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.     

细胞质及线粒体ferritin与铁代谢

铁是机体代谢所必需的微量元素之一。近年来,铁在机体内的代谢越来越受到人们的重视。维持体内铁的平衡,对保证机体的正常生理功能显得极为重要。胞质铁蛋白（cytosolic ferrifin,CFt）是细胞内重要的调节铁平衡的因子之一。而近年发现的线粒体铁蛋白（mitochondrial ferritin,MtFt）是一种定位在线粒上、和铁代谢密切相关的蛋白,具有组织受限性表达的特点,它在结构和功能上与胞质铁蛋白相比有一定的相似性,但是由于其mRNA上没有铁调控元件,它的表达不直接受铁调节蛋白调控,所以其确切功能及表达机制还未完全明了,因此,近年来有不少人开展了这方面的研究。对线粒体铁蛋白的深入研究将极大地丰富人们对铁在亚细胞水平上的代谢机制和功能的认识。文章介绍了细胞质铁蛋白的调控机制以及线粒体铁蛋白的结构、功能、表达及与铁代谢的关系。     

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。     

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Ca~(2 )转运与细胞代谢调节

线粒体具有一套完整的Ca2 转运系统 ,包括两条摄取途径和三条释放途径。生理条件下 ,它们在细胞胞质与线粒体钙稳态维持以及细胞能量代谢中起重要作用 ,线粒体从胞质摄取的Ca2 可激活某些Ca2 敏感的呼吸酶和代谢过程。病理条件下 ,线粒体Ca2 转运发生紊乱 ,通过线粒体通透性转换导致细胞坏死或凋亡     

线粒体Ca^2＋转运与细胞代谢调节

线粒体具有一套完整的Ｃａ＾２＋转运系统,包括两条摄取途径和三条释放途径。生理条件下,它们在细胞胞质与线粒体钙稳态维持以及细胞能量代谢中起重要作用,线粒体从胞质摄取的Ｃａ＾２＋可激活某些Ｃａ＾２＋敏感的呼吸酶和代谢过程。病理条件下,线粒体Ｃａ＾２＋转运发生紊乱,通过线粒体通透性转换导致细胞坏死或凋亡。     

线粒体钙单向转运体和线粒体应激关系的研究进展

钙离子(calcium ion,Ca~(2+))在线粒体功能障碍及细胞损伤凋亡过程中发挥重要的细胞信号作用。近些年来关于Ca~(2+)通道以及其调控蛋白的研究越来越多,其中,线粒体单向转运体(uniporter)复合物的结构组成及其相关蛋白的分布特点成为主要研究热点。作为uniporter复合物中关键的通道蛋白,线粒体钙单向转运蛋白(mitochondrial calcium uniporter,MCU)可顺电化学梯度摄入Ca~(2+),将Ca~(2+)从胞质转运到线粒体基质并控制转运速率,其在胞内Ca~(2+)信号转导、Ca~(2+)稳态、线粒体能量代谢以及细胞凋亡方面具有重要意义。识别调控线粒体内Ca~(2+)信号的MCU及其相关蛋白可深入阐明线粒体应激在相关疾病中的发生发展,并为进一步的疾病治疗提供理论依据。     

MicroRNA:线粒体功能调控的新机制

线粒体是真核生物进行能量代谢的主要场所,在自由基产生、细胞凋亡、衰老等生理病理活动中也起到重要作用。线粒体功能受核基因和线粒体基因共同调控,microRNA(miRNA)介导的基因转录后调控是重要机制之一。核基因编码的miRNA不仅可以通过调控核基因编码的线粒体相关蛋白的表达影响线粒体结构和功能,而且可以进入线粒体并调控线粒体基因的表达。另一方面,线粒体基因也可能编码miRNA,直接调控线粒体基因表达或转运至胞质调控核基因的表达。     

铁硫蛋白的生物合成及相关疾病

铁硫蛋白是以铁硫簇为辅基,相对分子质量较小的一类蛋白质．它广泛存在于各种生物体内,参与电子传递、能量代谢以及基因表达调控等重要生理过程．其生物合成过程复杂,并且从细菌到人类高度保守．在真核细胞内,铁硫蛋白的组装由线粒体铁硫簇组装系统（mitochondrial iron sulfur cluster assembly system,mitochondrial ISC assembly system）和细胞质铁硫簇组装器（cytosolic iron sulfur cluster assembly,CIA）完成．研究发现,铁硫蛋白的合成异常可导致弗里德赖希共济失调(friedreich ataxia,FRDA)、遗传性肌病和铁粒幼细胞性贫血等多种罕见疾病,这些疾病严重影响个体的生活质量和寿命．因此,深入了解铁硫蛋白的结构和生物合成过程,对研究其生物学功能与相关疾病的诊断和治疗有重要意义．     

