三碘甲腺原氨酸不能与蛋白质二硫键异构酶特异结合

 <正> 蛋白质二硫键异构酶(PDI)是由两个相同亚基组成的蛋白质,分子量约107000,能够催化蛋白质天然二硫键的形成。Edman等在1985年已阐明了鼠PDI的一级结构,近二、三年,又相继报道了三种蛋白的一级结构,它们是脯氨酰-4-羟化酶的β亚基、三碘甲腺原氨酸(T_3)结合蛋白(T_3BP)和蛋白质糖基化部位结合蛋白(GSBP),在氨基酸水平上与PDl分     

玉米蛋白质二硫键异构酶（PDI）基因的特征和表达

蛋白质二硫键异构酶（PDI）对蛋白的折叠和二硫键的形成起重要的作用.此外,PDI还执行许多其他的生物功能,是1个多功能酶. 本文通过研究玉米中1个PDI基因的特征和表达,探讨它的功能作用. 玉米中的PDI基因编码513个氨基酸.同源分析表明,该基因和水稻、小麦的PDI基因聚为一类,有很高的蛋白相似性.蛋白结构分析表明,该基因具有明显的PDI基因的结构特点,包括硫氧还蛋白活性位点(CGHC)以及内质网定位信号(KDEL).Northern杂交分析显示,该基因在发育种子的表达量高,同时受干旱、冷、ABA和盐等逆境胁迫诱导表达.PDI与GFP融合表达研究基因的亚细胞定位,表明该基因定位在除细胞膜外的细胞质和细胞器上.     

蛋白质二硫键异构酶家族的结构与功能

蛋白质二硫键异构酶（protein disulfide isomerase,PDI）家族是一类在内质网中起作用的巯基-二硫键氧化还原酶.它们通常含有CXXC（Cys-Xaa-Xaa-Cys,CXXC）活性位点,活性位点的两个半胱氨酸残基可催化底物二硫键的形成、异构及还原.所有PDI家族成员包含至少一个约100个氨基酸残基的硫氧还蛋白同源结构域.PDI家族的主要职能是催化内质网中新生肽链的氧化折叠,另外在内质网相关的蛋白质降解途径（ERAD）、蛋白质转运、钙稳态、抗原提呈及病毒入侵等方面也起重要作用.     

重组人蛋白质二硫键异构酶活性片段的克隆与表达纯化策略

蛋白质二硫键异构酶(protein disulfide somerase,PDI)在体内或体外均可以非特异地催化其它蛋白质的二硫键的形成、还原和异构化,进而辅助蛋白的折叠和复性。近来发现PDI还具有非ATP依赖的分子伴侣的功能。鉴于PDI具有的重要功能,拟利用PDI来辅助基因工程产品的复性,如包含体蛋白(尤其是含有二硫键的蛋白),这将为基因工程产品的下游纯化复性,开拓新的思路。PDI含有两个硫氧还蛋白(thioredoxin,TRX)同源区a区和a′区,各含有一个CGHC活性位点,与TRX的CGPC位点同源。a区和a′区各具有50％的二硫键异构     

二硫键异构酶

天然二硫键的形成是许多蛋白正确折叠中的限速步骤,在稳定蛋白质构象和保持蛋白质活性方面起重要作用。讨论的二硫键异构酶是内质网中一种重要的蛋白折叠催化剂,它催化蛋白二硫键的形成和错误配对二硫键的重排,并有抑制错误折叠蛋白聚集的分子伴侣活性。PDI广泛应用于基因工程上提高外源蛋白表达水平。     

支持向量机方法预测蛋白质结构中的二硫键

在蛋白质结构预测的研究中,一个重要的问题就是正确预测二硫键的连接,二硫键的准确预测可以减少蛋白质构像的搜索空间,有利于蛋白质3D结构的预测,本文将预测二硫键的连接问题转化成对连接模式的分类问题,并成功地将支持向量机方法引入到预测工作中。通过对半胱氨酸局域序列连接模式的分类预测,可以由蛋白质的一级结构序列预测该蛋白质的二硫键的连接。结果表明蛋白质的二硫键的连接与半胱氨酸局域序列连接模式有重要联系,应用支持向量机方法对蛋白质结构的二硫键预测取得了良好的结果。     

