title: 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 (PrP(C)) to the infectious scrapie form (PrP(Sc)). However, the mechanism that exogenous PrP(Sc) infects cells and where pathologic conversion of PrP(C) to the PrP(Sc) 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 PrP(C) to the pathologic PrP(Sc) form in N2a cells exposed to strain RML PrP(Sc) infected brain homogenates, suggesting that a critical determinant of PrP(C) conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP(Sc) infects cells by lipid raft dependent, macropinocytosis.
authors.affiliation: Department of Cellular and Molecular Medicine, University of California San Diego School of Medicine, La Jolla, California, United States of America.
authors.forename: Jehangir S
authors.initials: JS
authors.lastname: Wadia
authors.forename: Monica
authors.initials: M
authors.lastname: Schaller
authors.forename: R Anthony
authors.initials: RA
authors.lastname: Williamson
authors.forename: Steven F
authors.initials: SF
authors.lastname: Dowdy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D003871:Dermatan Sulfate / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D006493:Heparin / Q000378:metabolism; D019426:Integrases / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D010873:Pinocytosis; D018621:PrPC Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018620:PrPSc Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011995:Recombination, Genetic; D013997:Time Factors
chemical_list: D018621:PrPC Proteins; D018620:PrPSc Proteins; D011993:Recombinant Fusion Proteins; D003871:Dermatan Sulfate; D006493:Heparin; C045073:Cre recombinase; D019426:Integrases