Ppp5c 和TTC16 基因的TPR 结构域与Hsp70、Hsp90 蛋白 的相互作用及对细胞周期的影响

目的:研究Ppp5c及TTC16基因的TPR(tetratricopeptide repeat)结构域短片段和Hsp70及Hsp90家族蛋白的相互做用,及其过表达对细胞周期的影响。方法:通过生物信息学的分析及PCR的方法,克隆Ppp5c及TTC16基因的TPR结构域以及HSPA1A、HSP90AA1的全长基因,并连入酵母双杂交载体,通过ClonTech的酵母双杂交实验体系研究蛋白和蛋白之间的相互作用。把Ppp5c及TTC16基因的TPR结构域克隆入真核表达载体,构架稳定表达Ppp5c及TTC16基因的TPR结构域的MCF-7细胞系,并通过流式细胞实验观察细胞周期。结果:Ppp5c及TTC16基因的TPR结构域能与HSPA1A或HSP90AA1发生相互作用。Ppp5c及TTC16基因的TPR结构域在MCF-7中的过表达能严重影响细胞周期,引起细胞凋亡和S期阻滞。结论:本实验初步揭示了不同蛋白的TPR结构域在与Hsp70及Hsp90蛋白的相互作用性质的异同点以及其过表达对细胞周期的影响,为全面理解TPR结构域的功能、Ppp5c以及TTC16蛋白在细胞内的功能奠定了前期实验基础。     

酵母双杂交筛选心脏cDNA文库中与人热激蛋白70相互作用的蛋白质

目的:利用酵母双杂交系统从人心肌cDNA文库中筛选与热激蛋白70(HSP70)相互作用的蛋白质。方法:从人心脏cDNA文库扩增Hsp70基因,克隆于pGBKT7载体上,酶切鉴定及序列分析,并检测pGBKT7-Hsp70酵母细胞AH109中的自激活活性;将构建的酵母表达诱饵质粒载体pGBKT7-Hsp70转化AH109酵母细胞,与转化有人心脏cDNA文库的酵母Yl87进行交配实验,筛选与HSP70相互作用的蛋白质,通过一对一的回复杂交实验排除假阳性,对阳性克隆进行序列测定和生物信息学分析。结果:构建了"诱饵"质粒栽体pGBKT7-Hsp70,并证明其在酵母双杂交系统中无自激活活性,筛选得到多个与Hsp70相互作用的阳性转化子,并最终得到HSP70的1个相互作用蛋白质HIP。结论:应用酵母双杂交系统筛选出与HSP70相互作用的1个蛋白质,它们的相互作用可能与HSP70发挥细胞分子伴侣作用有关。     

Tec家族蛋白酪氨酸激酶Itk PH结构域与OS-9蛋白的相互作用

用酵母双杂交技术筛选与ItkPH结构域相互作用的蛋白分子 ,以了解Itk的功能及其在T细胞信号转导中的位置与作用 .Itk的PH结构域扩增后克隆入酵母双杂交系统的pLexA载体 ,转化酵母细胞EGY4 8(p8op lacZ) ,经检测PH结构域无自激活作用 ,且对酵母细胞无毒性作用 .用PH结构域作为“钓饵”蛋白 ,在酵母双杂交系统中筛选构建于AD载体的T细胞cDNA文库 .将PH结构域及筛库所得基因片段分别进行融合表达 ,用于体外结合实验 ,进一步证实二者的相互作用 .经营养缺陷选择、诱导筛选和鉴定确证 ,筛库所得的插段约 15 0 0bp的文库质粒为一真阳性克隆 .经blast比较分析为骨肉瘤、横纹肌肉瘤等肿瘤组织中高表达的os 9基因 .体外结合实验也表明 ,ItkPH结构域可与该基因表达产物结合 .Itk的PH结构域可与OS 9蛋白相互作用 .二者结合的意义有待进一步研究     

