Study On The Structure And Aggregation Mechanism Of Prion Protein Amyloid Fibrils

Prion disease and Parkinson's dementia are two common protein conformational diseases that threaten the healthy of human beings.Although the two diseases have been studied over the decades,we have not found effective therapeutic methods for them yet,mainly due to the facts that we know little about the structures and pathogenic molecular mechanisms of their infectious agents.The pathological processes of prion disease and Parkinson's dementia are shown to be very complicated,which invole with proteins with multiple aggregates(monomers,oligomers and fibrils)that would interact mutually.Routine analytical techniques can hardly be applied in such complicated protein aggregates to get enough structural information,and thus cannot uncover the pathogenic molecular mechanisms of the emergence,development and propagation of those diseasesSolid state nuclear magnetic resonance(ssNMR)is powerful technique that can probe three-dimensional(3D)structures and interactions of complicated protein aggregates at an atomic level.By mainly using ssNMR combining with molecular dynamic simulation,we have investigated the 3D structure of the prion fibril related to prion disease,and represented the molecular mechanism of prion cross-species propagation.We have also investigated the interactions between ?-synuclein and A?40 in different aggregation states that are related to Parkinson's dementia,and analyzed the possible pathogenic molecular mechanisms in preliminary.This paper falls into four parts,of which the first three parts include the studies of prion fibrils and the forth part includes the study of the interactions between ?-synuclein(monomers or fibrils)and A?40(monomers or fibrils)Firstly,in order to study the prion disease,we have obtained prion proteins using bacterial gene expression,and prepared three kinds of mis-folded prion samples,including the prion mis-folded intermediates,oligomers and fibrils under different in vitro conditions.To reveal the aggregating mechanism of mis-folded prions,we have tested the structures of these prion samples using ssNMR and circular dichroism,and screened out one prion fibril homogeneous in a molecular level.The resulting fibril sample is then optimized to offer ssNMR spectra with high resolution and high sensitivity,which lays the basis of structural determination of prion fibrils by ssNMR.Secondly,we have acquired and analyzed a series of multi-dimensional ssNMR spectra.From these spectra,we have assigned the chemical shifts of 54 residues in the 167-223 segment,which form the fibrillar core with five ?-strands,and extracted intra-molecular and inter-molecular distance restraints,which confirm that the prion monomers are stacked in the style of the parallel in-register intermolecular ?-sheet.We have then presented a 3D structure model of the prion fibril from the above information.Based on the structural model and further kinetic data on cross-seeded fibrillation of prion(truncated)mutants,we conclude that the N-terminal 23-88 segment of prion is the main factor affecting the fibrillization and propose a molecular mechanism of the self-assembly of prion fibrils.Thirdly,we have designed prions and their mutants from different species according to the 3D structural model,in order to simulate the corresponding pathological process in vitro.We have analyzed the fibrillization of those prions and mutants induced by the mouse mPrP23-230 fibril in detail.According to the molecular dynamic simulation of the interactions between the mPrP23-230 fibril and other prions,we regard the steric effect of the 165th residue and the side-chain hydrogen of the 167thresidue as the main factor that affect the cross-species propagation of prion diseases.We then propose a molecular mechanism of the cross-species propagation of prion diseases,in which the 165th and167th resiudes work as the starting point of conformational transformation of prion protein monomers.At last,we have preliminarily investigated the molecular mechanism of interactions between ?-synuclein and A?40,which are related to Parkinson's dementia.Based on the data of NMR and fibrillation experiments,we demonstrate that,?-synuclein monomers do not interact with A?40 monomers in solution,?-synuclein monomers inhibit the elongation of AP40 fibrils,and ?-synuclein fibrils not only facilitate the elongation of A?40 fibrils but also affect the structures of A?40 fibrils.