Design of bioelectronic interfaces by exploiting hinge-bending motions in proteins

We report a flexible strategy for transducing ligand-binding events into electrochemical responses for a wide variety of proteins. The method exploits ligand-mediated hinge-bending motions, intrinsic to the bacterial periplasmic binding protein superfamily, to establish allosterically controlled interactions between electrode surfaces and redox-active, Ru(II)-labeled proteins. This approach allows the development of protein-based bioelectronic interfaces that respond to a diverse set of analytes. Families of these interfaces can be generated either by exploiting natural binding diversity within the superfamily or by reengineering the specificity of individual proteins. These proteins may have numerous medical, environmental, and defense applications.     

The MerR metalloregulatory protein binds mercuric ion as a tricoordinate, metal-bridged dimer

Bacterial MerR proteins are dimeric DNA-binding proteins that mediate the Hg(II)-dependent induction of mercury resistance operons. Site-directed mutagenesis of the Bacillus sp. RC607 MerR protein reveals that three of four Cys residues per monomer are required for Hg(II) binding at the single high-affinity binding site. Inactive mutant homodimers can exchange subunits to form heterodimers active for Hg(II) binding. Studies of a heterodimer retaining only three of eight cysteine residues per dimer reveal that Cys79 in one subunit and Cys114 and Cys123 in the second subunit are necessary and sufficient for high-affinity Hg(II) binding in an asymmetric, subunit bridging coordination complex.     

蜡梅金属硫蛋白基因的克隆与原核表达

金属硫蛋白(metallothionein,MT)是一类富含Cys,能够结合重金属的低分子量蛋白质,广泛分布于生物界。在构建好的蜡梅花cDNA文库并进行EST分析的基础上,通过随机克隆测序得到了1个蜡梅金属硫蛋白的cDNA,命名为CpMetallothionein(CpMT)。CpMTcDNA全长为1083bp,基因内部含有一长度为240bp的开放阅读框,可编码79个氨基酸残基。将CpMT插入原核表达载体pET-32a,并转化Origami2感受态细胞。诱导表达产物经SDS-PAGE结果显示,目的蛋白约为30kD。表达的融合蛋白以包涵体和可溶性蛋白2种形式存在,用His-Bind蛋白纯化回收试剂盒对其进行纯化回收,得到了高纯度蛋白,为今后研究奠定基础。     

黑曲霉内切β-木聚糖酶三维结构模建及分子进化分析

内切β-木聚糖酶是一类木聚糖降解酶,对黑曲霉来源的内切β-木聚糖酶核酸序列进行序列比对,构建进化树;采用同源模建方法模建内切β-木聚糖酶的三级结构,并预测内切β-木聚糖酶的二级结构。进化树将内切β-木聚糖酶分成3个组：第1组和第2组的3-D结构相似,而第3组的3-D结构和前2者差别较大。第1组合有1个α-螺旋和13个β-折叠二级结构,二硫键位于Cysf119和Cysm之间;第2组合有1个α-螺旋和13个β-折叠二级结构;第3组合有13个α-螺旋、3个3,10-螺旋和9个β-折叠二级结构,二硫键位于Cys281和cys287之间,而且第3组还含有一个复杂的β-α-β结构域。该研究为黑曲霉内切β-木聚糖酶的结构,功能和蛋白质工程研究提供了一定的理论基础。     

菜粉蝶气味结合蛋白和化学感受蛋白生物信息学分析

气味结合蛋白(odorant-binding proteins,OBPs)和化学感受蛋白(chemosensory proteins,CSPs)在菜粉蝶(Pieris rapae)气味识别的过程中发挥着重要作用。通过本地BLAST等方法在菜粉蝶基因组中找到了推测的OBPs和CSPs蛋白序列,采用多序列比对、半胱氨酸模式序列识别、进化发育树构建等方法,鉴定出了24个PrOBPs和38个PrCSPs基因。其中,PrOBP1-PrOBP17这17个OBPs属于Classic OBPs蛋白家族,PrOBP19-PrOBP24属于Minus-C OBPs蛋白家族。鉴定出的38个PrCSPs都具有鳞翅目CSPs的半胱氨酸模式识别序列。本实验通过对菜粉蝶基因组中OBPs和CSPs基因的查找鉴定,丰富了昆虫OBPs和CSPs基因数据库,可为菜粉蝶气味识别和化学感受相关实验和应用提供参考。     

The Genome sequence of the SARS-associated coronavirus

We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development.     