神经病靶标酯酶调控结构域结合蛋白筛选及其在COS-7细胞中的表达

本研究采用酵母双杂交系统探寻与神经病靶标酯酶（NTE）调控结构域相互作用的蛋白因子,揭示与NTE信号转导相关的可能机制。通过构建含有NTE调控结构域的诱饵蛋白载体筛选胎脑文库,并将筛选得到的阳性克隆在酵母中进行了验证,随后在哺乳动物细胞中表达了该蛋白。生物信息学分析显示：该阳性克隆为前列腺素受体结合蛋白54（ARA54）,具有泛素连接酶活性,提示细胞可能存在依赖于细胞周期的NTE活性调节机制,为阐明NTE生理功能创造了条件[动物学报51（5）：840—844,2005]。     

HSP90高表达细胞株的建立及细胞增殖性状的改变

目的:建立稳定高表达热休克蛋白90(HSP90)细胞株,研究其对细胞增殖的影响.方法:含人HSP90 13全长基因的重组质粒pSmycHSP经亚克隆、纯化、酶切鉴定后,用电穿孔法转染到小鼠成纤维细胞系NIH-3T3细胞内.经G418筛选、克隆分离培养,用免疫细胞化学、免疫印迹鉴定阳性克隆.以转染空质粒的NIH-3T3细胞为对照,用MTT法、流式细胞术测定,分析HSP90高表达对细胞增殖和细胞周期的影响.结果:转染pSmycHSP的NIH-3T3细胞HSP90染色增强,生长速度减慢,S期DNA含量降低.结论:己建立稳定高表达热休克蛋白90(HSP90)NIH-3T3细胞株;转染pSmycHSP的NIH-3T3细胞能够有效地表达HSP90,影响细胞周期,使细胞增殖迟滞.     

Prelamin A相互作用蛋白Narf的鉴定和表达分析

目的:探讨A型核纤层蛋白前体( prelamin A)在细胞内堆积造成细胞早老的机理,筛选了prelamin A相互作用蛋白并研究其在早老细胞中的表达情况.方法:以prelamin A的C末端区域为诱饵蛋白,采用酵母双杂交方法从人骨骼肌cDNA文库中筛选prelamin A相互作用蛋白.构建了prelaminA识别因子(Narf)与绿色荧光蛋白融合表达载体pEGFP - Narf,与红色荧光蛋白- prelamin A融合表达质粒pDsRed - PLA共转染HEK293细胞,激光共聚焦显微观察共定位情况.Western blotting检测Narf在衰老表型HEK293PLA细胞的表达情况.结果:筛选得到包括Narf在内的7个候选相互作用蛋白.Narf与prelamin A能相互作用并共定位于核纤层,在prelamin A过表达的HEK293PLA细胞中Narf表达没有升高.结论:Narf在细胞内与prelamin A相互作用,且表达量不受后者影响.     

用酵母双杂交体系确定病毒特异性蛋白与宿主蛋白的相互作用

用B型流行性感冒病毒BM2蛋白开放阅读框架片段,构建酵母双杂交体系中的"饵”载体,明确其在酵母细胞中的表达,排除自身激活作用,并用酵母双杂交法从人cDNA基因库中筛选出与BM2蛋白相互作用的蛋白.结果表明B型流感病毒BM2蛋白与宿主细胞的某一蛋白有相互作用.应用酵母双杂交体系,可以非常有效地从人cDNA基因库中确定与"饵”蛋白相互作用的蛋白基因.     

