. Using proximity ligation assays, we show intracellular localization of FPN dimers and the interaction between FPN and hepcidin proteins as well. These results have important implications in the field of iron metabolism and add to our knowledge about FPN membrane topology and physiology of iron transport. This will be of importance in understanding the clinical implications of FPN mutations and of interest to future research aimed at targeting FPN expression to modulate iron homeostasis.
Publisher: Elsevier BV
Date: 07-2009
DOI: 10.1016/J.BBAGEN.2008.09.002
Abstract: The severity of liver disease and its presentation is thought to be influenced by many host factors. Prominent among these factors is the level of iron in the body. The liver plays an important role in coordinating the regulation of iron homeostasis and is involved in regulating the level of iron absorption in the duodenum and iron recycling by the macrophages. Iron homeostasis is disturbed by several metabolic and genetic disorders, including various forms of hereditary hemochromatosis. This review will focus on liver disease and how it is affected by disordered iron homeostasis, as observed in hereditary hemochromatosis and due to HFE mutations. The types of liver disease covered herein are chronic hepatitis C virus (HCV) infection, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatocellular carcinoma (HCC) and porphyria cutanea tarda (PCT).
Publisher: Wiley
Date: 18-11-2015
DOI: 10.1111/HIV.12188
Publisher: Informa UK Limited
Date: 26-02-2013
DOI: 10.4161/CC.23879
Publisher: Elsevier
Date: 2019
DOI: 10.1016/BS.VH.2019.01.003
Abstract: Since its discovery in 2001, there have been a number of important discoveries and findings that have increased our knowledge about the functioning of hepcidin. Hepcidin, the master iron regulator has been shown to be regulated by a number of physiological stimuli and their associated signaling pathways. This chapter will summarize our current understanding of how these physiological stimuli and downstream signaling molecules are involved in hepcidin modulation and ultimately contribute to the regulation of systemic or local iron homeostasis. The signaling pathways and molecules described here have been shown to primarily affect hepcidin at a transcriptional level, but these transcriptional changes correlate with changes in systemic iron levels as well, supporting the functional effects of hepcidin regulation by these signaling pathways.
Publisher: Elsevier BV
Date: 08-2013
DOI: 10.1016/J.PLACENTA.2013.05.005
Abstract: Normal fetal neurological development depends on a regulated supply of maternal thyroid hormone (TH). We have previously demonstrated that transthyretin (TTR) a TH binding protein, is synthesized, secreted and internalized by trophoblast cells and may provide a route for the transfer of TH from mother to fetus. Our objective was to determine if a member of the low-density lipoprotein receptor family mediates TTR or TTR-TH internalization. TTR-TH internalization by JEG-3 cells is reduced in the presence of receptor associated protein (RAP) or albumin suggesting that TTR-TH is internalized through an LDL-receptor dependent endocytic process.
Publisher: The Company of Biologists
Date: 15-03-2005
DOI: 10.1242/JCS.01723
Abstract: The TRAPP complex identified in yeast regulates vesicular transport in the early secretory pathway. Although some components of the TRAPP complex are structurally conserved in mammalian cells, the function of the mammalian components has not been examined. We describe our biochemical and functional analysis of mammalian Bet3, the most conserved component of the TRAPP complex. Bet3 mRNA is ubiquitously expressed in all tissues. Antibodies raised against recombinant Bet3 specifically recognize a protein of 22 kDa. In contrast to yeast Bet3p, the majority of Bet3 is present in the cytosol. To investigate the possible involvement of Bet3 in transport events in mammalian cells, we utilized a semi-intact cell system that reconstitutes the transport of the envelope glycoprotein of vesicular stomatitis virus (VSV-G) from the ER to the Golgi apparatus. In this system, antibodies against Bet3 inhibit transport in a dose-dependent manner, and cytosol that is immunodepleted of Bet3 is also defective in this transport. This defect can be rescued by supplementing the Bet3-depleted cytosol with recombinant GST-Bet3. We also show that Bet3 acts after COPII but before Rab1, α-SNAP and the EGTA-sensitive stage during ER-Golgi transport. Gel filtration analysis demonstrates that Bet3 exists in two distinct pools in the cytosol, the high-molecular-weight pool may represent the TRAPP complex, whereas the other probably represents the monomeric Bet3.
Publisher: Wiley
Date: 10-03-2014
DOI: 10.1111/LIV.12494
Abstract: Mammalian target of rapamycin and angiotensin-converting enzyme inhibition has been shown to have antifibrotic activity in models of liver fibrosis. The aim of our study was to determine the efficacy of rapamycin, everolimus, irbesartan and captopril, alone and in combination, as antifibrotic agents in the Mdr2(-/-) model of cholestasis both in early injury and established disease. Mdr2(-/-) mice were treated for 4 weeks with vehicle, rapamycin (1 mg/kg) or everolimus (5 mg/kg) every second day or with captopril (30 mg/kg/day), irbesartan (10 mg/kg/day) or vehicle. Further groups of 3-week-old Mdr2(-/-) mice were treated with rapamycin and irbesartan in combination (1 mg/kg/day and 10 mg/kg/day) or with rapamycin (2 mg/kg/day) for 4 weeks. Liver injury and fibrosis were compared between treated and untreated animals. There were no significant improvements in liver injury, histology, hepatic hydroxyproline or profibrogenic gene expression following treatment with rapamycin, everolimus, captopril or irbesartan at any time point studied. Likewise, there were no improvements in liver histology or profibrogenic gene expression following combination therapy or high-dose rapamycin treatment. The antifibrotic effects of rapamycin, everolimus, captopril and irbesartan seen in other models of fibrosis were not replicated in the Mdr2(-/-) model in this study. This highlights the clear need to test specific antifibrotic agents in a number of different animal models. We believe this animal model is ideal to study usefulness of antifibrotic agents in cholestatic liver disease because of the similarity in genetics and hepatic histopathology to human cholestatic liver disease.
Publisher: Baishideng Publishing Group Inc.
Date: 2017
Publisher: Elsevier BV
Date: 09-2009
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1038/GIM.2015.140
Abstract: The prevalence of HFE-related hereditary hemochromatosis (HH) among European populations has been well studied. There are no prevalence data for atypical forms of HH caused by mutations in HFE2, HAMP, TFR2, or SLC40A1. The purpose of this study was to estimate the population prevalence of these non-HFE forms of HH. A list of HH pathogenic variants in publically available next-generation sequence (NGS) databases was compiled and allele frequencies were determined. Of 161 variants previously associated with HH, 43 were represented among the NGS data sets an additional 40 unreported functional variants also were identified. The predicted prevalence of HFE HH and the p.Cys282Tyr mutation closely matched previous estimates from similar populations. Of the non-HFE forms of iron overload, TFR2-, HFE2-, and HAMP-related forms are predicted to be rare, with pathogenic allele frequencies in the range of 0.00007 to 0.0005. Significantly, SLC40A1 variants that have been previously associated with autosomal-dominant ferroportin disease were identified in several populations (pathogenic allele frequency 0.0004), being most prevalent among Africans. We have, for the first time, estimated the population prevalence of non-HFE HH. This methodology could be applied to estimate the population prevalence of a wide variety of genetic disorders.Genet Med 18 6, 618-626.
Publisher: American Physiological Society
Date: 2010
DOI: 10.1152/AJPCELL.00621.2008
Abstract: Ferroportin disease is a heterogeneous iron release disorder resulting from mutations in the ferroportin gene. Ferroportin protein is a multitransmembrane domain iron transporter, responsible for iron export from cells, which, in turn, is regulated by the peptide hormone hepcidin. Mutations in the ferroportin gene may affect either regulation of the protein's transporter function or the ability of hepcidin to regulate iron efflux. We have used a combination of functional analysis of epitope-tagged ferroportin variants coupled with theoretical modeling to dissect the relationship between ferroportin mutations and their cognate phenotypes. Myc epitope-tagged human ferroportin expression constructs were transfected into Caco-2 intestinal cells and protein localization analyzed by immunofluorescence microscopy and colocalization with organelle markers. The effect of mutations on iron efflux was assessed by costaining with anti-ferritin antibodies and immunoblotting to quantitate cellular expression of ferritin and transferrin receptor 1. Wild-type ferroportin localized mainly to the cell surface and intracellular structures. All ferroportin disease-causing mutations studied had no effect on localization at the cell surface. N144H, N144T, and S338R mutant ferroportin retained the ability to transport iron. In contrast, A77D, V162Δ, and L170F mutants were iron transport defective. Surface staining experiments showed that both ends of the protein were located inside the cell. These data were used as the basis for theoretical modeling of the ferroportin molecule. The model predicted phenotypic clustering of mutations with gain-of-function variants associated with a hypothetical channel through the axis of ferroportin. Conversely, loss-of-function variants were located at the membrane/cytoplasm interface.
