含硒酶与非酶作用机制

在微生物、植物和动物体内,硒的功能形式多种多样,但其作用机制可归纳为酶与非酶两个方面,含硒酶的作用主要有：谷胱甘肽过氧化物酶（GPx）家族催化超氧化物还原,防止细胞膜的氧化损伤;脱磺酶（ID）家族调节甲状腺激素代谢,硫氧还蛋白还原酶（TDR）家族催化硫氧还蛋白（Trx）还原,TDR／Trx系统为细胞的生长和分化所必需,硒的非酶化学保护作用体现在：可诱导一些蛋白激酶的富半胱氨酸结构域发生氧化还原修饰,增强免疫功能等作用,硒在植物中的作用机制具有许多特殊性。     

单增李斯特菌氧化还原蛋白系统研究进展

单增李斯特菌是重要的食源性病原微生物,抗氧化应激是李斯特菌生存和致病的关键机制之一。活性氧(reactive oxygen species,ROS)浓度升高会破坏氧化还原平衡,使机体处于氧化胁迫的应激状态,进而导致生物大分子如蛋白质的损伤。蛋白质中半胱氨酸等含硫氨基酸对ROS尤其敏感,半胱氨酸残基脱氢氧化生成二硫键,可以稳定蛋白质空间构象,增加蛋白质的半衰期,进而使蛋白质免受损坏。抗氧化修复通常指的是对半胱氨酸残基的氧化还原过程,即二硫键的形成与打开。硫氧还蛋白家族包含硫氧还蛋白、谷氧还蛋白和Dsb-样蛋白系统,是生物体中常见的氧化还原修复系统。本文根据现有的文献报道,结合本课题组的研究进展,对单增李斯特菌硫氧还蛋白家族进行综述,以期为完善单增李斯特菌硫氧还蛋白调控系统提供参考。     

硫氧还蛋白还原酶缺陷的大肠杆菌宿主促进含半胱氨酸残基的重组蛋白可溶性表达

以人工设计的,不含半胱氨氨酸残基的三元蛋白,六聚和八聚鲑鱼降钙素融合蛋白和人尿激酶原等不同半胱氨酸残基含量的外源蛋白质为例,利用大肠杆菌硫氧还蛋白还原酶基因缺陷菌GH980（DE3 trxB^-）,探索把以包涵体形式表达的外源蛋白质变为可溶性表达的可能性及其规律。研究表明：由于硫氧还蛋白还原酶基因的缺陷所引超的细胞质氧化还原态势的变化,使一些在普通大肠杆菌宿主中以包涵 形式表达,含有半胱氨酸残基的重组蛋白,在GJ980中能在一定程度上以可溶性蛋白质形式表达;不含有半胱氨酸残基的重组蛋白在GJ980中仍以包涵体形式表达,推测重组蛋白在GJ980细胞质中形成二硫键对其正确构象的形成具有一定的作用。     

叶绿体硫氧还蛋白系统的调节机制

硫氧还蛋白(Trx)属于巯基-二硫键氧化还原酶家族, 通过作用于底物蛋白侧链2个半胱氨酸残基之间的二硫键(还原、异构和转移)来调控胞内蛋白的结构和功能。叶绿体Trx系统包括Trx及Trx类似蛋白、铁氧还蛋白(Fd)依赖的硫氧还蛋白还原酶(FTR)和还原型烟酰腺嘌呤二核苷磷酸(NADPH)依赖的硫氧还蛋白还原酶C (NTRC)。除了基质蛋白酶类活性变化及叶绿体蛋白的转运受Trx系统调控之外, 在叶绿体中还存在1条跨类囊体膜的还原势传递途径, 把基质Trx的还原势经跨膜转运蛋白介导, 最终传递给类囊体腔蛋白。FTR和NTRC共同作用维持叶绿体的氧化还原平衡。该文对叶绿体硫氧还蛋白系统的调节机制进行了综述, 同时讨论了叶绿体硫氧还蛋白系统对维持植物光合效率的重要意义。     

S-谷胱甘肽化修饰的研究进展

S-谷胱甘肽化(S-glutathionylation)是谷胱甘肽和靶蛋白半胱氨酸残基之间形成混合二硫化物的过程.由于其能调节靶蛋白功能,因此也属于蛋白质翻译后修饰.与其相对应,蛋白质的去谷胱甘肽化可由谷氧还蛋白(Grx)催化.因此,S-谷胱甘肽化修饰也被认为是一种防止蛋白质半胱氨酸巯基发生不可逆修饰的保护机制.由于该修饰还会改变含有巯基的氧化还原敏感型蛋白的结构与功能,因此也属于蛋白质功能调节的重要方式.哺乳动物细胞中S-谷胱甘肽化水平的改变与许多病理机制有关,但S-谷胱甘肽化在植物中的研究还处于起步阶段.本文综述了蛋白质的S-谷胱甘肽化的反应机制、检测方法、生理作用的相关研究进展,最后还提出今后研究中要解决的重要问题.     

谷氧还蛋白系统及其对细胞氧化还原态势的调控

细胞内氧化还原调控主要是由谷氧还蛋白系统和硫氧还蛋白系统完成。谷氧还蛋白属于硫氧还蛋白超家族,广泛分布在各种生物体内。作为一种巯基转移酶,它能够催化巯基．二硫键交换反应或者还原蛋白质谷胱甘肽二硫化物,以维持胞内的氧化还原态势。谷氧蛋白系统参与氧化胁迫、蛋白修饰、信号转导、细胞调亡和细胞分化等多种生物过程。对其体内作用靶蛋白的研究,有助于阐明谷氧还蛋白在整个细胞氧化还原网络的重要调控作用。     

硫氧还蛋白在凋亡途径中的作用机制

硫氧还蛋白（Trx）是体内广泛存在的氧化还原蛋白,其家族中两种重要的硫氧还蛋白：硫氧还蛋白1（thioredoxin1,Trx1）和硫氧还蛋白2（thioredoxin2,Trx2）都含有保守的-Cys-Gly-Pro-Cys-还原序列。由于Trx具有调节细胞生长增殖和抗凋亡的作用,因此Trx在凋亡途径中的作用机制就成为了对抗肿瘤的研究热点。     

NADPH依赖性硫氧还蛋白还原酶C在植物质体中的作用

硫氧还蛋白的氧化还原调节作用在生物界中普遍存在。它能够还原目标蛋白的二硫键,而自身的活性位点则被氧化。因此,对于新的催化循环,则需要由相应的还原酶将其再次还原成活性形式。硫氧还蛋白对维持高等植物的光合效率同样具有重要意义。叶绿体中的硫氧还蛋白分别由铁氧还蛋白依赖性硫氧还蛋白还原酶和NADPH依赖性硫氧还蛋白还原酶C(NTRC)两种酶还原。NTRC的本质是一种黄素蛋白,除了具有还原酶活性外,还整合了一个硫氧还蛋白结构域,在叶绿体和淀粉体的氧化还原调节中处于核心地位。这种特殊的双功能酶在卡尔文-本森循环、氧化戊糖磷酸途径、抗过氧化、四吡咯代谢、ATP和淀粉合成、生长素和光周期调控中扮演了多重角色。本综述总结了NTRC的生理功能,并讨论了该蛋白质对植物质体氧化还原稳态的调节机制。     

硫氧还蛋白结合蛋白-2的表观遗传学修饰及其与肿瘤的相关性

表观遗传学研究的是稳定的遗传信息的修饰,这种修饰在不改变DNA序列的情况下引起基因表达和功能的改变。肿瘤发生过程中,经常伴有抑癌基因的表观遗传学修饰,如DNA甲基化、组蛋白去乙酰化等。硫氧还蛋白结合蛋白-2是硫氧还蛋白的结合蛋白质,它可以和还原型的硫氧还蛋白相结合,与肿瘤发生密切相关。本文论述了有关肿瘤表观遗传学、硫氧还蛋白结合蛋白-2的表观遗传学修饰及其与肿瘤的相关性。     

ThiS可能作为一个原核翻译后修饰分子通过二硫键结合细胞蛋白

泛素及其相关蛋白是真核细胞中广泛存在的结构高度保守的一类小分子蛋白,参与蛋白翻译后修饰. 尽管在少数原核种属含有Pupylation这样的翻译后修饰,在原核细胞中尚未发现通用的泛素样修饰系统. ThiS是原核细胞广泛存在的泛素样小蛋白分子,它作为硫转运蛋白参与辅助因子的合成. 当与靶蛋白融合重组表达时,ThiS可降低靶蛋白在大肠杆菌中的稳定性. 本研究旨在探讨ThiS是否可能在原核细胞中参与翻译后修饰. ThiS在大肠杆菌中重组表达时,它可与细胞蛋白游离巯基发生共价结合,但与真核细胞泛素修饰不同,ThiS是通过12位半胱氨酸的游离巯基与蛋白形成二硫键,而不是通过C端活化的硫代羧基的转化过程发生共价结合. 在细胞内,氧化应激可诱导ThiS与蛋白的共价结合. 结果提示,ThiS在大肠杆菌中与细胞蛋白的结合,可能与真核细胞泛素化修饰在功能上存在进化联系;原核细胞中这种ThiS的结合形式可能代表一种古老的原核泛素样修饰方式.     

