稀土等离子与铁生物调控

本文在简介转铁蛋白、转铁蛋白受体的铁（Ⅲ）调控作用研究进展的基础上,综述了稀土离子、镓与转铁蛋白的作用及稀土离子、镓代谢与铁（Ⅲ）调控系统的关系。     

铜/镍金属伴侣蛋白的研究进展

含铜/镍金属酶的成熟需要一系列的铜/镍金属伴侣蛋白,这些铜/镍金属伴侣蛋白分别参与铜或者镍的转运,对维持细胞体内铜/镍金属平衡至关重要,同时金属酶完成金属催化活性中心的组装也依赖于这类伴侣蛋白。近年来关于铜/镍金属蛋白的研究取得可喜的进展,这些研究为进一步认识体内铜/镍平衡体系提供了重要依据。本文首先简要地介绍铜的摄取和细胞内平衡体系,接着着重介绍三个重要的铜转运蛋白Atox1、Cox17和CCS关于结构和功能的进展,以及这些铜转运蛋白和药物相互作用的机理。然后详细介绍在氢化酶和脲酶成熟路径中参与了镍的摄取、调节、转运和存储,维持细胞内镍金属平衡的镍伴侣蛋白,并介绍了脲酶、氢化酶这两条成熟路径之间的联系。     

葡萄糖转运蛋白4功能调节信号通路及其在药物研发中的应用

葡萄糖转运蛋白4(glucose transporter 4,GLUT4)是胰岛素响应组织骨骼肌和脂肪组织内负责葡萄糖吸收的转运蛋白,它与生物体糖代谢过程密切相关.在肥胖或以胰岛素抵抗为特征的2型糖尿病等代谢性疾病中,GLUT4功能受损;反之,GLUT4功能的变化也能影响整体的糖代谢水平.本文概述了GLUT4的功能、组织分布、功能调节方式以及调控GLUT4功能的小分子化合物的研究进展,讨论了GLUT4在其他疾病中的应用,并展望了其未来研究方向.     

结构生物学技术在线粒体钙离子单向转运体结构功能研究中的运用

线粒体钙离子单向转运体是一种位于线粒体内膜上的转运蛋白,具有选择和传导钙离子的功能,参与多种细胞的生理过程。目前已经确定了该单向转运体的组成成员,其中包括离子转运蛋白MCU、MCU的负调控亚基MCUb、2个Ca²⁺结合蛋白MICU1、MICU2以及MCU的调控亚基EMRE蛋白。近十多年来已经有大量的研究提出并构建了多种线粒体通过该单向转运体调节Ca²⁺摄取的模型。最近对其研究的重点转向单向转运体单个组分的结构以及对它们复合体的结构表征。这些问题的解决得益于结构生物学中核磁共振技术、X射线衍射技术的持续发展和冷冻电镜技术的突破。回顾了结构生物学技术在有关单向转运体结构问题过程中的综合运用。这一内容将有助于我们了解核磁共振、X射线晶体学以及冷冻电镜技术各自的优缺点,同时深入探究线粒体钙离子单向转运体各部分的结构及其发挥功能的分子机制。     

螯合亲和色谱法分离转铁蛋白的4种分子形式

在ＴＳＫｇｅｌ－Ｃｈｅｌａｔｅ－５ＰＷ柱上用固定Ｃｕ２~（２＋）离子的螯合亲和色谱法（ＨＰ－ＩＭＡＣ）研究了Ｆｅ（Ⅲ）同去铁转铁蛋白的结合,讨论了所固定的几种金属离子对保留时间和柱寿命的影响;在生理ｐＨ条件下分离出了非饱和铁转铁蛋白的４种分子形式：ＦｅＮＴｆ,ＴｆＦｅｃ,ＦｅＮＴｆＦｅｃ（或Ｆｅ２Ｔｆ）和Ａｐｏ－Ｔｆ。     

铜转运蛋白C端金属结合域与Ag~+及Hg~(2+)的相互作用

铜转运蛋白(CTR1)不仅参与铜的细胞摄取,而且在其它重金属离子的摄取过程中也发挥重要作用.本文采用紫外-可见(UV-Vis)光谱,核磁共振(NMR)和质谱(MS)的方法,研究了人源CTR1(hCTR1)的C端金属结合域(C8)与Ag+和Hg2+的相互作用.研究表明,Ag+和Hg2+都能与C8结合,但二者与C8的结合机制明显不同.每个C8分子可以结合两个Ag+离子,但一个Hg2+却可以与两个C8形成桥联.此外,Ag+离子与C8的配位是一个中等速度的交换过程,而Hg2+离子则为快速交换过程.C8的半胱氨酸残基是两种离子的重要结合位点,同时组氨酸残基也参与两种金属离子的配位,其中Ag+优先结合组氨酸,而Hg2+则对半胱氨酸的结合具有显著的优势.虽然HCH基序对C8与金属配位至关重要,一些远端的其它氨基酸也可以参与C8与银离子的配位,这可能与CTR1在摄取Ag+过程中的金属转移机制相关.这些结果为理解hCTR1蛋白摄取重金属离子的作用机制提供了必要的信息.     

铕(III)离子与人血清脱铁转铁蛋白结合的紫外差光谱研究

在pH7.4,温度为25℃的条件下,用紫外吸收差光谱进行了Eu^3^+对人血清脱铁转铁蛋白的滴定。结果表明Eu^3^+与人血清脱铁转铁蛋白结合后其差光谱在245nm和296nm处出现吸收峰,在245nm处,Eu^3^+-脱铁转铁蛋白配合物的摩尔吸光系数是(2.2±0.1)×10^4cm^-^1.mol^-^1.dm^3,Eu^3^+可占据脱铁转铁蛋白的2个金属离子结合部位,Eu^3^+优先占据脱铁转铁蛋白的C端结合部位,条件平衡常数是logK~C=8.42±0.12,logK~N=6.03±0.42。Eu^3^+与R~E^3^+(R~E=Nd,Sm,Gd和Tb)间的线性自由能关系表明,稀土离子占据脱铁转铁蛋白的C端结合部位时受离子大小的影响。     

铕(III)离子与人血清脱铁转铁蛋白结合的紫外差光谱研究

在pH7.4,温度为25℃的条件下,用紫外吸收差光谱进行了Eu^3^+对人血清脱铁转铁蛋白的滴定。结果表明Eu^3^+与人血清脱铁转铁蛋白结合后其差光谱在245nm和296nm处出现吸收峰,在245nm处,Eu^3^+-脱铁转铁蛋白配合物的摩尔吸光系数是(2.2±0.1)×10^4cm^-^1.mol^-^1.dm^3,Eu^3^+可占据脱铁转铁蛋白的2个金属离子结合部位,Eu^3^+优先占据脱铁转铁蛋白的C端结合部位,条件平衡常数是logK~C=8.42±0.12,logK~N=6.03±0.42。Eu^3^+与R~E^3^+(R~E=Nd,Sm,Gd和Tb)间的线性自由能关系表明,稀土离子占据脱铁转铁蛋白的C端结合部位时受离子大小的影响。     

高效液相色谱-柱后同位素稀释质谱法定量分析人血清中转铁蛋白及白蛋白

建立了高效液相色谱(HPLC)与电感耦合等离子体质谱(ICP-MS)联用,同时结合柱后同位素稀释法,通过直接测定硫及铁元素,实现蛋白质绝对定量的分析方法。选取分子量不同的4种标准蛋白:转铁蛋白、β乳球蛋白、肌红蛋白和溶菌酶作为混合蛋白,34S或54Fe同位素稀释剂与液相色谱洗脱液经三通混合后,在线进入ICP-MS检测。根据同位素稀释法公式及蛋白中硫、铁的含量计算混合蛋白中每种蛋白的浓度,与天平称量结果一致,对方法进行了验证。将方法应用到人血清转铁蛋白、白蛋白的定量分析,针对两种蛋白含量差别大导致的信号差异,通过改变同位素稀释剂的流速,成功测定了人血清中转铁蛋白、白蛋白的含量。采用34S及54Fe同位素稀释剂分别定量的血清中转铁蛋白浓度为(2.35±0.01)g/L和(2.22±0.13)g/L,结果基本吻合。方法精密度RSD均小于10%,可用于基体样品中含硫蛋白及金属蛋白的定量分析。     

内质网硒蛋白的研究进展

硒是哺乳动物必需的一种微量营养元素,主要以硒代半胱氨酸的形式存在于各种硒蛋白中,硒的主要生物功能通过硒蛋白实现.在25种哺乳动物硒蛋白中,有7种硒蛋白位于内质网,分别为2型脱碘酶、15-kDa硒蛋白、硒蛋白M、硒蛋白T、硒蛋白K、硒蛋白S和硒蛋白N.除了2型脱碘酶外,对其余内质网硒蛋白知之甚少.最近一些研究显示,一些内质网硒蛋白在氧化还原平衡调节、蛋白质折叠质量控制、错误折叠蛋白从内质网逆向转运至胞质、Ca2+稳态调节、内质网应激调节及炎症调节等过程中发挥作用.本文介绍了每种内质网硒蛋白的结构、功能及其生理和病理作用的一些最新研究进展,并对未来需要研究的内容进行了展望.     

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.     

Functional organization of the outer membrane of escherichia coli: phage and colicin receptors as components of iron uptake systems.

The functional interaction of outer membrane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 and ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all 3 colicins. The interaction of the ton A, ton B, and feu functions apparently permits quite different "substrates" to overcome the permeability barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of 3H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferri-ferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source was used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: 1) ton A; 2) ton B mutants; 3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; 4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; 5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000-83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene...     

Small-molecule screening identifies the selanazal drug ebselen as a potent inhibitor of DMT1-mediated iron uptake

HEK293T cells overexpressing divalent metal transporter-1 (DMT1) were established to screen for small-molecule inhibitors of iron uptake. Using a fluorescence-based assay, we tested 2000 known bioactive compounds to find 3 small molecules that potently block ferrous iron uptake. One of the inhibitors, ebselen, is a seleno compound used in clinical trials as a protective agent against ischemic stroke. Ebselen inhibited Fe(II) uptake (IC(50) of approximately 0.22 microM), but did not influence Fe(III) transport or DMT1-mediated manganese uptake. An unrelated antioxidant, pyrrolidine dithiobarbamate (PDTC), also inhibited DMT1 activity (IC(50) of approximately 1.54 microM). Both ebselen and PDTC increased cellular levels of reduced glutathione. These observations indicate that Fe(II) transport by DMT1 can be modulated by cellular redox status and suggest that ebselen may act therapeutically to limit iron-catalyzed damage due to transport inhibition.     

Binding of Chromium(III) to Transferrin Could Be Involved in Detoxification of Dietary Chromium(III) Rather than Transport of an Essential Trace Element

Cr^III binding to transferrin (Tf; the main Fe^III transport protein) has been postulated to mediate cellular uptake of Cr^III to facilitate a purported essential role for this element. Experiments using HepG2 (human hepatoma) cells, which were chosen because of high levels of the transferrin receptor, showed that Cr^III binding to vacant Fe^III‐binding sites of human Tf effectively blocks cellular Cr^III uptake. Through bio‐layer interferometry studies of the Tf cycle, it was found that both exclusion and efflux of Cr₂^IIITf from cells was caused by 1) relatively low Cr₂Tf affinity to cell‐surface Tf receptors compared to Fe₂Tf, and 2) disruption of metal release under endosomal conditions and post‐endosomal Tf dissociation from the receptor. These data support mounting evidence that Cr^III is not essential and that Tf binding is likely to be a natural protective mechanism against the toxicity and potential genotoxicity of dietary Cr through blocking Cr^III cellular accumulation.     

Dissection the endocytic routes of viral capsid proteins-coated upconversion nanoparticles by single-particle tracking

Real-time exploring the cellular endocytic pathway of viral capsid proteins (VCPs) functionalized nanocargos at the single-particle level can provide deep insight into the kinetic information involved in virus infection. In this work, porcine circovirus type 2 (PCV2) VCPs with different functions are modified onto the surface of upconversion nanoparticles (VCPs-UCNPs) to investigate the cellular internalization process in real-time. Clathrin-mediated endocytosis is found to be the essential uptake mechanism for these VCPs-UCNPs. Besides, it is verified that P₁-UCNPs (PCV2 VCPs with nuclear localization signal, namely P₁) can be easily assembled close to the perinuclear area, which is different from that of P₂-UCNPs (PCV2 VCPs without nuclear localization signal, namely P₂). Interestingly, multistep entry processes are observed. Particularly, confined diffusion is observed during the transmembrane process. The intracellular transport of VCPs-UCNPs is dependent on microtubules toward the cell interior. During this process, P₁-UCNPs display increased velocities with active transport, while diffusion much faster around the perinuclear area. But for P₂-UCNPs, there are only two phases involved in their endocytosis process. This study presents distinct dynamic mechanisms for the nanocargos with different functions, which would make a useful contribution to the development of robust drug delivery systems.     

Structural rearrangement of Neisseria meningitidis transferrin binding protein A (TbpA) prior to human transferrin protein (hTf) binding

Gram-negative bacterium Neisseria meningitidis, responsible for human infectious disease meningitis, acquires the iron (Fe³⁺) ion needed for its survival from human transferrin protein (hTf). For this transport, transferrin binding proteins TbpA and TbpB are facilitated by the bacterium. The transfer cannot occur without TbpA, while the absence of TbpB only slows down the transfer. Thus, understanding the TbpA-hTf binding at the atomic level is crucial for the fight against bacterial meningitis infections. In this study, atomistic level of mechanism for TbpA-hTf binding is elucidated through 100 ns long all-atom classical MD simulations on free (uncomplexed) TbpA. TbpA protein underwent conformational change from ‘open’ state to ‘closed’ state, where two loop domains, loops 5 and 8, were very close to each other. This state clearly cannot accommodate hTf in the cleft between these two loops. Moreover, the helix finger domain, which might play a critical role in Fe³⁺ ion uptake, also shifted downwards leading to unfavorable Tbp-hTf binding. Results of this study indicated that TbpA must switch between ‘closed’ state to ‘open’ state, where loops 5 and 8 are far from each other creating a cleft for hTf binding. The atomistic level of understanding to conformational switch is crucial for TbpA-hTf complex inhibition strategies. Drug candidates can be designed to prevent this conformational switch, keeping TbpA locked in ‘closed’ state.     

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2-DE) were used to identify iron-responsive proteins in the white-rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron-sufficient and iron-deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS-PAGE for P. chrysosporium and twelve for L. edodes. In 2-DE, the numbers of iron-restricted induced proteins were 12 and 9, respectively, in a resolution range of 15-60 kDa and pI 4.5-8.1. SDS-PAGE for the plasma membrane protein did not show differences, whereas the outer-membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron-complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.