Molecular Mechanism For Prion Protein Misfolding And Posttranslational Modifications Of Prion Protein

Amyloid fibrils associated with neurodegenerative diseases such as Alzheimer disease, prion disease, and amyotrophic lateral sclerosis (ALS) can be considered biologically relevant failures of cellular quality control mechanism. It is known that in vivo human prion protein (PrP) and its pathogenic mutants, human copper, zinc superoxide dismutase (SOD1) pathogenic mutants have the tendency to form fibril deposits in a variety of tissues and they are associated with prion disease, and ALS, respectively, while the rabbit PrP and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. Furthermore, misfolded SOD1accumulating in ALS can cause aggregation of their native counterparts in crowded physiological environments through a mechanism similar to the infectious prion protein PrPSc causing aggregation of its cellular isoform PrPC. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins including prion proteins from human and rabbit. As revealed by assays based on thioflavin T binding and sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1mutant A4V. The presence of crowding agents greatly accelerates the exposure of hydrophobic regions of A4V, especially PEG20000. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at300g/1is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll70and dextran70at200g/1. We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed. Information obtained from the present study can enhance our understanding of the molecular mechanisms of neurodegenerative diseases, and should lead to a better understanding of how proteins misfold and how proteins avoid misfolding in crowded physiological environments.Most studies of prion protein aggregation in vitro are using proteins expressed and purified from E. coli without any posttranslational modifications. But more and more evidence shows that glycosylation and glycosylphosphatidylinositol (GPI) anchoring could play an essential role in the coversion of PrPc into PrPSc and the mechanisms of how such posttranslational modifications regulate the transmission of prion diseases are still not fully discovered. In order to get glycosylated human prion proteins, we have established the baculavirus-insect cell expression system and expressed human prion protein and its two pathogenic mutants V180I and F198S. The results of Western blot shows that more than three bands of human prion protein between25-50kDa exist because of the heterogeneity of glycosylation. The treatment of PIPLC and Triton X-114phase dispersion confirms that human prion protein is GPI anchored. The results of glycosylation of human prion protein and its mutants detected by LTQ Orbitrap MS shows that both wild-type and V1801are glycosylated at181and197sites, yet glycosylation at181site of F198S is not revealed. Moreover, wild-type human prion protein and V180I locate on the membrane while F198S and Q217R mainly appear in the cytosol of RK13cells. We thus suggest that some pathogenic mutations could influence the posttranslational modifications of prion proteins which could have an effect on their localization in cells. More efforts need to be exerted in order to explicit the mechamisms.