• The active motif of Zot, AT1002, increases ZO-1 and Myosin 1 Beta serine phosphorylation, their interaction, and intestinal permeability

      Thakar, Manjusha; Goldblum, Simeon E.; Not, Tarcisio; Fasano, Alessio (2006)
      Background: As we have previously reported, Zonula occludens toxin (Zot) elaborated by Vibrio cholerae anchors to the bacterial outer membrane and undergoes a Vibrio-specific cleavage at amino acid residue 288, with subsequent release of its C-terminal fragment in the intestinal micromilieau. The N-terminus of the cleaved Zot fragment contains a conserved 6-mer protease activated receptor (PAR)-activating peptide (AP) motif. We synthesized the 6-mer and named it AT1002. Aims: 1.To determine whether AT1002 modulates tj both in vivo and in vitro. 2. To establish whether AT1002 signaling affect ZO-1 phosphorylation. 3. To study ZO-1 interaction with partner and scaffolding proteins in presence of AT1002. Methods: 1. Transepithelial electrical resistance (TEER) was monitored either in presence or absence of AT1002 added to the mucosal aspect of rat small intestine mounted in Ussing chambers. 2. The in vivo intestinal permeability of mouse intestine was studied by dual sugar test with HPLC technique. 3. Phosphorylation of ZO-1 induced by AT1002 was analyzed by Western immunoblotting. 4. The effect of AT1002 on protein-protein interaction of ZO-1 with partner proteins and scaffold proteins were investigated by co-immunoprecipitation analysis. Conclusions: 1.AT1002 caused the tight junctions disassembly as shown by decrease in TEER in rat tissues mounted in Ussing chambers. 2.AT1002 was biologically active both in vivo and in vitro as established by experiments performed on intestinal tissues in Ussing chambers and dual sugar test permeability test. 3.AT1002 induced ZO-1 serine phosphorylation that temporarily preceded tight junction disassembly. 4.AT1002 increased serine phosphoralytion of myosin 1 beta and increased association of myosin 1 beta with ZO-1. 5. 6. AT1002 didn’t alter the association of ZO-1 and ZO-2.
    • Zonula Occludens Toxin Induces a Decreased Expression of the Tight Junction Protein Occludin

      Di Pierro, Mariarosaria; Drago, Sandro; Thakar, Manjusha; Lu, Ruiliang; Maimone, Francesco; Fasano, Alessio (2002)