Mechanisms Of Prion Protein Misfolding In Crowded Cell-like Environment

To understand the role of crowded physiological environment in the pathogenesis of neurodegenerative diseases, we studied amyloid formation of human prion protein in macromolecular crowding using ThT fluorescence, ANS fluorescence, UV turbidity and TEM. From the results, we have found that macromolecular crowding dramatically accelerates amyloid formation by human prion protein. A sigmoidal equation has been used to fit these kinetic data, yielding lag times and apparent rate constants for the growth of fibrils for this amyloidogenic protein. These biochemical data indicate that crowded cell-like environments significantly accelerate the nucleation step of fibril formation of human prion protein (a significant decrease in the lag time) as well as the elongation step. These results can in principle be predicted based on some known data concerning protein concentration effects on fibril formation both in vitro and in vivo. Furthermore, macromolecular crowding causes human prion protein to form short fibrils and non-fibrillar particles with lower conformational stability and higher protease resistance activity, compared with those formed in dilute solutions. Our data demonstrate that a crowded physiological environment could play an important role in the pathogenesis of neurodegenerative diseases by accelerating amyloidogenic protein misfolding and inducing human prion fibril fragmentation which is considered to be an essential step in prion replication.Then, we studied amyloid formation of rabbit prion protein in the presence of macromolecular crowding agent. We have found that rabbit prion protein does form amyloid fibrils in the absence of a crowding agent but the process was strongly inhibited by the presence of high concentrations of crowding agent. Morphology images from TEM and AFM suggest that macromolecular crowding could play negative role in rabbit prion protein amyloid formation reactions by trapping the rabbit prion protein in oligomers or prefinril state.We have also expressed and purified human prion mutant (S173N/L137I/G99N) and rabbit prion mutant (N174S/I138L/N100G). Macromolecular crowding agents significant accelerate amyliod formation of rabbit prion protein mutant but have no obvious change to the morphology of fibrils. Moreover, although the nucleation of fibrillization of human prion mutant is significantly delayed in dilute solutions, macromolecular crowding still promotes amyloid formation of such a mutant to some extent.