Roles Of Disulfide Bond In Biochemical And Functional Properties Of Human Prion Protein

This thesis is focused on roles of disulfide bond in properties of human prion protein.Firstly, the expressed recombinant human wild-type PrP protein was purified for using in anestablished redox process for the reduction and oxidation of the ethanethiol group within PrP.Then we tested susceptibilities of N-termi octapeptide repeat mutant protein to fibrillizationand aggregation induced by formation of disulfide bond. Sedimentation tests illustrated thatredox process remarkably promoted the aggregation of recombinant PrP, but not the deletionand insertion octapeptide mutants. Thioflavin T binding assay revealed an enhancedfibrillization of the recombinant human PrP after redox process, but not a significantdifference for mutants. Far-UV circular dichroism demonstrated that the wild type proteintreated with redox process showed a significant β-sheet rich structure, and a β-sheet decreasein mutants. Furthermore, PrP-specific Western blots identified that the recombinant PrP afterredox possessed stronger proteinase K-resistance, and mutants showed a more increasedproteinase K-resistance. Those data indicates that the formation of the disulfide bridgeinduces the alteration of the secondary structure and enhances the progresses of aggregationand fibrillization of PrP protein, and sequence of five octapeptide repeats seems to be anecessarity to a stable transformation of prion protein. To further investigate roles of thedisulfide bond, we built site-directed mutants of recombinant human prion proteinpQE30-C214G, pQE30-C179G/C214G and pQE30-M166C/E221C. For undergoing andfuture experiment, we continue performing thiol-disulfide redox process after proteinpurification, then followed with assays mentioned above. Moreover, a transfection usingplasmids pcDNA3.1-C179G/C214G and pcDNA3.1-M166C/E221C in eukaryotic cell culturewill be done to test cell toxicity or protection if possible.