嗜酸氧化亚铁硫杆菌ISC铁硫簇系统的合成

【目的】铁硫簇是最古老的一种氧化还原中心,它普遍存在于所有生命体内,在光合作用、呼吸作用和固氮作用这三个地球生命最基本的代谢途径中扮演着重要的角色。【方法】以嗜酸氧化亚铁硫杆菌(A.ferrooxidans ATCC 23270)基因组为模板,克隆表达其ISC铁硫簇组装的3个核心蛋白,IscS(半胱氨酸脱硫酶蛋白)、IscU(支架蛋白)和IscA(铁供体蛋白)。【结果】研究发现IscS能催化半胱氨酸脱硫,为铁硫簇的组装提供硫,支架蛋白IscU不具备结合铁的能力,IscA具有较强的铁结合能力。【结论】铁硫簇体外组装证明Fe-IscA在体外能将结合的铁传递给IscS,并在IscU上进行铁硫簇的组装。     

隐花色素蛋白和铁硫簇蛋白相互作用及其在褐飞虱发育与生殖中的作用

[目的]在真核生物中验证隐花色素蛋白(Cryptochrome)/铁硫簇蛋白(Iron-sulfur cluster assembly 1)的相互作用,研究2种蛋白在褐飞虱Nilaparvata lugens发育和生殖过程中的作用,为二者在磁感受机制中的协同作用及磁场对昆虫生理状态的调控机制提供依据和新的研究方向.[方...     

生物磁受体蛋白MagR/IscA研究进展

铁硫簇蛋白是一类重要的线粒体功能蛋白,在细胞能量代谢、电子传递、底物结合与激活、铁/硫存储、酶促反应、基因表达调控等诸多过程中均发挥了关键作用.铁硫簇蛋白质组装及转运过程一旦发生障碍,必将严重影响细胞内铁的稳态及铁硫蛋白的功能.分子质量约11 ku的铁硫簇蛋白IscA,是铁硫蛋白亚家族hesB高保守性成员之一,能结合铁离子及[2Fe-2S]簇,参与铁硫簇蛋白质合成,因此IscA在铁硫簇组装蛋白与级联反应系统中具有重要的作用.更值得关注的是,2015年谢灿和张生家两个研究组发现IscA1具有磁受体(MagR/MAR)作用,此外,谢灿课题组揭示MagR能与Cry形成磁感应复合物行使磁感应器(magnetic sensor,MagS)功能.尤为重要的是,体内实验表明通过外磁场刺激活化MagR能调控相关磁基因表达,影响神经活动及行为定位.鉴于MagR磁受体的独特功能,张生家等将磁受体的基因定位与远程磁刺激相结合,发明了一种非损伤性的神经调控方法,称之为磁遗传学.本文简要介绍MagR/IscA及其同源基因的始初发现与鉴定历程、进化保守性、独特的生理生物学功能,并凝练出磁遗传假说机制调控模型,以解释MagR/IscA的磁遗传学功能.     

铜对线粒体中铁硫蛋白功能的毒性机制研究

该文探讨了铜对线粒体内铁硫蛋白的毒性机理。通过包装慢病毒将Hep G2细胞中铜转运蛋白ATP7B(ATPase copper transporting beta)基因敲低,并用铜离子处理构建高铜细胞模型。通过免疫印迹、胶内酶活、紫外–可见光分光光度法检测细胞线粒体内铁硫蛋白、非铁硫蛋白及铁硫簇组装蛋白量和活性的改变;用电镜观察高铜模型中线粒体的形态改变;用海马能量代谢分析仪检测铜离子对细胞能量代谢的影响。结果发现,高铜细胞模型线粒体内铁硫簇组装蛋白ISCA2(ironsulfur cluster assembly 2)及ISCU(iron-sulfur cluster assembly enzyme)水平下降,抑制了铁硫簇的组装,并进一步影响了线粒体内[2Fe-2S]型及[4Fe-4S]型铁硫蛋白功能,但并不影响非铁硫蛋白。高铜状态也影响了呼吸链复合体活性及线粒体能量代谢,并导致线粒体形态发生改变。这些结果表明,异常累积的铜离子也会通过抑制线粒体中铁硫簇的组装,影响线粒体内铁硫蛋白的功能。     

铁硫簇的结构、功能及生物合成研究进展

铁硫簇是普遍存在于生物体中的最古老的生命物质之一.铁硫簇基本结构单元有[2Fe-2S]、[3Fe-4S]、[4Fe-4S]及.[8Fe-7S]等几种形式,不同结构的铁硫簇具有不同的生物学功能,主要包括参与电子传递、底物的结合与激活、铁/硫的存储、基因表达的调控、酶活的调控等.铁硫簇既可在生物体内合成,也可在体外进行人工组装.铁硫簇的生物合成主要和NIF、ISC、SUF这三个系统有关.研究已确定了参与铁硫簇合成的关键蛋白,但对它们分子水平上的机制及如何进行相互作用在体内外合成铁硫簇的认识尚待进一步研究.     