Crystal structures of two functionally different thioredoxins in spinach chloroplasts

Thioredoxins are small ubiquitous proteins which act as general protein disulfide reductases in living cells. Chloroplasts contain two distinct thioredoxins ( f and m) with different phylogenetic origin. Both act as enzyme regulatory proteins but have different specificities towards target enzymes. Thioredoxin f (Trx f), which shares only low sequence identity with thioredoxin m (Trx m) and with all other known thioredoxins, activates enzymes of the Calvin cycle and other photosynthetic processes. Trx m shows high sequence similarity with bacterial thioredoxins and activates other chloroplast enzymes. The here described structural studies of the two chloroplast thioredoxins were carried out in order to gain insight into the structure/function relationships of these proteins. Crystal structures were determined for oxidized, recombinant thioredoxin f (Trx f-L) and at the N terminus truncated form of it (Trx f-S), as well as for oxidized and reduced thioredoxin m (at 2.1 and 2.3 A resolution, respectively). Whereas thioredoxin f crystallized as a monomer, both truncated thioredoxin f and thioredoxin m crystallized as non-covalent dimers. The structures of thioredoxins f and m exhibit the typical thioredoxin fold consisting of a central twisted five-stranded beta-sheet surrounded by four alpha-helices. Thioredoxin f contains an additional alpha-helix at the N terminus and an exposed third cysteine close to the active site. The overall three-dimensional structures of the two chloroplast thioredoxins are quite similar. However, the two proteins have a significantly different surface topology and charge distribution around the active site. An interesting feature which might significantly contribute to the specificity of thioredoxin f is an inherent flexibility of its active site, which has expressed itself crystallographically in two different crystal forms.     

硫氧还蛋白与心血管疾病

硫氧还蛋白是细胞内最重要的二硫键还原酶,对维持细胞内蛋白质的还原状态并正常发挥功能着重要的作用,此外。硫氧还蛋白、硫氧还蛋白还原酶和硫氧还蛋白过氧化物酶组成了细胞内最重要的抗氧化系统之一,在对抗细胞的氧化应激上起着重要作用。心血管疾病是威胁人类健康的主要疾病,它与炎症反应和氧化应激有着密切的联系。文章将从硫氧还蛋白的抗氧化、抗炎、抗细胞凋亡,调控与炎症基因表达有关的核转录因子的转录活性,以及调节细胞内蛋白质的亚硝基化等诸多方面阐述硫氧还蛋白在防御心血管疾病方面可能具有的生物学功能。     

Localization of the thioredoxin system in normal rat kidney

Components of the thioredoxin system were localized in normal rat kidney using immunoperoxidase techniques at the light microscopic level and immunogold techniques at the ultrastructural level. Results from both methods were similar. Thioredoxin, thioredoxin reductases, and peroxiredoxins showed cell-type-specific localization, with the same cell types (proximal and distal tubular epithelial, papillary collecting duct, and transitional epithelial cells) previously identified as having high amounts of antioxidant enzyme immunoreactive proteins and oxidative damage products also having high levels of proteins of the thioredoxin system. In addition, peroxiredoxins II and IV were found in high levels in the cytoplasm of red blood cells, identified in kidney blood vessels. While thioredoxin and thioredoxin reductase 1 were found in all subcellular locations in kidney cells, thioredoxin reductase 2 was found predominantly in mitochondria. Thioredoxin reductase 1 was identified in rat plasma, suggesting it is a secreted protein. Peroxiredoxins often had specific subcellular locations, with peroxiredoxins III and V found in mitochondria and peroxiredoxin IV found in lysosomes. Our results emphasize the complex nature of the thioredoxin system, demonstrating unique cell-type and organelle specificity.     

Role of the second immunoglobulin-like loop of nectin in cell-cell adhesion

We investigated whether and how rat liver thioredoxin reductase spares alpha-tocopherol in biomembranes. Purified hydroperoxides of beta-linoleoyl-gamma-palmitoylphosphatidylcholine were decreased 35% by treatment with thioredoxin reductase and 54% by thioredoxin reductase plus E. coli thioredoxin. Thioredoxin reductase also halved the amount of hydroperoxides that had been formed during photoperoxidation of liposomes composed of beta-linoleoyl-gamma-palmitoylphosphatidylcholine, and of emulsions of both cholesterol and cholesteryl linolenate. In erythrocyte ghosts, thioredoxin reductase spared alpha-tocopherol from oxidation by both soybean lipoxygenase and ferricyanide. Thioredoxin reductase also decreased F(2)-isoprostanes in ghosts oxidized by ferricyanide, suggesting that its ability to spare alpha-tocopherol relates to reduction of lipid hydroperoxides.     