海南产桶形芋螺蛋白质二硫键异构酶的鉴定

蛋白质二硫键异构酶(PDI)是内质网新生肽链折叠中一个重要的折叠酶.在热 带药用海洋生物芋螺的毒液中富含PDI酶,该酶对于毒液中芋螺毒素神经肽的体内 氧化折叠至关重要.本研究主要采用凝胶过滤层析和制备型Rotofor液相等电聚焦 电泳等多种方法,从海南产桶形芋螺（Conus betulinus Linnaeus）毒管中分离 纯化天然的PDI酶蛋白,经电泳和MALDI-TOF MS质谱鉴定分析确证获得了高纯度 的桶形芋螺PDI酶,建立了天然芋螺PDI酶分离纯化的技术方法. 以芋螺毒素线性 肽K412为底物进行了PDI酶活性鉴定.结果表明,该分离纯化的PDI酶能够促进K412 的氧化折叠.由于芋螺毒素的氧化折叠非常复杂,且氧化折叠后具有正确二硫键连 接方式的芋螺毒素才具有各种药理活性,因此,本研究结果为后续PDI酶在种类繁 多的芋螺毒素氧化折叠中的应用及其作用机制研究提供了重要的物质基础.     

蛋白质二硫键异构酶的纯化和活力测定

 从500g新鲜牛肝制得蛋白质二硫键异构酶(PDI,EC 5.3.4.1)98mg。该酶制剂在SDS-聚丙烯酰胺凝胶电泳中表现为亚基分子量62,000的均一条带。在260nm追踪,因二硫键错接而失活的牛胰核糖核酸酶A,经PDI作用使其二硫键重排恢复活力,从而催化酵母RNA的水解来测定PDI活力。这种单波长法比文献中介绍的追踪A_(260)—A_(280)的双波长法更为灵敏方便。酶的克分子消光系数ε_M=1.03×10~5(pH7.5),其比活性为1400单位/克蛋白质。     

二硫键与蛋白质的结构

二硫键是肽链上2个半胱氨酸残基的巯基基团发生氧化反应形成的共价键．具有链内二硫键和链间二硫键2种形式。与氨基酸的氨基氮原子之间形成的稳定共价键不同．二硫键容易被还原而断裂,断裂后可再次氧化重新形成二硫键,因而是可以动态变化的化学键。二硫键是参与一级结构也是形成高级结构的重要化学键,对蛋白质折叠和高级结构的形成与维持十分重要。讨论了二硫键的形成和特征及其与蛋白质结构和功能之间的关系,并讨论了生物学教学中关于二硫键的一些疑问．     

几种南海芋螺二硫键异构酶的克隆及进化分析

目的:从来自中国南海的4种芋螺中克隆出包含完整3’和5’非翻译区的蛋白质二硫键异构酶(PDI)全基因序列,并对其进行序列及进化分析。方法:根据各种生物PDI基因的保守区域设计引物,利用3’和5’cDNA末端快速扩增(RACE)方法克隆出PDI全基因序列,并通过生物信息学方法对各芋螺PDI序列进行分析。结果与结论:从中国南海玉女芋螺、黑星芋螺、堂皇芋螺、桶形芋螺cDNA中克隆出包含有完整3’和5’非翻译区的PDI全基因序列;分析结果表明各芋螺之间的同源性大于90%,而与对虾、人类、酿酒酵母的同源性均小于60%;各芋螺PDI与其他生物的2个活性位点序列高度保守,而底物结合位点具有物种特异性,进化树显示各芋螺PDI的特征可能受其捕食食性影响。     

Catalysis of thiol/disulfide exchange. Glutaredoxin 1 and protein-disulfide isomerase use different mechanisms to enhance oxidase and reductase activities

