The Analysis Of The Effects Of PrP On Microtubule Polymerization In Vitro And Sensitive Detection Of PrP^Sc By Western Blot Assay Based On Streptomycin Sulfate Precipitation

Transmissible spongiform encephalopathies(TSEs),prion diseases,are rare degenerative neurological disorders that afflict human beings,including Creutzfeldt-Jakob disease(CJD),Gerstmann-Str(a｜¨)ussler-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 of the effect of PrP on microtublule polymerization,the analyses of sensitive detection of PrP^Sc by Western blot assay based on streptomycin sulfate precipitation and the study of molecular interaction between prion protein and GFAP both in native and recombinant forms in vitro.PartⅠ:Analysis of the effects of PrP on microtubule polymerization in VitroUsing pull-down and co-immunoprecipitation assay,a remarkable interaction between the full-length His-PrP23-231 and tubulin was identified.We further mapped the regions within PrP binding with tubulin with pull-down and co-immunoprecipitation assay,and demonstrated that the N terminus peptides containing PrP 23-91,PrP23-50 and PrP51-91 could interact with tublin,while the C terminus peptide PrP91-231 failed.It demonstrated that the interaction regions within PrP with tublin located at the N terminus peptide 23-50 aa and octapeptide repeat region 51-91 aa.To test whether the interaction between PrP and tubulin influenced the assembling of microtubules from tubulin in vitro,microtubule assembly assay, sedimentation test and morphological observation of transmission electron microscopy were performed.Our results showed that only PrP in the context of full-length peptide is concerned with the modulation of microtubule dynamics as a tubulin-sequestering protein,while its N- or C-terminal segments seem not to affect microtubule polymerization in vitro.To evaluate the binding activities with tubulin among various PrPs with different numbers of octapeptide repeats,various PrPs were employed into a tubulin-coated ELISA.Compared with that of PG5,the reactions of PG9 and PG12 showed stronger binding activities with tubulin,whereas that of PG0 was obviously weaker.The results indicated that the numbers of octapeptide repeats within PrP directly affected the binding activity with tubulin and the binding activity became stronger along with the increase of numbers of octapeptide repeats.To investigate the effectiveness of the numbers of octapeptide repeats within PrP on microtubule dynamics,microtubule assembly assay,sedimentation test and morphological observation of transmission electron microscopy were performed. Compared with PG5,PG9 and PG12 induced much higher turbidities,showing a number-dependent manner.Interestingly,although PG0 showed weaker binding activity with tubulin,an unexpectedly strong increase of solution turbidity was repeatedly observed in the preparation of PG0,which was even higher than that of PG9 and PG12.Thus,the numbers of octapeptide repeats may not only be associated with binding activity of PrP to tubulin,but also play an important role in regulating inhibitive effect of PrP on microtubule polymerization.Only the correct numbers of octapeptide repeats can efficiently balance the regulating activity of PrP to the tubulin polymerization.PartⅡ:Sensitive detection of PrP^Sc by Western blot assay based on streptomycin sulfate precipitationWe optimized the Western blot assay for PrP^Sc with a precipitation procedure of streptomycin sulfate.In order to probe the optimal working concentration of streptomycin sulfate,different quantities of streptomycin sulfate were subjected into the preparations containing PrP^Sc stock sample.Western blot analyses identified a clearly streptomycin sulfate dose-dependant manner of PrP^Sc distribution in fractions of the pellets.In addition,we evaluated the influence of pH value on the effectiveness of streptomycin precipitation for PrP.The results showed that the precipitating activity of streptomycin for PrP was quite efficient and stable under neutral or weakly acidic condition,but dropped down along with the increase of pH value.To seek whether treatment of PrP^Sc with streptomycin sulfate increased the identifying sensitivity in Western blots,different amounts of hamster PrP^Sc stock sample were incubated with streptomycin sulfate in lysis buffer.The results demonstrated that streptomycin precipitation increased markedly the detective sensitivity of PrP^Sc,regardless in low concentration or in large volume.To address the precipitating activities of streptomycin sulfate on PrP^Sc in brain tissue or body fluids,hamster PrP^Sc stock sample was spiked into hamster PrP^C stock sample,CSF and urine,respectively.Contrast to the preparations without streptomycin sulfate that no positive signal was observed,the specific PrP^Sc reactive signals were obviously detected in all preparations.In addition,the PrP^Sc from a human brain tissue of familiar Creutzfeldt Jacob Disease(fCJD) was efficiently precipitated with streptomycin sulfate. PartⅢ:Molecular interaction between prion protein and GFAP both in native and recombinant forms in vitroIn the present study,remarkable GFAP-PrP^Sc or GFAP-PrP^C complexes were separately detected in the brain homogenates of 263K(Scrapie)-infected or normal hamsters by co-immunoprecipitation assay.To get more exact molecular evidences for interaction between prion protein(PrP) and GFAP,various recombinant PrP or GFAP proteins were expressed using prokaryotic-expressing and in-vitro translation system.Using pull down and co-immunoprecipitation assays,reliable molecular interaction between PrP and GFAP was observed,and proteinase K(PK)-digested PrP^Sc molecules were confirmed to be able to bind the recombinant GFAP specifically as well.The region within PrP that was responsible for interaction with GFAP was narrowed to PK-resistant core of PrP(i.e.aa91-231).