含14-3-3蛋白抑制肽R18重组腺病毒制备及其在心肌细胞中的表达

目的:构建并鉴定含14-3-3蛋白抑制肽R18的重组腺病毒,为研究14-3-3蛋白的功能提供基础工具。方法:用同源重组方法构建含14-3-3蛋白抑制肽R18的复制缺陷型腺病毒载体(AdR18),并加以鉴定、扩增,以获得高滴度AdR18病毒液,体外感染乳大鼠心肌细胞,检测目的基因表达。结果:将构建的重组腺病毒载体AdR18感染乳大鼠心肌细胞并表达48h后,蛋白印迹结果显示AdR18感染组有明显R18的表达,对照组无表达。结论:腺病毒载体可高效率导入外源基因在心肌细胞中高表达。     

木薯14-3-3蛋白家族成员MeGRF3基因的原核表达及多克隆抗体制备

14-3-3蛋白是植物体内重要的信号转导调节分子,在碳代谢、逆境胁迫响应、生长发育等过程中发挥重要调控作用。为了深入解析14-3-3蛋白家族在木薯中的生物学功能,该研究采用酶切连接方法构建木薯14-3-3蛋白家族成员MeGRF3的原核表达载体,用热激法转化大肠杆菌Rosetta(DE3)株系,诱导表达带有MeGRF3的融合蛋白;使用纯化后的MeGRF3融合蛋白免疫新西兰公兔制备多克隆抗体,并检测抗体的效价和特异性。结果显示:(1)成功构建了木薯MeGRF3的原核表达载体pET-30a-MeGRF3,并诱导表达得到带有MeGRF3和6个His标签的融合蛋白。(2)MeGRF3融合蛋白主要以包涵体的形式存在,相对分子质量约为33 kD。(3)间接酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)测定的抗体效价为1024000。(4)Western blot分析显示,木薯叶片、茎尖、茎皮和块根中均检测到与MeGRF3蛋白大小一致的条带,说明MeGRF3在这4个组织器官中均表达,但主要是在茎尖和块根中大量积累,表明该研究成功制备的MeGRF3多克隆抗体的特异性较好。     

人GST-14-3-3σ融合蛋白的原核表达、纯化及活性检测

目的:构建人14-3-3σ基因的原核表达载体,获得其原核表达产物,并对融合蛋白进行纯化及活性检测。方法:采用PCR技术从人乳腺文库中扩增14-3-3σ基因编码序列,将其克隆到pGEX-KG载体中,重组质粒转化大肠杆菌Rossate后表达重组蛋白,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,并通过SDS-PAGE和Western印迹检测融合蛋白的表达,采用GST pull-down技术检测已纯化的蛋白与已知体外相互作用蛋白AKT之间的相互作用。结果:从人乳腺文库中扩增获得约750 bp的DNA片段,并成功克隆至pGEX-KG载体上,经双酶切鉴定得到与预期片段大小相符的外源基因插入片段,测序与目的基因序列完全一致;在Rossate菌株中诱导表达出相对分子质量约52 000的目的蛋白,SDS-PAGE和Western印迹结果表明融合蛋白表达成功,并纯化得到GST-14-3-3σ融合蛋白;通过GST pull-down技术检测证实GST-14-3-3σ融合蛋白可以和AKT在体外结合,并证实其具有生物学活性。结论:获得了原核表达的活性较好的GST-14-3-3σ蛋白,为后续研究细胞周期蛋白调控机制奠定了实验基础。     

14-3-3蛋白研究进展

14-3-3蛋白是高度保守的、所有真核生物细胞中都普遍存在的、在大多数生物物种中由一个基因家族编码的一类蛋白调控家族。它几乎参与生命体所有的生理反应过程,人们在各种组织细胞中发现了各种不同的14-3-3蛋白。作为与磷酸丝氨酸／苏氨酸结合的第一信号分子,14-3-3蛋白在细胞的信号转导中起着至关重要的作用,尤其是它直接参与调节蛋白激酶和蛋白磷酸化酶的活性,被称为蛋白质与蛋白质相互作用的”桥梁蛋白”;它可以与转录因子结合形成复合体,调节相关基因的表达。一些研究表明,14-3-3蛋白调控机制的紊乱可以直接导致疾病的发生,在临床上14-3-3蛋白常常可以作为诊断的标志物。     

14-3-3蛋白在细胞周期调节中的作用

14-3-3是一个在真核细胞中广泛表达、功能复杂的蛋白家族,主要通过磷酸化依赖的方式与靶蛋白结合,从而发挥其调控作用。细胞周期的调节对维持基因组的稳定性至关重要。近年来的研究发现,14-3—3蛋白可以和越来越多的细胞周期调节蛋白相互作用,调节G2／M期和G1／S期转换,从而对细胞周期起调控作用。简要综述了14—3—3蛋白在细胞周期调节中的作用。     