An NADP/thioredoxin system in leaves: purification and characterization of NADP-thioredoxin reductase and thioredoxin h from spinach

An NADP/thioredoxin system, consisting of NADPH, NADP-thioredoxin reductase (NTR), and its thioredoxin, thioredoxin h, has been previously described for heterotrophic plant tissues, i.e., wheat seeds and cultured carrot cells. Until now there was no evidence for this system in green leaves. Here, we report the identification of protein components of the NADP/thioredoxin system in leaves of several species. Thioredoxin h and NTR, which were both recovered in the extrachloroplastic fraction, were purified to apparent homogeneity from spinach leaves. This represents the first time that NTR has been characterized from a plant source. Similar to that from bacterial and mammalian sources, spinach leaf NTR was a flavoprotein (Mr 68,000) composed of two subunits of identical molecular mass (Mr 33,000) that resembled Escherichia coli NTR immunologically. Spinach thioredoxin h existed in two forms (Mr of 13,500 and 12,000) and was highly specific for plant NTR. Thioredoxin h and NTR partially purified from spinach roots showed properties similar to their counterparts from leaves. Spinach cytosolic thioredoxin h differed from chloroplast thioredoxin m or f from the same source but was similar to thioredoxin h from wheat seed in immunological properties.     

Release of thioredoxin from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with environmental stimuli: solubilization of thioredoxin with ethanol

Thioredoxin is crucial for the maintenance of the redox status of cells of all types. Mammalian thioredoxin is secreted from various types of cells, although the mechanism underlying has not yet been clarified. Previously, we demonstrated that thioredoxin was released from Saccharomyces cerevisiae after treatment with ethanol. In this paper, we show that as well as ethanol, low-pH shock and hypoosmotic shock release thioredoxin. Low-molecular-weight proteins in yeast cells were preferentially released by treatment with ethanol and low-pH shock. A cell wall integrity pathway seems partially involved in the hypoosmotic shock-induced release of thioredoxin. Considerable amounts of thioredoxin were present in the insoluble fractions of the cells, a portion of which was associated with lipid microdomains that are resistant to nonionic detergent at 4°C. The intracellular localization of thioredoxin may influence the efficiency of its release from yeast cells with ethanol.     

An immunocytochemical study of fetal cells at the maternal-placental interface using monoclonal antibodies to keratins,vimentin and desmin

Summary Thioredoxin and thioredoxin reductase (NADPH-oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) have been proposed to be involved in several thioldependent reduction-oxidation reactions in cells. Both proteins have been immunohistochemically demonstrated in the periphery of the cytoplasm and in cytoplasmic granules of acinar and islet cells in mouse pancreas. In animals fed ad libitum, the staining for thioredoxin was more intense in the exocrine acinar cells than in the islet cells, whereas that for thioredoxin reductase was more intense in the endocrine than in the exocrine pancreas. In the islets of fed mice all endocrine cell types showed about the same staining intensity for thioredoxin, while thioredoxin reductase was greatly enriched in the somatostatin-containing D cells. Starvation overnight caused an increased staining for both proteins in the acinar cells as well as in the islets. Under conditions of starvation, thioredoxin reductase, in contrast to thioredoxin, appeared to increase preferentially in the islet B cells, as compared with the D cells. Cysteamine treatment reduced the staining for somatostatin and for thioredoxin reductase in the D cells without any obvious effect on the other pancreatic cells. The results are compatible with a role for thioredoxin and thioredoxin reductase in secretion.     

Different cellular distribution of thioredoxin and subunit M1 of ribonucleotide reductase in rat tissues

The cellular distribution of thioredoxin and protein M1 of ribonucleotide reductase in adult rat tissues was investigated with immunohistochemical techniques using specific antisera. Tissues with high or low frequency of either mitotic or meiotic cell divisions were compared. Thioredoxin was demonstrated in many cells types that showed no detectable protein M1 of ribonucleotide reductase. A few cell types with protein M1 immunoreactivity also contained immunoreactive thioredoxin. However, in most cells no such co-localization could be demonstrated. This lack of correlation between cells containing subunit M1 of ribonucleotide reductase and the thioredoxin indicates that thioredoxin is not the physiologist hydrogen donor for ribonucleotide reductase in rat tissues and that the expression of two enzymes is differently regulated.     

Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.     

硫氧还蛋白的结构及在生物抗氧化中的功能

硫氧还蛋白(thioredoxin,Trx)是广泛存在于原核与真核生物体内的氧化还原调节蛋白。Trx通过对目标蛋白质进行还原,从而调节机体的氧化还原平衡。Trx与硫氧还蛋白还原酶(thioredoxin reductase,TrxR)及NADPH共同组成硫氧还蛋白系统参与众多生理过程。细胞中的活性氧是导致生物氧化胁迫的一个主要方面。Trx可以通过对细胞内被氧化的二硫键的还原来修复机体的氧化损伤,并通过这种方式防止机体衰老。同时,Trx系统可以与其它氧化还原系统如谷胱甘肽(GSH)系统协调配合,并消除体内过多的活性氧。