植物中钼的吸收转运及钼辅因子与钼酶的研究进展

钼是植物生长发育所必需的微量元素,只有和蛋白质或者蝶呤结合形成钼辅因子才能产生生物活性。自然界存在2种钼辅因子:以铁硫簇为基础的铁钼辅因子(Fe Moco)和以钼蝶呤为基础的钼辅因子(MPT/Moco)。植物对钼的吸收有2种转运蛋白系统,一种是专一性转运蛋白,如MOTl和MOT2;另一种是共转运蛋白,如磷酸盐转运蛋白(PHT)和硫酸盐转运蛋白(SULTR)。最近研究发现一种钼酶——线粒体氨肟还原蛋白(m ARC)。本文综述了近年来植物体内钼的吸收与转运机制、钼辅因子的合成过程以及钼酶的研究进展,并提出了今后的重点研究方向。     

AtSufE is an essential activator of plastidic and mitochondrial desulfurases in Arabidopsis

Iron-sulfur (Fe-S) clusters are vital prosthetic groups for Fe-S proteins involved in fundamental processes such as electron transfer, metabolism, sensing and signaling. In plants, sulfur (SUF) protein-mediated Fe-S cluster biogenesis involves iron acquisition and sulfur mobilization, processes suggested to be plastidic. Here we have shown that AtSufE in Arabidopsis rescues growth defects in SufE-deficient Escherichia coli. In contrast to other SUF proteins, AtSufE localizes to plastids and mitochondria interacting with the plastidic AtSufS and mitochondrial AtNifS1 cysteine desulfurases. AtSufE activates AtSufS and AtNifS1 cysteine desulfurization, and AtSufE activity restoration in either plastids or mitochondria is not sufficient to rescue embryo lethality in AtSufE loss-of-function mutants. AtSufE overexpression induces AtSufS and AtNifS1 expression, which in turn leads to elevated cysteine desulfurization activity, chlorosis and retarded development. Our data demonstrate that plastidic and mitochondrial Fe-S cluster biogenesis shares a common, essential component, and that AtSufE acts as an activator of plastidic and mitochondrial desulfurases in Arabidopsis.     

Biogenesis of iron-sulfur proteins in eukaryotes: components, mechanism and pathology

Iron-sulfur (Fe-S) clusters are ubiquitous co-factors of proteins that play an important role in metabolism, electron-transfer and regulation of gene expression. In eukaryotes mitochondria are the primary site of Fe-S cluster biogenesis. The organelles contain some ten proteins of the so-called iron-sulfur cluster (ISC) assembly machinery that is well-conserved in bacteria and eukaryotes. The ISC assembly machinery is responsible for biogenesis of Fe-S proteins within mitochondria. In addition, this machinery is involved in the maturation of extra-mitochondrial Fe-S proteins by cooperating with mitochondrial proteins with an exclusive function in this process. This review summarizes recent developments in our understanding of the biogenesis of cellular Fe-S proteins in eukaryotes. Particular emphasis is given to disorders in Fe-S protein biogenesis causing human disease.     

Frataxin-mediated iron delivery to ferrochelatase in the final step of heme biosynthesis

Human ferrochelatase, a mitochondrial membrane-associated protein, catalyzes the terminal step of heme biosynthesis by insertion of ferrous iron into protoporphyrin IX. The recently solved x-ray structure of human ferrochelatase identifies a potential binding site for an iron donor protein on the matrix side of the homodimer. Herein we demonstrate Hs holofrataxin to be a high affinity iron binding partner for Hs ferrochelatase that is capable of both delivering iron to ferrochelatase and mediating the terminal step in mitochondrial heme biosynthesis. A general regulatory mechanism for mitochondrial iron metabolism is described that defines frataxin involvement in both heme and iron-sulfur cluster biosyntheses. In essence, the distinct binding affinities of holofrataxin to the target proteins, ferrochelatase (heme synthesis) and ISU (iron-sulfur cluster synthesis), allows discrimination between the two major iron-dependent pathways and facilitates targeted heme biosynthesis following down-regulation of frataxin.     

Mrs3p, Mrs4p, and frataxin provide iron for Fe-S cluster synthesis in mitochondria

Yeast Mrs3p and Mrs4p are evolutionarily conserved mitochondrial carrier proteins that transport iron into mitochondria under some conditions. Yeast frataxin (Yfh1p), the homolog of the human protein implicated in Friedreich ataxia, is involved in iron homeostasis. However, its precise functions are controversial. Anaerobically grown triple mutant cells (Deltamrs3/4/Deltayfh1) displayed a severe growth defect corrected by in vivo iron supplementation. Because anaerobically grown cells do not synthesize heme, and they do not experience oxidative stress, this growth defect was most likely due to Fe-S cluster deficiency. Fe-S cluster formation was assessed in anaerobically grown cells shifted to air for a brief period. In isolated mitochondria, Fe-S clusters were detected on newly imported yeast ferredoxin precursor and on endogenous aconitase by means of [35S]cysteine labeling and native gel separation. New cluster formation was dependent on iron addition to mitochondria, and the iron concentration dependence was shifted dramatically upward in the Deltamrs3/4 mutant, indicating a role of Mrs3/4p in iron transport. The frataxin mutant strain lacked protein import capacity because of low mitochondrial membrane potential, although this was partially restored by growth in the presence of high iron. Under these conditions, a kinetic defect in new Fe-S cluster formation was still noted. Import of frataxin into frataxin-minus isolated mitochondria promptly corrected the Fe-S cluster assembly defect without further iron addition. These findings show that Mrs3/4p transports iron into mitochondria, whereas frataxin makes iron already within mitochondria available for Fe-S cluster synthesis.     

Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria.

The import of metals, iron in particular, into mitochondria is poorly understood. Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins. We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria. By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import. Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP. Only reduced iron is functional in generating heme. Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane. Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining why in vivo these ions are not incorporated into porphyrin. The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron. Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria. Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.     

GTP is required for iron-sulfur cluster biogenesis in mitochondria

Iron-sulfur (Fe-S) cluster biogenesis in mitochondria is an essential process and is conserved from yeast to humans. Several proteins with Fe-S cluster cofactors reside in mitochondria, including aconitase [4Fe-4S] and ferredoxin [2Fe-2S]. We found that mitochondria isolated from wild-type yeast contain a pool of apoaconitase and machinery capable of forming new clusters and inserting them into this endogenous apoprotein pool. These observations allowed us to develop assays to assess the role of nucleotides (GTP and ATP) in cluster biogenesis in mitochondria. We show that Fe-S cluster biogenesis in isolated mitochondria is enhanced by the addition of GTP and ATP. Hydrolysis of both GTP and ATP is necessary, and the addition of ATP cannot circumvent processes that require GTP hydrolysis. Both in vivo and in vitro experiments suggest that GTP must enter into the matrix to exert its effects on cluster biogenesis. Upon import into isolated mitochondria, purified apoferredoxin can also be used as a substrate by the Fe-S cluster machinery in a GTP-dependent manner. GTP is likely required for a common step involved in the cluster biogenesis of aconitase and ferredoxin. To our knowledge this is the first report demonstrating a role of GTP in mitochondrial Fe-S cluster biogenesis.     

The essential role of mitochondria in the biogenesis of cellular iron-sulfur proteins

Iron-sulfur (Fe/S) proteins play an important role in electron transfer processes and in various enzymatic reactions. In eukaryotic cells, known Fe/S proteins are localised in mitochondria, the cytosol and the nucleus. The biogenesis of these proteins has only recently become the focus of investigations. Mitochondria are the major site of Fe/S cluster biosynthesis in the cell. The organelles contain an Fe/S cluster biosynthesis apparatus that resembles that of prokaryotic cells. This apparatus consists of some ten proteins including a cysteine desulfurase producing elemental sulfur for biogenesis, a ferredoxin involved in reduction, and two chaperones. The mitochondrial Fe/S cluster synthesis apparatus not only assembles mitochondrial Fe/S proteins, but also initiates formation of extra-mitochondrial Fe/S proteins. This involves the export of sulfur and possibly iron from mitochondria to the cytosol, a reaction performed by the ABC transporter Atm1p of the mitochondrial inner membrane. A possible substrate of Atm1p is an Fe/S cluster that may be stabilised for transport. Constituents of the cytosol involved in the incorporation of the Fe/S cluster into apoproteins have not been described yet. Many of the mitochondrial proteins involved in Fe/S cluster formation are essential, illustrating the central importance of Fe/S proteins for life. Defects in Fe/S protein biogenesis are associated with the abnormal accumulation of iron within mitochondria and are the cause of an iron storage disease.     

RNA silencing of mitochondrial m-Nfs1 reduces Fe-S enzyme activity both in mitochondria and cytosol of mammalian cells

In prokaryotes and yeast, the general mechanism of biogenesis of iron-sulfur (Fe-S) clusters involves activities of several proteins among which IscS and Nfs1p provide, through cysteine desulfuration, elemental sulfide for Fe-S core formation. Although these proteins have been well characterized, the role of their mammalian homolog in Fe-S cluster biogenesis has never been evaluated. We report here the first functional study that implicates the putative cysteine desulfurase m-Nfs1 in the biogenesis of both mitochondrial and cytosolic mammalian Fe-S proteins. Depletion of m-Nfs1 in cultured fibroblasts through small interfering RNA-based gene silencing significantly inhibited the activities of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II) of the respiratory chain, as well as aconitase of the Krebs cycle, with no alteration in their protein levels. Activity of cytosolic xanthine oxidase, which holds a [2Fe-2S] cluster, was also specifically reduced, and iron-regulatory protein-1 was converted from its [4Fe-4S] aconitase form to its apo- or RNA-binding form. Reduction of Fe-S enzyme activities occurred earlier and more markedly in the cytosol than in mitochondria, suggesting that there is a mechanism that primarily dedicates m-Nfs1 to the biogenesis of mitochondrial Fe-S clusters in order to maintain cell survival. Finally, depletion of m-Nfs1, which conferred on apo-IRP-1 a high affinity for ferritin mRNA, was associated with the down-regulation of the iron storage protein ferritin.