Understanding the structure/activity relationships of the iron regulatory peptide hepcidin

The peptide hormone hepcidin is a key homeostatic regulator of iron metabolism and involved in pathological regulation of iron in response to infection, inflammation, hypoxia, and anemia. It acts by binding to the iron exporter ferroportin, causing it to be internalized and degraded; however, little is known about the structure/activity relationships of the interaction of hepcidin with ferroportin. We show that there are key residues in the N-terminal region of hepcidin that influence its interaction with ferroportin, and we explore the structure/function relationships at these positions. A series of hepcidin mutants in which disulfide bonds were replaced with diselenide bonds showed no change in activity compared to native hepcidin. These results identify important constraints for the development of hepcidin congeners for the treatment of hereditary iron overload.     

Brain iron metabolism and its perturbation in neurological diseases

Abstract  

Enormous advances have been made in the last decade in understanding iron metabolism and iron homeostasis at both the cellular and the systemic level. This includes the identification of genes and proteins involved in iron transport, such as the ferric reductase DcytB, the proton-coupled ferrous (divalent) iron transporter DMT1, the iron exporter ferroportin and the membrane-bound ferroxidase hephaestin. The modulation of their translation by the iron regulatory protein (IRP) system has also been identified together with the impressive signalling cascades involved in regulating the chef d’orchestre of systemic iron homeostasis, hepcidin. However, exactly how the brain regulates fluxes and storage of iron between neurons, oligodendrocytes, astrocytes and microglial cells remains an enigma. In this review we discuss the possible mechanisms which may be involved in the transfer of iron across the blood–brain barrier, together with the possible role played by astrocytes. The consequences of iron deficiency and iron excess on brain function are described. Finally, various neurodegenerative diseases, where accumulation of iron may be important in the pathogenesis, are presented as well as the possible use of iron chelators to diminish disease progression.     

Quantification of hepcidin using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

Hepcidin is known to be a key systemic iron‐regulatory hormone which has been demonstrated to be associated with a number of iron disorders. Hepcidin concentrations are increased in inflammation and suppressed in hemochromatosis. In view of the role of hepcidin in disease, its potential as a diagnostic tool in a clinical setting is evident. This study describes the development of a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) assay for the quantitative determination of hepcidin concentrations in clinical samples. A stable isotope labeled hepcidin was prepared as an internal standard and a standard quantity was added to urine samples. Extraction was performed with weak cation‐exchange magnetic nanoparticles. The basic peptides were eluted from the magnetic nanoparticles using a matrix solution directly onto a target plate and analyzed by MALDI‐TOF MS to determine the concentration of hepcidin. The assay was validated in charcoal stripped urine, and good recovery (70–80%) was obtained, as were limit of quantitation data (5 nmol/L), accuracy (RE <10%), precision (CV <21%), within ‐day repeatability (CV <13%) and between‐day repeatability (CV <21%). Urine hepcidin levels were 10 nmol/mmol creatinine in healthy controls, with reduced levels in hereditary hemochromatosis (P < 0.000005) and elevated levels in inflammation (P < 0.0007). In summary a validated method has been developed for the determination of hepcidin concentrations in clinical samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitation of hepcidin in serum using ultra‐high‐pressure liquid chromatography and a linear ion trap mass spectrometer

Hepcidin is a peptide hormone that functions as a key regulator of mammalian iron metabolism. Biological levels are increased in end‐stage renal disease and during inflammation but suppressed in hemochromatosis. Thus hepcidin levels have diagnostic importance. This study describes the development of an analytical method for the quantitative determination of the concentration of hepcidin in clinical samples. The fragmentation of hepcidin was investigated using triple quadrupole and linear ion trap mass spectrometers. A standard quantity of a stable isotopically labelled hepcidin internal standard was added to serum samples. Extraction was performed by protein precipitation and weak cation‐exchange magnetic nanoparticles. Chromatography was carried out on sub 2 µm particle stationary phase, using ultra‐high‐pressure liquid chromatography and a linear ion trap for quantitation. The lower limit of quantitation was 0.4 nmol/L with less than 20% accuracy and precision. The mean hepcidin concentration in sera for controls was 4.6 ± 2.7 nmol/L, in patients with sickle cell disease, 7.0 ± 8.9 nmol/L; in patients with end‐stage renal disease, 30.5 ± 15.7 nmol/L; and patients with penetrant hereditary hemochromatosis, 1.4 ± 0.8 nmol/L. Copyright © 2010 John Wiley & Sons, Ltd.     

