title: GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro.
abstract: Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane. It is widely recognized that LF, and the related fusion protein LFnDTA, must be completely denatured in order to transit through the PA63 formed pore and enter the eukaryotic cell cytosol. We demonstrate by protease protection assays that the molecular chaperone GRP78 mediates the unfolding of LFnDTA and LF at neutral pH and thereby converts these proteins from a trypsin resistant to sensitive conformation. We have used immunoelectron microscopy and gold-labelled antibodies to demonstrate that both GRP78 and GRP94 chaperones are present in the lumen of endosomal vesicles. Finally, we have used siRNA to demonstrate that knock-down of GRP78 results in the emergence of resistance to anthrax lethal toxin and oedema toxin action.
authors.affiliation: Section of Molecular Medicine, Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Alfred G
authors.initials: AG
authors.lastname: Tamayo
authors.forename: Louise
authors.initials: L
authors.lastname: Slater
authors.forename: Julian
authors.initials: J
authors.lastname: Taylor-Parker
authors.forename: Ajit
authors.initials: A
authors.lastname: Bharti
authors.forename: Robert
authors.initials: R
authors.lastname: Harrison
authors.forename: Deborah T
authors.initials: DT
authors.lastname: Hung
authors.forename: John R
authors.initials: JR
authors.lastname: Murphy
mesh_terms: D000818:Animals; D000881:Anthrax / Q000378:metabolism / Q000382:microbiology; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D001408:Bacillus anthracis / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D003600:Cytosol / Q000201:enzymology; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D051379:Mice; D021381:Protein Transport / Q000502:physiology*; D058767:Protein Unfolding; D034622:RNA Interference; D034741:RNA, Small Interfering; D014357:Trypsin / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; C030325:anthrax toxin; C053387:endoplasmin; D000090802:rab7 GTP-Binding Proteins; C000716465:rab7 GTP-binding proteins, mouse; D014357:Trypsin; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins