热休克蛋白70在苯并（a）芘所致DNA损伤修复中的作用和交互作用蛋白分析

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英文题名：Study of the Function and Interacting Proteins of Hsp70 in the Repair of BaP-induced Damages 作者：段燕英 论文级别：博士 学科专业名称：劳动卫生与环境卫生学 中文关键词：热休克蛋白70 ; DNA修复 ; BaP ; 彗星实验 ; 宿主细胞再活化实验 ; 免疫共沉淀 ; 激光共聚焦 ; 高效液相色谱-电喷雾-串联质谱分析 英文关键词：Heat shock protein 70 ; DNA repair ; BaP ; comet assay ; host cell reactivation ; immunoprecipitaion ; confocal microscopy analysis ; liquid chromatography electrospray ionisation tandem mass spectrometry 学位年度：2008 导师：邬堂春 学科代码：100402 学位授予单位：华中科技大学

摘要

真核生物和原核生物在物理、化学和生物应激因素(如高温、多环芳烃、病毒感染等)的作用下,机体会迅速启动高度程序化的热休克反应(heat shock response,HSR),诱导合成一组高度保守的热休克蛋白(heat shock proteins, HSPs)。HSPs是生物界(包括从细菌到人类)经过长期进化仍保留下来的、对体内外不良因素起保护作用的一类蛋白质。根据HSPs在SDS-PAGE上的结果,按其分子量大小将HSPs分为如下几个家族:大分子HSPs (≥100 KD),HSP90 (81 - 99 KD),HSP70(65 - 80 KD),HSP60(55 - 64 KD),HSP40(35 - 54 KD),小分子HSPs(≤34 KD),其中最为主要和重要的是HSP70家族的Hsp70。许多研究提示了其可能的作用和功能如下:1) Hsp70保护细胞或生物免遭各种应激因素的损害,赋予它们从各种应激中恢复的能力;其中,表现最为明显的是热耐受能力的形成,即当细胞或生物接触亚致死温度后,表现对致死温度的存活率明显增加,并且对其他性质的致死性应激的抵抗能力也可能增强;2) Hsp70对细胞内重要遗传物质DNA可能具有重要保护作用,且在生物生长、发育和分化过程中也起着重要作用;3) Hsp70与体内许多重要生物活性物质(如类固醇、肿瘤坏死因子等)有着密切的联系,参与体内许多调节过程;4)更重要的是,Hsp70作为分子伴侣,促进蛋白质的合成、折叠、装配和运输,还参与变性蛋白质的清除,这种重要功能可能与热耐受、毒物耐受有关。
     苯并(a)芘(BaP)是一类来源于有机物不完全燃烧、有致癌性的多环芳烃,可以和DNA结合形成加合物,这样的损伤经由核苷酸切除修复途径修复。此外,在BaP代谢过程中产生的反应活性氧(ROS)也能对生物大分子造成损害。ROS对DNA造成的损伤经由碱基切除修复途径修复。本实验在人群和细胞水平的研究结果显示Hsp70的表达水平和残留的BaP导致的DNA损伤负相关,提示Hsp70可能在对BaP所致DNA损伤的修复方面发挥了一定作用。Hsp70正常时主要分布在细胞浆,而有害因素应激时转移入核,且分布于染色体周围,这个重要现象也提示了Hsp70在DNA保护中的可能作用与意义。DNA修复在肿瘤形成和治疗方面是一个重要的研究方向,而既往的大多数研究从不同的DNA修复途径分别研究它们的重要作用与机制。而多数情况下,环境有害物质对DNA造成的损伤需要多种修复途径共同进行修复,需要同时考虑多种蛋白质在DNA修复中的作用。Hsp70是重要的分子伴侣,参与蛋白质的合成、折叠、装配、运输和变性蛋白质的清除等,无疑它也可能与DNA修复相关蛋白有关。
     本研究通过构建高表达和低表达Hsp70两种细胞模型,用BaP处理细胞,利用彗星实验、宿主细胞再活化实验来观察Hsp70表达水平不同细胞中修复能力的差异,同时用免疫共沉淀和高效液相色谱-电喷雾-串联质谱检测在DNA修复过程中和Hsp70结合存在的各种蛋白,从中筛选出可能的Hsp70作用底物。并对筛选出来的Hsp70交互作用蛋白利用免疫共沉淀、激光共聚焦和放射性自显影进行进一步的确证,为阐述Hsp70在BaP染毒修复过程中的作用机制提供科学依据。本研究共分四部分。
     第一部分高和低表达Hsp70细胞模型的建立
