以蛋自质为基础模拟谷胱甘肽过氧化物酶

硒元素作为一种生命体中必须的微量元素,与人类的健康和疾病息息相关.硒元素主要以硒代半胱氨酸的形式存在于至少25种硒蛋白中,执行着多种生物功能.在这20多种硒蛋白中,谷胱甘肽过氧化物酶( GPx)作为一种主要的抗氧化酶,能够有效地利用谷胱甘肽还原氢过氧化物以防止机体的氧化损伤.这里,我们主要介绍以蛋白质为骨架构筑GPx模...     

具有谷胱甘肽过氧化物酶(GPx)活力的含硒四肽(SecRGD)

以精氨酸-甘氨酸-天冬氨酸(RGD)序列为基础, 在N-端引入硒代半胱氨酸(Sec)设计了SecRGD序列模拟谷胱甘肽过氧化物酶(GPx), 利用Fmoc固相合成法合成了SecRGD. 采用ESI-MS质谱和氢化物原子荧光光谱法对硒肽进行表征, 采用酶偶联法进行GPx活力测定和酶动力学分析, 用噻唑蓝(MTT)比色法评价了硒肽的抗氧化效果. 结果表明, 该硒肽的存在形式为SecRGD的二聚体. 该硒肽具有GPx活力, 其催化谷胱甘肽(GSH)还原H2O2的GPx活力为5.54 U/μmol, 高于经典的GPx模拟物Ebselen. 稳态动力学分析结果表明, 该硒肽的催化机制为乒乓机制. 该硒肽具有分子量小, 易溶于水, 毒性低及可有效保护Vero细胞免受氧化损伤的优点, 具有作为抗氧化药物的应用前景.     

人工硒酶设计新策略

作为人体内重要的抗氧化酶,谷胱甘肽过氧化物酶受到人们越来越多的关注.为探索其催化机制,并开发极具潜力的抗氧化药物,国际上广泛开展了人工模拟谷胱甘肽过氧化物酶的研究工作.基于化学、生物学、超分子科学以及纳米科学及其深度交叉,人们研究构建了从小分子到大分子,再到纳米等不同结构的谷胱甘肽过氧化物酶模拟物.本文主要综述本研究组和其他研究组基于以大分子为骨架设计人工谷胱甘肽过氧化物酶的研究思路和策略.研究体系包括合成的大分子硒酶模型、自组装大分子硒酶模型和生物大分子硒酶模型等.     

硒与端粒酶

硒是人体必需微量元素,它参与抗氧化酶谷胱甘肽过氧化物酶和多种硒蛋白的组成,在体内发挥消除自由基、抗氧化物等作用。硒的抗衰老与抑制肿瘤细胞生长的作用是通过它对端粒酶的作用而实现的。研究端粒酶的调控及其与细胞凋亡的关系已成为人类种瘤研究的新热点。     

硒蛋白的抗氧化性研究与第21个氨基酸的发现

硒是人体必需的微量元素,以硒代半胱氨酸(Sec)的形式存在于蛋白质中作为硒酶的活性中心发挥作用,其生物功能主要是抗氧化。由于硒与人体健康具有十分密切的关系,所以硒蛋白的研究有着重要的理论和实际意义。本文以第一个硒蛋白细胞谷胱甘肽过氧化物酶为例,结合作者自己的工作,重点对该硒酶的结构、催化机制和模拟进行了综述,并就TGA编码Sec致第21个氨基酸的发现以及基于硒代半胱氨酸插入元件(SECIS)的特征寻找新硒蛋白的研究进展进行了介绍。     

仿硒酶研究进展

硒是人体中必需的微量元素,它与各种疾病和人类健康息息相关。硒在生物体内以硒代半胱氨酸形式表现其生理活性和功能。为了探索硒在硒蛋白中结构和功能关系并可能发展成硒相关的适用药物,人们付出许多努力来发展硒蛋白模拟化学。由于硒酶—谷胱甘肽过氧化物酶（GPX）重要的抗氧化作用以及潜在的药用价值,国际上广泛开展了对它的人工模拟研究。本文对近年来硒酶模拟化学和生物学相关研究进展进行了综述。     

