Study Of The Function And Interacting Proteins Of Hsp70 In The Repair Of BaP-induced Damages

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 levels16HBE 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 damagesTo 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 treatmentResults 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 treatmentLots 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.