线粒体铁代谢与人类疾病的研究进展

线粒体铁代谢的研究主要包括两个方面：铁在胞质和线粒体之间的转运和调控;铁硫簇和血红素在线粒体内的合成与转运。目前认为线粒体铁的转入主要是与mitoferrinl／2（MFRNl和MFRN2）和ABCBl0有关,运出可能与ABCB6和／或ABCB7有关,转运和调控的具体机制不是很清楚,推测与某种含有铁硫簇的信号分子有关。哺乳动物铁硫簇的合成可以发生在胞质和线粒体内,但以线粒体为主;真核生物中与铁硫簇合成相关的蛋白达二十多种,其中FXN、ISCS、ISDll和ISCU及其同系物被认为是核心组分。血红素的合成起始和终止发生在线粒体内,终止步骤为亚铁螯合酶将铁插入原卟啉IX,该酶活性又依赖于铁硫簇。因此,铁硫簇的合成与调控是线粒体铁代谢的核心,也是整个细胞铁运作的核心。本文主要围绕线粒体铁代谢特别是铁硫簇的合成异常引起的疾病做一简单的综述。     

真核细胞中铁硫簇的组装机制及相关铁硫蛋白疾病

铁硫簇是一类古老而功能众多的蛋白质辅基,在细胞中参与电子传递过程、酶促反应及感知内环境的变化而调节基因的表达等。虽然铁硫簇的组成元素和结构都较为简单,但是铁硫簇的组装是需多种组装蛋白参与、有序进行的催化反应。直至近几年,人们才逐渐阐明了在生命体中铁硫簇是如何组装并结合到未成熟的铁硫蛋白中的。如果线粒体中铁硫簇组装及转运过程发生障碍,将严重影响细胞内铁的稳态及铁硫蛋白的功能,由此可见,线粒体中铁硫簇的组装功能使得线粒体成为细胞中必不可少的一类细胞器。该文重点概述了近十年来真核生物中铁硫簇组装机制的研究进展并阐述线粒体铁硫簇组装在人体中的重要作用及其组装障碍所引起的疾病。     

大肠杆菌YacG锌指结构的金属结合及功能特性

YacG蛋白是一种能够抑制大肠杆菌促旋酶（E.coli gyrase）活性的内源性小分子蛋白质,仅由65 个氨基酸残基组成。核磁共振(NMR)研究发现,YacG结构中含有1个Cys-X2-Cys-X15-Cys-X3-Cys序列的锌指结构域,然而其作用并不清楚。本研究发现,在添加外源锌或者铁的M9基础培养基中,表达并纯化得到分别含有锌和铁的YacG蛋白,而在同时添加铁和L-半胱氨酸的M9基础培养基中可以纯化得到含有铁硫簇的蛋白质。这表明,YacG不仅是一个锌指蛋白,也是铁结合或铁硫簇结合蛋白。定点突变实验发现,YacG锌指结构中的4个半胱氨酸残基突变后,其结合的锌、铁、铁硫簇的含量都显著下降。这提示,锌结合、铁结合以及铁硫簇结合的位点均位于锌指结构域中的4个半胱氨酸残基。体内YacG过表达实验显示,用IPTG在大肠杆菌体内诱导表达野生型YacG蛋白会导致其生长明显受到抑制,而过表达突变体蛋白（YacG-C12/28S）对其生长的抑制作用将会减弱。体外实验进一步发现,锌结合、铁结合以及铁硫簇结合形式的YacG蛋白对E.coli gyrase促DNA螺旋活性的抑制作用没有明显差别,但是锌指结构突变体蛋白（YacG-C12/28S）对gyrase活性的抑制作用显著减弱。这说明,完整的锌指结构对YacG抑制gyrase活性的功能具有重要作用。此研究有可能为gyrase抑制剂类抗生素药物的研发提供有用的线索。     