Regeneration of peroxiredoxins by p53-regulated sestrins, homologs of bacterial AhpD

Acting as a signal, hydrogen peroxide circumvents antioxidant defense by overoxidizing peroxiredoxins (Prxs), the enzymes that metabolize peroxides. We show that sestrins, a family of proteins whose expression is modulated by p53, are required for regeneration of Prxs containing Cys-SO(2)H, thus reestablishing the antioxidant firewall. Sestrins contain a predicted redox-active domain homologous to AhpD, the enzyme catalyzing the reduction of a bacterial Prx, AhpC. Purified Hi95 (sestrin 2) protein supports adenosine triphosphate-dependent reduction of overoxidized PrxI in vitro, indicating that unlike AhpD, which is a disulfide reductase, sestrins are cysteine sulfinyl reductases. As modulators of peroxide signaling and antioxidant defense, sestrins constitute potential therapeutic targets.     

广西猪瘟病毒E2基因克隆及序列分析

[目的]了解广西猪瘟病毒(CSFV)流行毒株的变异规律及其与疫苗株的基因差异,为正确选择猪瘟疫苗和防制广西猪瘟提供参考依据.[方法]以广西本地的阳性猪瘟病料为研究对象,应用RT-PCR扩增CSFV的E2基因,经克隆、测序后用DNASTAR软件对其序列进行比对分析,并绘制遗传进化树.[结果]从41份疑似猪瘟病料中共扩增出4个阳性样品的E2基因(1140 bp),经序列比对分析发现,扩增获得的4株CSFV毒株(GXGG1、GXLC1、GXLC2和GXNN1)与兔化弱毒株(HCLV)、Shimen强毒株的核苷酸同源性为82.1%~82.3%和82.9%~83.4%、其推导氨基酸同源性为88.4%~89.2%和88.9%~90.0%,4株毒株间的E2基因核苷酸及其推导氨基酸同源性分别为90.8%~99.6%和94.2%~99.5%,且均属于基因群Ⅱ;E2基因推导的氨基酸表明,E2蛋白空间结构及抗原结构的氨基酸位点693Cys、737Cys、792Cys、818Cys、828Cys、856Cys、833Pro、834Thr、837Arg均未发生变异,但其单抗识别位点G713E、N729D、K734R发生变异.[结论]近年来广西CSFV流行毒株的变异未出现较大差异.与HCLV株、Shimen强毒株亲缘关系较远,但与Paderborn株、GXWZ02株亲缘关系较近.     

广西巴马小型猪MCP-1基因克隆及序列分析

[目的]研究广西巴马小型猪单核细胞趋化蛋白-1（MCP-1）基因CDs区的序列特点,为建立广西巴马小型猪动脉粥样硬化模型及揭示动脉粥样硬化的发生、发展机理提供理论依据.[方法]从广西巴马小型猪动脉血管组织中扩增MCP-1基因CDs区全长序列,与人类及其他易建立动脉粥样硬化模型动物的序列进行比对分析,并预测其蛋白质信号肽位点及结构域.[结果]广西巴马小型猪MCP-1基因CDs区全长300 bp,编码99个氨基酸;与人类MCP-1基因CDs区（S71513.1）的同源性最高,为84.4％.广西巴马小型猪MCP-1蛋白的前23位氨基酸为信号肽,后76位氨基酸为成熟肽,等电点（pI）9.51,蛋白质分子量10976.04 kDa,信号肽位置有一段跨膜结构;广西巴马小型猪与人类MCP-1蛋白在形成二级结构时,保守半胱氨酸参与形成二硫键的位置一致,分别在第11、12、36和52号（除信号肽片段）位上排列为Cys-Cys,且是第11号位与第36号位形成二硫键,第12号位与第52号位形成二硫键.[结论]以广西巴马小型猪构建动脉粥样硬化模型时,MCP-1基因表达量可作为判断动脉血管疾病的一个辅助指标.     

水稻OsLEA2基因的克隆及表达分析

利用RT-PCR技术从水稻中克隆到OsLEA2的cDNA。序列分析表明,OsLEA2基因的读码框为312 bp,编码一个由103个氨基酸残基组成的蛋白,富含Ala、Lys、Glu、His、Val和Arg,不含Asn、Cys、Phe和Trp。OsLEA2蛋白的1～73位氨基酸残基形成LEA₁结构域。OsLEA2蛋白的二级结构有3个α-螺旋构象区域,没有伸展的β-片层构象,为亲水蛋白。进化树分析表明OsLEA2属于第4组LEA蛋白的4A亚组,OsLEA2与4A亚组LEA蛋白的氨基酸一致性为29%～45%。实时定量RT-PCR分析表明表明OsLEA2基因在水稻的不同生长发育时期的不同组织中都能表达,在完熟期的根和穗中,OsLEA2基因的表达明显升高。     

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses

Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys(358). This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys(358) to Ser(358) oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.     