Publisher: Springer Science and Business Media LLC
Date: 11-09-2015
DOI: 10.1038/SREP13986
Abstract: BMP-SMAD signalling plays a crucial role in numerous biological processes including embryonic development and iron homeostasis. Dysregulation of the iron-regulatory hormone hepcidin is associated with many clinical iron-related disorders. We hypothesised that molecules which mediate BMP-SMAD signalling play important roles in the regulation of iron homeostasis and variants in these proteins may be potential genetic modifiers of iron-related diseases. We examined the role of endofin, a SMAD anchor and show that knockdown of endofin in liver cells inhibits basal and BMP-induced hepcidin expression along with other BMP-regulated genes, ID1 and SMAD7 . We show for the first time, the in situ interaction of endofin with SMAD proteins and significantly reduced SMAD phosphorylation with endofin knockdown, suggesting that endofin modulates hepcidin through BMP-SMAD signalling. Characterisation of naturally occurring SNPs show that mutations in the conserved FYVE domain result in mislocalisation of endofin, potentially affecting downstream signalling and modulating hepcidin expression. In conclusion, we have identified a hitherto unrecognised link, endofin, between the BMP-SMAD signalling pathway and the regulation of hepcidin expression and iron homeostasis. This study further defines the molecular network involved in iron regulation and provides potential targets for the treatment of iron-related disorders.
Publisher: Wiley
Date: 10-09-2013
DOI: 10.1111/JGH.12365_6
Publisher: Wiley
Date: 10-09-2013
DOI: 10.1111/JGH.12365_7
Publisher: Wiley
Date: 10-09-2013
DOI: 10.1111/JGH.12365_8
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-02-2017
DOI: 10.1002/HEP.29002
Publisher: American Society for Cell Biology (ASCB)
Date: 06-1998
DOI: 10.1091/MBC.9.6.1549
Abstract: Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant α-SNAP fused to glutathioneS-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway.
Publisher: Elsevier BV
Date: 10-2016
Publisher: Wiley
Date: 2004
DOI: 10.1002/GENE.20023
Abstract: Transferrin Receptor 2 (TfR2) is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 lead to type 3 hemochromatosis in humans. We have developed mice with a targeted deletion of TfR2. The Cre-recombinase:loxP system used to create the mice allows both full deletion and tissue-specific deletion of TfR2. The development of these mice will provide new models for type 3 hemochromatosis and assist in determining the role of TfR2 in iron metabolism.
Publisher: Springer Science and Business Media LLC
Date: 09-10-2020
DOI: 10.1007/S00392-019-01557-0
Abstract: Patients with HF are at a higher risk of rehospitalisation and, as such, significant costs to our healthcare system. A non-invasive method to collect body fluids and measure Gal-3 could improve the current management of HF. In this study, we investigated the potential prognostic utility of salivary Galectin-3 (Gal-3) in patients with heart failure (HF). We collected saliva s les from patients with HF (n = 105) either at hospital discharge or during routine clinical visits. Gal-3 concentrations in saliva s les were measured by ELISA. The Kaplan-Meier survival curve analysis and Cox proportional regression model were used to determine the potential prognostic utility of salivary Gal-3 concentrations. The primary end point was either cardiovascular death or hospitalisation. Salivary Gal-3 concentrations were significantly higher (p 172.58 ng/mL had a significantly (p 172.58 ng/mL demonstrated a higher cumulative risk of the primary outcome compared to those with lower Gal-3 levels, even after adjusting for other variables. Confirming our findings in a larger multi-centre clinical trial in the future would enable salivary Gal-3 measurements to form part of routine management for patients with HF.
Publisher: Elsevier BV
Date: 09-2012
Publisher: Rockefeller University Press
Date: 12-1997
Abstract: Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.
Publisher: Elsevier BV
Date: 02-2003
Publisher: Informa UK Limited
Date: 06-2008
Publisher: Elsevier BV
Date: 10-2005
DOI: 10.1016/J.JHEP.2005.02.047
Abstract: Hepcidin is a liver-expressed peptide which plays an important role in the regulation of iron metabolism. It is a negative regulator of iron absorption and release of iron from cells. The aims of this study were to analyse the expression and localisation of prohepcidin in liver and cell lines. We generated antibodies against recombinant mouse prohepcidin and studied its expression in cell lines, primary hepatocytes and livers of normal mice and mice with abnormalities in iron metabolism. Prohepcidin localised to the secretory pathway, primarily the Golgi apparatus in liver cells and tissues. Hfe and beta2-microglobulin knockout mice have similar levels of prohepcidin protein expression as compared to wild-type mice despite increased iron stores. Sex-linked anaemia mice have iron deficiency and no prohepcidin expression in the liver. Prohepcidin protein is present in the secretory pathway of liver cells. Despite iron loading, mouse models of haemochromatosis have comparatively normal levels of prohepcidin expression whereas mice with iron deficiency have no prohepcidin expression.
Publisher: Elsevier BV
Date: 02-2015
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 22-08-2008
DOI: 10.1002/HEP.22597
Abstract: Lymphotoxin-beta (LTbeta) is a proinflammatory cytokine and a member of the tumor necrosis factor (TNF) superfamily known for its role in mediating lymph node development and homeostasis. Our recent studies suggest a role for LTbeta in mediating the pathogenesis of human chronic liver disease. We hypothesize that LTbeta co-ordinates the wound healing response in liver injury via direct effects on hepatic stellate cells. This study used the choline-deficient, ethionine-supplemented (CDE) dietary model of chronic liver injury, which induces inflammation, liver progenitor cell proliferation, and portal fibrosis, to assess (1) the cellular expression of LTbeta, and (2) the role of LTbeta receptor (LTbetaR) in mediating wound healing, in LTbetaR(-/-) versus wild-type mice. In addition, primary isolates of hepatic stellate cells were treated with LTbetaR-ligands LTbeta and LTbeta-related inducible ligand competing for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT), and mediators of hepatic stellate cell function and fibrogenesis were assessed. LTbeta was localized to progenitor cells immediately adjacent to activated hepatic stellate cells in the periportal region of the liver in wild-type mice fed the CDE diet. LTbetaR(-/-) mice fed the CDE diet showed significantly reduced fibrosis and a dysregulated immune response. LTbetaR was demonstrated on isolated hepatic stellate cells, which when stimulated by LTbeta and LIGHT, activated the nuclear factor kappa B (NF-kappaB) signaling pathway. Neither LTbeta nor LIGHT had any effect on alpha-smooth muscle actin, tissue inhibitor of metalloproteinase 1, transforming growth factor beta, or procollagen alpha(1)(I) expression however, leukocyte recruitment-associated factors intercellular adhesion molecule 1 and regulated upon activation T cells expressed and secreted (RANTES) were markedly up-regulated. RANTES caused the chemotaxis of a liver progenitor cell line expressing CCR5. This study suggests that LTbetaR on hepatic stellate cells may be involved in paracrine signaling with nearby LTbeta-expressing liver progenitor cells mediating recruitment of progenitor cells, hepatic stellate cells, and leukocytes required for wound healing and regeneration during chronic liver injury.
