Epigenetic regulation of iron homeostasis in Arabidopsis

Iron (Fe) is one of the most important microelement required for plant growth and development because of its unique property of catalyzing oxidation/reduction reactions. Iron deficiency impairs fundamental processes which could lead to a decrease in chlorophyll production and pollen fertility, thus influencing crop productivity and quality. However, iron in excess is toxic to the cell and is harmful to the plant. To exactly control the iron content in all tissues, plants have evolved many strategies to regulate iron homeostasis, which refers to 2 successive steps: iron uptake at the root surface, and iron distribution in vivo. In the last decades, a number of transporters and regulatory factors involved in this process have been isolated and identified. To cope with the complicated flexible environmental conditions, plants apply diverse mechanisms to regulate the expression and activity of these components. One of the most important mechanisms is epigenetic regulation of iron homeostasis. This review has been presented to provide an update on the information supporting the involvement of histone modifications in iron homeostasis and possible future course of the field.     

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model

Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.     

Phylogenetic and sequence analyses of insect transferrins suggest that only transferrin 1 has a role in iron homeostasis

Iron is essential to life,but surprisingly little is known about how iron is managed in nonvertebrate animals.In mammals,the well-characterized transferrins bind iron and are involved in iron transport or immunity,whereas other members of the transferrin family do not have a role in iron homeostasis.In insects,the functions of transferrins are still poorly understood.The goals of this project were to identify the transferrin genes in a diverse set of insect species,resolve the evolutionary relationships among these genes,and predict which of the transferrins are likely to have a role in iron homeostasis.Our phylogenetic analysis of transferrins from 16 orders of insects and two orders of noninsect hexapods demonstrated that there are four orthologous groups of insect transferrins.Our analysis suggests that transferrin 2 arose prior to the origin of insects,and transferrins/,i,and 4 arose early in insect evolution.Primary sequence analysis of each of the insect transferrins was used to predict signal peptides,carboxyl-terminal transmembrane regions,GPI-anchors,and iron binding.Based on this analysis,we suggest that transferrins 2,and 4 are unlikely to play a major role in iron homeostasis.In contrast,the transferrin 1 orthologs are predicted to be secreted,soluble,iron-binding proteins.We conclude that transferrin 1 orthologs are the most likely to play an important role in iron homeostasis.Interestingly,it appears that the louse,aphid,and thrips lineages have lost the transferrin 1 gene and,thus,have evolved to manage iron without transferrins.     

Single-Cell Transcriptome Analysis Maps the Developmental Track of the Human Heart

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The Bradyrhizobium japonicum fsrB gene is essential for utilization of structurally diverse ferric siderophores to fulfill its nutritional iron requirement

In Bradyrhizobium japonicum, iron uptake from ferric siderophores involves selective outer membrane proteins and non-selective periplasmic and cytoplasmic membrane components that accommodate numerous structurally diverse siderophores. Free iron traverses the cytoplasmic membrane through the ferrous (Fe²⁺) transporter system FeoAB, but the other non-selective components have not been described. Here, we identify fsrB as an iron-regulated gene required for growth on iron chelates of catecholate- and hydroxymate-type siderophores, but not on inorganic iron. Utilization of the non-physiological iron chelator EDDHA as an iron source was also dependent on fsrB. Uptake activities of ⁵⁵Fe³⁺ bound to ferrioxamine B, ferrichrome or enterobactin were severely diminished in the fsrB mutant compared with the wild type. Growth of the fsrB or feoB strains on ferrichrome were rescued with plasmid-borne E. coli fhuCDB ferrichrome transport genes, suggesting that FsrB activity occurs in the periplasm rather than the cytoplasm. Whole cells of an fsrB mutant are defective in ferric reductase activity. Both whole cells and spheroplasts catalyzed the demetallation of ferric siderophores that were defective in an fsrB mutant. Collectively, the data support a model whereby FsrB is required for reduction of iron and its dissociation from the siderophore in the periplasm, followed by transport of the ferrous ion into the cytoplasm by FeoAB.     

