铁调素调节蛋白(HJV)———一个新的铁代谢调节蛋白

铁调素调节蛋白 (hemojuvelin,HJV) 是最近发现的一种重要的铁代谢调节蛋白. HJV基因突变是年轻型血色素沉着症 (Juvenile hemochromatosis,JH ) 的重要原因之一. 研究显示,HJV可能是一种极为重要的铁调素 (hepcidin) 表达的调节蛋白,通过参与铁调素表达的调节从而在铁代谢中发挥重要作用.     

风疹病毒衣壳蛋白与宿主细胞相互作用蛋白的初步筛选及分析

风疹病毒(Rubella virus,RV)的衣壳蛋白(Capsid protein,CP)不仅是病毒颗粒的重要组成部分,而且还可以通过与宿主蛋白之间的相互作用来调控病毒的转录和复制.为了系统研究衣壳蛋白与宿主蛋白之间的相互作用关系,我们从RV基因组中克隆获得衣壳蛋白基因序列,将该序列导入含有eXact-6His串联亲和纯化标签的慢病毒表达载体中,并构建了稳定表达eXact-6His-Capsid融合蛋白的293T细胞系.通过eXact和6His标签的两次亲和纯化获得衣壳蛋白相互作用蛋白复合物,质谱检测并筛选后发现22个可能与衣壳蛋白相互作用的宿主蛋白.随后构建衣壳蛋白的相互作用网络并进行功能学分析,发现其相互作用蛋白主要参与病毒感染、RNA剪切、细胞凋亡及酶相关通路等过程.     

骨形态发生蛋白6对机体铁代谢调控的机制

骨形态发生蛋白6(BMP6)为TGF-β超家族中的一员,已有的研究表明BMP6具有成骨和成软骨作用,最新的研究发现了它对机体的铁代谢也具有重要的调控作用,通过调节铁调素(hepcidin)的表达,在调节机体铁稳态过程中扮演着重要角色。     

鼠伤寒沙门菌感染巨噬细胞铁稳态相关基因mRNA表达谱

用荧光定量PCR法检测鼠RAW264.7巨噬细胞感染与未感染鼠伤寒沙门菌后18种铁穗态相关基因的表达,评估宿主与病原体相互作用中铁稳态效应。研究显示,活的鼠伤寒沙门菌感染巨噬细胞1 h后可以诱导转铁蛋白受体表达,引起细胞内动态铁池相关基因的mRNA水平上长。基因表达分析显示,沙门菌通过诱导铁氧还原酶(Steap3)、铁膜转运蛋白(Dmt1)、铁调节因子Tfr2/Hfe以及铁调节蛋白(Irp1和Irp2)的表达主动吸收铁,而经铁转运蛋白(Fpn1)的铁外流并无明显改变。沙门菌在感染后1h积极地驱动了转铁蛋白介导的铁吸收程序。     

毕赤酵母工程菌人hepcidin的纯化及部分铁代谢活性研究

采用摇瓶培养重组毕赤酵母(Pichia pastoris)表达并分泌重组人hepcidin至胞外,经等电沉淀,凝胶过滤纯化,电泳检测样品纯度,通过Western blot检测小鼠内皮细胞中hepcidin对GFP-FPN1及TfR1表达的影响.研究发现发酵hepcidin产量达150 mg/L,纯化后经Tricine-SDS-PAGE检测为单一条带,分子质量与理论质量一致,具有抗菌活性,转染内皮细胞证实重组hepcidin可影响内皮细胞GFP-FPN1及TfR1的表达,具有调节铁代谢活性,对研究hepcidin与铁代谢的相关分子吸收机制及药物开发应用奠定了基础,对潜在的医学诊断治疗具有重要意义.     

Hepcidin的生物学特性及其研究进展

Hepcidin是一种由肝脏合成的富含半胱氨酸的小分子肽。近几年的研究证实hepcidin对于调节机体铁离子的代谢平衡发挥着重要的作用,其可抑制肠道铁吸收和单核巨噬细胞系统铁释放。此外,除了机体铁状况,感染、炎症、贫血和缺氧等原因也会改变hepcidin的表达水平。通过对hepcidin的分子生物学特点、表达调控及生物活性、医学及药用价值等方面研究进展的概述,对采用基因工程的方法生产hepcidin进行了评述及展望。     

荧光能量转移技术在生物学研究中的应用

荧光能量转移(FRET)是指两个携带不同荧光基团的大分子在相互间距离足够近时(10～100A)所发生的能量非放射性地由一个荧光基团向另一个荧光基团转移的现象。结合绿色荧光蛋白的发现,FRET技术可用于检测生物大分子中不同亚基的位置和生物大分子间的相互作用。近年来,FRET技术在生物学研究中的突破性进展是在活体细胞中实时监测生物大分子之间的相互作用。本文就绿色荧光蛋白的发现,FRET技术的原理、研究进展和应用前景作简要综述。     