Publisher: Elsevier BV
Date: 03-2017
Publisher: Elsevier BV
Date: 11-2006
DOI: 10.1016/J.CGH.2006.07.009
Abstract: Two major mutations are defined within the hemochromatosis gene, HFE. Although the effects of the C282Y substitution have been well characterized, the clinical significance of the C282Y/H63D state remains unclear. This study assessed the phenotypic expression in C282Y/H63D subjects as compared with C282Y homozygotes. Data were obtained from 91 C282Y/H63D probands, 158 C282Y/H63D subjects identified through family screening, and 483 C282Y homozygotes. Subjects underwent clinical evaluation, genotyping, biochemical assessment, and liver biopsy examination where clinically indicated. C282Y/H63D probands had significantly less clinical and biochemical expression than C282Y homozygotes. Biochemical expression was higher in C282Y/H63D probands than in C282Y/H63D subjects identified through family screening (P < .001). Of the C282Y/H63D subjects with serum ferritin levels greater than 1000 mug/L, all had known comorbid factors that could have contributed to the increased ferritin level. Of the 51 C282Y/H63D subjects who underwent liver biopsy examination, significantly increased iron stores were present in 9 subjects and hepatic fibrosis was present in 13. Twelve of the 13 had evidence of hepatic steatosis, excess alcohol consumption, or diabetes. The mobilizable iron level was significantly higher in C282Y homozygous males than in compound heterozygous males (P < .001). Genetic screening of C282Y/H63D first-degree relatives detected 5 C282Y homozygotes. C282Y/H63D subjects referred for assessment had a high prevalence of increased iron indices but did not develop progressive clinical disease without comorbid factors such as steatosis, diabetes, or excess alcohol consumption. When fibrosis was seen, 1 or more comorbid factors almost always were present. Thus, phlebotomy therapy is warranted and cascade screening of relatives should be performed because expressing C282Y homozygotes may be detected.
Publisher: American Physiological Society
Date: 02-2016
Abstract: Iron is an essential element, since it is a component of many macromolecules involved in erse physiological and cellular functions, including oxygen transport, cellular growth, and metabolism. Systemic iron homeostasis is predominantly regulated by the liver through the iron regulatory hormone hepcidin. Hepcidin expression is itself regulated by a number of proteins, including transferrin receptor 2 (TFR2). TFR2 has been shown to be expressed in the liver, bone marrow, macrophages, and peripheral blood mononuclear cells. Studies from our laboratory have shown that mice with a hepatocyte-specific deletion of Tfr2 recapitulate the hemochromatosis phenotype of the global Tfr2 knockout mice, suggesting that the hepatic expression of TFR2 is important in systemic iron homeostasis. It is unclear how TFR2 in macrophages contributes to the regulation of iron metabolism. We examined the role of TFR2 in macrophages by analysis of transgenic mice lacking Tfr2 in macrophages by crossing Tfr2 f/f mice with LysM-Cre mice. Mice were fed an iron-rich diet or injected with lipopolysaccharide to examine the role of macrophage Tfr2 in iron- or inflammation-mediated regulation of hepcidin. Body iron homeostasis was unaffected in the knockout mice, suggesting that macrophage TFR2 is not required for the regulation of systemic iron metabolism. However, peritoneal macrophages of knockout mice had significantly lower levels of ferroportin mRNA and protein, suggesting that TFR2 may be involved in regulating ferroportin levels in macrophages. These studies further elucidate the role of TFR2 in the regulation of iron homeostasis and its role in regulation of ferroportin and thus macrophage iron homeostasis.
Publisher: American Physiological Society
Date: 04-2011
Abstract: Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder resulting in a myriad of abnormalities, including progressive neurodegeneration and cancer predisposition. At the cellular level, A-T is a disease of chronic oxidative stress (OS) causing damage to proteins, lipids, and DNA. OS is contributed to by pro-oxidative transition metals such as iron that catalyze the conversion of weakly reactive oxygen species to highly reactive hydroxyl radicals. Iron-associated OS has been linked to neurodegeneration in Alzheimer's and Parkinson's diseases and development of lymphoid tumors (which afflict ∼30% of A-T patients). To investigate iron regulation in A-T, iron indexes, regulatory genes, and OS markers were studied in livers of wild-type and Ataxia telangiectasia mutated ( Atm) null mice on control or high-iron diets. Atm −/− mice had increased serum iron, hepatic iron, and ferritin and significantly higher Hepcidin compared with wild-type mice. When challenged with the high-iron diet, Bmp6 and Hfe expression was significantly increased. Atm −/− mice had increased protein tyrosine nitration and significantly higher Heme Oxygenase (decycling) 1 levels that were substantially increased by a high-iron diet. Ferroportin gene expression was significantly increased however, protein levels were unchanged. We demonstrate that Atm −/− mice have a propensity to accumulate iron that is associated with a significant increase in hepatic OS. The iron-induced increase in hepcidin peptide in turn suppresses ferroportin protein levels, thus nullifying the upregulation of mRNA expression in response to increased OS. Our results suggest that increased iron status may contribute to the chronic OS seen in A-T patients and development of disease pathology.
Publisher: SAGE Publications
Date: 05-2006
DOI: 10.1258/000456306776865197
Abstract: A 14-year-old boy who presented with debilitating lethargy was shown to have an elevated serum ferritin of 572 μg/L and a C282Y homozygous HFE genotype. Liver iron concentration was measured non-invasively by magnetic resonance imaging, which revealed a liver iron concentration of 59 μmol/g dry weight (children's reference range ). The early phenotypic expression was further investigated by screening genomic DNA for the presence of co-inherited mutations in genes responsible for non- HFE haemochromatosis. Coding regions and splice sites in genes encoding hepcidin and haemojuvelin were sequenced and previously described mutations in ferroportin 1 and transferrin receptor 2 genes were screened. Although no mutations were found, the most likely cause for the early expression is the presence of novel mutations or gene(s).
Publisher: Springer Science and Business Media LLC
Date: 03-2010
DOI: 10.1038/ONC.2010.37
Abstract: The proper function of the spindle is crucial to the high fidelity of chromosome segregation and is indispensable for tumor suppression in humans. Centrobin is a recently identified centrosomal protein that has a role in stabilizing the microtubule structure. Here we functionally characterize the defects in centrosome integrity and spindle assembly in Centrobin-depleted cells. Centrobin-depleted cells show a range of spindle abnormalities including unfocused poles that are not associated with centrosomes, S-shaped spindles and mini spindles. These cells undergo mitotic arrest and subsequently often die by apoptosis, as determined by live cell imaging. Co-depletion of Mad2 relieves the mitotic arrest, indicating that cells arrest due to a failure to silence the spindle checkpoint in metaphase. Consistent with this, Centrobin-depleted metaphase cells stained positive for BubR1 and BubR1 S676. Staining with a panel of centrosome markers showed a loss of centrosome anchoring to the mitotic spindle. Furthermore, these cells show less cold-stable microtubules and a shorter distance between kinetochore pairs. These results show a requirement of Centrobin in maintaining centrosome integrity, which in turn promotes anchoring of mitotic spindle to the centrosomes. Furthermore, this anchoring is required for the stability of microtubule-kinetochore attachments and biogenesis of tension-ridden and properly functioning mitotic spindle.
Publisher: American Society for Cell Biology (ASCB)
Date: 08-2000
Abstract: Transitional endoplasmic reticulum (tER) consists of confluent rough and smooth endoplasmic reticulum (ER) domains. In a cell-free incubation system, low-density microsomes (1.17 g cc − 1 ) isolated from rat liver homogenates reconstitute tER by Mg 2+ GTP- and Mg 2+ ATP-hydrolysis–dependent membrane fusion. The ATPases associated with different cellular activities protein p97 has been identified as the relevant ATPase. The ATP depletion by hexokinase or treatment with either N-ethylmaleimide or anti-p97 prevented assembly of the smooth ER domain of tER. High-salt washing of low-density microsomes inhibited assembly of the smooth ER domain of tER, whereas the readdition of purified p97 with associated p47 promoted reconstitution. The t-SNARE syntaxin 5 was observed within the smooth ER domain of tER, and antisyntaxin 5 abrogated formation of this same membrane compartment. Thus, p97 and syntaxin 5 regulate assembly of the smooth ER domain of tER and hence one of the earliest membrane differentiated components of the secretory pathway.