SPARC基因过表达对卵巢癌淋巴结高转移细胞生物学特性的影响

该研究旨在探讨富含半胱氨酸的酸性分泌蛋白基因(secreted protein acidic and rich in cysteine gene,SPARC)过表达对卵巢癌淋巴结高转移细胞(SKOV3-PM4)生物学特性的影响。构建SPARC基因的慢病毒表达载体并转染SKOV3-PM4细胞,Real-time PCR和Western blot验证转染后的表达效率,激光共聚焦免疫荧光进行蛋白的细胞定位,细胞计数法和集落形成实验测定细胞增殖能力,流式细胞仪检测细胞周期,Transwell小室实验测定细胞体外侵袭、迁移能力。实验结果显示,SPARC蛋白存在于核周及胞质;过表达SPARC基因后,SKOV3-PM4细胞增殖受到明显抑制(P0.05);细胞周期检测结果显示,各期改变无明显差异;体外侵袭、迁移实验结果显示,SKOV3-PM4细胞侵袭、迁移能力显著降低(P0.05)。实验结果表明,SPARC基因在卵巢癌淋巴结转移中可能发挥抑癌基因的生物学作用。     

早幼粒细胞白血病基因结构域诱饵载体的构建及鉴定

目的研究早幼粒细胞白血病基因（promyelocytic leukemia,PML）中含环指／B—BOX结构与含coiled—coil结构的两个结构域的功能,构建含其结构域序列的诱饵表达载体,为进一步应用酵母双杂交系统筛选与之相互作用的蛋白建立实验基础。方法PCR扩增PML的两个结构域序列,克隆人诱饵载体pG—BKT7中,经测序鉴定后,将诱饵载体转化到酵母细胞AH109中,检测诱饵蛋白有无毒性,渗漏和自激活作用,同时利用蛋白印迹法分析诱饵蛋白的表达。结果成功扩增了PML两个结构域的基因片段,并正确克隆入pGBKT7中。诱饵载体成功转化到酵母细胞AH109中,其中一个诱饵蛋白BD—PML—B无毒性,但具有渗漏和自激活作用,另一个诱饵蛋白BD—PML—C无毒性,渗漏和自激活作用,蛋白印迹法分析证实酵母细胞表达诱饵蛋白。结论含环指／B—BOX的结构域具有转录因子活性,全长PML的转录活性与之有关;成功构建了含coiled—coil结构的PML结合域的酵母诱饵表达载体,为运用酵母双杂交技术筛选与之作用的蛋白并探讨其功能奠定了基础。     

肠道病毒71型3D聚合酶转录激活域的界定

EV71是导致手足口病的主要病原体之一,其3D聚合酶作为依赖于RNA的RNA聚合酶,在病毒基因组转录及复制过程中发挥重要作用。当前,3D与宿主细胞的相互作用鲜有研究。酵母双杂交实验是研究蛋白质相互作用成熟有效的方法。本文将3D基因克隆至pGBKT7载体,构建了用于酵母双杂交实验的诱饵质粒,鉴定正确后转化酵母细胞AH109,依次检测了3D蛋白的表达、细胞毒性和自激活能力,结果表明3D基因可在AH109菌株中表达,对后者生长无显著影响,但融合蛋白具有自激活能力。进一步构建一系列含3D蛋白截短体的诱饵质粒,通过自激活实验界定了3D的最小转录激活域(1~94aa),为后续利用酵母双杂交技术研究与3D相互作用的细胞蛋白奠定了基础。     

Expression of the small heat shock protein gene, hsp30, in Rana catesbeiana fibroblasts

In the present study, we examined the expression of the Rana catesbeiana small heat shock protein gene, hsp30, in an FT fibroblast cell line. Northern and western blot analyses revealed that hsp30 mRNA or HSP30 protein was not present constitutively but was strongly induced at a heat shock temperature of 35 degrees C. However, treatment of FT cells with sodium arsenite at concentrations that induced hsp gene expression in other amphibian systems caused cell death. Non-lethal concentrations of sodium arsenite (10 microM) induced only minimal accumulation of hsp30 mRNA or protein after 12 h. Immunocytochemical analyses employing laser scanning confocal microscopy detected the presence of heat-inducible HSP30, in a granular or punctate pattern. HSP30 was enriched in the nucleus with more diffuse localization in the cytoplasm. The nuclear localization of HSP30 was more prominent with continuous heat shock. These heat treatments did not alter FT cell shape or disrupt actin cytoskeletal organization. Also, HSP30 did not co-localize with the actin cytoskeleton.     

