Cingulin binds to the ZU5 domain of scaffolding protein ZO-1 to promote its extended conformation, stabilization, and tight junction accumulation.

Publisher: Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Country of Publication: United States NLM ID: 2985121R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1083-351X (Electronic) Linking ISSN: 00219258 NLM ISO Abbreviation: J Biol Chem Subsets: MEDLINE

Publication: 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology
Original Publication: Baltimore, MD : American Society for Biochemistry and Molecular Biology

Cytoskeletal Proteins*/chemistry ; Cytoskeletal Proteins*/genetics ; Cytoskeletal Proteins*/metabolism ; Tight Junctions*/metabolism ; Zonula Occludens-1 Protein*/chemistry ; Zonula Occludens-1 Protein*/genetics ; Zonula Occludens-1 Protein*/metabolism; Cytoskeleton/metabolism ; Gene Knockdown Techniques ; Phosphoproteins/genetics ; Phosphoproteins/metabolism ; Protein Domains ; Protein Folding ; Protein Stability ; Protein Structure, Quaternary

Zonula occludens-1 (ZO-1), the major scaffolding protein of tight junctions (TJs), recruits the cytoskeleton-associated proteins cingulin (CGN) and paracingulin (CGNL1) to TJs by binding to their N-terminal ZO-1 interaction motif. The conformation of ZO-1 can be either folded or extended, depending on cytoskeletal tension and intramolecular and intermolecular interactions, and only ZO-1 in the extended conformation recruits the transcription factor DbpA to TJs. However, the sequences of ZO-1 that interact with CGN and CGNL1 and the role of TJ proteins in ZO-1 TJ assembly are not known. Here, we used glutathione-S-transferase pulldowns and immunofluorescence microscopy to show that CGN and CGNL1 bind to the C-terminal ZU5 domain of ZO-1 and that this domain is required for CGN and CGNL1 recruitment to TJs and to phase-separated ZO-1 condensates in cells. We show that KO of CGN, but not CGNL1, results in decreased accumulation of ZO-1 at TJs. Furthermore, ZO-1 lacking the ZU5 domain showed decreased accumulation at TJs, was detectable along lateral contacts, had a higher mobile fraction than full-length ZO-1 by fluorescence recovery after photobleaching analysis, and had a folded conformation, as determined by structured illumination microscopy of its N-terminal and C-terminal ends. The CGN-ZU5 interaction promotes the extended conformation of ZO-1, since binding of the CGN-ZO-1 interaction motif region to ZO-1 resulted in its interaction with DbpA in cells and in vitro. Together, these results show that binding of CGN to the ZU5 domain of ZO-1 promotes ZO-1 stabilization and accumulation at TJs by promoting its extended conformation.
Competing Interests: Conflict of interest J. R. T. is a founder and shareholder of Thelium Therapeutics and has served as a consultant for Entrinsic, Immunic, and Kallyope. S. C. has served as a consultant for Immunic. All other authors declare that they have no conflicts of interest with the contents of this article.
(Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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R01 DK061931 United States DK NIDDK NIH HHS; R01 DK068271 United States DK NIDDK NIH HHS

Keywords: ZO-1; cingulin; paracingulin; tight junctions

0 (Cytoskeletal Proteins)
0 (Phosphoproteins)
0 (Zonula Occludens-1 Protein)

Date Created: 20220308 Date Completed: 20220426 Latest Revision: 20220716

20231215

PMC9010756

10.1016/j.jbc.2022.101797

35259394