     对于Hsp70高表达细胞模型的建立,我们通过转染16HBE细胞pcDNA3.1/pcDNA3.1-hsp70重组质粒,并用G418筛选稳定的Hsp70高表达细胞株(16HBE/hsp70)。用转染空载体质粒pcDNA3.1的16HBE细胞作为转染对照(16HBE /pcDNA)。
     对于Hsp70低表达细胞模型的建立我们采用了槲皮素抑制Hsp70表达和RNA干扰技术两种方法,通过对Hsp70的表达水平的验证对进行两种方法的效果进行比较。
     槲皮素(QCT)是一种广泛存在的生物黄酮类物质,可以抑制某些肿瘤细胞Hsp70的合成,使Hsp70表达降低。我们首先采用不同浓度槲皮素(50、100、150、200μM)处理16HBE细胞6h,监测细胞存活率的同时用Western-blot技术检测槲皮素处理后,16HBE细胞中Hsp70蛋白的表达。结果发现,随着槲皮素浓度的增加,16HBE细胞的Hsp70的表达呈下降趋势。50μM槲皮素处理时,Hsp70表达即观察到显著下降(P<0.01),这样一直到200μM。而细胞存活率在浓度小于150μM时,均大于90%,在QCT浓度为150μM时,细胞存活率为87%。因此,结合Western Blot和细胞存活率实验,最终确定100μM槲皮素处理6h作为抑制Hsp70表达的模型,0.01%的DMSO处理作为溶剂对照(16HBE/DMSO)。
     RNA干扰(RNAi)是近年来发展起来的一种研究基因功能的新方法。RNA干扰是指在进化过程中高度保守的、由双链RNA诱发的、同源mRNA高效特异性降解的现象。近几年来RNAi研究取得了突破性进展,被《Science》杂志评为2001年的十大科学进展之一,并名列2002年十大科学进展之首。由于使用RNAi技术可以特异性剔除或关闭特定基因的表达,该技术已被广泛用于探索基因的功能。运用这项技术时,可以通过转染编码shRNA的质粒、小干扰RNA等达到特异性降低目的基因的目的。在本研究中,采用转染质粒的方法,在瞬时转染48小时后,对Hsp70的表达进行检测,从而对沉默效果进行评估。
     最后我们对高表达和低表达Hsp70的细胞模型进行了鉴定。利用细胞免疫荧光技术和Western-blot技术检测各组细胞中Hsp70的表达。细胞免疫荧光结果发现RNAi组细胞(16HBE/RNAi)的荧光强度明显低于正常培养的16HBE组细胞,而高表达Hsp70组(16HBE/hsp70)细胞的胞浆内荧光强度明显高于正常培养的16HBE组细胞;Western-blot检测发现,与16HBE组相比,16HBE /RNAi组Hsp70表达降低了42% (P﹤0.01),16HBE /hsp70组Hsp70表达增加了78% (P﹤0.01)。16HBE /DMSO、16HBE/HK和16HBE /pcDNA组Hsp70的表达未见明显变化(P > 0.05)。槲皮素浓度为100μM时,对Hsp70的表达抑制了53%,稍高于沉默组,但是由于槲皮素对Hsp70的抑制是非特异性的,因而采用RNA干扰技术进行后续的相关研究。
     第二部分Hsp70在B(a)P、BPDE所致DNA损伤修复中的作用
     为比较B(a)P染毒后,不同Hsp70表达水平的16HBE细胞DNA修复能力的差异,本部分首先用彗星试验检测16μM BaP染毒16HBE细胞2h,恢复不同时间(0,2,4,8,24h),Hsp70表达水平不同的细胞对DNA损伤的修复差异。所设八组细胞Control、16HBE/HK、16HBE /RNAi、16HBE /hsp70、16HBE/pcDNA、DMSO、S9、NC(normal cultured)在处理后存活率均大于80%,并且在DMSO、S9和NC组中,存活率大于90%,均符合毒理学要求。彗星实验结果表明随着恢复时间的延长,各组细胞OTMs均下降,说明残留DNA损伤的减少。在恢复前2h内,降低迅速,提示这个时间段为修复最活跃阶段。在2h到8h,速度减慢。24h后,OTMs基本达到正常水平(NC,normal cultured,显示正常培养时的OTMs),提示修复过程基本完成。高表达Hsp70组在修复最活跃的前2h相对于对照组,OTMs值降低更迅速,说明高表达Hsp70组修复进行得更快。并且在恢复的各个时间点,高表达Hsp70组残留的OTMs值均小于对照组,且差异有极显著性(P<0.01)。而在低表达Hsp70组中,在修复最活跃的前2h内OTMs的降低速度和对照组相比明显减慢(P<0.01)。由此可见,在修复最活跃的前2h的结果提示,Hsp70参与了BaP造成的DNA损伤的修复过程,它可以促进细胞对这种损伤的修复。这可能与作为分子伴侣的Hsp70可以帮助变性受损的蛋白质复性或降解有关,高表达的Hsp70增强了分子伴侣作用,保持了蛋白质的稳态,减轻了细胞的损伤。