Glutaredoxin (Grx) and protein-disulfide isomerase (PDI) are members of the thioredoxin superfamily of thiol/disulfide exchange catalysts. Thermodynamically, rat PDI is a 600-fold better oxidizing agent than Grx1 from Escherichia coli. Despite that, Grx1 is a surprisingly good protein oxidase. It catalyzes protein disulfide formation in a redox buffer with an initial velocity that is 30-fold faster than PDI. Catalysis of protein and peptide oxidation by the individual catalytic domains of PDI and by a Grx1-PDI chimera show that differences in active site chemistry are fundamental to their oxidase activity. Mutations in the active site cysteines reveal that Grx1 needs only one cysteine to catalyze rapid substrate oxidation, whereas PDI requires both cysteines. Grx1 is a good oxidase because of the high reactivity of a Grx1-glutathione mixed disulfide, and PDI is a good oxidase because of the high reactivity of the disulfide between the two active site cysteines. As a protein disulfide reductase, Grx1 is also superior to PDI. It catalyzes the reduction of nonnative disulfides in scrambled ribonuclease and protein-glutathione mixed disulfides 30-180 times faster than PDI. A multidomain structure is necessary for PDI to catalyze effective protein reduction; however, placing Grx1 into the PDI multidomain structure does not enhance its already high reductase activity. Grx1 and PDI have both found mechanisms to enhance active site reactivity toward proteins, particularly in the kinetically difficult direction: Grx1 by providing a reactive glutathione mixed disulfide to supplement its oxidase activity and PDI by utilizing its multidomain structure to supplement its reductase activity.     

Protein disulfide isomerase: the structure of oxidative folding

Cellular functions hinge on the ability of proteins to adopt their correct folds, and misfolded proteins can lead to disease. Here, we focus on the proteins that catalyze disulfide bond formation, a step in the oxidative folding pathway that takes place in specialized cellular compartments. In the endoplasmic reticulum of eukaryotes, disulfide formation is catalyzed by protein disulfide isomerase (PDI); by contrast, prokaryotes produce a family of disulfide bond (Dsb) proteins, which together achieve an equivalent outcome in the bacterial periplasm. The recent crystal structure of yeast PDI has increased our understanding of the function and mechanism of PDI. Comparison of the structure of yeast PDI with those of bacterial DsbC and DsbG reveals some similarities but also striking differences that suggest directions for future research aimed at unraveling the catalytic mechanism of disulfide bond formation in the cell.     

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.     

Substrate recognition by the protein disulfide isomerases

Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds. Their primary function is to stabilize the folded structure of the protein, although disulfide bond formation can also play a regulatory role. Native disulfide bond formation is not trivial, so it is often the rate-limiting step of protein folding both in vivo and in vitro. Complex coordinated systems of molecular chaperones and protein folding catalysts have evolved to help proteins attain their correct folded conformation. This includes a family of enzymes involved in catalyzing thiol-disulfide exchange in the endoplasmic reticulum, the protein disulfide isomerase (PDI) family. There are now 17 reported PDI family members in the endoplasmic reticulum of human cells, but the functional differentiation of these is far from complete. Despite PDI being the first catalyst of protein folding reported, there is much that is still not known about its mechanisms of action. This review will focus on the interactions of the human PDI family members with substrates, including recent research on identifying and characterizing their substrate-binding sites and on determining their natural substrates in vivo.     

Catalysis of creatine kinase refolding by protein disulfide isomerase involves disulfide cross-link and dimer to tetramer switch

Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 microm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 microm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.     

The catalytic activity of protein-disulfide isomerase requires a conformationally flexible molecule

Protein-disulfide isomerase (PDI) catalyzes the formation of the correct pattern of disulfide bonds in secretory proteins. A low resolution crystal structure of yeast PDI described here reveals large scale conformational changes compared with the initially reported structure, indicating that PDI is a highly flexible molecule with its catalytic domains, a and a', representing two mobile arms connected to a more rigid core composed of the b and b' domains. Limited proteolysis revealed that the linker between the a domain and the core is more susceptible to degradation than that connecting the a' domain to the core. By restricting the two arms with inter-domain disulfide bonds, the molecular flexibility of PDI, especially that of its a domain, was demonstrated to be essential for the enzymatic activity in vitro and in vivo. The crystal structure also featured a PDI dimer, and a propensity to dimerize in solution and in the ER was confirmed by cross-linking experiments and the split green fluorescent protein system. Although sedimentation studies suggested that the self-association of PDI is weak, we hypothesize that PDI exists as an interconvertible mixture of monomers and dimers in the endoplasmic reticulum due to its high abundance in this compartment.     

