• 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.
    • Proteinase-Activated Receptor 2 (PAR-2) Involvement In The Zot/Zonulin-Mediated Regulation Of Intestinal Tight Junctions

      Clemente, Maria Grazia; Vogel, Stefanie N.; Hollenberg, Morley D., 1942-; Fasano, Alessio (2004-05)
    • The Vibrio Cholera-generated Zonulin occludens Toxin (Zot) N-terminal Cleavage Site Contains a Protease-activated Receptor Activating Peptide (PAR-AP) That Retains Biological Activity on Intestinal Tight Junctions

      Thakar, Manjusha; Vogel, Stefanie N.; Kao, Joseph P. Y.; Fasano, Alessio (2005)
      Background : We have previously demonstrated that Zonulin occludens toxin (Zot) elaborated by Vibrio Cholera anchors to the bacterial outer membrane through its single spanning domain and undergoes to a Vibrio-specific cleavage at amino acid residue 288. The resulting 12 Kda c-terminal fragment is then released in the intestinal micromilieu were exerts its permeating effect on intercellular tight junctions (tj). The N-terminus of th cleaved Zot fragment contains a conserved 6-mer protease activated receptor (PAR)-activating peptide (AP) motif. Aim: We have previously reported that Zot/Zonulin receptor is similar to PAR2, we elected to establish whether the six-mer motif (called AT1002) retains the Zot biological activity on tj. Methods: Rat small intestine was mounted in ussing chambers and AT1002-induced changes in transepithelial electrical resistance TEER) monitored Rat epithelial cells (IEC6) were used to study the intracellular signaling, including Ca2+ release,changes in tj protein-protein interactions, and phosphorylation of tj proteins. Results: Rat small intestine exposed to AT1002 showed a significant reduction in TEER as compared to the negative control starting at 30 minutes and reaching a plateau after 120 minutes. At a time point coincident with the effect con TEER (30 min) AT1002 induced an increment in ZO-1 phosphorylation that was associated to a decrease in ZO1-occludin interaction and an increase in ZO1-ZO2 interaction. Addition of AT1002 to IEC6 cells did not cause any increase in intracellular Ca2+, while the PAR-AP caused a dose-dependent increased in intracellular Ca2+ that reached a plateau at 50 fym. Conclusions: The 6-mer synthetic peptide At1002 retained the Zot biological activity an intercellular tj and caused a decrease in TEER. This effect was not associated to Ca2+ release, however was related to changes in protein-protein interaction of tj elements following Z)1 phosphorylation.
    • 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)