人蛋白二硫键异构酶的纯化及其抗血清制备

人蛋白二硫键异构酶的纯化及其抗血清制备吴秉毅,冯桂湘,吕静,张帆,冯永清,王福琴（第一军医大学珠江医院广州５１０２８０）关键词蛋白二硫键异构酶,分离与纯化,抗血清制备二硫键在维持蛋白质的空间结构、生物学活性中起重要作用。二硫键的形成是蛋白质翻译后修饰...     

氯霉素和四环素阻碍细菌蛋白分泌研究进展

氯霉素和四环素发挥活性的一个途径就是阻碍细菌蛋白质的分泌,其分泌功能是由其氨基端的信号序列决定的,该序列能将蛋白质引导到由SecY,E,G和A组成的转运蛋白复合体上。蛋白的转运还取决于融合蛋白的折叠特点,蛋白质转运到周质后的错误折叠可导致毒素聚集体形成,快速折叠还会使转运复合体发生拥堵,使所有的蛋白质分泌都受到抑制,导致细胞死亡。抗生素氯霉素和四环素处理细菌后会导致转运复合体中SecY的降解,造成致命的蛋白拥堵。现就抗生素氯霉素和四环素的干扰细菌蛋白质合成的作用机制以及导致SecY的降解来发挥阻碍细菌蛋白质分泌活性的一个新模式进行概述,以期为探讨新的靶向细菌的治疗方法提供科学依据。     

蛋白质复性研究进展

许多蛋白在大肠杆菌中高效表达时,其产物常以无活性的包含体形式存在,包含体蛋白的复性往往是制备这些蛋白的关键步骤之一,蛋白复性包括肽链折叠和分子内二硫键的氧化这两个互相影响的过程,本文综述了蛋白折叠过程的研究进展,及促进蛋白折叠和二硫键氧化的方法。     

大肠杆菌二硫键形成蛋白A（DsbA）研究进展

二硫键形成蛋白A(DisulfidebondformationproteinA,DsbA)是存在于大肠杆菌周质胞腔内的一种参与新生蛋白质折叠过程中催化二硫键形成的折叠酶。综述了DsbA三维结构、进化过程、协助蛋白质体内外复性方面的研究进展。DsbA比硫氧还原蛋白具有更强的氧化性,其强氧化性来自于Cys30残基异常低的pKa值和不稳定的氧化型结构,通过定点突变的研究表明了Cys30残基是DsbA活性中心最关键的氨基酸残基之一。DsbA不论在体内与目标蛋白融合表达还是在体外以折叠酶形式添加,都能有效地催化蛋白质的折叠复性,同时DsbA还具有部分分子伴侣的活性。     

大肠杆菌二硫键形成蛋白A（DsbA）研究进展

二硫键形成蛋白A（Disulfide bond formation protein A,DsbA）是存在于大肠杆菌周质胞腔内的一种参与新生蛋白质折叠过程中催化二硫键形成的折叠酶。综述了DsbA三维结构、进化过程、协助蛋白质体内外复性方面的研究进展。DsbA比硫氧还原蛋白具有更强的氧化性,其强氧化性来自于Cys³⁰残基异常低的pKa值和不稳定的氧化型结构,通过定点突变的研究表明了Cys³⁰残基是DsbA活性中心最关键的氨基酸残基之一。DsbA不论在体内与目标蛋白融合表达还是在体外以折叠酶形式添加,都能有效地催化蛋白质的折叠复性,同时DsbA还具有部分分子伴侣的活性。     

重组蛋白的体外再折叠

重组蛋白的再折叠是基因工程下游处理中非常重要的环节。本文在分析了蛋白体外折叠的机制后,指出了重组蛋白再折叠的一般策略,并综述了近年来的主要新方法,包括：分析伴侣介导的再折叠去污剂协助的再折叠,反向微团中的蛋白再折叠,折叠促进剂的添加以及再折地促进二硫键形成的方法 。     

蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究,蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中,大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠,这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥,因为后者仅仅是进行链内二硫键重排的化学反应所必需,与控制肽链折叠无直接关系。3根据对BPTI的研究,二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用,既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明,天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成,更具有普遍意义。4在氧化重折叠的早期,二硫键的形成基本上是一个随机过程,随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外,在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述,在折叠的后期,二硫键的形成决定于折叠中间体的构象,类天然、有柔性的结构有利于天然二硫键形成和正确折叠,具有这类结构的分子为有效的折叠中间体,最终都能转变为天然产物;而无效折叠中间体往往具有稳定的结构,使巯基、二硫键内埋妨碍二硫键重排,并因能垒的障碍不利于进一步折叠。因此,降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则,实验证明,碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外,这里还就氧化重折叠的基础和应用研究的前景进行了讨论。     

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     

热休克蛋白在阿尔茨海默病中的研究

热休克蛋白（heat shock protein,HSP）是一种重要的分子伴侣,它们参与辅助蛋白质合成、折叠、转运以及定位等过程,并且在协调蛋白质水解、阻止蛋白质错误折叠和聚积方面发挥重要作用。阿尔茨海默病（Alzheimer＇s disease,AD）是最常见的神经退行性疾病,以神经细胞内过度磷酸化的tau蛋白异常聚积形成神经原纤维缠结以及细胞外β淀粉样蛋白（β-amyloid,Aβ）异常折叠形成淀粉样斑为主要病理特征。研究表明HSP不但对tau蛋白的聚积／降解发挥重要作用,并且可抑制Aβ相关的毒性作用。这些研究结果提示了分子伴侣有可能成为AD治疗的新靶点,现对该方面的研究进展进行综述。     