Domain architecture of protein-disulfide isomerase facilitates its dual role as an oxidase and an isomerase in Ero1p-mediated disulfide formation

Native disulfide bond formation in eukaryotes is dependent on protein-disulfide isomerase (PDI) and its homologs, which contain varying combinations of catalytically active and inactive thioredoxin domains. However, the specific contribution of PDI to the formation of new disulfides versus reduction/rearrangement of non-native disulfides is poorly understood. We analyzed the role of individual PDI domains in disulfide bond formation in a reaction driven by their natural oxidant, Ero1p. We found that Ero1p oxidizes the isolated PDI catalytic thioredoxin domains, A and A' at the same rate. In contrast, we found that in the context of full-length PDI, there is an asymmetry in the rate of oxidation of the two active sites. This asymmetry is the result of a dual effect: an enhanced rate of oxidation of the second catalytic (A') domain and the substrate-mediated inhibition of oxidation of the first catalytic (A) domain. The specific order of thioredoxin domains in PDI is important in establishing the asymmetry in the rate of oxidation of the two active sites thus allowing A and A', two thioredoxin domains that are similar in sequence and structure, to serve opposing functional roles as a disulfide isomerase and disulfide oxidase, respectively. These findings reveal how native disulfide folding is accomplished in the endoplasmic reticulum and provide a context for understanding the proliferation of PDI homologs with combinatorial arrangements of thioredoxin domains.     

Peptide disulfide RKCGCFF facilitates oxidative protein refolding by mimicking protein disulfide isomerase

This communication reports a new design of peptide disulfide, RKCGCFF, for facilitating oxidative protein refolding. The new design mimics the properties of protein disulfide isomerase (PDI) by introducing hydrophobic and positively charged patches into the two terminals of disulfide CGC. RKCGCFF was found more effective than the traditional oxidant oxidized glutathione (GSSG) as well as its counterpart, RKCGC, in facilitating the oxidative refolding of lysozyme. More importantly, RKCGCFF could improve lysozyme refolding yield at a high concentration (0.7 mg/mL). The research proved that incorporation of hydrophobic and charged patches into the CGC disulfide made the oxidant more similar to PDI in structure and properties.     

Both PDI and PDIp can attack the native disulfide bonds in thermally-unfolded RNase and form stable disulfide-linked complexes

Protein disulfide isomerase (PDI) and its pancreatic homolog (PDIp) are folding catalysts for the formation, reduction, and/or isomerization of disulfide bonds in substrate proteins. However, the question as to whether PDI and PDIp can directly attack the native disulfide bonds in substrate proteins is still not answered, which is the subject of the present study. We found that RNase can be thermally unfolded at 65°C under non-reductive conditions while its native disulfide bonds remain intact, and the unfolded RNase can refold and reactivate during cooling. Co-incubation of RNase with PDI or PDIp during thermal unfolding can inactivate RNase in a PDI/PDIp concentration-dependent manner. The alkylated PDI and PDIp, which are devoid of enzymatic activities, cannot inactivate RNase, suggesting that the inactivation of RNase results from the disruption of its native disulfide bonds catalyzed by the enzymatic activities of PDI/PDIp. In support of this suggestion, we show that both PDI and PDIp form stable disulfide-linked complexes only with thermally-unfolded RNase, and RNase in the complexes can be released and reactivated dependently of the redox conditions used. The N-terminal active site of PDIp is essential for the inactivation of RNase. These data indicate that PDI and PDIp can perform thiol-disulfide exchange reactions with native disulfide bonds in unfolded RNase via formation of stable disulfide-linked complexes, and from these complexes RNase is further released.     

Catalysis of protein disulfide bond isomerization in a homogeneous substrate

Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a beta hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N and C terminus contain a fluorescence donor (tryptophan) and acceptor (N(epsilon)-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E(o') = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys-Gly-His-Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/K(M) = 1.7 x 10(5) M(-1) s(-1), which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude that catalysis of disulfide bond isomerization by PDI does not necessarily involve a cycle of substrate reduction/oxidation.