菠菜14-3-3蛋白基因的克隆及硝酸盐胁迫下的表达分析

利用RT-PCR和RACE技术,从菠菜中首次获得了14-3-3蛋白基因的全长cDNA序列(GenBank登录号JX952165),命名为So14-3-3.该基因全长1 166 bp,开放阅读框801 bp,编码266个氨基酸.序列比对发现So143 3蛋白与其他植物14-3-3蛋白氨基酸序列一致性高达77.6％～84.7％.半定量RT-PCR表明,随NO3-胁迫处理时间的延长和浓度的增加,菠菜根和叶中So14-3-3基因的表达增强.实验构建了pGEX4T-So14-3-3原核表达载体,并通过IPTG诱导后获得分子量约为56 kD的蛋白.进一步的蛋白质印迹检测结果表明,随着NO3处理时间的延长和浓度的增加,So14-3-3蛋白表达也增加.该实验结果为进一步研究So14 3-3蛋白功能提供了基本的实验基础.     

14-3-3θ基因过表达载体的构建及其过表达乳腺癌细胞株的建立

目的:为进一步研究14-3-3θ基因对乳腺癌生物学行为的影响,构建14-3-3θ基因真核细胞过表达重组载体并建立其过表达的乳腺癌细胞株。方法:合成的14-3-3θ基因重组在p ENTR 3C DUAL载体中,课题组采用DNA重组技术将14-3-3θ基因剪切分离后,插入真核表达质粒载体pc DNA4.0中,重组得到真核过表达载体pc DNA4.0-14-3-3θ,并用其转染293T细胞后用western blot进行验证,将经过验证的真核过表达载体pc DNA4.0-14-3-3θ转染乳腺癌细胞后用zeocin筛选,筛选出的乳腺癌细胞经过western-blot和PCR验证确为稳定过表达14-3-3θ的乳腺癌细胞。结果:成功构建14-3-3θ基因过表达真核载体并建立其过表达乳腺癌细胞株,揭示14-3-3θ基因可能在乳腺癌的早期诊断和预后预测中具有重要作用。     

ADAM22蛋白与14-3-3β蛋白之间的相互作用

ADAM家族是一类具有去整合域和金属蛋白酶域的跨膜蛋白, 广泛参与各种重要的生理过程, 如精卵结合、神经系统发育、成肌细胞融合以及炎症反应等. 该家族蛋白具备潜在的粘连和蛋白酶活性. adam22在脑部高度表达, 基因剔除小鼠则出现严重的共济失调, 并在断奶前死亡, 但其作用机制不详. 用酵母双杂合系统筛选到与ADAM22相互作用的蛋白质14-3-3b, 离体结合实验、免疫共沉淀进一步证实了ADAM22与14-3-3β蛋白相互作用的专一性. ADAM22胞内部分系列缺失实验表明, C端第864~892氨基酸残基是14-3-3β的结合基序. 14-3-3β在脑部大量表达, 具有介导细胞的扩散、迁移及调节细胞周期等重要功能. 因此, 本实验证实这两种蛋白质之间存在相互作用,为阐明ADAM22在神经系统发育中的功能奠定了基础.     

植物中14-3-3蛋白的主要功能

14-3-3蛋白家族广泛存在于真核生物中,序列高度保守。主要以同源或异源二聚体形式存在,可以同时与两个靶蛋白或者与一个靶蛋白的两个结构域相互作用,通过与靶蛋白上的一小段共有序列的磷酸化丝氨酸/苏氨酸残基结合来发挥其调控功能。本文综述了植物中的14-3-3蛋白及其主要功能,并重点综述了14-3-3蛋白对植物基本碳、氮代谢的调控。     

人14-3-3蛋白家族的生物信息学分析

目的:分析人14-3-3蛋白家族的同源性及分子进化关系。方法:利用已公布的人基因组数据库,采用BLASTN程序检索人14-3-3蛋白家族各成员的编码基因和假基因,并利用DNAMAN软件进行序列联配,绘制其分子进化树。结果:该家族半数成员具有多个假基因序列,为返转座类型假基因。进化分析表明该家族有共同的祖先,可归为3个亚类。结论:人14-3-3蛋白家族每个成员长期进化所形成的多样性提示其功能具有独特性。     

Regulation of Dyrk1A kinase activity by 14-3-3

Dual-specificity tyrosine(Y) regulated kinase 1A (DYRK1A) is a serine/threonine protein kinase implicated in mental retardation resulting from Down syndrome. In this study, we carried out yeast two-hybrid screening to find proteins regulating DYRK1A kinase activity. We identified 14-3-3 as a Dyrk1A interacting protein, which is consistent with the previous finding of the interaction between the yeast orthologues Yak1p and Bmh1/2p. We showed the interaction between Dyrk1A and 14-3-3 in vitro and in vivo. The binding required the N-terminus of Dyrk1A and was independent of the Dyrk1A phosphorylation status. Functionally, 14-3-3 binding increased Dyrk1A kinase activity in a dose dependent manner in vitro. In vivo, a small peptide inhibiting 14-3-3 binding, sc138, decreased Dyrk1A kinase activity in COS7. In summary, these results suggest that DYRK1A kinase activity could be regulated by the interaction of 14-3-3.     