保守GTP酶ObgE的分子伴侣活性研究

分子伴侣(molecular chaperone)能够帮助新生多肽链或错误折叠的蛋白质形成天然构象,但本身又不是成熟蛋白质的组成成分。蛋白质需要分子伴侣的帮助,才能够从核糖体合成的新生肽链折叠成有生物活性的大分子。E.coli的ObgE蛋白是保守的GTP酶,ObgE蛋白参与信号转导、蛋白运输和细胞周期调控,并与E.coli在氨基酸饥饿下的应激反应有关。本实验通过分子克隆,将E.coli ObgE蛋白的基因克隆到表达载体pET-28a中,转化到E.coli BL21进行蛋白表达纯化。纯化后的ObgE蛋白通过柠檬酸合成酶变复性实验、α-葡萄糖苷酶变复性实验、牛碳酸酐酶变复性实验,检测ObgE蛋白的分子伴侣活性,发现ObgE具有一定的分子伴侣活性,为该蛋白的研究应用奠定了基础。     

蛋白质中二硫键附近肽段识别规律研究

本文对蛋白质中二硫键附近的残基进行了计算机统计分析,结果发现平行和反平行残基间存在着特异的配对规律。这种残基间的相互作用或识别,可能与蛋白质折叠过程中正确地形成二硫键有关。该结果有助于蛋白质工程设计。     

Acceleration of disulfide-coupled protein folding using glutathione derivatives

Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding of proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives - glutamyl-cysteinyl-arginine (ECR) and arginyl-cysteinyl-glycine (RCG) - and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (K(RCG) = 3.69 × 10(-3) s(-1)) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide-exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.     

Protein disulfide isomerase

During the maturation of extracellular proteins, disulfide bonds that chemically cross-link specific cysteines are often added to stabilize a protein or to join it covalently to other proteins. Disulfide formation, which requires a change in the covalent structure of the protein, occurs as the protein folds into its three-dimensional structure. In the eukaryotic endoplasmic reticulum and in the bacterial periplasm, an elaborate system of chaperones and folding catalysts ensure that disulfides connect the proper cysteines and that the folding protein does not make improper interactions. This review focuses specifically on one of these folding assistants, protein disulfide isomerase (PDI), an enzyme that catalyzes disulfide formation and isomerization and a chaperone that inhibits aggregation.     

1H, 13C and 15N resonance assignments of the bb′ domains of human protein disulfide isomerase

Protein disulfide isomerase (PDI) participates in protein folding and catalyses formation of disulfide bonds. The b′ domain of human PDI contributes to binding unfolded proteins; its structure is stabilized by the b domain. Here, we report NMR chemical shift assignments for the bb′ fragment.     

Probing protein folding and stability using disulfide bonds

Disulfide bonds are required to stabilize the folded conformations of many proteins. The rates and equilibria of processes involved in disulfide bond formation and breakage can be manipulated experimentally and can be used to obtain important information about protein folding and stability. A number of experimental procedures for studying these processes, and approaches to interpreting the resulting data, are described here.     

Glutathione is required to regulate the formation of native disulfide bonds within proteins entering the secretory pathway

The formation of native disulfide bonds is an essential event in the folding and maturation of proteins entering the secretory pathway. For native disulfides to form efficiently an oxidative pathway is required for disulfide bond formation and a reductive pathway is required to ensure isomerization of non-native disulfide bonds. The oxidative pathway involves the oxidation of substrate proteins by PDI, which in turn is oxidized by endoplasmic reticulum oxidase (Ero1). Here we demonstrate that overexpression of Ero1 results in the acceleration of disulfide bond formation and correct protein folding. In contrast, lowering the levels of glutathione within the cell resulted in acceleration of disulfide bond formation but did not lead to correct protein folding. These results demonstrate that lowering the level of glutathione in the cell compromises the reductive pathway and prevents disulfide bond isomerization from occurring efficiently, highlighting the crucial role played by glutathione in native disulfide bond formation within the mammalian endoplasmic reticulum.     

Redox signaling loops in the unfolded protein response

The endoplasmic reticulum (ER) is the first compartment of secretory pathway. It plays a major role in ER chaperone-assisted folding and quality control, including post-translational modification such as disulfide bond formation of newly synthesized secretory proteins. Protein folding and assembly takes place in the ER, where redox conditions are distinctively different from the other organelles and are favorable for disulfide formation. These reactions generate the production of reactive oxygen species (ROS) as a byproduct of thiol/disulfide exchange reaction among ER oxidoreductin 1 (Ero1), protein disulfide isomerase (PDI) and ER client proteins, during the formation of disulfide bonds in nascent or incorrectly folded proteins. When uncontrolled, this phenomenon perturbs ER homeostasis, thus aggravating the accumulation of improperly folded or unfolded proteins in this compartment (ER stress). This results in the activation of an adaptive mechanism named the unfolded protein response (UPR). In mammalian cells, the UPR is mediated by three ER-resident membrane proteins (PERK, IRE1 and ATF6) and regulates the expression of the UPR target genes, which themselves encode ER chaperones, folding enzymes, pro-apoptotic proteins and antioxidants, with the objective of restoring ER homeostatic balance. In this review, we will describe redox dependent activation (ER) and amplification (cytosol) loops that control the UPR and the consequences these regulatory loops have on cell fate and physiology.     

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.