     BPDE是BaP的代谢终产物,可以与DNA的亲核位点,即鸟嘌呤的外环胺基端共价结合形成BPDE-DNA加合物,导致特异性突变。这样的加合物损伤可以通过核苷酸切除修复(NER)途径修复,如果没有得到及时有效的修复就会导致肿瘤的形成。Hsp70是HSPs家族中的重要成员,多项研究显示在DNA毒性应激因素作用下,Hsp70的表达水平和残留的DNA损伤负相关。有研究发现这种伴侣蛋白在碱基切除机制修复(BER)和错配修复(MMR)中都发挥了一定的作用,但在NER方面,真核生物方面尚缺乏相关研究。故本部分拟在这方面作一些探讨,研究Hsp70是否参与了对BPDE所致加合物损伤的核苷酸切除修复。宿主细胞再活化实验(HCR)是通过瞬时转染被不同浓度BPDE(10、20、30、40μM)损伤的萤火虫荧光素酶报告基因的pCMVluc质粒,40h后检测宿主细胞对损伤质粒DNA的修复情况,根据宿主细胞表达荧光物质的含量来评估宿主细胞的DNA整体修复能力。高表达Hsp70组相对对照组,荧光素酶的表达明显提高,说明对加合物损伤的修复能力的增强,且在10μM时,P<0.01,在20μM时,P<0.05。低表达Hsp70组相对对照组,荧光素酶的表达显著降低,提示其修复能力的减弱,且在10μM时,P<0.05。
     第三部分BaP作用下Hsp70交互作用蛋白的鉴定
     本研究第二部分的研究结果显示,Hsp70表达不同的16HBE细胞在对BaP、BPDE所致DNA损伤的修复中,表现不同的修复能力。这样的结果提示Hsp70可能在这一过程中发挥了作用。Hsp70作为一种分子伴侣,可以帮助蛋白质维持正确的构象、对变性蛋白进行修复或者降解,因此,我们猜想可能存在一些蛋白底物,Hsp70通过和这些蛋白底物作用而在DNA修复过程中发挥一定的作用。在本部分中,16μM B(a)P染毒16HBE细胞2h恢复4h后,运用免疫共沉淀把在修复过程中和Hsp70有交互作用的所有蛋白沉淀下来,并对其进行高效液相色谱电喷雾串联质谱分析(HPLC ESI MS/MS)。样本中总共有730种蛋白被检测,我们对其中含量较高的84种蛋白进行了分析,根据swiss-prot database蛋白质数据库(http://www.expasy.org)提供了蛋白质的各种说明,包括蛋白名称、起源、功能、交互作用、结果等,把这84种蛋白分成了13个功能组,分别是细胞结构和移动(14%)、蛋白质代谢和修饰(12%)、DNA稳定性(6%)、细胞内和细胞间信号(7%)、细胞分化和增殖(74%)、蛋白交互作用(6%)、凋亡(5%)、核酸代谢(5%)、生理功能的执行(6%),细胞损伤保护(2%)、能量代谢(1%),未知部分和其他部分(27%)。从结果可见,在修复过程中,Hsp70不仅和参与生理功能的蛋白质有交互作用,而且和维持DNA稳定、抵抗细胞损伤的蛋白质之间也有交互作用,Hsp70可能是通过复杂的功能网络参与BaP所致损伤的修复,这为进一步阐述Hsp70在修复过程中所发挥的多重功能提供了线索。
     第四部分BaP作用下Hsp70和CKII的交互作用及Hsp70对CKII活性的影响
     多种研究结果提示Hsp70和DNA修复关系密切。Bases首先发现Hsp70能促进人白血病细胞对射线照射的修复,参与了氧化性损伤的碱基切除修复过程。而后有研究者报道Hsp70通过促进碱基切除修复过程中关键酶的活性,如APE和polyβ而促进修复。原核生物中的Hsp70,即DnaK也参与了核苷酸切除修复过程。在前一部分的结果中的酪蛋白激酶II(CKII),它是恢复阶段和Hsp70相互作用的重要蛋白。CKII本身是一种丝氨酸/苏氨酸激酶,可以通过磷酸化XRCC1、XRCC4和APE而促进氧化性损伤的修复,此外,CKII也和核苷酸切除修复途径的重要修复酶XPB有关。由于Hsp70和CKII在DNA修复方面都参与了NER和BER,在BER过程中有共同底物的APE,而我们上部分的结果发现两者在修复过程中是复合存在的。故在本部分中,运用免疫共沉淀和激光共聚焦技术对两者的交互作用进行深入研究,并用放射性自显影技术检测Hsp70是否对CKII的活性有影响。。
     Hsp70和CKII免疫共沉淀结果显示,在兔抗人Hsp70抗体沉淀下来的复合物中,有CKII的存在。逆向免疫共沉淀即在羊抗人CKII抗体沉淀下来的复合物中也检测到了Hsp70的存在。在16HBE细胞BaP染毒和不染毒时,我们得到了相同的结果。考虑到DNA修复是发生在细胞核内的,我们进一步用激光共聚焦技术来检测这两种蛋白质的位置关系,结果发现Hsp70和CKII在不染毒时主要分布在细胞浆,两者大部分重合。在BaP染毒后这两种蛋白均有一部分进入了细胞核,并且在某些位置明显重合。而测定不同Hsp70表达水平的细胞中的CKII活性结果显示高表达Hsp70细胞CKII活性比对照组增高(P<0.05)而低表达Hsp70细胞中CKII活性则降低。
