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。     

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.     

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.     

通过快速定点突变表征人源含硒单链抗体酶的底物结合部位

为了对人源含硒单链抗体酶Se-scFv-B3的底物结合部位和催化基团进行研究, 在理论预测的基础上, 通过快速定点突变法分别在2个理论预测的底物结合部位(位点1和位点2)内选定Ala180和Ala44定点突变为丝氨酸(Ser). 2个突变体蛋白经化学修饰将Ser转变成谷胱甘肽过氧化物酶(GPX)的催化基团硒代半胱氨酸(Sec)后, 前者的GPX活力达到了Se-scFv-B3的2倍多, 而后者的GPX活力没有明显提高, 这表明位点1可能是主要的底物结合部位, 与理论预测的结果一致.     

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.     

硒-甲基硒代半胱氨酸的合成与拆分研究

天然硒-甲基硒代半胱氨酸为L型含硒氨基酸,是第21种人体必须氨基酸———硒代半胱氨酸的甲基化衍生物。1960年首次由Trelease S·F·等从美国黄芪中分离得到[1],它还广泛存在于大蒜、洋葱和椰菜等植物以及富硒酵母中。硒-甲基硒代半胱氨酸具有补硒、防治癌症、抗氧化、抗衰老、     

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...     

以蛋白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模拟物的一些策略和方法,以期望于能够更好的理解硒蛋白的生物学性质,甚至开拓更为有效的技术去模拟这种抗氧化酶.     

微波水解-液相色谱-原子荧光光谱法测定硒蛋白中的硒代氨基酸

针对酶解法测定硒代氨基酸操作复杂,时间长,水解不完全且水解产物不稳定等问题,建立一种微波水解-液相色谱-原子荧光光谱仪测定硒蛋白中硒代氨基酸的方法。样品经HCl(6 mol/L)微波水解后,调节pH值至7.5,采用阴离子色谱柱,pH=6.0的(NH_(4))_(2)HPO_(4)(40 mmol/L)溶液为流动相,流速0.6 mL/min,等度洗脱12 min的条件对硒代胱氨酸(SeCys_(2))、甲基-硒代半胱氨酸(MeSeCys)和硒代蛋氨酸(SeMet)进行分离,用原子荧光光谱仪测定其含量。微波水解条件采用星点设计-响应面法进行优化,优选最佳水解条件为:称样量20 mg,水解温度150℃,水解时间40 min。SeCys_(2)、MeSeCys和SeMet均在0~100 ng浓度范围内呈线性关系,相关系数均大于0.999,检出限分别为0.07、0.03和0.14 mg/kg,RSD分别为5.6%、4.6%、3.7%(n=6)。用来测定硒代氨基酸,耗时短,成本低,水解完全,操作简单,水解产物稳定,能够科学评价硒蛋白中硒代氨基酸的组成,对硒蛋白产品的质量控制和溯源提供了科学方法,值得推广应用。     

AE-HG-AFS测定长期汞暴露人群补硒后尿中硒的形态

建立了一种利用阴离子交换高效液相色谱与氢化物发生原子荧光光谱联用同时测定四、六价硒及硒代半胱氨酸(SeCys)形态的方法。优化了六价硒的还原条件及仪器检测参数,以不同浓度的柠檬酸铵作为流动相,在10 min内同时分离了四、六价硒及硒代半胱氨酸(SeCys)。采用加标法定量,加标回收率在90%～119%之间,相对标准偏差为1.6%～3.1%(100μg/L),四、六价硒及硒代半胱氨酸(SeCys)的检出限分别为0.32μg/L、0.47μg/L和0.44μg/L(进样量为100μL)。应用该法对长期汞暴露人群补硒后尿中的小分子硒的形态进行了分析,仅检测到硒代半胱氨酸(SeCys)。     

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。     

A Facile Method for Producing Selenocysteine‐Containing Proteins

Selenocysteine (Sec, U) confers new chemical properties on proteins. Improved tools are thus required that enable Sec insertion into any desired position of a protein. We report a facile method for synthesizing selenoproteins with multiple Sec residues by expanding the genetic code of Escherichia coli. We recently discovered allo‐tRNAs, tRNA species with unusual structure, that are as efficient serine acceptors as E. coli tRNA^Ser. Ser‐allo‐tRNA was converted into Sec‐allo‐tRNA by Aeromonas salmonicida selenocysteine synthase (SelA). Sec‐allo‐tRNA variants were able to read through five UAG codons in the fdhF mRNA coding for E. coli formate dehydrogenase H, and produced active FDH_H with five Sec residues in E. coli. Engineering of the E. coli selenium metabolism along with mutational changes in allo‐tRNA and SelA improved the yield and purity of recombinant human glutathione peroxidase 1 (to over 80 %). Thus, our allo‐tRNA^UTu system offers a new selenoprotein engineering platform.     