Problems of iron metabolism with special reference to biochemical, physiological, and clinical aspects

A brief account of the functions and chemical structures of the principal iron compounds found in the human body is followed by a discussion of the physiology of iron metabolism, starting with its resorption, continuing with its transport and assimilation by the body cells, and concluding with its elimination. Special attention is paid to the problems of intracellular and extracellular transport of iron. A number of diseases with characteristic disturbances of the iron metabolism are also briefly described.     

与缺铁性贫血有关的维生素及微量元素

主要介绍了维生素及微量元素与缺铁性贫血之间的关系。缺铁性贫血(IDA)是世界范围内普遍存在的营养性疾病，是因人体储存铁缺乏导致血红蛋白合成量减少而形成的一种贫血。许多营养素都与IDA有关，与IDA有关的维生素有核黄素、维生素C、维生素A、维生素E、叶酸及维生素B12，与IDA有关的微量元素有铁、铜、锌、镉、钒、铅等。     

Development of a nano-liquid chromatography on chip tandem mass spectrometry method for high-sensitivity hepcidin quantitation

Microfluidic LC systems present undeniable advantages over classical LC in terms of sensitivity. Hepcidin, a peptide marker of clinical disorders linked to iron metabolism, was used as model to demonstrate peptide quantification potentialities of LC-chip coupled to a nanoelectrospray source ion trap mass spectrometer in an aqueous sample. First, stable isotope labelled hepcidin was chosen as internal standard and gradient as well as sample compositions were optimised using design of experiments as development tool. The method was then prevalidated using accuracy profiles in order to select the most appropriate response function and to confirm the ability of the technique to quantify low hepcidin concentration. A reliable and very sensitive quantitation method was finally obtained using this integrated microfluidic technology. Indeed, good results with respect to accuracy, trueness and precision were achieved, as well as a very low limit of quantitation (0.07 ng/ml). Method suitability of nano-LC on chip tandem mass spectrometry for hepcidin quantitation was also demonstrated in complex media such as human plasma.     

Aluminium and iron in the brain—prospects for chelation

Aluminium and iron both accumulate in the brain in the course of ageing. We first briefly review how aluminium may interfere with iron metabolism through its interaction with iron homeostatic mechanisms. Then we present comparative data on the chelation of brain aluminium and iron in appropriate animal models of loading with the two metals. With both desferrioxamine (DFO) B and hydroxypyridone derivatives, brain iron is much more difficult to chelate than brain aluminium. This probably reflects the localisation of the former in ferritin and haemosiderin, and the latter in a more labile, non-protein form. The potential of long-term chelation therapy in the prevention of a number of neurodegenerative diseases associated with ageing is discussed.     

Impact of graphene-based nanomaterials (GBNMs) on the structural and functional conformations of hepcidin peptide

Graphene-based nanomaterials (GBNMs) are widely used in various industrial and biomedical applications. GBNMs of different compositions, size and shapes are being introduced without thorough toxicity evaluation due to the unavailability of regulatory guidelines. Computational toxicity prediction methods are used by regulatory bodies to quickly assess health hazards caused by newer materials. Due to increasing demand of GBNMs in various size and functional groups in industrial and consumer based applications, rapid and reliable computational toxicity assessment methods are urgently needed. In the present work, we investigate the impact of graphene and graphene oxide nanomaterials on the structural conformations of small hepcidin peptide and compare the materials for their structural and conformational changes. Our molecular dynamics simulation studies revealed conformational changes in hepcidin due to its interaction with GBMNs, which results in a loss of its functional properties. Our results indicate that hepcidin peptide undergo severe structural deformations when superimposed on the graphene sheet in comparison to graphene oxide sheet. These observations suggest that graphene is more toxic than a graphene oxide nanosheet of similar area. Overall, this study indicates that computational methods based on structural deformation, using molecular dynamics (MD) simulations, can be used for the early evaluation of toxicity potential of novel nanomaterials.     