     综上所述,本研究的结果如下:
     (1)确定了100μM的槲皮素为抑制16HBE细胞Hsp70表达的最佳剂量,采用含hsp70基因的cDNA重组质粒进行细胞转染并经筛选建立了稳定的Hsp70高表达细胞株。
     (2)Hsp70可以增强BaP、BPDE所致DNA损伤的修复。
     (3)在B(a)P染毒恢复阶段,Hsp70与维持DNA稳定、细胞结构和移动等多种功能的蛋白质结合存在。
     (4) Hsp70和修复密切相关的蛋白CKII在未染毒时在胞浆结合,染毒后均进入胞核并结合存在,并且高表达的Hsp70可以增强CKII的活性。
     本研究的创新之处在于:(1)同时使用了高表达和低表达Hsp70的细胞模型,为探讨Hsp70的功能提供有力的依据,可以从正反两个方面同时验证Hsp70的功能;(2)考虑到了Hsp70作为分子伴侣的性质,从对其底物的研究入手以阐述它在DNA修复中的具体作用。
     本课题有待深入研究的方面有:(1) Hsp70和CKII的相互关系尚需进一步研究,特别是Hsp70参与DNA修复的具体机制还有待深入的阐述;(2) DNA整体修复能力的测定如何应用于人群现场流行病学调查,如何应用于职业性有害因素对机体DNA损伤修复的评价,还需要进一步的探讨。
Heat shock proteins (Hsps) are highly conserved proteins which are triggered in all organisms exposed to environmental stressors such as elevated temperature, nicotinamide, carbon monoxide, heavy metals, ionization, ischemia and hypoxia. Based upon their apparent molecular weight, HSPs are divided into many groups such as high-molecular-mass HSPs (≥100 kD), HSP90 (81 to 99 kD), HSP70 (65 to 80 kD), HSP60 (55 to 64 kD), HSP40 (35 to 54 kD), and small HSPs (≤34 kD). The HSP70 family have been extensively studied which are found to function as molecular chaperones, assisting nascent polypeptides proper configuration and facilitating the misfolding peptides degradation. Their overexpressions greatly change the tolerance and sensitivity of organism to physical, chemical and biological harmful stimuli. Hsp70 was mainly in cytoplasm under physiological condition but moved to nucleus when organism was under stress, besides, two previous research in our lab found that Hsp70 level was inversely correlated to residual DNA damage, which both hint that Hsp70 may be involved in DNA repair. However, many related research was base on a relation between Hsp70 and DNA repair and that whether Hsp70 plays a role in the DNA repair remains unknowed. Considering the molecular chaperone essence of Hsp70, there may be some protein substrates, through which Hsp70 modulate the DNA repair process.
