The Mechanism Researches On Prion Self-assembly

Obviously,prion could be seen as a natural existed protein which was closely related to human neurodegenerative diseases,such as Parkinson's disease,Alzheimer's disease.The self-assembly of prion had complicated mechanism.That would play important roles in understanding of the process for tackling prion related serious diseases.Meanwhile,efficient control of self-assembly would be advanced in finding functional materials.The self-assembly process appeared many kinds of intermediates.Until now,one could not capture the important intermediates,which were possessed different functions and compositions.The states of prion aggregates and structures maintained obscure.The yeast prion,Sup35,was chosen as model in self-assembly process researches.The transfection of prion aggregates into cells and the mechanisms of genetic stability should also be elaborated.We started the researches for prion self-assembly from in vitro prepared and in vivo expressed Sup35p-NM.This thesis centered on the structures,functions and mechanisms of prion aggregates.The following research findings were completed.We are the first to take the bottom-up and top-down kinetic strategies by size exclusion chromatography.de novo assembly of Sup35-NM can be separated two intermediates,41 min species and 46 min species.41 min species accelerated the de novo assembly process.46 min species were spherical and be seen as the assembly units in de novo assembly.The prion assembly process had two periods,the primary process and the aging process.We found the trimer-elements in aging aggregates,which were came from the primary process and possessed SDS and boiling resistance.The aging aggregates could be dissociated trimer,dimer of trimer or trimer of trimer one by one from the ends through SDS and boiling condition.The aging kinetics of dissociation proven that the dissociated species were all based on trimer and had different interface interactions.The Sup35 purified from E.coli were mainly contained non-aggragated-prone monomers which were bounded by other proteins.Wertem blots,BN-PAGE and mass spectrums all demonstrated the impurity protein were Heat-shock protein 90(HSP90),Dna K(HSP70),30 S subunit of ribosome,ATPase beta subuints and Omp F porin,whose bounding strengths can only broken up by SDS-boiling under PAGE condition.We had built up the transfection approaches for yeast protoplasts from in vitro prepared Sup35p-NM assembly.We successful got the[PSI+]yeast prion phenotypes by yeast protoplasts methods.We screened the chemical,2-DQ which could kill the[PSI+]white phenotype cells.This process had no relationship with protein glycosylation in the[PSI+]white phenotypes.What's more,we found that high concentrations of glucose in medium and the common galatose medium had the opposite effects on the[PSI+]white phenotypes.These works in yeast[PSI+]testified that there was close relationship between prion replication and the metabolic process of sugars.