铁代谢与铁调素hepcidin

铁是机体必需的营养元素。然而,铁过载则导致细胞的损伤。由于生物体缺少排泄铁的机制,因而,肠铁吸收的调控便成为维持机体铁稳态的关键。新近研究发现hepcidin对机体铁稳态的调节起着至关重要的作用,被人们称为铁调节激素。Hepcidin主要在肝细胞中合成,之后分泌至血液将体内铁需要的信号传至小肠,调控肠铁的吸收。这一过程主要通过调节小肠铁转运相关蛋白的表达而实现。任何影响hepcidin表达的因素都可能破坏体内的铁平衡,造成铁代谢相关疾病。     

单核巨噬细胞铁代谢相关蛋白的表达调控

人类机体的铁代谢表现为受限制的对外界铁的吸收和有效的机体内的铁的再循环利用,单核巨噬细胞系统通过吞噬衰老的红细胞,储存和释放铁,在机体铁的循环再利用方面起到了重要的作用。因此,单核巨噬细胞系统对整个机体铁稳态的维持非常重要。近年来,随着转铁蛋白受体1(transferrin receptor1,TfR1)、铁蛋白(ferritin,Fn)、二价金属离子转运蛋白1(divalent metal transporter1,DMT1)、膜铁转运蛋白1(ferroportin1,FPN1),以及铁调素(hepcidin)等在单核巨噬细胞系统中功能和调控机制研究的不断深入,日益加深了人们对单核巨噬细胞系统的铁代谢过程和调控机制的了解。该文综述了铁水平、NO以及炎症等因素对单核巨噬细胞系统TfR1、Fn、DMT1、FPN1、hepcidin等蛋白表达的调控及其机制研究的最新进展。     

生物大分子相互作用检测技术新进展———三色荧光级联荧光共振能量转移技术

荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET),是指能量从一种受激发的荧光基团(fluorophore)以非辐射的方式转移到另一种荧光基团的物理现象.FRET的能量转移效率是两个荧光基团间距离的函数,并对此距离十分敏感,它的有效响应距离一般在1～10nm之间,因而可被用于测定原子间及分子间的距离.这一特点使FRET技术在大分子构象变化、大分子之间相互作用、细胞信号通路等研究中发挥重要作用,成为生物医学研究中的重要方法.但细胞内的生物学过程常常涉及多于两个的大分子间相互作用,二色荧光基团的FRET技术不能满足这种生物学研究的需求.最近,两个研究小组在这方面取得突破,建立了分别基于共聚焦显微镜和流式细胞仪的三色荧光级联FRET技术.这一技术的出现将会极大地促进生物学及相关研究领域的发展.     

The iron regulatory hormone hepcidin reduces ferroportin 1 content and iron release in H9C2 cardiomyocytes

Iron plays a key pathophysiological role in a number of cardiac diseases. Studies on the mechanisms of heart iron homeostasis are therefore crucial for understanding the causes of excessive heart iron. In addition to iron uptake, cellular iron balance in the heart also depends on iron export. We provided evidence for the existence of iron exporter ferroportin 1 (Fpn1) in the heart in a recent study. The presence of hepcidin, a recently discovered iron regulatory hormone, was also confirmed in the heart recently. Based on these findings and the inhibiting role of hepcidin on Fpn1 in other tissues, we speculated that hepcidin might be able to bind with, internalize and degrade Fpn1 and then decrease iron export in heart cells, leading to an abnormal increase in heart iron and iron mediated cell injury. We therefore investigated the effects of hepcidin on the contents of Fpn1 and iron release in H9C2 cardiomyocyte cell line. We demonstrated that hepcidin has the ability to reduce Fpn1 content as well as iron release in this cell. The similar regulation patterns of hepcidin on the Fpn1 and iron release suggested that the decreased iron release resulted from the decreased content of Fpn1 induced by hepcidin. We also found that hepcidin has no significant effects on ceruloplasmin (CP) and hephaestin (Heph) — two proteins required for iron release from mammalian cells. The data imply that Fpn1, rather than Heph and CP, is the limited factor in the regulation of iron release from heart cells under physiological conditions.     

The role of cellular iron deficiency in controlling iron export

BackgroundIron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover.MethodsWe ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency.Conclusions/General significanceWe show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.     

