【作者】 李晓丽；

【导师】 董小平；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2010， 博士

【摘要】 可传播性海绵状脑病(Transmissible spongiform encephalopathies, TSEs)即朊病毒病(prion diseases),是一类罕见的致死性神经系统退行性疾病,包括人类的克雅氏病(Creutzfeldt-Jakob disease, CJD)、GSS综合症(Gerstmann-Straussler-Scheinker syndrome, GSS)、Kuru病、家族性致死性失眠症(fatal familial insomnia, FFI)、绵羊和山羊的瘙痒病(scrapie),牛及其它动物的海绵状脑病等,根据疾病的来源不同可分为自发形成(散发型)、遗传型和医源型三种形式。本文分为三部分,第一部分我们利用原核表达系统表达了全长人PrP以及各种PrP八肽重复区突变体,采用蛋白酶消化、沉淀实验、聚集实验以及圆二色谱(CD)等方法探讨了锰离子对野生型PrP以及各中八肽重复突变体生物学特性的影响。第二部分采用pull-down实验和免疫共沉淀方法探讨了PrP51-91和微管蛋白之间的相互作用。并且我们通过共沉淀法、比浊法及免疫荧光检测PrP51-91与铜离子对微管蛋白聚合的影响。此外,再通过共聚焦显微镜以及MTT法检测PrP51-91与铜离子对细胞中微管以及细胞毒性的影响。第三部分我们在分子水平证实了细胞内PrP (CytoPrP)与微管间的相互作用,并进一步确定CytoPrP对微管的影响能导致的细胞凋亡。第一部分：锰离子对PrP生化特性的影响研究锰离子可能在朊病毒的发病过程中起一定作用。我们利用锰离子处理野生型PrP和PrP八肽重复序列突变体后。通过PK消化、透射电镜、沉淀实验以及圆二色谱法分析锰离子对各种重组PrP生化特性的影响情况。为了检测锰离子能否影响野生型PrP和PrP八肽重复序列突变体的PK抗性,我们利用不同浓度的PK酶对经锰离子处理后的PrP进行消化,通过Western blot进行PK抗性检测。结果显示,经锰离子处理后,野生型PrP和PrP突变体的PK抗性均增强,并且PrP八肽重复序列突变体增强程度要强于野生型PrP。为了检测锰离子能否影响野生型PrP和PrP八肽重复序列突变体聚集,我们利用透射电镜、比浊法以及沉淀实验对其进行检测。结果显示,经锰离子处理后,野生型PrP和PrP突变体更容易发生聚集,并且PrP八肽重复序列突变体比野生型PrP更容易发生聚集。为了检测锰离子对野生型PrP和PrP八肽重复序列突变体二级结构的影响。我们利用圆二色谱法对其进行观察。结果显示,经锰离子处理后,野生型PrP和PrP突变体p-折叠的含量明显增加,并且PrP八肽重复序列突变体p-折叠的含量增加幅度明显高于野生型PrP增加的幅度。这些结果提示,锰离子能增强PrP的PK抗性,使其更容易发生聚集,使其由以α螺旋为主变为以β-折叠为主,而且锰离子对PrP的作用强度与八肽重复区的数目有关。锰离子可能作为一种辅助因子参与PrPC到PrPSc的转变。第二部分：PrP51-91促进微管聚集并拮抗铜离子的微管解聚作用我们通过免疫共沉淀以及pull down实验显示原核表达的PrP八肽重复序列能与微管蛋白形成复合物,提示PrP八肽序列能与微管蛋白发生相互作用。为了检测PrP八肽重复序列与tubulin的相互作用能否影响微管的聚集,我们利用体外微管聚集实验、共沉淀实验对其进行检测。结果显示,PrP八肽重复序列能促进微管发生聚集。为了检测PrP八肽重复序列能否抵抗铜离子导致的微管解聚作用,我们利用比浊法、免疫荧光对其进行检测。结果显示,铜离子能使微管发生解聚作用,PrP八肽重复序列则能抵抗铜离子所引起的微管解聚作用。为了检测PrP八肽重复序列以及铜离子对细胞中微管以及细胞活性的影响,我们利用共聚焦显微镜以及MTT法对其进行观察。结果显示,铜离子能使细胞中微管发生解聚,而PrP八肽重复序列则能抵抗铜离子所引起的微管解聚。此外,铜离子对细胞的毒性可能与其促进微管解聚有关,PrP八肽重复序列先于或者同时与铜离子作用细胞能拮抗铜离子引起的细胞毒性作用。第三部分：CytoPrP能使微管发生解聚而导致细胞凋亡我们将HeLa细胞转染CytoPrP,利用免疫共沉淀实验确定在细胞中CytoPrP能与微管发生相互作用。我们再利用共聚焦显微镜进一步确定CytoPrP能与微管在细胞中能发生共定位。为了检测CytoPrP与tubulin的相互作用能否影响微管的聚集,我们利用共聚焦显微镜以及Western blot对其进行检测。结果显示,CytoPrP能抑制细胞内微管发生聚集,并且使细胞内微管蛋白含量减少。为了检测CytoPrP所引起的细胞凋亡是通过微管解聚引起的,我们利用MTT以及Annexin V/PI双染方法对其进行检测。结果显示,CytoPrP能使细胞发生凋亡并且使其发生凋亡是通过促进微管解聚引起的。这些结果提示,CytoPrP与微管发生相互作用后,促进微管发生解聚,从而导致细胞凋亡。更多还原