CE‐MS method development for peptides analysis,especially hepcidin,an iron metabolism marker

A method for the resolution of a peptides mixture including hepcidin‐25, an iron metabolism marker, was developed by CE‐ESI‐MS. Several strategies were tested to optimize peptide separation, such as the addition of cyclodextrins or organic solvents in the BGE or the use of coated capillaries. Best results in terms of resolution, symmetry and efficiency were obtained with a BGE made of 500 mM ammonium acetate pH 4.5/ACN 70:30 v/v. Using the methodology of experimental design, BGE concentration, sheath liquid composition and MS‐coupling parameters were then optimized in order to obtain the best signal intensity for hepcidin. Finally, a 225 mM BGE and a sheath liquid composed of isopropanol/water 80:20 v/v containing 0.5% v/v formic acid were selected as it constitutes the best compromise for selectivity, peak shape and sensitivity.     

Comparison of copper labeling followed by liquid chromatography-inductively coupled plasma mass spectrometry and immunochemical assays for serum hepcidin-25 determination

Hepcidin-25 has been defined as the key biomarker in iron metabolism. This peptide binds to the iron transporter ferroportin to cause its degradation. Therefore, the need for specific, accurate and precise methods for the quantification of hepcidin-25 in biological fluids is dramatically increasing. In this regard, the use of rapid immunochemical methods that provide low limit of quantification is desired for routine clinical use. However, such fast methodologies should be first analytically evaluated and compared with alternative strategies to check for their advantages and limitations. Here we compare the use of a commercial immunochemical assay for hepcidin determination with a novel analytical approach based on Cu-labeling of the peptide followed by Cu determination using liquid chromatography (HPLC) and plasma mass spectrometry (ICP-MS). The figures of merit of both systems reveal similar analytical characteristics and both seem to be adequate for the determination of the peptide at biologically relevant concentrations in human serum samples. The analysis of a larger number of samples (n = 50) by both techniques showed a good agreement in the concentrations found. Such finding permits to address the hepcidin recovery in the sample preparation procedure necessary for the HPLC-ICP-MS analysis in human serum that turn out to be 76–85%. Additionally, limitations due to cross-reactivity issues of the ELISA method could be addressed in some of the samples by using LC-ICP-MS and were confirmed by LC-Electrospray-MS.     

Nutritional genomics for the characterization of the effect of bioactive molecules in lipid metabolism and related pathways

Cardiovascular disease and cancer are the main causes of morbidity and mortality worldwide. Thus, investigators have focused their efforts on gaining insight into understanding the mechanisms involved in the development and evolution of these diseases. In the past decade, and with the contribution of the -omics technologies, strong evidence has supported an essential role of gene-nutrient interactions in these processes, pointing at natural bioactive molecules as promising complementary agents that are useful in preventing or mitigating these diseases. In addition, alterations in lipid metabolism have recently gained strong interest since they have been described as a common event required for the progression of both diseases. In the present review, we give an overview of lipid metabolism, mainly focusing on lipoprotein metabolism and the mechanisms controlling lipid homeostasis. In addition, we review the modulation of lipid metabolism by bioactive molecules, highlighting their potential use as therapeutic agents in preventing, and treating chronic diseases such as cardiovascular disease and cancer. Finally, we report the usefulness of the -omics technologies in nutritional research, focusing on recent findings, within nutritional genomics, in the interaction of bioactive components from foods with several genes that are involved in the development and progression of these diseases.     

