Inhibition of ubiquitin-proteasome pathway–mediated IκBα degradation by a naturally occurring antibacterial peptide
Youhe Gao, … , Alfred L. Goldberg, Michael Simons
Youhe Gao, … , Alfred L. Goldberg, Michael Simons
Published August 1, 2000
Citation Information: J Clin Invest. 2000;106(3):439-448. https://doi.org/10.1172/JCI9826.
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Article

Inhibition of ubiquitin-proteasome pathway–mediated IκBα degradation by a naturally occurring antibacterial peptide

Abstract

Induction of NF-κB–dependent gene expression plays an important role in a number of biological processes including inflammation and ischemia-reperfusion injury. However, few attempts aimed at selective regulation of this transcription factor have been successful. We report here that a naturally occurring antibacterial peptide PR39 reversibly binds to the α7 subunit of the 26S proteasome and blocks degradation of NF-κB inhibitor IκBα by the ubiquitin-proteasome pathway without affecting overall proteasome activity. IκBα phosphorylation and ubiquitination occur normally after PR39 treatment, and binding of valosin-containing proteins is not impaired. The inhibition of IκBα degradation abolishes induction of NF-κB–dependent gene expression in cell culture and in mouse models of acute pancreatitis and myocardial infarction, including upregulation of endothelial adhesion proteins VCAM-1 and ICAM-1. In the latter model, sustained infusion of PR39 peptide resulted in significant reduction of myocardial infarct size. PR39 and related peptides may provide novel means to regulate cellular function and to control of NF-κB–dependent gene expression for therapeutic purposes.

Authors

Youhe Gao, Stewart Lecker, Mark J. Post, Antti J. Hietaranta, Jian Li, Rudiger Volk, Min Li, Kaori Sato, Ashok K. Saluja, Michael L. Steer, Alfred L. Goldberg, Michael Simons

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Figure 5

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PR39 does not affect known steps in IκBα processing. (a) The effect of P...
PR39 does not affect known steps in IκBα processing. (a) The effect of PR39 on IκBα phosphorylation in cell culture. U937 cells cultured in the absence (–) or presence (+) of pretreatment with 10 μM PR39 were exposed to 1 ng/mL TNF-α; 45 minutes later the cells were lysed and subjected to SDS-PAGE and Western blotting with anti-IκBα Ab. Note increased amount of the typical-appearing phosphorylated IκBα band in PR39-treated cells. (b) The effect of PR39 on IκBα ubiquitination in vitro. In vitro ubiquitination of phosphorylated IκBα protein was carried out using crude HeLa-cell extract in the presence of control peptides (lanes 3 and 4), PR39 (lanes 1 and 2), or in the absence of both (lane 5). Note that the addition of either PR39 at two different concentrations or of a control (random) peptide failed to affect IκBα ubiquitination. At the same time, no ubiquitination of phosphorylation site IκBα mutants (lanes 6 and 7) or unphosphorylated IκBα (weight IκBα, lane 8) was detected. (c) The effect of PR39 on IκBα ubiquitination in cell culture. HA-Ub–expressing ECV304 cells were exposed to 1 ng/mL TNF-α in the presence (+) or absence (–) of 10 μM PR39 pretreatment; 45 minutes later the cells were lysed and subjected to IκBα immunoprecipitation followed by Western blotting of the pellet material with anti-HA antibody. Note multiple ubiquitinated IκBα intermediaries in PR39-treated, but not control cells. (d) PR39 does not inhibit IκBα-VCP binding. Wild-type ECV304 cells treated as described in c were subjected to immunoprecipitation with anti-IκBα antibody (IκBα-IP) followed by Western blotting with the anti–VCP-3 Ab. Note the presence of 90-kD VCP band in the presence or absence of PR39 treatment.