转录因子AtWRKY35序列分析及原核表达载体的构建

WRKY转录因子是一超级基因家族,数目众多,广泛存在于高等植物中。该研究通过对WRKY基因家族的一员WRKY35进行生物信息学分析,结果发现,在其5-存在一个WRKYGQK核心结构域,一个Cys2His2或Cys2His/Cys锌指型结构,无跨膜结构域;同时通过PCR扩增、酶切、连接等将WRKY35基因片段连接到原核表达载体PET28上,这可为WRKY35基因的蛋白质表达及其后续基因功能鉴定奠定基础。     

大丽轮枝菌分泌蛋白提取方法比较

【目的】采用超滤法（UF）、三氯乙酸沉淀法（TCA）、离子交换富集法（IEX）分别从大丽轮枝菌（Verticillium dahliae）高致病性菌株VdG1培养上清液中提取分泌蛋白,对提取样品进行质谱鉴定。寻找适合大丽轮枝菌分泌蛋白的提取方法,为大丽轮枝菌分泌蛋白组深入研究奠定基础。挖掘大丽轮枝菌分泌蛋白中潜在的致病相关蛋白,为其深入功能验证及致病机理研究提供平台。【方法】3种方法提取大丽轮枝菌VdG1分泌蛋白,shot-gun方法进行质谱鉴定。利用生物信息学软件WoLFPSORT、SignalP、TMHMM、PHOBIUS对其中经典分泌蛋白进行预测。通过CAZy和PHI数据库进行经典分泌蛋白中碳水化合物水解酶（CAZy）和病原菌寄主互作因子（PHI）注释,BLASTp软件进行RxLx、LysM、Cys Pattern等模体蛋白比对分析。提取的蛋白样品接种感病棉种（军棉1号）进行样品生物学活性及致病性检测。【结果】TCA法、IEX法和UF法制备大丽轮枝菌分泌蛋白,其提取效率分别为1.18、0.96和0.56 mg?L-1。SDS-PAGE电泳检测显示UF法提取的蛋白样品条带少而模糊。而TCA法和IEX法提取的蛋白样品条带较多,样品的纯度也较高;经生物信息学软件预测分析,提取的蛋白样品中含有经典分泌蛋白分别为124、112和75个;质谱鉴定的分泌蛋白中含有潜在致病因子分别为98、85和57个;另外,IEX法制备大丽轮枝菌分泌蛋白效果较好,具有小分子偏好性,且能够保持样品的生物学活性,该蛋白样品接种棉花叶片能够引起萎蔫、坏死的症状。【结论】3种方法提取大丽轮枝菌分泌蛋白,UF法提取效率最低,纯度最差,蛋白数量最少,蛋白样品中含有潜在的毒素蛋白数量也最少,而且对于大体积处理相当耗时。TCA法和IEX法都适合大丽轮枝菌分泌蛋白的制备,但是TCA法制备过程中不能保持样品的生物学活性,后续适合于2-D电泳检测等试验。IEX法首次应用于病原菌分泌蛋白的制备,操作简单、方便,全部过程均由仪器系统完成。制备的大丽轮枝菌分泌蛋白具有生物学活性,能够引起棉花叶片的萎蔫、坏死病症。另外,数据库及软件比对分析发现大丽轮枝菌分泌蛋白组中含有CAZy酶类61个、PHI相关蛋白38个、RxLx模体蛋白13个、LysM模体蛋白1个、Cys Pattern的模体蛋白15个、VdNEP蛋白家族2个。该结果将为致病相关蛋白的筛选和深入功能验证提供重要依据。     