PRAS40与14-3-3蛋白各亚型相互作用的酵母双杂交系统检测

PRAS40是近几年新发现的Akt作用底物,14-3-3结合蛋白。为确定PRAS40与14-3-3蛋白7种亚基间相互作用关系,利用gateway方法构建用于酵母双杂交系统的诱饵质粒pEG-PRAS40及转录激活质粒pJG-PRAS40,将PRAS40和14-3-3各亚型质粒分别作为诱饵蛋白质粒及转录激活质粒共转化酵母细胞EGY48,通过氨基酸营养缺陷生长实验及β-半乳糖苷酶显色反应分析两种蛋白相互作用程度。酶切鉴定证实成功地构建了pEG-PRAS40和pJG-PRAS40质粒,酵母双杂交实验结果显示PRAS40可以和14-3-3亚型tau,beta,zeta及epsilon相结合,epsilon较强,beta和zeta次之,tau较弱。此结果将为深入研究PRAS40与14-3-3蛋白生物学功能及发现药物靶标奠定基础。     

The role of 14-3-3 proteins in cell signalling pathways and virus infection

14-3-3 proteins are highly conserved in species ranging from yeast to mammals and regulate numerous signalling pathways via direct interactions with proteins carrying phosphorylated 14-3-3–binding motifs. Recent studies have shown that 14-3-3 proteins can also play a role in viral infections. This review summarizes the biological functions of 14-3-3 proteins in protein trafficking, cell-cycle control, apoptosis, autophagy and other cell signal transduction pathways, as well as the associated mechanisms. Recent findings regarding the role of 14-3-3 proteins in viral infection and innate immunity are also reviewed.     

14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如:新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

Interaction of 14-3-3 proteins with the insulin-like growth factor I receptor (IGFIR): evidence for a role of 14-3-3 proteins in IGFIR signaling

We have extended our previous yeast two-hybrid findings to show that 14-3-3beta also interacts with the insulin-like growth factor I receptor (IGFIR) in mammalian cells overexpressing both proteins and that the interaction involves serine 1283 and is dependent on receptor activation. Treatment of cells with the phorbol ester PMA stimulates the interaction of 14-3-3beta with the IGFIR in the absence of receptor tyrosine phosphorylation, suggesting that receptor activation leads to activation of an endogenous protein kinase that catalyzes the phosphorylation of serine 1283. To investigate the role of 14-3-3 proteins in IGF signal transduction, IGFIR structure-function studies were performed. Mutation of serine 1283 alone (S1283A) (a mutation that decreases but does not abolish the interaction of the IGFIR with 14-3-3) did not affect anchorage-independent growth of NIH 3T3 fibroblasts overexpressing the mutant receptor. However, the simultaneous mutation of this residue and the truncation of the C-terminal 27 residues of the receptor (Delta1310/S1283A) abolished the interaction of the receptor with 14-3-3 and reversed the enhanced colony formation observed with the IGFIR truncation mutation alone (Delta1310). The difference between the Delta1310 and Delta1310/S1283A transfectants in the soft agar assay was confirmed by tumorigenesis experiments. These findings suggest that 14-3-3 proteins interact with the IGFIR in vivo and that this interaction may play a role in a transformation pathway signaled by the IGFIR.     

14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK)

Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC₅₀=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants.     

Interaction between ACC synthase 1 and 14-3-3 proteins in rice: a new insight

In this study, the interaction between rice 14-3-3 protein and 1-aminocyclopropane carboxylic acid synthase (ACS) was observed in yeast cells using yeast two-hybrid assays. Given the fact that 14-3-3 proteins generally bind to their target proteins in a phosphorylation-dependent manner, a hypothesis regarding the regulatory role of 14-3-3 proteins in the activation of ACS is proposed in which 14-3-3 proteins may bind to the phosphorylated C-terminal tails of ACSs and help them to escape from their fated degradation when ethylene biosynthesis is needed. It is reasonable to believe that 14-3-3 protein may play an important role in regulating ACS activity. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 9, pp. 1231–1237.