硒蛋白与糖尿病——硒的两面性

糖尿病是危害人类健康的全球性重大疾病,胰岛素抵抗是诱发2型糖尿病的重要因素。微量必需元素硒与人体健康密切相关,通过硒蛋白发挥多种重要生物学功能。近年来硒与糖尿病的关系引人关注,早期研究表明硒具有类胰岛素作用,可望用于防治糖尿病,但近来的人群试验和动物研究却表明硒在糖尿病发生发展中的作用具有两面性,长期补充一定剂量的硒反而增加了胰岛素抵抗和2型糖尿病的发病率。而且,硒在糖尿病发生发展中的两面性被证实与几种硒蛋白密切相关,包括谷胱甘肽过氧化物酶1(GPx1)、硒蛋白S(SelS)和硒蛋白P(SelP)等。本文结合本课题组的工作介绍了硒在糖尿病中的两面性以及硒蛋白在糖尿病发生发展中的作用,并对未来的研究方向进行了展望。     

具有谷胱甘肽过氧化物酶活力的含硒三肽

合成了由硒代半胱氨酸(U)、谷氨酰胺(Q)和色氨酸(W)组成的QUW,QWU,WQU,WUQ,UWQ和UQW 6个具有谷胱甘肽过氧化物酶(GPx)活力的含硒三肽;采用双酶偶联法进行了GPx活力测定和稳态动力学分析;通过噻唑蓝(MTT)比色法、划痕愈合实验和Western blot技术表征了含硒三肽对肝癌Hep G2细胞生长和迁移能力的影响.结果表明,当U位于氨基端时,含硒三肽的GPx活力高于U位于中间位置或者羧基端时.UWQ催化谷胱甘肽(GSH)还原H2O2的活力最高,其催化机制为乒乓机制.UWQ可使Hep G2细胞运动能力减弱,降低肝癌细胞的浸润转移能力.     

含硒杂环化合物的合成和对超氧阴离子作用的ESR研究

引言硒作为人体必需的微量元素,其主要生化作用与谷胱甘肽过氧化物酶密切相关.在体内,该酶可以催化许多过氧化物的分解,并且与谷胱甘肽一起组成一个强有力的细胞防御体系,发挥其抗氧化作用.近几年来,人们发现了一种小分子有机硒化物2-苯基-1,2-苯并异硒唑酮-3(简称Ebselen)具有类似谷胱甘肽过氧化物酶的活性.它能有效地抑制活性氧自由基的产生而引起的细胞损伤,具有良好的抗炎活性,且毒性极低(大鼠LD_(50)>6180mg/os).因而,普遍认为Ebselen及其类似物是一类具有广谱抗炎活性的潜在药物,在医药领域具有广阔的应用前景.     

内质网硒蛋白的研究进展

硒是哺乳动物必需的一种微量营养元素,主要以硒代半胱氨酸的形式存在于各种硒蛋白中,硒的主要生物功能通过硒蛋白实现.在25种哺乳动物硒蛋白中,有7种硒蛋白位于内质网,分别为2型脱碘酶、15-kDa硒蛋白、硒蛋白M、硒蛋白T、硒蛋白K、硒蛋白S和硒蛋白N.除了2型脱碘酶外,对其余内质网硒蛋白知之甚少.最近一些研究显示,一些内质网硒蛋白在氧化还原平衡调节、蛋白质折叠质量控制、错误折叠蛋白从内质网逆向转运至胞质、Ca2+稳态调节、内质网应激调节及炎症调节等过程中发挥作用.本文介绍了每种内质网硒蛋白的结构、功能及其生理和病理作用的一些最新研究进展,并对未来需要研究的内容进行了展望.     