蛋白质的 SUMO 化修饰及其在玉米中的研究进展

小泛素化修饰（Small ubiquitin-like modification,SUMOylation）是一种重要的蛋白质翻译后修饰,参与植物多种生命活动。在这个修饰过程中,SUMO 蛋白通过共价键与靶蛋白结合,影响其功能和活性。该修饰过程涉及多种酶类,包括 SUMO 特异性蛋白酶、SUMO 活化酶、SUMO 结合酶和 SUMO 连接酶。植物体内存在多种 SUMO 蛋白和酶类,且其结构和功能存在差异。然而,目前对于不同 SUMO 蛋白与修饰过程中涉及到的酶组合间的作用关系尚未完全明确。因此,厘清这些 SUMO 蛋白及其酶类的特点,对进一步的研究极为重要。此外,SUMOylation 底物鉴定也是研究中的一个挑战。虽然已有一些鉴定 SUMOylation 底物的方法,如质谱分析和酵母双杂交等,但仍存在局限性。特别是在作物中的研究力度更浅,包括玉米。因此,需要开发更多的鉴定方法来识别 SUMOylation 底物,并进一步揭示 SUMOylation 在玉米生长发育和逆境应答中的作用机制。由此,为推动 SUMOylation 研究,综述 SUMO 蛋白、SUMOylation 相关酶类、蛋白质的 SUMOylation 过程、SUMOylation 底物的鉴定方法和玉米蛋白质的 SUMOylation 研究进展。旨在通过了解 SUMOylation 的规律模式和当前的研究技术,为玉米育种和其他植物研究提供重要指导,并有助于相关人员深入理解 SUMOylation 在植物生长发育和逆境胁迫的响应规律。     

一种新型具有GPx和SOD的 含硒抗氧化肽制备

本研究人工设计合成了具有SOD 和GPx 氨基酸一级序列的小肽,将目的蛋白中活性部 位的化学修饰位点设计成Cys,同时将其它位点的Cys 变成不影响结构的其他氨基酸获得人工合 成的76 个氨基酸的抗氧化肽序列,尝试利用E.coli 的营养缺陷型表达系统进行76 肽模拟物的表 达,并对表达的肽活性进行鉴定,预期为抗氧化模拟物的研究提供一些理论参数。     

Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia

Yersinia is the genus of bacteria that is the causative agent in plague or the black death, and on several occasions this organism has killed a significant portion of the world's population. An essential virulence determinant of Yersinia was shown to be a protein tyrosine phosphatase. The recombinant 50-kilodalton Yersinia phosphatase had a specificity for removal of phosphate from Tyr-containing as opposed to Ser/Thr-containing phosphopeptides and proteins. Site-directed mutagenesis was used to show that the Yersinia phosphatase possesses an essential Cys residue required for catalysis. Amino acids surrounding an essential Cys residue are highly conserved, as are other amino acids in the Yersinia and mammalian protein tyrosine phosphatases, suggesting that they use a common catalytic mechanism.     

Pathogenicity determinants in smut fungi revealed by genome comparison

Biotrophic pathogens, such as the related maize pathogenic fungi Ustilago maydis and Sporisorium reilianum, establish an intimate relationship with their hosts by secreting protein effectors. Because secreted effectors interacting with plant proteins should rapidly evolve, we identified variable genomic regions by sequencing the genome of S. reilianum and comparing it with the U. maydis genome. We detected 43 regions of low sequence conservation in otherwise well-conserved syntenic genomes. These regions primarily encode secreted effectors and include previously identified virulence clusters. By deletion analysis in U. maydis, we demonstrate a role in virulence for four previously unknown diversity regions. This highlights the power of comparative genomics of closely related species for identification of virulence determinants.     

Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis

Cryptochromes (CRY) are photolyase-like blue-light receptors that mediate light responses in plants and animals. How plant cryptochromes act in response to blue light is not well understood. We report here the identification and characterization of the Arabidopsis CIB1 (cryptochrome-interacting basic-helix-loop-helix) protein. CIB1 interacts with CRY2 (cryptochrome 2) in a blue light-specific manner in yeast and Arabidopsis cells, and it acts together with additional CIB1-related proteins to promote CRY2-dependent floral initiation. CIB1 binds to G box (CACGTG) in vitro with a higher affinity than its interaction with other E-box elements (CANNTG). However, CIB1 stimulates FT messenger RNA expression, and it interacts with chromatin DNA of the FT gene that possesses various E-box elements except G box. We propose that the blue light-dependent interaction of cryptochrome(s) with CIB1 and CIB1-related proteins represents an early photoreceptor signaling mechanism in plants.     

拟南芥NiNJA基因酵母双杂诱饵载体构建及互作蛋白的筛选

JAZ蛋白是植物茉莉酸信号途径的重要负调控因子,JAZ与NiNJA形成蛋白复合体抑制茉莉酸下游转录因子的转录活性,NiNJA蛋白是联系JAZ蛋白与下游转录因子的重要因子.为了研究NiNJA调控的下游基因,首先构建了NiNJA基因的诱饵载体pGBKT7-NiNJA,然后转化酵母Y2H Gold感受态,通过自转录激活实验,发现诱饵载体pGBKT7-NiNJA没有自转录激活活性.在此基础上,从拟南芥“Mate&PlateTM”Library 进行酵母双杂筛选,获得若干个与NiNJA互作的蛋白,为下一步鉴定NiNJA的互作蛋白及茉莉酸的信号调控途径打下基础.