tailieunhanh - Báo cáo khoa học: Role of receptor-mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity

Using thein situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment, and then undergoes endosomal proteoly-sis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin A, Authier F (2005)FEBS J272, 4385– 4397]. Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma | ễFEBS Journal Role of receptor-mediated endocytosis endosomal acidification and cathepsin D in cholera toxin cytotoxicity Tatiana El Hage1 2 Clemence Merlen1 2 Sylvie Fabrega1 2 and Francois Authier1 2 1 INSERM U756 Chatenay-Malabry France 2 Universite Paris-Sud Faculte de Pharmacie Chatenay-Malabry France Keywords acidification cathepsin D cholera toxin endosome G protein Correspondence F. Authier INSERM U756 Universite Paris-Sud Faculte de Pharmacie 5 rue JeanBaptiste Clement 92296 Chatenay-Malabry France Fax 33 1 46835844 Tel 33 1 46835528 E-mail These authors contributed equally to this work Received 19 December 2006 revised 7 March 2007 accepted 20 March 2007 doi Using the in situ liver model system we have recently shown that after cholera toxin binding to hepatic cells cholera toxin accumulates in a low-density endosomal compartment and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin-D Merlen C Fayol-Messaoudi D Fabrega S El Hage T Servin A Authier F 2005 FEBS J 272 43854397 . Here we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma. Following administration of a saturating dose of cholera toxin to rats rapid endocytosis of both cholera toxin subunits was observed coincident with massive internalization of both the 45 kDa and 47 kDa Gsa proteins. These events coincided with the endosomal recruitment of ADP-ribosylation factor proteins especially ADP-ribosylation factor-6 with a time course identical to that of toxin and the A subunit of the stimulatory G protein Gsa translocation. After an initial lag phase of 30 min these constituents were linked to NAD-dependent ADP-ribosyla-tion of endogenous Gsa with maximum accumulation observed at 30-60 min postinjection. Assessment of the subsequent postendosomal fate of internalized Gsa revealed sustained endolysosomal transfer