Abstract
Occludin is an integral membrane protein that has been suggested to play a role in the organization and dynamic function of the epithelial tight junction (TJ). A number of other proteins have also been described to localize to the TJ. We have used a novel bait peptide method to investigate potential protein-protein interactions of the putative coiled-coil domain of occludin with some of these other TJ proteins. A 27-amino acid peptide of the human occludin sequence was synthesized, biotinylated at the N terminus, and modified to contain a photoactive moiety at either its hydrophobic or hydrophilic surface. These bait peptides were alpha-helical in solution, characteristic of coiled-coil structures. Photoactivation studies in the presence and absence of control peptides were used to assess the potential interactions in polarized sheets of a human intestinal cell line T84. Although a large number of proteins associated with the TJ or that are known to be involved in regulatory events of epithelial cells failed to be specifically labeled, occludin itself, ZO-1, protein kinase C-zeta, c-Yes, the regulatory subunit of phosphatidylinositol 3-kinase, and the gap junction component connexin 26 were specifically labeled. Our data demonstrate the potential of one specific domain of occludin, contained within 27 amino acids, to coordinate the binding of proteins that have been previously suggested to modulate TJ structure and function.
Highlights
Over the last decade a number of proteins have been identified that localize to the tight junction (TJ)1 structures of epithelial cells
Disruption of TJ function mediated by the constitutive activation of Raf-1 is associated with down-regulation of occludin and claudin-1 expression, an effect that can be reversed by the reintroduction of occludin expression that in turn restores claudin-1 protein levels [9]
The SH3, PSD-95, DlgA, and ZO-1-like, and proline-rich regions appear to be capable of interacting with other intracellular proteins, such as occludin, that localize to epithelial TJs
Summary
Peptide Bait Synthesis—Peptides (see Table I) were synthesized on an automated Pioneer Peptide Synthesizer (PE/ABI) with Fmoc-pro-. Standard one-letter amino acid codes are used except where F* indicates benzoyl phenylalanine and K* indicates lysine modified by N-hydroxysuccinimidyl-4-azidobenzoate used for photo-activated covalent attachment. All peptides were amidated at the C terminus. Control peptides included both peptides having photoactive residues but lacking biotin and peptides lacking photoactive residues and biotin
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