光谱技术研究固氮酶铁硫簇的理化特性

Ｎ－甲基甲酰胺碱度是提取高质量固氮酶铁钼辅基的关键因素之一。过量的亚甲蓝能氧化并分解铁铜铺基为含双相铁硫簇和铁硫簇固氮酶铁钼辅基和在紫外可见光谱区中均无特征吸收峰,而在３２０ｎｍ处却呈弱吸收峰,棕色固氮菌固氮酶和该菌的突变菌侏ＵＷ４５固氮酶（缺铁钼辅基）中的非含钼的铁硫簇在紫外可见光谱区３２０ｎｍ和４０５ｎｍ处均含有特征吸收峰．     

铁硫簇的生物合成

铁硫簇在细胞的生物学过程中起着重要的作用,可参与电子传递、代谢控制和基因调节等过程。研究显示铁硫簇具有多样性,它的合成依赖于ISC和SUF系统,固氮酶中还需要NIF系统的参与。ISC系统由iscSUA-hscBA-fdx基因串编码,合成的是一类“管家”蛋白,适于在正常条件下表达。SUF系统由基因串sufABCDSE编码,常在恶劣环境如氧化应激和铁饥饿条件下表达。NIF系统由nifSU基因编码,适于固氮酶(厌氧条件下起作用)铁硫簇的合成。     

Interplay of IscA and IscU in biogenesis of iron-sulfur clusters

Increasing evidence suggests that sulfur in ubiquitous iron-sulfur clusters is derived from L-cysteine via cysteine desulfurases. In Escherichia coli, the major cysteine desulfurase activity for biogenesis of iron-sulfur clusters has been attributed to IscS. The gene that encodes IscS is a member of an operon iscSUA, which also encodes two highly conserved proteins: IscU and IscA. Previous studies suggested that both IscU and IscA may act as the iron-sulfur cluster assembly scaffold proteins. However, recent evidence indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in IscU (Ding, H., Harrison, K., and Lu, J. (2005) J. Biol. Chem. 280, 30432-30437). To further elucidate the function of IscA in biogenesis of iron-sulfur clusters, we evaluate the iron-sulfur cluster binding activity of IscA and IscU under physiologically relevant conditions. When equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS, L-cysteine and dithiothreitol, iron-sulfur clusters are assembled in IscU, but not in IscA, suggesting that IscU is a preferred iron-sulfur cluster assembly scaffold protein. In contrast, when equal amounts of IscA and IscU are incubated with an equivalent amount of ferrous iron in the presence of IscS and dithiothreitol but without L-cysteine, nearly all iron is bound to IscA. The iron binding in IscA appears to prevent the formation of the biologically inaccessible ferric hydroxide under aerobic conditions. Subsequent addition of L-cysteine efficiently mobilizes the iron center in IscA and transfers the iron for the iron-sulfur cluster assembly in IscU. The results suggest an intriguing interplay between IscA and IscU in which IscA acts as an iron chaperon that recruits "free" iron and delivers the iron for biogenesis of iron-sulfur clusters in IscU under aerobic conditions.     

Deletion of the Proposed Iron Chaperones IscA/SufA Results in Accumulation of a Red Intermediate Cysteine Desulfurase IscS in Escherichia coli

In Escherichia coli, sulfur in iron-sulfur clusters is primarily derived from l-cysteine via the cysteine desulfurase IscS. However, the iron donor for iron-sulfur cluster assembly remains elusive. Previous studies have shown that, among the iron-sulfur cluster assembly proteins in E. coli, IscA has a unique and strong iron-binding activity and that the iron-bound IscA can efficiently provide iron for iron-sulfur cluster assembly in proteins in vitro, indicating that IscA may act as an iron chaperone for iron-sulfur cluster biogenesis. Here we report that deletion of IscA and its paralog SufA in E. coli cells results in the accumulation of a red-colored cysteine desulfurase IscS under aerobic growth conditions. Depletion of intracellular iron using a membrane-permeable iron chelator, 2,2′-dipyridyl, also leads to the accumulation of red IscS in wild-type E. coli cells, suggesting that the deletion of IscA/SufA may be emulated by depletion of intracellular iron. Purified red IscS has an absorption peak at 528 nm in addition to the peak at 395 nm of pyridoxal 5′-phosphate. When red IscS is oxidized by hydrogen peroxide, the peak at 528 nm is shifted to 510 nm, which is similar to that of alanine-quinonoid intermediate in cysteine desulfurases. Indeed, red IscS can also be produced in vitro by incubating wild-type IscS with excess l-alanine and sulfide. The results led us to propose that deletion of IscA/SufA may disrupt the iron delivery for iron-sulfur cluster biogenesis, therefore impeding sulfur delivery by IscS, and result in the accumulation of red IscS in E. coli cells.     

CpNifS-dependent iron-sulfur cluster biogenesis in chloroplasts

Iron-sulfur (Fe-S) clusters are important prosthetic groups in all organisms. The biosynthesis of Fe-S clusters has been studied extensively in bacteria and yeast. By contrast, much remains to be discovered about Fe-S cluster biogenesis in higher plants. Plant plastids are known to make their own Fe-S clusters. Plastid Fe-S proteins are involved in essential metabolic pathways, such as photosynthesis, nitrogen and sulfur assimilation, protein import, and chlorophyll transformation. This review aims to summarize the roles of Fe-S proteins in essential metabolic pathways and to give an overview of the latest findings on plastidic Fe-S assembly. The plastidic Fe-S biosynthetic machinery contains many homologues of bacterial mobilization of sulfur (SUF) proteins, but there are additional components and properties that may be plant-specific. These additional features could make the plastidic machinery more suitable for assembling Fe-S clusters in the presence of oxygen, and may enable it to be regulated in response to oxidative stress, iron status and light.     

Formation of iron-sulfur clusters in bacteria: an emerging field in bioinorganic chemistry

Biological iron-sulfur clusters are chemically versatile inorganic structures that are attached to many proteins. These clusters are intimately involved in the functions of their partner proteins and they are required to sustain life on earth. Recent work has demonstrated that, in spite of their simple structures, the assembly and insertion of iron-sulfur clusters into their protein partners is a complex biological process. This complexity is probably related to the cellular toxicity of iron and sulfur in their free forms.     

IscA mediates iron delivery for assembly of iron-sulfur clusters in IscU under the limited accessible free iron conditions

Increasing evidence suggests that IscS, a cysteine desulfurase, provides sulfur for assembly of transient iron-sulfur clusters in IscU. IscU appears to act as a scaffold and eventually transfers the assembled clusters to target proteins. However, the iron donor for the iron-sulfur cluster assembly largely remains elusive. Here we find that Escherichia coli IscU fails to assemble iron-sulfur clusters when the accessible "free" iron in solution is limited by an iron chelator sodium citrate. Remarkably, IscA, an iron-sulfur cluster assembly protein with an iron association constant of 3.0 x 10(19) m(-1), is able to overcome the iron limitation due to sodium citrate and deliver iron for the IscS-mediated iron-sulfur cluster assembly in IscU. Substitution of the invariant cysteine residues Cys-99 or Cys-101 in IscA with serine completely abolishes the iron binding activity of the protein. The IscA mutants that fail to bind iron are unable to mediate iron delivery for the iron-sulfur cluster assembly in IscU under the limited accessible "free" iron conditions. The results suggest that IscA is capable of recruiting intracellular iron and providing iron for the iron-sulfur cluster assembly in IscU in cells in which the accessible "free" iron content is probably restricted.     

Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis

Ferredoxins are iron-sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron-sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2--formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron-sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron-sulfur clusters, but not steroidogenesis. To further assess the role of the FDX-FDXR system in mammalian iron-sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron-sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron-sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron-sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron-sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.     

Iron-sulfur cluster biogenesis and human disease

Iron-sulfur (Fe-S) clusters are essential for numerous biological processes, including mitochondrial respiratory chain activity and various other enzymatic and regulatory functions. Human Fe-S cluster assembly proteins are frequently encoded by single genes, and inherited defects in some of these genes cause disease. Recently, the spectrum of diseases attributable to abnormal Fe-S cluster biogenesis has extended beyond Friedreich ataxia to include a sideroblastic anemia with deficiency of glutaredoxin 5 and a myopathy associated with a deficiency of a Fe-S cluster assembly scaffold protein, ISCU. Mutations within other mammalian Fe-S cluster assembly genes could be causative for human diseases that manifest distinctive combinations of tissue-specific impairments. Thus, defects in the iron-sulfur cluster biogenesis pathway could underlie many human diseases.