One-Pot Chemical Protein Synthesis Utilizing Fmoc-Masked Selenazolidine to Address the Redox Functionality of Human Selenoprotein F**

Human SELENOF is an endoplasmic reticulum (ER) selenoprotein that contains the redox active motif CXU (C is cysteine and U is selenocysteine), resembling the redox motif of thiol-disulfide oxidoreductases (CXXC). Like other selenoproteins, the challenge in accessing SELENOF has somewhat limited its full biological characterization thus far. Here we present the one-pot chemical synthesis of the thioredoxin-like domain of SELENOF, highlighted by the use of Fmoc-protected selenazolidine, native chemical ligations and deselenization reactions. The redox potential of the CXU motif, together with insulin turbidimetric assay suggested that SELENOF may catalyze the reduction of disulfides in misfolded proteins. Furthermore, we demonstrate that SELENOF is not a protein disulfide isomerase (PDI)-like enzyme, as it did not enhance the folding of the two protein models; bovine pancreatic trypsin inhibitor and hirudin. These studies suggest that SELENOF may be responsible for reducing the non-native disulfide bonds of misfolded glycoproteins as part of the quality control system in the ER.     

Preparation and characterization of nylon 6/Cu2+-exchanged and Fe3+-exchanged montmorillonite nanocomposite

Nylon 6/Cu²⁺-exchanged and Fe³⁺-exchanged montmorillonite nanocomposites have been prepared by a melt intercalation technique directly from Cu²⁺-exchanged and Fe³⁺-exchanged montmorillonite. Hexadecyltrimethylammonium bromide was chosen as the clay/matrix reactive compatibilizer. The intercalation spacing and the degree of dispersion were determined by X-ray diffraction and transmission electron microscopy. Also the thermal character of the nanocomposites prepared was analyzed by thermogravimetric analysis.     

Mass spectrometric determination of selenenylsulfide linkages in rat selenoprotein P

The reversible formation of a selenenylsulfide linkage in mammalian thioredoxin reductase was identified as having a key role in its activity. Identification of selenenylsulfide and/or diselenide linkages is therefore critical to the determination of the structure and function of selenoproteins. A selenopeptide, (298)SGSAITUQCAENLPSLCSUQGLFAEEK(324) (U=selenocysteine), was isolated from a tryptic digest of rat selenoprotein P. Its two cysteine residues and two selenocysteine (Sec) residues were determined to be present in oxidized form by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The selenopeptide was subjected to partial reduction by dithiothreitol with immediate alkylation by iodoacetamide. This process was monitored by MALDI-TOFMS to determine the number of alkylations that had taken place. The partially reduced and alkylated peptides were then analyzed by nano-electrospray ionization tandem mass spectrometry and the results indicated that selenenylsulfide linkages Sec304-Cys314 and Cys306-Sec316 were present. It is concluded that selenoprotein P contains these two selenenylsulfide bonds.     

Studies on the reaction between reduced riboflavin and selenocystine

Selenocysteine (Sec) is a crucial component of mammalian thioredoxin reductase (TrxR) where it serves as a nucleophile for disulfide bond rupture in thioredoxin (Trx). Generation of the reduced state of Sec in TrxR requires consecutive two electron transfer steps, namely: (i) from NADPH to flavin adenine dinucleotide, (ii) from reduced flavin to the disulfide bond Cys⁵⁹‐S‐S‐Cys⁶⁴, and finally (iii) from Cys⁵⁹ and Cys⁶⁴ to the selenosulfide bond Cys⁴⁹⁷‐S‐Se‐Sec⁴⁹⁸. In this work, we studied the reaction between reduced riboflavin (RibH2) and selenocystine (Sec‐Sec), an oxidized form of Sec. The interaction between RibH2 and Sec‐Sec proceeded relatively slowly in comparison with its reverse reaction, that is, reduction of riboflavin (Rib) by Sec. The rate constant for the reaction between RibH2 and Sec‐Sec was (7.9 ± 0.1) × 10^?2 M^?1 s^?1 (pH 7.0, 25.0°C). The reaction between Rib and Sec proceeded via two steps, namely, a rapid reversible binding of Rib to Sec having a protonated selenol group to form a Sec‐Rib complex, followed by nucleophilic attack of Sec‐Rib by a second Sec molecule harboring a deprotonated selenol group. The equilibrium constant for the overall reduction process of Rib by Sec is (1.2 ± 0.1) × 10⁶ M^?1 (25.0°C). The finding that the interaction of RibH2 with oxidized selenol is reversible with its equilibrium favored toward the reverse reaction provides an additional explanation for the exceptional mechanism of the mammalian Trx/TrxR system involving transient reduction of a disulfide bond.     