Brachypodium distachyon as a new model system for understanding iron homeostasis in grasses: phylogenetic and expression analysis of Yellow Stripe-Like (YSL) transporters

Background and Aims

Brachypodium distachyon is a temperate grass with a small stature, rapid life cycle and completely sequenced genome that has great promise as a model system to study grass-specific traits for crop improvement. Under iron (Fe)-deficient conditions, grasses synthesize and secrete Fe(III)-chelating agents called phytosiderophores (PS). In Zea mays, Yellow Stripe1 (ZmYS1) is the transporter responsible for the uptake of Fe(III)–PS complexes from the soil. Some members of the family of related proteins called Yellow Stripe-Like (YSL) have roles in internal Fe translocation of plants, while the function of other members remains uninvestigated. The aim of this study is to establish brachypodium as a model system to study Fe homeostasis in grasses, identify YSL proteins in brachypodium and maize, and analyse their expression profiles in brachypodium in response to Fe deficiency.

Methods

The YSL family of proteins in brachypodium and maize were identified based on sequence similarity to ZmYS1. Expression patterns of the brachypodium YSL genes (BdYSL genes) were determined by quantitative RT–PCR under Fe-deficient and Fe-sufficient conditions. The types of PS secreted, and secretion pattern of PS in brachypodium were analysed by high-performance liquid chromatography.

Key Results

Eighteen YSL family members in maize and 19 members in brachypodium were identified. Phylogenetic analysis revealed that some YSLs group into a grass-specific clade. The Fe status of the plant can regulate expression of brachypodium YSL genes in both shoots and roots. 3-Hydroxy-2′-deoxymugineic acid (HDMA) is the dominant type of PS secreted by brachypodium, and its secretion is diurnally regulated.

Conclusions

PS secretion by brachypodium parallels that of related crop species such as barley and wheat. A single grass species-specific YSL clade is present, and expression of the BdYSL members of this clade could not be detected in shoots or roots, suggesting grass-specific functions in reproductive tissues. Finally, the Fe-responsive expression profiles of several YSLs suggest roles in Fe homeostasis.     

家蚕免疫稳态调控分子的鉴定和表达模式分析

昆虫免疫稳态的维持有赖于准确地激活和有效地抑制Toll或IMD信号通路中的关键转录因子-- Dorsal/Dif或Relish。在果蝇等昆虫中, 已报道了多种降低转录因子稳定性和活性的免疫稳态调控分子, 突变或敲除这类分子导致免疫系统的过度激活。对家蚕Bombyx mori免疫信号通路的研究中, 至今为止尚无对这类分子的探索。本研究通过比较基因组学, 在家蚕基因组中鉴定了多个可能参与免疫稳态调控的分子, 包括Wnt家族成员、 Ubc9、 FAF和POSH等; 并通过检测家蚕被微生物感染后这些分子在多种免疫器官中的诱导表达模式, 发现这些分子的表达水平在微生物感染后普遍呈下降趋势, 虽然在某些组织中表达量有明显的升高（>1.5倍）, 但此高表达水平均不能维持且迅速下降; 而且免疫稳态调控分子和受其调控的信号通路的对应关系在不同组织中表现出差异。本研究是首次对家蚕免疫稳态调控分子的报道, 为深入研究家蚕免疫负调控的分子机制提供了参考。     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes.     

