Modulation of SCFβ-TrCP-dependent IκBα Ubiquitination by Hydrogen Peroxide

Sami Banerjee,Jaroslaw W Zmijewski,Emmanuel Lorne,Gang Liu,Yonggang Sha,Edward Abraham

doi:10.1074/jbc.m109.060822

Sami Banerjee, Jaroslaw W Zmijewski + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.060822

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Abstract

Reactive oxygen species are known to participate in the regulation of intracellular signaling pathways, including activation of NF-kappaB. Recent studies have indicated that increases in intracellular concentrations of hydrogen peroxide (H(2)O(2)) have anti-inflammatory effects in neutrophils, including inhibition of the degradation of I kappaB alpha after TLR4 engagement. In the present experiments, we found that culture of lipopolysaccharide-stimulated neutrophils and HEK 293 cells with H(2)O(2) resulted in diminished ubiquitination of I kappaB alpha and decreased SCF(beta-TrCP) ubiquitin ligase activity. Exposure of neutrophils or HEK 293 cells to H(2)O(2) was associated with reduced binding between phosphorylated I kappaB alpha and SCF(beta-TrCP) but no change in the composition of the SCF(beta-TrCP) complex. Lipopolysaccharide-induced SCF(beta-TrCP) ubiquitin ligase activity as well as binding of beta-TrCP to phosphorylated I kappaB alpha was decreased in the lungs of acatalasemic mice and mice treated with the catalase inhibitor aminotriazole, situations in which intracellular concentrations of H(2)O(2) are increased. Exposure to H(2)O(2) resulted in oxidative modification of cysteine residues in beta-TrCP. Cysteine 308 in Blade 1 of the beta-TrCP beta-propeller region was found to be required for maximal binding between beta-TrCP and phosphorylated I kappaB alpha. These findings suggest that the anti-inflammatory effects of H(2)O(2) may result from its ability to decrease ubiquitination as well as subsequent degradation of I kappaB alpha through inhibiting the association between I kappaB alpha and SCF(beta-TrCP).

Highlights

Chronic obstructive pulmonary disease, sepsis, and ischemiareperfusion injury, that are characterized by activation of neutrophils, macrophages, and other cell populations to release cytokines and other proinflammatory mediators, many of which are under the regulatory control of the transcription factor NF-␬B [2,3,4,5]
Inhibitory Effects of Hydrogen Peroxide on TLR4-induced I␬B␣ Degradation—In recent studies, we demonstrated that increased intracellular levels of H2O2 were associated with diminished degradation of I␬B␣ and reduced 26 S proteasomal activity in LPS-stimulated neutrophils [6, 8]
Because phosphorylation of I␬B␣ is required for its polyubiquitination and subsequent degradation by the 26 S proteasome, we hypothesized that a mechanism by which H2O2 might diminish TLR4-induced degradation of I␬B␣ was through inhibiting I␬B␣ phosphorylation

Summary

Introduction

Chronic obstructive pulmonary disease, sepsis, and ischemiareperfusion injury, that are characterized by activation of neutrophils, macrophages, and other cell populations to release cytokines and other proinflammatory mediators, many of which are under the regulatory control of the transcription factor NF-␬B [2,3,4,5]. A and B, exposure of neutrophils or 293-hTLR4/MD2-CD14 cells to LPS resulted in increased ubiquitination of I␬B␣, an effect that was enhanced by blockade of 26 S proteasomal activity following the addition of the proteasomal inhibitor MG132 to the cultures.

Results

Conclusion