     In this study, we investigated the possible roles of Hsp70 in DNA repair in 16HBE cells by either knocking-down or overexpressing Hsp70 expression level under BaP treatment. The repair of BaP-induced damage, assessed by residual DNA damage, was measured by comet assay and the repair of DNA adducts was assessed by host cell reactivation assay. Later, immunoprecipitation(IP) and high performance liquid chromatography electrospray ionisation tandem mass spectrometry (HPLC ESI MS/MS) were further applied to detect the Hsp70-interacting proteins, among which casein kinase II(CKII), a Ser/Thr protein kinase, was an important protein involved in DNA repair. IP assay, confocal microscopy analysis and autoradiography were further performed to characterize the interaction between Hsp70 and CKII.
     PartⅠEstablishment of 16HBE cell models with over-expressed and knocked-down Hsp70 levels
     16HBE cells were transfected with recombinant plasmid pcDNA3.1/hsp70. And the positive clones appeared after a selection for 2 weeks by 800μg/ml G418 (neomycin). Positive clones were expanded and analyzed with the expression of Hsp70. The control group was transfected with pcDNA3.1 plasmids containing the neomycin resistance gene but not hsp70 cDNA. We developed stably transfected 16HBE cell lines with overexpressed Hsp70 (16HBE/hsp70) or with neomycin resistance gene but not hsp70 cDNA (16HBE /pcDNA).
     To inhibit the expression of Hsp70, 16HBE cells were treated with different concentrations of quercetin (QCT) (50, 100, 150, 200μM) for 6h, then water bathing for 1h at 42°C. The leves of Hsp70 in different dose groups were assessed by western blot. Compared with the control group, there was a significant decrease of Hsp70 in 50μM group (P < 0.01) and in the following dose groups (P < 0.01). As for cell survival rate, it was above 90% when the concentration of QCT was less than 150μM and it decreased to 87% when the concentration of QCT was 150μM. According to both cell survival rate and Hsp70 levels caused by different concentrations of QCT, 100μM QCT, which can inhibit Hsp70 level to 47%, were chosen to inhibit Hsp70 expression of 16HBE cells in QCT treatment model.
     RNA interference (RNAi) is the technique employing double-stranded RNA to target the destruction of homologous messenger RNAs. RNAi utilizes short double-stranded RNA to selectively inhibit gene expression of complementary RNA nucleotide sequences after transcription, but prior to translation. It has gained wide usage in genetics, having the potential for many practical applications. Because of the great progress made during the development, RNAi was estimated the top 10 scientific advancement in 2001. We could transfect plasmid coding small hairpin RNA (shRNA) or small interference RNA (siRNA) to specifically knock down certein gene, in this study, we selected the former to knock down the expression of Hsp70. Forty-eight hour after transfection, we appraised the expression levels of Hsp70 with western blot. The result showed that Hsp70 expression was decreased by approximately 50%. And we determined to apply RNAi to knock down Hsp70 level in the following study.