Gd3+掺杂TiO2纳米复合材料的可见光响应性能

采用溶胶-凝胶技术,在钛酸四丁酯(TBOT)的水解过程中,加入硝酸钆(Gd(NO₃)₃),得到具有可见光响应活性的Gd³⁺/TiO₂复合材料。应用TEM、XRD、TG-DTA和UV-Vis等手段对纳米TiO₂复合材料进行了表征。当Gd³⁺的掺杂量为0.5%时,Gd³⁺/TiO₂复合材料在550 nm附近产生宽强吸收带。Gd³⁺进入TiO₂晶格中,形成了新的掺杂能级(E_g=1.27 eV)。适量Gd³⁺掺杂的纳米TiO₂复合材料的光催化性能优于纯TiO₂粉体材料。     

Selenoproteins: the key factor in selenium essentiality. State of the art analytical techniques for selenoprotein studies

Selenium is an essential element for human health. The benefits of selenium are many including protection against cancer, heart diseases and other cardiovascular and muscle disorders. Selenium is also helpful in controlling gastrointestinal disorders, enhancing immunity of the human body and reducing age-related diseases. The health-promoting properties of Se are due to vital functions of selenoproteins in which selenium is present as selenocysteine, the 21st amino acid. To date, dozens of selenoprotein families have been described though many have roles that have not been fully elucidated. Selenoproteins research has attracted tremendous interest from different scientific areas. Analytical chemists have not remained indifferent to the attractive features of these unique proteins. Different analytical techniques, such as multidimensional chromatography–inductively coupled plasma mass spectrometry (ICPMS), electrospray (tandem) mass spectrometry (ESI-MS/MS), matrix-assisted laser desorption ionization time-of flight (MALDI-TOF) and sodium dodecyl sulphate polyacrylamide gel electrophoresis–laser ablation inductively coupled plasma mass spectrometry (SDS-PAGE-LA-ICPMS), have been applied to the determination of selenoproteins and selenium-containing proteins. This review describes the best-characterized selenoproteins to date in addition to the major contributions of analytical chemistry to the field of selenoproteins. The article also highlights the challenges of combining elemental and molecular mass spectrometry for the determination of selenoproteins and selenium-containing proteins.     

Structural characterization of C-S-H and C-A-S-H samples—Part II: Local environment investigated by spectroscopic analyses

Spectroscopic studies (¹H, ²³Na and ²⁷Al MAS NMR and Raman spectroscopy) have been used to characterize three series of C-S-H samples (0.8<Ca/Si<1.7): one C-S-H series, one aluminum inserted C-S-H series (named C-A-S-H series), and one sodium and aluminum inserted C-S-H series (named C-N-A-S-H series). Previous Rietveld analyses have been performed on the two first series and have clearly shown that (1) a unique ‘tobermorite M defect’ structural model allows to describe the C-S-H structure whatever the Ca/Si ratio and (2) the insertion of aluminum into the C-S-H structure led to the degradation of the crystallinity and to a systematic increase of the basal spacing of about 2 Å regardless the Ca/(Si+Al) ratio (at a constant Al/Si ratio of 0.1). Spectroscopic investigations indicate that the main part of the Al atoms is readily incorporated into the interlayer region of the C-S-H structure. Al atoms are mainly inserted as four-fold coordinated aluminates in the dreierketten silicate chain (either in bridging or paired tetrahedra) at low Ca/Si ratio. Four-fold aluminates are progressively replaced by six-fold coordinated aluminates located into the interlayer region of the C-S-H structure and bonded to silicate chains. Investigation of the hydrogen bonding in C-S-H indicates that the main part of the hydrogen bonds is intra-main layer, and thus explains the low stacking cohesion of the C-S-H structure leading to its nanometric crystal size and the OD character of the tobermorite like structures.     

A note on the electronic structure of O 2 −

Summary The electronic spectrum of O ₂ ^– is reinvestigated using CASSCF and CI methods. In particular, a previously noted curious flattening of theA ² _u curve has been studied in detail. The present analysis disagrees with the previous one where this flattening was found to be a result of an avoided curve crossing between a valence and a Rydberg state of O ₂ ^– . A simple procedure is suggested to determine whether a wavefunction is of real Rydberg character or if the bound character of the state is just an artefact of the calculation.