The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis

Ferroportin (SLC40A1) is an iron transporter postulated to play roles in intestinal iron absorption and cellular iron release. Hepcidin, a regulatory peptide, binds to ferroportin and causes it to be internalized and degraded. If ferroportin is the major cellular iron exporter, ineffective hepcidin function could explain manifestations of human hemochromatosis disorders. To investigate this, we inactivated the murine ferroportin (Fpn) gene globally and selectively. Embryonic lethality of Fpn(null/null) animals indicated that ferroportin is essential early in development. Rescue of embryonic lethality through selective inactivation of ferroportin in the embryo proper suggested that ferroportin has an important function in the extraembryonic visceral endoderm. Ferroportin-deficient animals accumulated iron in enterocytes, macrophages, and hepatocytes, consistent with a key role for ferroportin in those cell types. Intestine-specific inactivation of ferroportin confirmed that it is critical for intestinal iron absorption. These observations define the major sites of ferroportin activity and give insight into hemochromatosis.     

The role of ubiquitination in hepcidin-independent and hepcidin-dependent degradation of ferroportin

The iron exporter ferroportin (Fpn) is essential to transfer iron from cells to plasma. Systemic iron homeostasis in vertebrates is regulated by the hepcidin-mediated internalization of Fpn. Here, we demonstrate a second route for Fpn internalization; when cytosolic iron levels are low, Fpn is internalized in a hepcidin-independent manner dependent upon the E3 ubiquitin ligase Nedd4-2 and the Nedd4-2 binding protein Nfdip-1. Retention of cell-surface Fpn through reductions in Nedd4-2 results in cell death through depletion of cytosolic iron. Nedd4-2 is also required for internalization of Fpn in the absence of ferroxidase activity as well as for the entry?of hepcidin-induced Fpn into the multivesicular?body. C.?elegans lacks hepcidin genes, and C.?elegans Fpn expressed in mammalian cells is not internalized by hepcidin but is internalized in response to iron deprivation in a Nedd4-2-dependent manner, supporting the hypothesis that Nedd4-2-induced internalization of Fpn is evolutionarily conserved.     

Suppressed hepcidin expression correlates with hypotransferrinemia in copper-deficient rat pups but not dams

Copper deficiency leads to anemia but the mechanism is unknown. Copper deficiency also leads to hypoferremia, which may limit erythropoiesis. The hypoferremia may be due to limited function of multicopper oxidases (MCO) hephaestin in enterocytes or GPI-ceruloplasmin in macrophages of liver and spleen whose function as a ferroxidase is thought essential for iron transfer out of cells. Iron release may also be limited by ferroportin (Fpn), the iron efflux transporter. Fpn may be lower following copper deficiency because of impaired ferroxidase activity of MCO. Fpn is also dependent on the liver hormone hepcidin as Fpn is degraded when hepcidin binds to Fpn. Anemia and hypoferremia both down regulate hepcidin by separate mechanisms. Current studies confirmed and extended earlier studies with copper-deficient (CuD) rats that suggested low hepicidin resulted in augmented Fpn. However, current studies in CuD dams failed to confirm a correlation that hepcidin expression was associated with low transferrin receptor 2 (TfR2) levels and also challenged the dogma that holotransferrin can explain the correlation with hepcidin. CuD dams exhibited hypoferremia, low liver TfR2, anemia in some rats, yet no depression in Hamp expression, the hepcidin gene. Normal levels of GDF-15, the putative erythroid cytokine that suppresses hepcidin, were detected in plasma of CuD and iron-deficient (FeD) dams. Importantly, FeD dams did display greatly lower Hamp expression. Normal hepcidin in these CuD dams is puzzling since these rats may need extra iron to meet needs of lactation and the impaired iron transfer noted previously.     

The iron regulatory hormone hepcidin inhibits expression of iron release as well as iron uptake proteins in J774 cells

The mechanism by which hepcidin controls cellular iron release protein ferroportin 1 (Fpn1) in macrophages has been well established. However, little is known about the effects of hepcidin on cellular iron uptake proteins. Here, we demonstrated for the first time that hepcidin can significantly inhibit the expression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 in addition to Fpn1, and therefore reduce transferrin-bound iron and non-transferrin-bound iron uptake and also iron release in J774 macrophages. Analysis of mechanisms using the iron-depleted cells showed that hepcidin has a direct inhibitory effect on all iron transport proteins we examined. Further studies demonstrated that the down-regulation of TfR1 induced by hepcidin is associated with cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA), probably being mediated by the cAMP–PKA pathway in J774 macrophages.     