Challenges in the accurate speciation analysis of selenium in humans: first report on indicative levels of selenoproteins in a serum certified reference material for total selenium (BCR-637)

Se is one of the most investigated essential trace elements in the past years, mostly due to its cancer prevention properties. Nevertheless, the accurate determination of its biologically active species, such as the selenoproteins (SeProt) in human serum, is currently a challenging task. This is because of the lack of appropriate quantification standards, certified reference materials (CRMs), and/or reference measurement methods. Additionally, most of the methods developed so far for the determination of SeProt were applied to the analysis of control (volunteers) serums, which are not available to other laboratories, therefore making methods inter-comparison virtually impossible. We present here for the first time indicative levels of SeProt in a commercially available human serum, namely the BCR-637 CRM with certified level of total Se. The concentrations of selenium associated with glutathione peroxidase (GPx), selenoprotein P (SelP) and selenoalbumin (SeAlb) in this serum were calculated using the results obtained by 13 different analytical methods (literature and non-published data) on the basis of (affinity) high-performance liquid chromatography (AF-HPLC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). The indicative levels of SeProt in the BCR-637 serum can be used for validation of methods dealing with the determination of these proteins in human serum.     

Thiol cofactors for selenoenzymes and their synthetic mimics

The importance of selenium as an essential trace element is now well recognized. In proteins, the redox-active selenium moiety is incorporated as selenocysteine (Sec), the 21st amino acid. In mammals, selenium exerts its redox activities through several selenocysteine-containing enzymes, which include glutathione peroxidase (GPx), iodothyronine deiodinase (ID), and thioredoxin reductase (TrxR). Although these enzymes have Sec in their active sites, they catalyze completely different reactions and their substrate specificity and cofactor or co-substrate systems are significantly different. The antioxidant enzyme GPx uses the tripeptide glutathione (GSH) for the catalytic reduction of hydrogen peroxide and organic peroxides, whereas the larger and more advanced mammalian TrxRs have cysteine moieties in different subunits and prefer to utilize these internal cysteines as thiol cofactors for their catalytic activity. On the other hand, the nature of in vivo cofactor for the deiodinating enzyme ID is not known, although the use of thiols as reducing agents has been well-documented. Recent studies suggest that molecular recognition and effective binding of the thiol cofactors at the active site of the selenoenzymes and their mimics play crucial roles in the catalytic activity. The aim of this perspective is to present an overview of the thiol cofactor systems used by different selenoenzymes and their mimics.     

A systematic approach to the accurate quantification of selenium in serum selenoalbumin by HPLC-ICP-MS

In this paper, two different methods are for the first time systematically compared for the determination of selenium in human serum selenoalbumin (SeAlb). Firstly, SeAlb was enzymatically hydrolyzed and the resulting selenomethionine (SeMet) was quantified using species-specific isotope dilution (SSID) with reversed phase-HPLC (RP-HPLC) hyphenated to (collision/reaction cell) inductively coupled plasma-quadrupole mass spectrometry (CRC ICP-QMS). In order to assess the enzymatic hydrolysis yield, SeAlb was determined as an intact protein by affinity-HPLC (AF-HPLC) coupled to CRC ICP-QMS. Using this approach, glutathione peroxidase (GPx) and selenoprotein P (SelP) (the two selenoproteins present in serum) were also determined within the same chromatographic run. The levels of selenium associated with SeAlb in three serum materials, namely BCR-637, Seronorm level 1 and Seronorm level 2, obtained using both methods were in a good agreement. Verification of the absence of free SeMet, which interferes with the SeAlb determination (down to the amino acid level), in such materials was addressed by analyzing the fraction of GPx, partially purified by AF-HPLC, using RP-HPLC (GPx only) and size exclusion-HPLC (SE-HPLC) coupled to CRC ICP-QMS. The latter methodology was also used for the investigation of the presence of selenium species other than the selenoproteins in the (AF-HPLC) SelP and SeAlb fractions; the same selenium peaks were detected in both control and BCR-637 serum with a difference in age of ca. 12 years. It is also for the first time that the concentrations of selenium associated with SeAlb, GPx and SelP species in such commercially available serums (only certified or having indicative levels of total selenium content) are reported. Such indicative values can be used for reference purposes in future validation of speciation methods for selenium in human serum and/or inter-laboratory comparisons.     

