Divalent Metal Ion Influences The Biological Characteristics Of PrP And CytoPrP Induces Apoptosis Of Cell By Disrupting Microtubule Assembly

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.