Publisher: American Physiological Society
Date: 15-01-2014
Abstract: Treatment for iron deficiency anemia can involve iron supplementation via dietary or parenteral routes that result in different cellular iron distributions. The effect of the administered iron on the iron regulatory system and hepcidin in the liver has not been well studied. Hepcidin, the liver-expressed central iron-regulatory peptide, is itself regulated through the bone morphogenetic protein (BMP)/SMAD signaling pathway. Specifically, Bmp6 expression is upregulated in response to iron and induces hepcidin through phosphorylation of Smad1/5/8. The hemochromatosis-associated proteins Hfe and transferrin receptor 2 (Tfr2) are known upstream regulators of hepcidin, although their precise roles are still unclear. To investigate the mechanisms of this regulation and the roles of the Hfe and Tfr2, we subjected wild-type, Hfe −/− , Tfr2 −/− , and Hfe −/− / Tfr2 −/− mice to iron loading via dietary or parenteral routes. Systematic analysis demonstrated that Tfr2 is required for effective upregulation of Bmp6 in response to hepatocyte iron, but not nonparenchymal iron. Hfe is not required for Bmp6 upregulation, regardless of iron localization, but rather, is required for efficient downstream transmission of the regulatory signal. Our results demonstrate that Hfe and Tfr2 play separate roles in the regulatory responses to iron compartmentalized in different cell types and further elucidates the regulatory mechanisms controlling iron homeostasis.
Publisher: Baishideng Publishing Group Inc.
Date: 2013
Publisher: Humana Press
Date: 24-11-2011
Publisher: Elsevier BV
Date: 2018
Publisher: Elsevier BV
Date: 08-2018
Publisher: American Association for the Advancement of Science (AAAS)
Date: 24-05-1996
DOI: 10.1126/SCIENCE.272.5265.1161
Abstract: Little is known about the integral membrane proteins that participate in the early secretory pathway of mammalian cells. The complementary DNA encoding a 28-kilodalton protein (p28) of the cis-Golgi was cloned and sequenced. The protein was predicted to contain a central coiled-coil domain with a carboxyl-terminal membrane anchor. An in vitro assay for endoplasmic reticulum-Golgi transport was used to show that p28 participates in the docking and fusion stage of this transport event. Biochemical studies established that p28 is a core component of the Golgi SNAP receptor (SNARE) complex.
Publisher: BMJ
Date: 04-2005
Publisher: MDPI AG
Date: 04-10-2022
DOI: 10.3390/MOLECULES27196581
Abstract: The protein HFE (homeostatic iron regulator) is a key regulator of iron metabolism, and mutations in HFE underlie the most frequent form of hereditary haemochromatosis (HH-type I). Studies have shown that HFE interacts with transferrin receptor 1 (TFR1), a homodimeric type II transmembrane glycoprotein that is responsible for the cellular uptake of iron via iron-loaded transferrin (holo-transferrin) binding. It has been hypothesised that the HFE/TFR1 interaction serves as a sensor to the level of iron-loaded transferrin in circulation by means of a competition mechanism between HFE and iron-loaded transferrin association with TFR1. To investigate this, a series of peptides based on the helical binding interface between HFE and TFR1 were generated and shown to significantly interfere with the HFE/TFR1 interaction in an in vitro proximity ligation assay. The helical conformation of one of these peptides, corresponding to the α1 and α2 helices of HFE, was stabilised by the introduction of sidechain lactam “staples”, but this did not result in an increase in the ability of the peptide to disrupt the HFE/TFR1 interaction. These peptides inhibitors of the protein–protein interaction between HFE and TFR1 are potentially useful tools for the analysis of the functional role of HFE in the regulation of hepcidin expression.
Publisher: Springer Science and Business Media LLC
Date: 26-01-2018
Publisher: Springer Science and Business Media LLC
Date: 10-1997
DOI: 10.1038/39923
Abstract: In eukaryotic cells, the Golgi apparatus receives newly synthesized proteins from the endoplasmic reticulum (ER) and delivers them after covalent modification to their destination in the cell. These proteins move from the inside (cis) face to the plasma-membrane side (trans) of the Golgi, through a stack of cisternae, towards the trans-Golgi network (TGN), but very little is known about how proteins are moved through the Golgi compartments. In a model known as the maturation model, no special transport process was considered necessary, with protein movement along the Golgi being achieved by maturation of the cisternae. Alternatively, proteins could be transported by vesicles or membrane tubules. Although little is known about membrane-tubule-mediated transport, the molecular mechanism for vesicle-mediated transport is quite well understood, occurring through docking of SNAREs on the vesicle with those on the target membrane. We have now identified a protein of relative molecular mass 27K which is associated with the Golgi apparatus. The cytoplasmic domain of this protein or antibodies raised against it quantitatively inhibit transport in vitro from the ER to the trans-Golgi/TGN, acting at a stage between the cis/medial- and the trans-Golgi/TGN. This protein, which behaves like a SNARE and has been named GS27 (for Golgi SNARE of 27K), is identical to membrin, a protein implicated earlier in ER-to-Golgi transport. Our results suggest that protein movement from medial- to the trans-Golgi/TGN depends on SNARE-mediated vesicular transport.
Publisher: Elsevier BV
Date: 04-2002
DOI: 10.1016/S0168-8278(01)00304-X
Abstract: HFE-related haemochromatosis is a common disorder of iron metabolism. Most affected in iduals are homozygous for the C282Y mutation of HFE. Some are compound heterozygotes for C282Y/H63D. A small proportion have neither of these genotypes. We have investigated the phenotype of compound heterozygotes for C282Y and another missense mutation S65C. Genotype for the S65C mutation was determined in 309 subjects heterozygous for C282Y and negative for H63D, referred because of increased serum iron indices or family screening. A control s le comprising 315 in iduals was also studied. Twelve in iduals were compound heterozygotes for C282Y and S65C. Seven, referred for family screening, had normal serum iron indices. Five subjects had elevated serum iron indices three of these had elevated hepatic iron and have received treatment for iron overload. Transferrin saturation was significantly elevated in C282Y/S65C compound heterozygotes compared with simple C282Y heterozygotes. Some C282Y/S65C compound heterozygotes have elevated serum iron indices and iron overload. The penetrance of this genotype is low and other genetic and environmental factors may influence the expression of iron loading. Screening for S65C may be useful in in iduals with iron overload who are not homozygous for C282Y or compound heterozygous for C282Y/H63D.
Publisher: American Physiological Society
Date: 09-2017
Abstract: The liver is one of the largest and most functionally erse organs in the human body. In addition to roles in detoxification of xenobiotics, digestion, synthesis of important plasma proteins, gluconeogenesis, lipid metabolism, and storage, the liver also plays a significant role in iron homeostasis. Apart from being the storage site for excess body iron, it also plays a vital role in regulating the amount of iron released into the blood by enterocytes and macrophages. Since iron is essential for many important physiological and molecular processes, it increases the importance of liver in the proper functioning of the body’s metabolism. This hepatic iron-regulatory function can be attributed to the expression of many liver-specific or liver-enriched proteins, all of which play an important role in the regulation of iron homeostasis. This review focuses on these proteins and their known roles in the regulation of body iron metabolism.
Publisher: American Society of Hematology
Date: 14-07-2016
Publisher: Public Library of Science (PLoS)
Date: 09-03-2010
Publisher: Wiley
Date: 20-06-2013
DOI: 10.1111/JGH.12222
Abstract: Hereditary hemochromatosis (HH) is a widely recognized and well-studied condition in European populations. This is largely due to the high prevalence of the C282Y mutation of HFE. Although less common than in Europe, HH cases have been reported in the Asia-Pacific region because of mutations in both HFE and non-HFE genes. Mutations in all of the currently known genes implicated in non-HFE HH (hemojuvelin, hepcidin, transferrin receptor 2, and ferroportin) have been reported in patients from the Asia-Pacific region. This review discusses the molecular basis of HH and the genes and mutations known to cause non-HFE HH with particular reference to the Asia-Pacific region. Challenges in the genetic diagnosis of non-HFE HH are also discussed and how new technologies such as next generation sequencing may be informative in the future.
Publisher: Elsevier BV
Date: 10-1997
Publisher: Springer Science and Business Media LLC
Date: 10-12-2020
DOI: 10.1007/S00392-019-01583-Y
Abstract: The original version of this article unfortunately contained a mistake.