Downregulation of ERK2 is essential for the inhibition of radiation-induced cell death in HSP25 overexpressed L929 cells

We previously reported that overexpression of HSP25 delayed cell growth, increased the level of p21(waf), reduced the levels of cyclin D1, cyclin A and cdc2, and induced radioresistance in L929 cells. In this study, we demonstrated that HSP25 induced-radioresistance was abolished by transfection with plasmids containing antisense hsp25 cDNA. Extracellular regulated kinase (ERK) and MAP kinase/ERK kinase (MEK) expressions as well as their activation (phospho-forms) were inhibited by hsp25 overexpression. Furthermore, when control vector transfected cells were treated with PD98059, MEK inhibitor, they became resistant to radiation, suggesting that inhibition of ERK1/2 activities was essential for radioresistance in L929 cells. To confirm the relationship between ERK1/2 and hsp25-mediated radioresistance, ERK1 or ERK2 cDNA was transiently transfected into the hsp25 overexpressed cells and their radioresistance was examined. HSP25-mediated radioresistance was abolished by overexpression of ERK2, but not by overexpression of ERK1. Alteration of cell cycle distribution and cell cycle related protein expressions (cyclin D, cyclin A and cdc2) by hsp25 overexpression were also recovered by ERK2 cDNA transfection. Increase in Bcl-2 protein by hsp25 gene transfection was also reduced by subsequent ERK2 cDNA-transfection. Taken together, these results suggest that downregulation of ERK2 is essential for the inhibition of radiation-induced cell death in HSP25 overexpressed cells.     

Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase-3 activation

Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.     

Examination of KNK437- and quercetin-mediated inhibition of heat shock-induced heat shock protein gene expression in Xenopus laevis cultured cells

We examined the effect of quercetin (3,3',4',5,7-pentahydroxyflavon) and KNK437 (N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolactam), a benzylidene lactam compound, on heat-induced heat shock protein (hsp) gene expression in Xenopus laevis A6 kidney epithelial cells. In previous studies, both quercetin and KNK437 inhibited heat shock factor activity resulting in a repression of hsp mRNA and protein accumulation in human cultured cells. In this first study of the effect of these hsp gene expression inhibitors in a non-mammalian cell line, we report that both quercetin and KNK437 reduced the heat shock-induced accumulation of hsp30, hsp47 and hsp70 mRNA in X. laevis cultured cells. However, these inhibitors had no effect on the relative level of a non-heat shock protein mRNA, ef1alpha, in either control or heat shocked cells. Western blot and immunocytochemical analyses revealed that quercetin partially inhibited HSP30 protein accumulation. In contrast, HSP30 protein was not detectable in KNK437-treated cells. Finally, treatment of A6 cells with KNK437 inhibited the heat shock-induced acquisition of thermotolerance, as determined by preservation of actin filaments and cellular morphology using immunocytochemistry and laser scanning confocal microscopy.     

Characterization of a heat-shock-inducible hsp70 gene of the green alga Volvox carteri

The green alga Volvox carteri possesses several thousand cells, but just two cell types: large reproductive cells called gonidia, and small, biflagellate somatic cells. Gonidia are derived from large precursor cells that are created during embryogenesis by asymmetric cell divisions. The J domain protein GlsA (Gonidialess A) is required for these asymmetric divisions and is believed to function with an Hsp70 partner. As a first step toward identifying this partner, we cloned and characterized V. carteri hsp70A, which is orthologous to HSP70A of the related alga Chlamydomonas reinhardtii. Like HSP70A, V. carteri hsp70A contains multiple heat shock elements (HSEs) and is highly inducible by heat shock. Consistent with these properties, Volvox transformants that harbor a glsA antisense transgene that is driven by an hsp70A promoter fragment express Gls phenotypes that are temperature-dependent. hsp70A appears to be the only gene in the genome that encodes a cytoplasmic Hsp70, so we conclude that Hsp70A is clearly the best candidate to be the chaperone that participates with GlsA in asymmetric cell division.     

