Basic–Alimentary TractA membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease☆,☆☆,★
Section snippets
Peptide synthesis
Peptides were synthesized using an automated Pioneer Peptide Synthesizer (Applied Biosystems, Foster City, CA) on Fmoc-PEG-PS-resin. D-Biotin (Sigma, St. Louis, MO) was incorporated into peptides at the amino terminus using HBTU-HOBt/DIPEA in Me2SO. Peptide resins were cleaved with a 1-hour exposure of a 95% trifluoroacetic acid/2.5% triisopropylsilane/2.5% H2O solvent mixture. Released peptides were purified by preparative reverse-phase C18 high-performance liquid chromatography, characterized
A linear oligopeptide can inhibit Caco-2 MLC kinase in vitro
In confluent Caco-2 cell monolayers, the 215-kilodalton isoform of MLC kinase27 accounts for more than 95% of MLC kinase activity.16 This activity is critical to the regulation of myosin, because phosphorylation of MLC at serine 19 leads to increased actomyosin contraction. The activity of smooth-muscle MLC kinase is regulated by intramolecular interactions between the catalytic domain and the calmodulin-binding inhibitory domain. In the presence of Ca++ and calmodulin, this intramolecular
Discussion
Intestinal paracellular permeability is precisely regulated by the epithelial TJ.31, 32 Although the mechanisms by which TJ permeability is regulated are incompletely defined, MLC within the perijunctional actomyosin ring does appear to be a critical regulator of TJ permeability.17, 33 To better understand the details of actomyosin-dependent TJ regulation, we sought to identify a specific agent capable of inhibiting epithelial MLC kinase. We chose an oligopeptide derived from the inhibitory
Acknowledgements
The authors thank Sean P. Colgan for helpful discussions.
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Cited by (0)
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Address requests for reprints to: Jerrold R. Turner, M.D., Ph.D., The University of Chicago, 5841 South Maryland Avenue, MC 1089, Chicago, Illinois 60637. e-mail: [email protected].
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The current address for R. J. Mrsny is: Welsh School of Pharmacy, Cardiff University, Wales, United Kingdom.
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Supported by grants from the National Institutes of Health (DK61931 and DK56121 to J.R.T. and DK50694 to G.H.) and a merit award from the Department of Veterans Affairs (to G.H.).