Publisher: BMJ
Date: 07-2005
Publisher: Elsevier BV
Date: 2006
DOI: 10.1016/J.BIOCHI.2005.07.003
Abstract: Hepcidin is a liver-expressed antimicrobial and iron regulatory peptide. A number of studies have indicated that hepcidin is important for the correct regulation of body iron homeostasis. The aims of this study were to analyse the expression, trafficking and regulation of human hepcidin in an in vitro cell culture system. Human hepcidin was transfected into human embryonic kidney cells. Immunofluorescence and confocal microscopy analysis revealed that recombinant hepcidin localised to the Golgi complex. Recombinant hepcidin is secreted from the cell within 1 h of its synthesis. Recombinant hepcidin was purified from the cell culture medium using ion-exchange and metal-affinity chromatography and was active in antimicrobial assays. Amino-terminal sequence analysis of the secreted peptide revealed that it was the mature 25 amino acid form of hepcidin. Our results show that recombinant myc-His tagged human hepcidin was expressed, processed and secreted correctly and biologically active in antimicrobial assays.
Publisher: Springer Science and Business Media LLC
Date: 16-12-2021
DOI: 10.1038/S41418-020-00698-4
Abstract: Alterations in the metabolism of iron and its accumulation in the substantia nigra pars compacta accompany the pathogenesis of Parkinson’s disease (PD). Changes in iron homeostasis also occur during aging, which constitutes a PD major risk factor. As such, mitigation of iron overload via chelation strategies has been considered a plausible disease modifying approach. Iron chelation, however, is imperfect because of general undesired side effects and lack of specificity more effective approaches would rely on targeting distinctive pathways responsible for iron overload in brain regions relevant to PD and, in particular, the substantia nigra. We have previously demonstrated that the Transferrin/Transferrin Receptor 2 (TfR2) iron import mechanism functions in nigral dopaminergic neurons, is perturbed in PD models and patients, and therefore constitutes a potential therapeutic target to halt iron accumulation. To validate this hypothesis, we generated mice with targeted deletion of TfR2 in dopaminergic neurons. In these animals, we modeled PD with multiple approaches, based either on neurotoxin exposure or alpha-synuclein proteotoxic mechanisms. We found that TfR2 deletion can provide neuroprotection against dopaminergic degeneration, and against PD- and aging-related iron overload. The effects, however, were significantly more pronounced in females rather than in males. Our data indicate that the TfR2 iron import pathway represents an amenable strategy to h er PD progression. Data also suggest, however, that therapeutic strategies targeting TfR2 should consider a potential sexual dimorphism in neuroprotective response.
Publisher: American Society of Hematology
Date: 15-07-2002
Abstract: Hemochromatosis is a common disorder characterized by excess iron absorption and accumulation of iron in tissues. Usually hemochromatosis is inherited in an autosomal recessive pattern and is caused by mutations in the HFE gene. Less common non-HFE–related forms of hemochromatosis have been reported and are caused by mutations in the transferrin receptor 2 gene and in a gene localized to chromosome 1q. Autosomal dominant forms of hemochromatosis have also been described. Recently, 2 mutations in theferroportin1 gene, which encodes the iron transport protein ferroportin1, have been implicated in families with autosomal dominant hemochromatosis from the Netherlands and Italy. We report the finding of a novel mutation (V162del) in ferroportin1 in an Australian family with autosomal dominant hemochromatosis. We propose that this mutation disrupts the function of the ferroportin1 protein, leading to impaired iron homeostasis and iron overload.
Publisher: Baishideng Publishing Group Inc.
Date: 2007
Abstract: Non-HFE hereditary haemochromatosis (HH) refers to a genetically heterogeneous group of iron overload disorders that are unlinked to mutations in the HFE gene. The four main types of non-HFE HH are caused by mutations in the hemojuvelin, hepcidin, transferrin receptor 2 and ferroportin genes. Juvenile haemochromatosis is an autosomal recessive disorder and can be caused by mutations in either hemojuvelin or hepcidin. An adult onset form of HH similar to HFE-HH is caused by homozygosity for mutations in transferrin receptor 2. The autosomal dominant iron overload disorder ferroportin disease is caused by mutations in the iron exporter ferroportin. The clinical characteristics and molecular basis of the various types of non-HFE haemochromatosis are reviewed. The study of these disorders and the molecules involved has been invaluable in improving our understanding of the mechanisms involved in the regulation of iron metabolism.
Publisher: Elsevier BV
Date: 11-2020
Publisher: American Physiological Society
Date: 02-2008
DOI: 10.1152/AJPCELL.00492.2007
Abstract: Transferrin receptor 2 (TfR2), a homologue of transferrin receptor 1 (TfR1), is a key molecule involved in the regulation of iron homeostasis. Mutations in TfR2 result in iron overload with similar features to HFE-associated hereditary hemochromatosis. The precise role of TfR2 in iron metabolism and the functional consequences of disease-causing mutations have not been fully determined. We have expressed wild-type and various mutant forms of TfR2 that are associated with human disease in a mouse liver cell line. Intracellular and surface analysis shows that all the TfR2 mutations analyzed cause the intracellular retention of the protein in the endoplasmic reticulum, whereas the wild-type protein is expressed in endocytic structures and at the cell surface. Our results indicate that the majority of mutations that cause type 3 hereditary hemochromatosis are a consequence of the defective localization of the protein.
Publisher: American Society for Cell Biology (ASCB)
Date: 1999
DOI: 10.1091/MBC.10.1.119
Abstract: Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST–syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.
Publisher: Portland Press Ltd.
Date: 19-05-2015
DOI: 10.1042/BSR20150014
Abstract: Iron, an essential nutrient, is required for many erse biological processes. The absence of a defined pathway to excrete excess iron makes it essential for the body to regulate the amount of iron absorbed a deficiency could lead to iron deficiency and an excess to iron overload and associated disorders such as anaemia and haemochromatosis respectively. This regulation is mediated by the iron-regulatory hormone hepcidin. Hepcidin binds to the only known iron export protein, ferroportin (FPN), inducing its internalization and degradation, thus limiting the amount of iron released into the blood. The major factors that are implicated in hepcidin regulation include iron stores, hypoxia, inflammation and erythropoiesis. The present review summarizes our present knowledge about the molecular mechanisms and signalling pathways contributing to hepcidin regulation by these factors.
Publisher: Elsevier BV
Date: 12-2021
DOI: 10.1016/J.BIOCEL.2021.106094
Abstract: Iron is an essential element for virtually all living things. Body iron levels are tightly controlled as both increased iron levels and iron deficiency are associated with many clinical conditions. Increased iron levels are associated with a worse prognosis in some cancers, so understanding the role of iron in cancer development has thus been an active area of research. Regulated forms of cell death are important in development and disease pathogenesis. In this Medicine in Focus review article, we discuss the role of iron in cancer, and ferroptosis, a new form of iron-regulated cell death triggered by increased iron and peroxidation of lipids. We also review the pathogenesis of cancer, potential therapeutics for targeting the increased requirement of iron, as well as how ferroptosis activation may have a role in treatment of cancers.
Publisher: Wiley
Date: 20-11-2018
Publisher: Wiley
Date: 03-1995
Publisher: SAGE Publications
Date: 05-2013
DOI: 10.3851/IMP2720
Abstract: Interferon stimulated chemokine CXCL-10, interleukin (IL)-12 (p70) and IL-21 have been associated with HBsAg and HBeAg loss following treatment of HBV monoinfection. The aim of this study was to determine whether these factors were also associated with HBsAg and HBeAg loss in HIV–HBV-coinfected patients following HBV-active combination antiretroviral therapy (cART). HIV–HBV-coinfected patients with HBeAg seroconversion ( n=12 seroconverters [SC]) were compared to patients who did not seroconvert ( n=13 non-seroconverters [NSC]). CXCL-10, IL-12 and IL-21 (Luminex Bead Array, Life Technologies, Grand Island, NY, USA) were measured in plasma prior to initiation of HBV-active cART (baseline), at the time of seroconversion (T0) and at the closest time point before (T-1) and after (T+1) seroconversion. Levels of CXCL-10 declined significantly in all patients following HBV-active cART ( P .05 for both SC and NSC Kruskall-Wallis, Dunn's post-test). There was no difference between SC and NSC in the level of CXCL-10, IL-12 and IL-21 at any time point. We found no evidence that CXCL-10, IL-12 or IL-21 were associated with HBeAg seroconversion following HBV-active cART. Other immunological determinants should be explored in this setting.