     Later we applied immunocytochemistry and western blot to assess the Hsp70 expression in five groups, 16HBE/hsp70, 16HBE/pcDNA, 16HBE/RNAi, 16HBE/HK and 16HBE. The signals in 16HBE/hsp70 group was obviously enhanced, while in 16HBE/RNAi group, relative weak signal was observed. There were almost similar signals among 16HBE, 16HBE/HK and 16HBE/pcDNA groups. We next validated the results of immunocytochemistry signals by western blot. Results demonstrated that 16HBE/RNAi group intensity was notably reduced while intensity was greatly enhanced in 16HBE/hsp70 group. The quantified results showed that compared with the 16HBE group, the Hsp70 expressions in 16HBE/RNAi group was decreased by approximately 50% and in 16HBE/hsp70 groups, it was nearly 200% both with statistical significance (P<0.01).
     PartⅡRoles of Hsp70 plays in the DNA repair of BaP- and BPDE-induced damages
     To evaluate the effect of Hsp70 on DNA repair, we exposed cells with knocked-down, normal and over-expressed Hsp70 to benzo a pyrene (BaP) and incubated cells for 0, 2, 4, 8 and 24h. The repair kinetics of the different groups, measured as a reduction of OTMs with time, was determined by alkaline comet assay. Survival rate was above 80% in all groups and was above 90% in DMSO, S9 and NC group determined by trypan blue exclusion experiment and an electronic counter, which accorded with the requirement of toxicology. The result of comet assay showed that the OTMs in all groups decreased with the repair time, indicating that the residual DNA damage decreased. The formation of DNA stand lesion induced by BaP appeared to be a rapid event. After BaP exposure, the extent of DNA migration in all groups was significantly increased (P< 0.01), compared with the NC group, indicative of the initial excision repair or lasting strand lesion. The DNA migration decreased promptly within 2h, appeared to be somewhat slower afterward and nearly came to the level similar to the NC group at 24 h, suggestive the almost completion of the repair at this time point. In overexpressed Hsp70 group, i.e. the 16HBE/hsp70 group, the OTMs in the early 2h decreased faster than those in the control group with statistical difference (P < 0.01), presenting an enhanced DNA repair capacity of 16HBE with a higher Hsp70 level. While in the knocked-down Hsp70 group, namely, the 16HBE/RNAi group, the OTMs in the early 2h decreased slower than those in the control group with statistically significant difference (P < 0.01), suggesting a reduced repair capacity of 16HBE with a lower Hsp70 level.
     BaP treatment induced very little late apoptosis which gived no false positive results in the comet assay. No statistical significances were found among control, HK, pcDNA3.1, S9 and DMSO groups at all the repair time points. Our resuts indicated that Hsp70 can promote the repair of damages induced by BaP, which to some extent, may result from the chaperone function of Hsp70.
     The ultimate metabolite of BaP, (t)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE) can also results in DNA lesion by forming covalent DNA adducts, which were repaired by nucleotide excision repair (NER) or resulted in cancer. Several studies showed that the expression of Hsp70 reversely related to the residual DNA damage.
     And other researchers reported that this chaperone was involved in base excision repair (BER) as well as mismatch repair (MMR). In NER, research related to Escherichia coli supported the involvement of DnaK (Hsp70) in NER, whereas in eukaryote, whether Hsp70 plays a role in DNA repair remains unknowed. In the following study, we plan to explore whether Hsp70 is involved in DNA adduct repair capacity. A host cell reactivation assay was conducted in which BPDE-damaged luciferase reporter plasmid was transiently transfected into cells for DNA repair and luciferase gene expression. The concentrations of BPDE were 10, 20, 30 and 40μM. As a control, undamaged luciferase plasmid was also transfected into cells for luciferase expression. Effective repair of the BPDE-caused adducts in the plasmid led to the expression of the luciferase reporter gene. If the adducts were not repaired, expression of the luciferase reporter gene would be blocked. Our result revealed that inhibition of Hsp70 expression by RNAi, indeed, led to a significant decrease in reactivation of the luciferase reporter plasmid damaged by various doses of BPDE. A statistically significant difference was observed in the 10μM group (P<0.05). Additionally, overexpression of Hsp70 resulted in a clear increase in reporter reactivation with statistically significant difference in 10μM group (P<0.01) and 20μM group (P<0.05). These results indicated that changes in Hsp70 expression level contributed to, at least in part, the changes in DNA adduct repair capacity in 16HBE cells. No statistical significance was observed among control, HK and pcDNA3.1 groups.