人肝中硒蛋白K相互作用蛋白的筛选与验证

以硒蛋白K(SelK)突变体为"诱饵", 采用酵母双杂交系统对人肝cDNA文库进行筛选, 得到一个与SelK相互作用的蛋白──环腺苷酸应答元件结合蛋白3(CREB3). 将SelK与CREB3共同转染酵母细胞, 验证了SelK与CREB3的相互作用; 并采用受体漂白、敏化发射和荧光寿命3种荧光共振能量转移方法进一步验证了二者间的相互作用, 发现其不受SelK中硒代半胱氨酸(Sec)的影响. 推测SelK可能通过其Sec之前的区域与CREB3发生作用, 参与CREB3介导的内质网相关降解过程, 影响相关癌症的转移和发展.     

Post-transcriptional control of selenoprotein biosynthesis

Selenoproteins are defined as proteins containing the 21st proteinogenic amino acid, selenocysteine (Sec). Sec is encoded by UGA (STOP) codons which are re-coded to Sec by the presence of a selenocysteine insertion sequence (SECIS) element in the 3'-untranslated region of selenoprotein mRNAs. The SECIS element is bound by several proteins, including SECIS-binding protein 2 (SBP2). Translation of selenoproteins critically depends on the integrity of the SECIS element - SBP2 interaction. Mutations in a SECIS element can abrogate expression of the respective selenoprotein. Mutations in SBP2 impinge on biosynthesis of a subset of selenoproteins and lead to a syndrome including hormonal, neurological, immunological symptoms as well as myopathy. Several other RNA-binding proteins are involved in selenoprotein translation and mediate the hierarchical response of selenoproteins to selenium deficiency. Global inhibition of selenoprotein translation is lethal in the mouse and hypomorphic mutations in selenocysteine synthase in humans leads to Progressive Cerebello Cerebral Atrophy, a neurodevelopmental and neurodegenerative disease in pediatric patients.     

硒蛋白的分子生物学及与疾病的关系*

硒蛋白是微量元素硒在体内存在和发挥生物功能的主要形式。因硒蛋白的活性中心硒代半胱氨酸由传统终止码TGA编码,故从基因组中预测硒蛋白以及用基因工程技术表达硒蛋白均很困难。有关硒抗氧化、对癌症、神经退行性疾病和病毒作用的报导较多,但结论并不一致。本文综述了硒蛋白基因预测、蛋白质表达调控以及硒和硒蛋白对癌症、神经退行性疾病和病毒的作用及机制等方面的近期进展,研究提高硒蛋白生物信息学预测准确率和基因工程表达量的方法,分析了解硒蛋白与疾病发生发展的关系和机制,探索不同硒蛋白作为预防药物开发、作为癌症治疗和药物筛选靶标的可能性。     

Loss of selenium from selenoproteins: conversion of selenocysteine to dehydroalanine in vitro

Characterization of reduced and alkylated rat selenoprotein P by mass spectrometry yielded selenopeptides from which one or more selenium atoms were missing. Predicted selenopeptide mass peaks were accompanied by peaks corresponding to the conversion of one or more selenocysteine residues to dehydroalanine(s). Experiments were carried out to determine whether this loss of selenium occurred in vitro. A selenopeptide was isolated that contained two selenocysteine residues that were both in selenide-sulfide linkages with cysteine residues. After the peptide had been reduced and alkylated, in addition to the predicted mass peak with both selenocysteine residues present, two mass peaks were detected at positions expected for conversion of one and two selenocysteine residues of this selenopeptide to dehydroalanine residues, which was confirmed by tandem mass spectrometry. Similar findings were obtained from a study of another selenoprotein, rat plasma glutathione peroxidase. These results indicate that selenium atoms are lost from selenoproteins during purification and characterization. The loss of selenium from selenoproteins is probably through the mechanism of oxidation of selenocysteine residue to selenoxide followed by syn-beta-elimination of selenenic acid during sample processing.