Publisher: American Physiological Society
Date: 11-2011
Abstract: The HFE protein plays a crucial role in the control of cellular iron homeostasis. Steatosis is commonly observed in HFE-related iron-overload disorders, and current evidence suggests a causal link between iron and steatosis. Here, we investigated the potential contribution of HFE mutations to hepatic lipid metabolism and its role in the pathogenesis of nonalcoholic fatty liver disease. Wild-type (WT) and Hfe knockout mice ( Hfe −/− ) were fed either standard chow, a monounsaturated low fat, or a high-fat, high-carbohydrate diet (HFD) and assessed for liver injury, body iron status, and markers of lipid metabolism. Despite hepatic iron concentrations and body weights similar to WT controls, Hfe −/− mice fed the HFD developed severe hypoxia-related steatohepatitis, Tnf-α activation, and mitochondrial respiratory complex and antioxidant dysfunction with early fibrogenesis. These features were associated with an upregulation in the expression of genes involved in intracellular lipid synthesis and trafficking, while transcripts for mitochondrial fatty acid β-oxidation and adiponectin signaling-related genes were significantly attenuated. In contrast, HFD-fed WT mice developed bland steatosis only, with no inflammation or fibrosis and no upregulation of lipogenesis-related genes. A HFD led to reduced hepatic iron in Hfe −/− mice compared with chow-fed mice, despite higher serum iron, decreased hepcidin expression, and increased duodenal ferroportin mRNA . In conclusion, our results demonstrate that Hfe −/− mice show defective hepatic-intestinal iron and lipid signaling, which predispose them toward diet-induced hepatic lipotoxicity, accompanied by an accelerated progression of injury to fibrosis.
Publisher: Public Library of Science (PLoS)
Date: 20-12-2010
Publisher: Portland Press Ltd.
Date: 08-2020
DOI: 10.1042/BSR20201508
Abstract: Exome sequencing has identified the glyceronephosphate O-acyltransferase (GNPAT) gene as a genetic modifier of iron overload in hereditary hemochromatosis (HH). Subjects with HFE (Homeostatic Iron Regulator) p.C282Y mutations and the GNPAT p.D519G variant had more iron loading compared with subjects without the GNPAT variant. In response to an oral iron challenge, women with GNPAT polymorphisms loaded more iron as compared with women without polymorphisms, reinforcing a role for GNPAT in iron homeostasis. The aim of the present study was to develop and characterize an animal model of disease to further our understanding of genetic modifiers, and in particular the role of GNPAT in iron homeostasis. We generated an Hfe/Gnpat mouse model reminiscent of the patients previously studied and studied these mice for up to 26 weeks. We also examined the effect of dietary iron loading on mice with reduced Gnpat expression. Gnpat heterozygosity in Hfe knockout mice does not play a role in systemic iron homeostasis Gnpat+/− mice fed a high-iron diet, however, had lower hepatic hepcidin (HAMP) mRNA expression, whereas they have significantly higher serum iron levels and transferrin saturation compared with wildtype (WT) littermates on a similar diet. These results reinforce an independent role of GNPAT in systemic iron homeostasis, reproducing in an animal model, the observations in women with GNPAT polymorphisms subjected to an iron tolerance test.
Publisher: BMJ
Date: 07-2005
Publisher: Elsevier BV
Date: 03-2011
DOI: 10.1016/J.JHEP.2010.07.048
Abstract: Hereditary iron overload associated with mutations in the ferroportin gene produces a dichotomy of phenotypes resulting from either increase or decrease in iron efflux capacity. In this study, we examined the molecular basis of iron overload in a family of Vietnamese origin, characterized the molecular and cellular defect, and correlated it with the clinical and pathological phenotype. We analyzed the ferroportin gene by DNA sequencing. The molecular characterization was performed by immunofluorescence microscopy analysis of transfected cells. We analyzed ferritin levels, in cells expressing wild-type and mutant ferroportin, to define the nature of the molecular defect in iron transport. We identified a G to A nucleotide change at position 238 in the ferroportin gene leading to the G80S substitution. Cellular analysis of the mutant protein indicates that this amino acid change does not affect the localization of the protein but does affect its ability to transport iron. The G80S mutation results in a mutated ferroportin associated with iron overload and is predicted to be defective in iron export.
Publisher: BMJ
Date: 08-2003
Abstract: A severe form of iron overload with the clinicopathological features of haemochromatosis inherited in an autosomal dominant manner has been described in the Solomon Islands. The genetic basis of the disorder has not been identified. The disorder has similarities to type 4 haemochromatosis, which is caused by mutations in ferroportin1. The aims of this study were to identify the genetic basis of iron overload in a patient from the Solomon Islands. Genomic DNA was isolated from peripheral blood leucocytes of a Solomon Islands man with severe iron overload. The entire coding region and splice sites of the ferroportin1 gene was sequenced. A novel missense mutation (431A>C N144T) was identified in exon 5 of the ferroportin1 gene. A novel restriction endonuclease based assay which identifies both the N144T and N144H mutations was developed which will simplify the diagnosis and screening of patients for iron overload in the Solomon Islands and other populations. This is the first identified mutation associated with haemochromatosis in the Solomon Islands population.
Publisher: Elsevier BV
Date: 12-2013
DOI: 10.1038/LABINVEST.2013.121
Abstract: Endoplasmic reticulum (ER) stress is an important pathogenic mechanism for alcoholic (ALD) and nonalcoholic fatty liver disease (NAFLD). Iron overload is an important cofactor for liver injury in ALD and NAFLD, but its role in ER stress and associated stress signaling pathways is unclear. To investigate this, we developed a murine model of combined liver injury by co-feeding the mildly iron overloaded, the hemochromatosis gene-null (Hfe(-/)) mouse ad libitum with ethanol and a high-fat diet (HFD) for 8 weeks. This co-feeding led to profound steatohepatitis, significant fibrosis, and increased apoptosis in the Hfe(-/-) mice as compared with wild-type (WT) controls. Iron overload also led to induction of unfolded protein response (XBP1 splicing, activation of IRE-1α and PERK, as well as sequestration of GRP78) and ER stress (increased CHOP protein expression) following HFD and ethanol. This is associated with a muted autophagic response including reduced LC3-I expression and impaired conjugation to LC3-II, reduced beclin-1 protein, and failure of induction of autophagy-related proteins (Atg) 3, 5, 7, and 12. As a result of the impaired autophagy, levels of the sequestosome protein p62 were most elevated in the Hfe(-/-) group co-fed ethanol and HFD. Iron overload reduces the activation of adenosine monophosphate protein kinase associated with ethanol and HFD feeding. We conclude that iron toxicity may modulate hepatic stress signaling pathways by impairing adaptive cellular compensatory mechanisms in alcohol- and obesity-induced liver injury.
Publisher: Elsevier BV
Date: 2007
DOI: 10.1053/J.GASTRO.2006.11.028
Abstract: Transferrin receptor 2 (TfR2) plays a key role in the regulation of iron metabolism. Mutations of TfR2 in humans cause type 3 hereditary hemochromatosis. Although highly expressed in liver, several studies have reported TfR2 expression in other tissues. To determine the contribution of liver expressed TfR2 in iron homeostasis, we have generated and characterized a liver-specific TfR2-knockout (KO) mouse. Liver-specific TfR2-KO mice were generated by crossing TfR2-floxed mice with transgenic albumin-Cre mice. Tissue and serum from homozygous TfR2-floxed mice with and without albumin-Cre were analyzed. Serum transferrin saturation, hepatic, and splenic iron concentrations were determined. The expression of iron-related mRNA transcripts was analyzed by real-time PCR. Levels of the iron-related proteins TfR1, TfR2, ferritin, and prohepcidin were analyzed by immunoblotting. Liver-specific TfR2-KO mice develop significant iron overload comparable to complete TfR2-KO mice. At all ages studied, transferrin saturation, hepatic iron concentration, and hepatic ferritin were significantly elevated. Hepatic TfR2 mRNA and protein were absent in the livers of liver-specific TfR2-KO mice, and TfR1 expression was reduced consistent with liver iron loading. At 5 weeks of age, hepcidin1 mRNA, and prohepcidin protein were decreased in liver-specific TfR2-KO compared to control mice. The significant iron loading and modulation of expression of iron-related genes in liver-specific TfR2-KO mice demonstrates that the liver is the primary site for TfR2 expression and activity and that liver-expressed TfR2 is required for the regulation of hepcidin1.