【Abstract】 Transmissible spongiform encephalopathies (TSEs), prion diseases, are rare degenerative neurological disorders that afflict human beings, including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), Kuru, and fatal familial insomnia (FFI), sheep and goat (scrapie), cattle (bovine spongiform encephalopathy, BSE), and other animals. They may have a sporadic, inherited or acquired origin. This study contains three individual parts, including investigation the effect of PrP on the biochemical characteristics of the recombinant wild-type and mutated PrPs, the analyses of PrP51-91 on microtubule polymerization and the study of the mechanism of apoptosis caused by CytoPrP.Part I:The effects of manganese on the biochemical characteristics of PrPManganese may play some roles in the pathogenesis of prion disease. In this study, recombinant wild-type PrP (WT-PrP) and PrP mutants with deleted or inserted octarepeats were exposed to manganese, and then their biochemical and biophysical characteristics were evaluated by proteinase K (PK) digestion, sedimentation experiments, transmission electron microscopy and circular dichroism (CD). To test whether manganese can influence the PK resistance of various PrPs, different concentrations of PK were used to digest the PrPs. Western blots showed that there were remarkable PrP-specific signals in Mn2+-treated PrPs after treated with higher doses of PK. Moreover, compared with WT-PrP, PrP mutants are more resistant to PK digestion after treatment with manganese. To see the effect of manganese on aggregation of various PrPs, sedimentation experiments, turbidimetry and transmission electron microscopy were performed. Our results showed that the treatment of manganese can efficiently induce aggregations of PrPs, moreover, PrP mutants seem to be easier to cause aggregation then the WT-PrP after incubation with manganese.To investigate the effect of manganese on the structures of various PrPs, the secondary structures of refolded Mn+-treated PrPs were comparably examined by CD analysis. Our results showed that an obvious conversion from a predominant a-helix to a more extensiveβ-sheet formation was observed in the CD spectrums of each PrP preparation treated with Mn2+. Moreover, PrP mutants can cause conformational conversion easier than the WT-PrP after treatment with manganese. Our study provides the evidence that the increase of PK-resistance of PrPs after incubation with Mn2+correlates well with its more aggregation and higherβ-sheet content. Moreover, the effect of manganese on PrP seems to associate with the octapeptide repeats number. It strongly highlights that manganese may play an important role in the conversion from PrPC to PrPSc and the pathogenesis of prion disease.PartⅡ:PrP51-91 enhances the formation of microtubule and antagonizes Cu2+-induced microtubule-disrupting activityUsing pull-down and co-immunoprecipitation assay, PrP51-91 can form complex with tubulin form hamster brain homogenate. It suggests that a remarkable interaction between the PrP51-91 and tubulin was identified. To test whether the interaction between PrP and tubulin influenced the assembling of microtubules from tubulin in vitro, microtubule assembly assay and sedimentation test were performed. Our results showed that PrP51-91 can induce tubulin polymerization. To see whether PrP51-91 can antagonize Cu2+-induced inhibition of microtubule assembly, microtubule assembly assay and immunofluorescence staining were performed. Our results showed that Cu2+has an inhibitive effect on microtubule formation, whereas PrP51-91 efficiently protects against Cu2+-induced microtubule-disrupting activity. To identify that PrP51-91 can protect against Cu2+toxicity on the culture cells and stabilizes cellular microtubules. Confocal microscopy and MTT assay were performed. Our results showed that Cu2+can inhibit microtubule of cell polymerization, whereas PrP51-91 can antagonize Cu2+-induced microtubule-disrupting activity. Moreover, PrP51-91 can efficiently protect against Cu2+-induced cytotoxicity.PartⅢ:Cytosolic PrP induces apoptosis of cell by disrupting microtubule assemblyIn the present study, the Hela cells were transfected with pcDNA3.1-CytoPrP. The molecular interaction between cytosolic PrP and tubulin was confirmed using immunoprecipitation. Moreover, the confocal microscopy was used to identify that CytoPrP can co-localize with tubulin in the HeLa cells. To test whether the interaction between CytoPrP and tubulin influenced the assembling of microtubules from tubulin, confocal microscopy and Western blot assay were performed. Our results showed that CytoPrP can inhibit the polymerization of tubulin and decrease the quality of microtubule protein. In order to identify that the inhibition of microtubule polymerization caused by cytosolic PrP was able to lead to the apoptosis of cells, immunofluorescent staining and the annexin V/PI double-staining assays were performed. Our results demonstrated that the inhibition of microtubule polymerization caused by cytosolic PrP was able to lead to the apoptosis of cells. The association of apoptosis with microtubule-disrupting activity caused by cytosolic PrP may further provide insight into the unresolved biological function of PrP in the neurons.更多还原