Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Abstract

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrPC) to the infectious scrapie form (PrPSc). However, the mechanism that exogenous PrPSc infects cells and where pathologic conversion of PrPC to the PrPSc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrPC to the pathologic PrPSc form in N2a cells exposed to strain RML PrPSc infected brain homogenates, suggesting that a critical determinant of PrPC conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrPSc infects cells by lipid raft dependent, macropinocytosis.

Beyond the protein-only hypothesis: A novel mineral-lipid hypothesis in prion and protein-misfolding disease

“…Conversion of PrP--C to PrP--Sc 439 may occur internally after uptake (14), and may occur on "endosome--like" vesicles 440 found intra--cellularly in prion--infected tissue (25). These data appear to be 441 congruent with the proposed hypothesis of mineral--induced vesicles.…”

“…(PrP--C) converts to an alternate and pathologic folding conformation, termed PrP--Sc 250 (14). The mammalian prion protein (PrP; major prion protein) is a C--terminally--GPI--251 anchored cell--surface protein.…”

“…It is widely expressed on cells, though at higher levels 252 on neurons, neuroendocrine cells, and cells of the lympho--reticular system (14). The 253 full--length form, as well as likely processed truncated forms of the protein, are 254 expressed on the cell surface (14). The physiologic function of the protein is not yet 255 fully understood (14).…”

“…The 253 full--length form, as well as likely processed truncated forms of the protein, are 254 expressed on the cell surface (14). The physiologic function of the protein is not yet 255 fully understood (14). In TSE, normal host wild--type PrP--C is converted to scrapie 256 prion protein (PrP--Sc), the protein of identical sequence but differing (likely beta 257 sheet) folding conformation, by an unknown mechanism.…”

Beyond the protein-only hypothesis: A novel mineral-lipid hypothesis in prion and protein-misfolding disease

“…The mammalian prion protein (PrP; major prion protein) is a C--terminally--GPI--251 anchored cell--surface protein. It is widely expressed on cells, though at higher levels 252 on neurons, neuroendocrine cells, and cells of the lympho--reticular system (14). The 253 full--length form, as well as likely processed truncated forms of the protein, are 254 expressed on the cell surface (14).…”