Publisher: American Society for Cell Biology (ASCB)
Date: 09-1998
DOI: 10.1091/MBC.9.9.2423
Abstract: cDNA clones encoding a novel protein (VAMP5) homologous to synaptobrevins/VAMPs are detected during database searches. The predicted 102–amino acid VAMP5 harbors a 23-residue hydrophobic region near the carboxyl terminus and exhibits an overall amino acid identity of 33% with synaptobrevin/VAMP1 and 2 and cellubrevin. Northern blot analysis reveals that the mRNA for VAMP5 is preferentially expressed in the skeletal muscle and heart, whereas significantly lower levels are detected in several other tissues but not in the brain. During in vitro differentiation (myogenesis) of C2C12 myoblasts into myotubes, the mRNA level for VAMP5 is increased ∼8- to 10-fold. Immunoblot analysis using antibodies specific for VAMP5 shows that the protein levels are also elevated ∼6-fold during in vitro myogenesis of C2C12 cells. Indirect immunofluorescence microscopy and immunoelectron microscopy reveal that VAMP5 is associated with the plasma membrane as well as intracellular perinuclear and peripheral vesicular structures of myotubes. Epitope-tagged versions of VAMP5 are similarly targeted to the plasma membrane.
Publisher: Wiley
Date: 12-06-2016
DOI: 10.1002/AJH.24417
Publisher: Elsevier BV
Date: 10-2020
Publisher: American Society of Hematology
Date: 10-2003
Publisher: Elsevier BV
Date: 10-2008
Publisher: Wiley
Date: 23-02-2015
DOI: 10.1111/JGH.12720
Abstract: Development of effective antifibrotic treatments that can be translated to clinical practice is an important challenge in contemporary hepatology. A recent report on β-thalassemia patients demonstrated that deferasirox treatment reversed or stabilized liver fibrosis independent of its iron-chelating properties. In this study, we investigated deferasirox in cell and animal models to better understand its potential antifibrotic effects. The LX-2 stellate cell line was treated with 5 μM or 50 μM deferasirox (Exjade, Novartis Pharmaceuticals Australia, North Ryde, NSW, Australia) for up to 120 h. Three-week-old multidrug resistance 2 null (Mdr2(-/-) ) mice received oral deferasirox or vehicle for 4 weeks (30 mg/kg/day). Cells and liver tissue were collected for assessment of fibrosis and fibrogenic gene expression. In LX-2 cells treated with 50 μM deferasirox for 12 h, α1(I)procollagen expression was decreased by 25%, with maximal reductions (10-fold) seen following 24-120 h of treatment. Similarly, α-smooth muscle actin (αSMA) expression was significantly lower. Alterations in matrix remodeling genes, specifically decreased expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2, were observed. There was no significant difference in hepatic hydroxyproline content in Mdr2(-/-) mice following deferasirox administration (vehicle: 395 ± 27 μg/g vs deferasirox: 421 ± 33 μg/g). Similarly, no changes in the expression of fibrogenic genes were observed. Despite reductions in α1(I)procollagen and αSMA expression and alterations in matrix degradation genes in LX-2 cells, deferasirox did not exhibit antifibrotic activity in Mdr2(-/-) mice. Given the positive outcomes seen in human trials, it may be appropriate to study deferasirox in other animal models of fibrosis and/or for a longer duration of therapy.
Publisher: Public Library of Science (PLoS)
Date: 14-10-2013
Publisher: Springer Science and Business Media LLC
Date: 2002
Publisher: Springer Science and Business Media LLC
Date: 28-04-2021
DOI: 10.1007/S10534-021-00312-1
Abstract: Iron is an essential component for multiple biological processes. Its regulation within the body is thus tightly controlled. Dysregulation of iron levels within the body can result in several disorders associated with either excess iron accumulation, including haemochromatosis and thalassaemia, or iron deficiency. In cases of excess body iron, therapy involves depleting body iron levels either by venesection, typically for haemochromatosis, or using iron chelators for thalassemia. However, the current chelation options for people with iron overload are limited, with only three iron chelators approved for clinical use. This presents an opportunity for improved therapeutics to be identified and developed. The aim of this study was to examine multiple compounds from within the Davis open access natural product-based library (512 compounds) for their ability to chelate iron. In silico analysis of this library initially identified nine catechol-containing compounds and two closely related compounds. These compounds were subsequently screened using an in vitro DNA breakage assay and their ability to chelate biological iron was also examined in an iron-loaded hepatocyte cellular assay. Toxicity was assessed in hepatocyte and breast cancer cell lines. One compound, RAD362 [N-(3-aminopropyl)-3,4-dihydroxybenzamide] was able to protect against DNA damage, likely through the prevention of free radicals generated via the Fenton reaction RAD362 treatment resulted in decreased ferritin protein levels in iron-loaded hepatocytes. Lastly, RAD362 resulted in significantly less cell death than the commonly used iron chelator deferoxamine. This is the first study to identify compound RAD362 as an iron chelator and potential therapeutic.
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.AJPATH.2012.06.025
Abstract: Hereditary hemochromatosis is characterized by tissue iron loading and associated organ damage. However, the phenotype can be highly variable. The relationship between iron loading of different organs and the temporal nature of its deposition is still not well understood. We examined the progression of tissue iron loading in three mouse models to advance our understanding of the natural history of iron deposition in hereditary hemochromatosis. Wild-type, Hfe(-/-), Tfr2(-/-), and Hfe(-/-)/Tfr2(-/-) mice were analyzed at 3, 5, 10, 26, and 52 weeks, respectively. Hepatic, splenic, cardiac, and pancreatic iron concentrations were determined. Expression of both iron-regulatory and fibrosis genes was determined by quantitative real-time PCR in livers and hearts of 52-week-old mice. In all models, hepatic iron increased rapidly, plateauing before 10 weeks at different levels, depending on the genotype. Iron deposition in the pancreas and heart occurred after maximal iron loading of the liver was reached and was most marked in the Hfe(-/-)/Tfr2(-/-) mice. Although a significant positive correlation was identified between pancreatic and cardiac iron in all models at 52 weeks, there was no correlation between hepatic and either pancreatic or cardiac iron. There is variability in the timing and extent of tissue iron loading within a genotype, suggesting that hepatic iron levels in hereditary hemochromatosis may not accurately predict the iron content of other organs.
Publisher: Elsevier BV
Date: 2017
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 15-03-2016
DOI: 10.1002/HEP.28479
Publisher: Oxford University Press (OUP)
Date: 07-01-2015
Abstract: The anaemia of chronic disease (ACD) results from inflammation-mediated up-regulation of the iron regulatory hormone hepcidin, with the consequent sequestration of iron limiting its availability for erythropoiesis. The inflammatory cytokine IL-6, a regulator of hepcidin, has been implicated in this process. Recent in vivo and in vitro studies indicate that IL-22 is also able to stimulate hepcidin expression. We aimed to determine if IL-22 had a role in causing the hypoferremia associated with the inflammatory response. Wild-type and Il22-knockout mice were subjected to an acute inflammatory stimulus via administration of LPS and the response of hepcidin and iron homeostasis was analysed. In the absence of IL-22, there was a response of hepcidin, resulting in a reduction in serum iron levels. However, the hypoferremic response to LPS was slightly blunted in mice lacking IL-22, suggesting that, during LPS-mediated inflammation, IL-22 may play a minor role in mediating the hypoferremic response. These results may have implications for the treatment and management of the ACD.