胞质型肽聚糖识别蛋白RfPGRP-L2在维持红棕象甲肠道菌群稳态中的作用

【目的】明确入侵害虫红棕象甲Rhynchophorus ferrugineus胞质型肽聚糖识别蛋白RfPGRP-L2在肠道菌群稳态的维持和调控过程中的作用,将为靶向破坏肠道菌群稳态的害虫控制新策略研发提供新的科学依据和作用靶标。【方法】利用生物信息学方法分析RfPGRP-L2的序列特征。利用RT-qPCR分析RfPGRP-L2在健康红棕象甲4龄幼虫不同组织(头、脂肪体、表皮、前肠、中-/后肠、血淋巴)以及大肠杆菌Escherichia coli DH5α和金黄色葡萄球菌Staphylococcus aureus经注射（注射1 μL OD₆₀₀=1.6的菌液）和喂食（取食涂抹1 mL OD₆₀₀=1.6的菌液的甘蔗薄片）两种不同方式分别感染后红棕象甲4龄幼虫肠道和脂肪体中的表达量;进行RfPGRP-L2原核表达,利用体外孵育方法检测重组蛋白RfPGRP-L2对大肠杆菌DH5α和金黄色葡萄球菌的凝集和抑菌活性;RNAi干扰RfPGRP-L2后,检测红棕象甲4龄幼虫血淋巴和肠道中大肠杆菌菌落数的变化;利用RT-qPCR分析RNAi干扰RfPGRP-L2后红棕象甲4龄幼虫脂肪体和肠道中抗菌肽基因表达量的变化;利用基于细菌16S rRNA的高通量测序分析RNAi干扰RfPGRP-L2对健康红棕象甲4龄幼虫肠道菌群结构组成的影响。【结果】SMART预测发现红棕象甲RfPGRP-L2基因编码的蛋白中无跨膜结构域也无信号肽,这表明RfPGRP-L2是一种胞质型肽聚糖识别蛋白。RT-qPCR检测发现,RfPGRP-L2主要在健康红棕象甲4龄幼虫血淋巴、肠道和脂肪体等免疫组织中表达;被注射感染大肠杆菌和金黄色葡萄球菌6 h和12 h后,红棕象甲4龄幼虫脂肪体中RfPGRP-L2的表达量分别显著上调;被喂食感染大肠杆菌6 h后,红棕象甲4龄幼虫肠道中RfPGRP-L2的表达量显著增加。重组表达蛋白RfPGRP-L2能引起大肠杆菌和金黄色葡萄球菌发生凝集反应,这说明RfPGRP-L2能够识别这两种细菌。当RfPGRP-L2被干扰后,红棕象甲4龄幼虫对肠道和血淋巴中感染EGFP标记的大肠杆菌的清除能力显著弱于对照组;肠道中抗菌肽基因RfCecropin的表达量显著降低;健康红棕象甲4龄幼虫肠道中细菌的菌落数量显著高于对照组,而且肠道菌群结构组成也发生了明显的变化。【结论】红棕象甲体内胞质型肽聚糖识别蛋白RfPGRP-L2能够通过识别细菌并激活肠上皮细胞中相应的免疫信号通路促进抗菌肽基因的表达,从而介导对肠道菌群稳态的调控。     

A comparison of hydraulic and laser capture microdissection methods for collection of single B cells,PCR, and sequencing of antibody VDJ

A new procedure for measuring ATPase activity in which gamma-(33)P-labeled inorganic orthophoshate is detected by addition of ammonium molybdate followed by selective adsorption of the resulting phosphomolybdate to scintillation proximity beads in the presence of cesium chloride is described. This method is shown to give accurate and reproducible results over a wide range of detection conditions and product concentrations. It requires no separation or filtration steps and is highly compatible with automated high-throughput screening. Rates of hydrolysis are easily and accurately determined over a wide range, and thus the method is useful for kinetic studies also. We show that this scintillation proximity assay is useful for the study of the E1 helicase of human papillomavirus, but it is a general procedure which could also be applied to any ATPase or other nucleotide triphosphate-hydrolyzing enzyme or any other enzyme which generates orthophosphate as a reaction product.     

Cryo-EM structure of the heptameric calcium homeostasis modulator 1 channel

Calcium homeostasis modulator 1 (CALHM1) is a voltage- and Ca²⁺-gated ATP channel that plays an important role in neuronal signaling. However, as the previously reported CALHM structures are all in the ATP-conducting state, the gating mechanism of ATP permeation is still elusive. Here, we report cryo-EM reconstructions of two Danio rerio CALHM1 heptamers with ordered or flexible long C-terminal helices at resolutions of 3.2 Å and 2.9 Å, respectively, and one D. rerio CALHM1 octamer with flexible long C-terminal helices at a resolution of 3.5 Å. Structural analysis shows that the heptameric CALHM1s are in an ATP-nonconducting state with a central pore diameter of approximately 6.6 Å. Compared with those inside the octameric CALHM1, the N-helix inside the heptameric CALHM1 is in the “down” position to avoid steric clashing with the adjacent TM1 helix. Molecular dynamics simulations show that as the N-helix moves from the “down” position to the “up” position, the pore size of ATP molecule permeation increases significantly. Our results provide important information for elucidating the mechanism of ATP molecule permeation in the CALHM1 channel.