     PartⅢIdentification of proteins interacting with Hsp70 under BaP treatment
     Results in partⅡshowed that 16HBE cell with different Hsp70 level had different repair capacity of damages caused by BaP or BPDE. Hsp70, an important molecular chaperone, can modulate protein maturation and repair misfolded proteins, so there may be some protein substrates through which Hsp70 affect the DNA repair.
     In this part, we identified proteins co-immunoprecipitated by anti-Hsp70 antibody in human bronchial epithelium (16HBE) exposed to BaP using combined one-dimensional SDS-polyacrylamide gel electrophoresisgels (SDS-PAGE) and high performance liquid chromatography electrospray ionisation tandem mass spectrometry (HPLC ESI MS/MS). Our results showed that approximately 730 proteins were combined with Hsp70 and 84 of them were analyzed, which were categorized into 13 functional groups based on the annotation in Swiss-Prot database (http://www.expasy.org), including cell structure and motility, protein metabolism and modification, DNA stability, intracellular and intercellular signals, cell differentiation and proliferation, protein interaction, apoptosis, nucleic acid metabolism, physiologic function executant, cell damage protection, and energy metabolism. In addition, some identified proteins were annotated as hypothetical proteins or short of annotations.
     PartⅣInteraction between Hsp70 and CKII and the effect of Hsp70 on the activity of CKII under BaP treatment
     Lots of reports reveal that Hsp70 is involved in DNA repair. Bases first reported that Hsp70 could enhance the repair of damages induced by ionizing radiation in human leukemia cells and play a role in BER. Afterward, some researchers reported Hsp70 could promote the activation of some critical enzymes in BER such as APE, polyβ, thus enhancing the repair capacity. In prokaryote, research related to Escherichia coli supported the involvement of DnaK (Hsp70) in NER. Casein kinase II(CKII), a Ser/Thr protein kinase, was among identified Hsp70-interacting proteins. Several lines of evidence validated that CKII was involved in DNA repair by phosphorylation of some critical repair enzymes such as XRCC1, XRCC4 and APE. Because of the functional overlapping of Hsp70 and CKII and the identical substrate protein, namely, APE, we applied immunoprecipitation and confocal microscopy analysis to further explore the interaction between these two proteins. We immunoprecipitated endogenous Hsp70 by rabbit anti-Hsp70 antibody from the 16HBE cells and examined the association of CKII in the immunoprecipitated complexes by western blot, followed by an inverse IP. Significantly, CKII was co-immunoprecipitated with Hsp70 and as expected Hsp70 was also co-immunoprecipitated with CKII with or without BaP treatment. The immunofluorescence results showed that Hsp70 and CKII presented a diffuse distribution throughout the cytoplasm at physiological condition.
     However, after BaP treatment, fraction of them was distributed in the cell nucleus as well as the cytoplasm. The overlapped figure of confocal fluorescence microscopic analysis presented that the majority of CKII co-localized with Hsp70 but only visible in cytoplasm at physiological condition. And this co-localization could be detected in nuclear region as well as in cytoplasm after cells were exposed to BaP. Furthermore, the result from autoradiography revealed that overexpression of Hsp70 may promote the activity of CKII.
     Perspectives:
     1, The optimal concentration of QCT suppressing Hsp70 expression was determined as 100μmol/L and we established stable 16HBE cell overexpressed Hsp70 by transfection plasmid harboring hsp70 gene and selection by G418 for 14 days.
     2, Hsp70 reduced the damage caused by BaP or BPDE.
     3, During the recovery period, Hsp70 interacted with many proteins related to DNA stability, cell structure and motion and so on..
     4, Hsp70 co-localized with CKII in cytoplasm at physiological condition and at DNA repair condition, their colocalization was detected both in nuclear region and in cytoplasm. Furthermore, over-expression of Hsp70 promotes the activity of CKII.

引文

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