The interaction between RPAP3 and TRBP reveals a possible involvement of the HSP90/R2TP chaperone complex in the regulation of miRNA activity

MicroRNAs silence mRNAs by guiding the RISC complex. RISC assembly occurs following cleavage of pre-miRNAs by Dicer, assisted by TRBP or PACT, and the transfer of miRNAs to AGO proteins. The R2TP complex is an HSP90 co-chaperone involved in the assembly of ribonucleoprotein particles. Here, we show that the R2TP component RPAP3 binds TRBP but not PACT. The RPAP3-TPR1 domain interacts with the TRBP-dsRBD3, and the 1.5 Å resolution crystal structure of this complex identifies key residues involved in the interaction. Remarkably, binding of TRBP to RPAP3 or Dicer is mutually exclusive. Additionally, we found that AGO(1/2), TRBP and Dicer are all sensitive to HSP90 inhibition, and that TRBP sensitivity is increased in the absence of RPAP3. Finally, RPAP3 seems to impede miRNA activity, raising the possibility that the R2TP chaperone might sequester TRBP to regulate the miRNA pathway.     

BTI基因转染诱导EC9706细胞凋亡及G_1阻滞

为了研究荞麦胰蛋白酶抑制剂(buckwheat trypsin inhibitor,BTI)对肿瘤细胞凋亡与细胞周期的影响,构建增强型绿色荧光蛋白(EGFP)与BTI融合蛋白真核表达质粒.将BTI基因成功克隆至pEGFP-N1中转染食管癌EC9706细胞后,激光共聚焦显微镜镜检显示,BTI-EGFP获得良好表达.表达的融合蛋白大部分分布于细胞核,在细胞质中有少量分布.Western印迹检测可见约27kD和36 kD的特异性条带.流式细胞术分析结果显示,BTI能够诱导EC9706细胞发生凋亡,并使细胞停滞于G0/G1期.     

Small heat-shock protein Hsp9 has dual functions in stress adaptation and stress-induced G2-M checkpoint regulation via Cdc25 inactivation in Schizosaccharomyces pombe

The small heat-shock protein Hsp9 from Schizosaccharomyces pombe was previously reported to be a homologue of Saccharomyces cerevisiae HSP12. Although Hsp9 is expressed in response to heat shock and nutritional limitation, its function is still not completely understood. Here, we explored the biological function of Hsp9 in S. pombe. The hsp9 gene might play a role in stress adaptation; hsp9 deletion caused heat sensitivity and overexpression induced heat tolerance. In addition, Hsp9 also contribute to cell cycle regulation in the nucleus. Δhsp9 cells grew more quickly and were shorter in length than wild-type cells. Moreover, Δhsp9 cells did not achieve checkpoint arrest under stress conditions, leading to cell death, and exhibited a short doubling time and short G2 phase. Overexpression of hsp9 induced cell cycle delay, increased the population of G2 phase cells, and rescued the phenotypes of cdc2-33, cdc25-22, Δrad24, and Δrad25 mutants, suggesting that Hsp9 probably regulates Cdc2 phosphorylation by modulating the Cdc25 activity. Indeed, immunoprecipitation experiments revealed that Hsp9 is associated with 14-3-3 and Cdc25. In Δhsp9 cells, the association of 14-3-3 with Cdc25 was weakened and Cdc2 phosphorylaton was reduced. Together, our data suggest that Hsp9 has dual functions in stress adaptation and regulating a G2-M checkpoint by the Cdc25 inactivation; this differs from S. cerevisiae HSP12, which maintains cell membrane stability under stress conditions.