Publisher: American Society of Hematology
Date: 10-03-2011
DOI: 10.1182/BLOOD-2010-08-303859
Abstract: The induction of the iron-regulatory peptide hepcidin by proinflammatory cytokines is thought to result in the withholding of iron from invading pathogens. Hfe and transferrin receptor 2 (Tfr2) are involved in the homeostatic regulation of hepcidin and their disruption causes hereditary hemochromatosis (HH). To determine whether either Hfe or Tfr2 is involved in the inflammatory pathway regulating hepcidin, we analyzed the effect of inflammation in 3 mouse models of HH. The inflammatory response and indicators of iron homeostasis were measured in wild-type, Hfe−/−, Tfr2−/−, and Hfe−/−/Tfr2−/− mice injected with lipopolysaccharide (LPS). The administration of LPS significantly reduced serum iron in wild-type and Hfe−/− mice, with smaller reductions in Tfr2−/− and Hfe−/−/Tfr2−/− mice. Low basal levels of hepcidin in the Hfe−/−/Tfr2−/− mice were increased in response to LPS, but remained significantly lower than in the other strains of mice. These results suggest that despite the absence of Hfe and Tfr2, hepcidin is responsive to inflammation however, the low basal expression and subsequent low levels of circulating hepcidin are insufficient to reduce serum iron effectively. This suggests that in HH, the iron-withholding response to invading pathogens may be inadequate, and this is especially the case in the absence of both Hfe and Tfr2.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 27-04-2018
DOI: 10.1002/HEP4.1190
Abstract: Rodent and cell‐culture models support a role for iron‐related adipokine dysregulation and insulin resistance in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) however, substantial human data are lacking. We examined the relationship between measures of iron status, adipokines, and insulin resistance in patients with NAFLD in the presence and absence of venesection. This study forms part of the Impact of Iron on Insulin Resistance and Liver Histology in Nonalcoholic Steatohepatitis (IIRON2) study, a prospective randomized controlled trial of venesection for adults with NAFLD. Paired serum s les at baseline and 6 months (end of treatment) in controls (n = 28) and patients who had venesection (n = 23) were assayed for adiponectin, leptin, resistin, retinol binding protein‐4, tumor necrosis factor α, and interleukin‐6, using a Quantibody, customized, multiplexed enzyme‐linked immunosorbent assay array. Hepatic iron concentration (HIC) was determined using MR FerriScan. Unexpectedly, analysis revealed a significant positive correlation between baseline serum adiponectin concentration and HIC, which strengthened after correction for age, sex, and body mass index (rho = 0.36 P = 0.007). In addition, there were significant inverse correlations between HIC and measures of insulin resistance (adipose tissue insulin resistance (Adipo‐IR), serum insulin, serum glucose, homeostasis model assessment of insulin resistance, hemoglobin A1c, and hepatic steatosis), whereas a positive correlation was noted with the insulin sensitivity index. Changes in serum adipokines over 6 months did not differ between the control and venesection groups. Conclusion: HIC positively correlates with serum adiponectin and insulin sensitivity in patients with NAFLD. Further study is required to establish causality and mechanistic explanations for these associations and their relevance in the pathogenesis of insulin resistance and NAFLD. ( Hepatology Communications 2018 :644‐653)
Publisher: American Institute of Mathematical Sciences (AIMS)
Date: 2017
Publisher: Elsevier BV
Date: 04-2016
DOI: 10.1016/J.BBAMCR.2016.01.026
Abstract: Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics.
Publisher: BMJ
Date: 04-04-2014
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 18-03-2015
DOI: 10.1002/HEP.27711
Abstract: To identify polymorphisms associated with variability of iron overload severity in HFE ‐associated hemochromatosis, we performed exome sequencing of DNA from 35 male HFE C282Y homozygotes with either markedly increased iron stores (n = 22 cases) or with normal or mildly increased iron stores (n = 13 controls). The 35 participants, residents of the United States, Canada, and Australia, reported no or light alcohol consumption. Sequencing data included 82,068 single‐nucleotide variants, and 10,337 genes were tested for a difference between cases and controls. A variant in the GNPAT gene showed the most significant association with severe iron overload ( P = 3 × 10 −6 P = 0.033 by the likelihood ratio test after correction for multiple comparisons). Sixteen of twenty‐two participants with severe iron overload had glyceronephosphate O‐acyltransferase ( GNPAT ) polymorphism p.D519G (rs11558492 15 heterozygotes, one homozygote). No control participant had this polymorphism. To examine functional consequences of GNPAT deficiency, we performed small interfering RNA–based knockdown of GNPAT in the human liver‐derived cell line, HepG2/C3A. This knockdown resulted in a ‐fold decrease in expression of the messenger RNA encoding the iron‐regulatory hormone, hepcidin. Conclusion: GNPAT p.D519G is associated with a high‐iron phenotype in HFE C282Y homozygotes and may participate in hepcidin regulation. (H epatology 2015 :429–439
Publisher: American Physiological Society
Date: 04-2015
DOI: 10.1152/AJPCELL.00264.2014
Abstract: Mutations in the TMPRSS6 gene are associated with severe iron-refractory iron deficiency anemia resulting from an overexpression of hepcidin, the key regulator of iron homeostasis. The matriptase (MT)-2 protein (encoded by the TMPRSS6 gene) regulates hepcidin expression by cleaving hemojuvelin [HJV/hemochromatosis type 2 (HFE2)], a bone morphogenetic protein (BMP) coreceptor in the hepcidin regulatory pathway. We investigated the functional consequences of five clinically associated TMPRSS6 variants and the role of MT-2 protein domains by generating epitope-tagged mutant and domain-swapped MT-2-MT-1 (encoded by the ST14 gene) chimeric constructs and expressing them in HepG2/C3A cells. We developed a novel cell culture immunofluorescence assay to assess the effect of MT-2 on cell surface HJV expression levels, compatible with HJV cleavage. The TMPRSS6 variants Y141C, I212T, G442R, and C510S were retained intracellularly and were unable to inhibit BMP6 induction of hepcidin. The R271Q variant, although it has been associated with iron-refractory iron deficiency anemia, appears to remain functional. Analysis of the chimeric constructs showed that replacement of sperm protein, enterokinase, and agrin (SEA), low-density-lipoprotein receptor class A (LDLRA), and protease (PROT) domains from MT-2 with those from MT-1 resulted in limited cell surface localization, while the complement C1r/C1s, Uegf, Bmp1 (CUB) domain chimera retained localization at the cell surface. The SEA domain chimera was able to reduce cell surface HJV expression, while the CUB, LDLRA, and PROT domain chimeras were not. These studies suggest that the SEA and LDLRA domains of MT-2 are important for trafficking to the cell surface and that the CUB, LDLRA, and PROT domains are required for cleavage of HJV.
Publisher: Elsevier BV
Date: 08-1998
Publisher: Elsevier BV
Date: 09-2018
Publisher: Portland Press Ltd.
Date: 29-11-2017
DOI: 10.1042/BSR20170195
Abstract: Red blood cell production (erythropoiesis) is the single largest consumer of iron in the body this need is satisfied by maintaining a sensitive regulation of iron levels. The level of erythropoietic demand regulates the expression of the iron hormone hepcidin and thus iron absorption. Erythropoiesis-mediated regulation of hepcidin is an area of increasing importance and recent studies have identified a number of potential regulatory proteins. This review summarizes our current knowledge about these candidate erythroid regulators of hepcidin and the relation between transferrin receptors and erythropoiesis.
Publisher: Baishideng Publishing Group Inc.
Date: 2016
Abstract: The mechanisms that promote liver injury in non-alcoholic fatty liver disease (NAFLD) are yet to be thoroughly elucidated. As such, effective treatment strategies are lacking and novel therapeutic targets are required. Iron has been widely implicated in the pathogenesis of NAFLD and represents a potential target for treatment. Relationships between serum ferritin concentration and NAFLD are noted in a majority of studies, although serum ferritin is an imprecise measure of iron loading. Numerous mechanisms for a pathogenic role of hepatic iron in NAFLD have been demonstrated in animal and cell culture models. However, the human data linking hepatic iron to liver injury in NAFLD is less clear, with seemingly conflicting evidence, supporting either an effect of iron in hepatocytes or within reticulo-endothelial cells. Adipose tissue has emerged as a key site at which iron may have a pathogenic role in NAFLD. Evidence for this comes indirectly from studies that have evaluated the role of adipose tissue iron with respect to insulin resistance. Adding further complexity, multiple strands of evidence support an effect of NAFLD itself on iron metabolism. In this review, we summarise the human and basic science data that has evaluated the role of iron in NAFLD pathogenesis.
Start Date: 01-2021
End Date: 11-2027
Amount: $4,969,663.00
Funder: Australian Research Council
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