HSP70 attenuates compression-induced apoptosis of nucleus pulposus cells by suppressing mitochondrial fission via upregulating the expression of SIRT3

Compression-induced apoptosis of nucleus pulposus (NP) cells plays a pivotal role in the pathogenesis of intervertebral disc degeneration (IVDD). Recent studies have shown that the dysregulation of mitochondrial fission and fusion is implicated in the pathogenesis of a variety of diseases. However, its role in and regulatory effects on compression-induced apoptosis of NP cells have not yet been fully elucidated. Heat shock protein 70 (HSP70) is a major cytoprotective heat shock protein, but its physiological role in IVDD, especially its effect on mitochondrial fission and fusion, is still unknown. Herein, we found that compression could induce mitochondrial fission, which ultimately trigger apoptosis of NP cells via the mitochondrial apoptotic pathway. In addition, we identified the cytoprotective effects of HSP70 on NP cells, and we found that promoting the expression of HSP70 could protect NP cells from abnormal mechanical loading in vitro and in vivo. Finally, we showed that HSP70 inhibited compression-induced mitochondrial fission by promoting SIRT3 expression, thereby attenuating mitochondrial dysfunction and the production of reactive oxygen species and ultimately inhibiting the mitochondrial apoptotic pathway in NP cells. In conclusion, our results demonstrated that HSP70 could attenuate compression-induced apoptosis of NP cells by suppressing mitochondrial fission via upregulating SIRT3 expression. Promoting the expression of HSP70 might be a novel strategy for the treatment of IVDD.Subject terms: Rheumatic diseases, Apoptosis     

含铁蛋白介导的铁转运分子机制

铁是生命体必需的微量元素,因为它是一些重要功能酶的协同因子。这些功能酶有着广泛的功能,从呼吸作用到核酸的复制。但是,当铁含量多于细胞稳态的时候,它将产生对机体有毒的羟基。生物体已经发展了自身的调控机制,包括铁的摄取,存储和输出来控制细胞内的铁处于平衡态。二价阳离子转运蛋白,铁输出蛋白和hephaestin参与小肠吸收,转铁蛋白和转铁蛋白受体参与铁的摄取和转运,铁蛋白可以存储铁,铁调控蛋白的功能是调节铁代谢。这篇文章综述着重阐述了含铁蛋白介导的铁传递机制。     

Protective effects of baicalin and quercetin on an iron-overloaded mouse: comparison of liver, kidney and heart tissues

Excessive accumulation of iron in the body can lead to organ injury. Some flavonoids which possess high affinity to iron can be used as iron chelators for curing iron overload diseases such as haemochromatosis. In this article, the effects of baicalin and quercetin on different mouse tissues (liver, kidney and heart) afflicted with iron overload-induced injury were studied. It was found that after administration of 500 mg kg?1 iron dextran for 45 days, the degree of oxidative injury in the three organs was different. When iron dextran-induced iron-overloaded mice were fed a diet containing baicalin or quercetin (1% w/w), both flavonoids showed significant effects in suppressing iron overload-induced injury. The efficiencies of both flavonoids on different tissues varied. Both flavonoids caused a significant decrease of iron content in liver and kidney tissues but not in the heart, whereas the flavonoids were more efficient in inhibiting the increase of carbonyl content and the thiobarbituric acid reactive substance levels in heart tissues. The results indicate that the protective effects of baicalin and quercetin on different tissues of iron-overloaded mice are different.     

Chelating agents for human diseases related to aluminium overload

The aim of this review is to give a general view on the current status of the role of aluminium in human health and disease. The main aspects of aluminium metabolism in humans are covered, summarizing the state of knowledge on the absorption, distribution, retention and excretion of aluminium, giving particular emphasis to the main metabolic pathways of this metal ion in different organs. Then the pathological consequences of aluminium overload in man, its role in neurodegenerative diseases and the emerging implication of this metal ions in different pathologies are treated. Finally, the function of different chelators utilized in clinical practice in the therapy of aluminium depending diseases is discussed and the latest studies on aluminium chelators are presented. Some emphasis is given to the parallelism between iron and aluminium chelators, and in particular interesting correlations between structural parameters of these two trivalent metal ions are presented.