Fasting up‐regulates ferroportin 1 expression via a Ghrelin/GHSR/MAPK signaling pathway

The significant positive correlation between ghrelin and iron and hepcidin levels in the plasma of children with iron deficiency anemia prompted us to hypothesize that ghrelin may affect iron metabolism. Here, we investigated the effects of fasting or ghrelin on the expression of hepcidin, ferroportin 1 (Fpn1), transferrin receptor 1 (TfR1), ferritin light chain (Ft‐L) proteins, and ghrelin, and also hormone secretagogue receptor 1 alpha (GHSR1α) and ghrelin O‐acyltransferase (GOAT) mRNAs in the spleen and/or macrophage. We demonstrated that fasting induces a significant increase in the expression of ghrelin, GHSR1α, GOAT, and hepcidin mRNAs, as well as Ft‐L and Fpn1 but not TfR1 proteins in the spleens of mice in vivo. Similar to the effects of fasting on the spleen, ghrelin induced a significant increase in the expression of Ft‐L and Fpn1 but not TfR1 proteins in macrophages in vitro. In addition, ghrelin was found to induce a significant enhancement in phosphorylation of ERK as well as translocation of pERK from the cytosol to nuclei. Furthermore, the increased pERK and Fpn1 induced by ghrelin was demonstrated to be preventable by pre‐treatment with either GHSR1α antagonist or pERK inhibitor. Our findings support the hypothesis that fasting upregulates Fpn1 expression, probably via a ghrelin/GHSR/MAPK signaling pathway.     

Copper deficiency has minimal impact on ferroportin expression or function

Interactions between copper and iron homeostasis have been known since the nineteenth century when anemia in humans was first described due to copper limitation. However, the mechanism remains unknown. Intestinal and liver iron concentrations are usually higher following copper deficiency (CuD). This may be due to impaired function of the multicopper oxidases hephaestin or ceruloplasmin (Cp), respectively. However, iron retention could be due to altered ferroportin (Fpn), the essential iron efflux transporter in enterocytes and macrophages. Fpn mRNA is controlled partially by intracellular iron and IRE dependence. CuD should augment Fpn based on iron level. Some argue that Fpn stability is controlled partially by membrane ferroxidase (GPI-Cp). CuD should result in lower Fpn since GPI-Cp expression and function is reduced. Fpn turnover is controlled by hepcidin. CuD results in variable Hamp (hepcidin) expression. Fpn mRNA and protein level were evaluated following dietary CuD in rats and mice. To correlate with Fpn expression, measurements of tissue iron were conducted in several rodent models. Following CuD there was little change in Fpn mRNA. Previous work indicated that under certain circumstances Fpn protein was augmented in liver and spleen following CuD. Fpn levels in CuD did not correlate with either total iron or non-heme iron (NHI), as iron levels in CuD liver were higher and in spleen lower than copper adequate controls. Fpn steady state levels appear to be regulated by a complex set of factors. Changes in Fpn do not explain the anemia of CuD.     

Duodenal Ferroportin Is Up-Regulated in Patients with Chronic Hepatitis C

Hepatitis C virus (HCV) infection is a leading cause of liver-related mortality. Chronic hepatitis C (CHC) is frequently associated with disturbances in iron homeostasis, with serum iron and hepatic iron stores being elevated. Accumulating evidence indicates that chronic HCV infection suppresses expression of hepatic hepcidin, a key mediator of iron homeostasis, leading to iron overload conditions. Since hepcidin mediates degradation of ferroportin, a basolateral transporter involved in the release of iron from cells, diminished hepcidin expression probably leads to up-regulation of ferroportin-1 (Fpn1) in patients with CHC. In this study, we determined the protein levels of duodenal Fpn1, and found that its expression was significantly up-regulated in patients with CHC. The expression of duodenal Fpn1 is negatively correlated with mRNA levels of hepcidin, and positively correlated with serum iron parameters. Although iron is a critical factor for growth of a variety of pathogenic bacteria, our results suggest that iron overload in blood does not increase the infection rate of bacteria in patients with CHC.     

Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy

Advances in molecular biology provide various methods to define the structure and function of the individual proteins that form the component parts of subcellular structures. The ability to see the dynamic behavior of a specific protein inside the living cell became possible through the application of advanced fluorescence resonance energy transfer (FRET) microscope techniques. The fluorophore molecule used for FRET imaging has a characteristic absorption and emission spectrum that should be considered for characterizing the FRET signal. In this article we describe the system development for the image acquisition for one- and two-photon excitation FRET microscopy. We also describe the precision FRET (PFRET) data analysis algorithm that we developed to remove spectral bleed-through and variation in the fluorophore expression level (or concentration) for the donor and acceptor molecules. The acquired images have been processed using a PFRET algorithm to calculate the energy transfer efficiency and the distance between donor and acceptor molecules. We implemented the software correction to study the organization of the apical endosome in epithelial polarized MDCK cells and dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). For these proteins, the results revealed that the extent of correction affects the conventionally calculated energy transfer efficiency (E) and the distance (r) between donor and acceptor molecules by 38 and 9%, respectively.