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Shigella IpaH0722 E3 ubiquitin ligase effector targets TRAF2 to inhibit PKC-NF-κB activity in invaded epithelial cells.

Ashida H, Nakano H, Sasakawa C - PLoS Pathog. (2013)

Bottom Line: Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation.These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway.IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

View Article: PubMed Central - PubMed

Affiliation: Division of Bacterial Infection Biology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

ABSTRACT
NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

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IpaH0722 targets TRAF2 for ubiquitination.(A) In vitro ubiquitination assay using TRAF2 and a mixture of E1, UbcH5b, ATP, and ubiquitin in the presence or absence of IpaH0722 or IpaH0722CA. (B) 293T cells were transfected with FLAG-TRAF2 with and without Myc-IpaH0722 or Myc-IpaH0722CA. After 24 h, the cells were treated with CHX (50 mg/ml) and cell lysates were prepared at the indicated time points. (C) 293T cells were transfected with FLAG-TRAF2 with and without FLAG-IpaH0722 or FLAG-IpaH0722CA. After 24 h, the cells were treated with DMSO or CHX (50 mg/ml) plus MG132 or E64D+pepstatin A. Cell lysates were prepared after 3 h and samples were subjected to immunoblotting. (D) Traf2/Traf5−/− MEFs and Traf2/Traf5−/− MEFs stably expressing Traf2 were infected with Shigella WT or ΔipaH0722. Cell lysates were harvested at the indicated time point and subjected to immunoblotting with anti- IκBα Anti-actin antibody was used as a loading control. (E) A model of this study. Shigella invasion induced phagosome membrane rupture of epithelial cells, which stimulates DAG–PKC–NF-κB activation. However, Shigella deliver IpaH0722 to inhibit PKC–NF-κB activation by ubiquitinating TRAF2 and promoting its proteasome degradation.
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ppat-1003409-g006: IpaH0722 targets TRAF2 for ubiquitination.(A) In vitro ubiquitination assay using TRAF2 and a mixture of E1, UbcH5b, ATP, and ubiquitin in the presence or absence of IpaH0722 or IpaH0722CA. (B) 293T cells were transfected with FLAG-TRAF2 with and without Myc-IpaH0722 or Myc-IpaH0722CA. After 24 h, the cells were treated with CHX (50 mg/ml) and cell lysates were prepared at the indicated time points. (C) 293T cells were transfected with FLAG-TRAF2 with and without FLAG-IpaH0722 or FLAG-IpaH0722CA. After 24 h, the cells were treated with DMSO or CHX (50 mg/ml) plus MG132 or E64D+pepstatin A. Cell lysates were prepared after 3 h and samples were subjected to immunoblotting. (D) Traf2/Traf5−/− MEFs and Traf2/Traf5−/− MEFs stably expressing Traf2 were infected with Shigella WT or ΔipaH0722. Cell lysates were harvested at the indicated time point and subjected to immunoblotting with anti- IκBα Anti-actin antibody was used as a loading control. (E) A model of this study. Shigella invasion induced phagosome membrane rupture of epithelial cells, which stimulates DAG–PKC–NF-κB activation. However, Shigella deliver IpaH0722 to inhibit PKC–NF-κB activation by ubiquitinating TRAF2 and promoting its proteasome degradation.

Mentions: Since IpaH0722 inhibited NF-κB activation in an E3 ubiquitin ligase-dependent manner, we tested whether IpaH0722 ubiquitinated TRAF2 using an in vitro ubiquitination assay. TRAF2, IpaH0722, or IpaH0722CA (purified from E. coli) were combined with E1, ATP, UbcH5b (an E2 ubiqitin conjugating enzyme) in assay medium and subjected to immunoblotting. TRAF2 was ubiquitinated by IpaH0722 but not IpaH0722CA (Fig. 6A). To confirm the fate of ubiquitinated TRAF2, we measured the stability of TRAF2 in 293T cells ectopically expressing IpaH0722 or IpaH0722CA and treated with cycloheximide (CHX; a protein synthesis inhibitor) for 2, 4, and 6 h. TRAF2 degradation was faster in IpaH0722 expressing cells compared to IpaH0722CA expressing cells, which confirmed that IpaH0722 targeted TRAF2 for ubiquitin-mediated protein degradation (Fig. 6B). We also measured TRAF2 degradation in 293T cells that were co-transfected with TRAF2 plus IpaH0722 or IpaH0722CA, and treated with MG132 (a proteasome inhibitor) or E64D plus pepstain A (lysosome inhibitors). As shown in Fig. 6C, MG132 treatment prevented TRAF2 degradation in the presence of IpaH0722, whereas E64D plus pepstatin A treatment did not prevent TRAF2 degradation. These data confirm that IpaH0722 targets TRAF2 for ubiquitination and proteasomal degradation.


Shigella IpaH0722 E3 ubiquitin ligase effector targets TRAF2 to inhibit PKC-NF-κB activity in invaded epithelial cells.

Ashida H, Nakano H, Sasakawa C - PLoS Pathog. (2013)

IpaH0722 targets TRAF2 for ubiquitination.(A) In vitro ubiquitination assay using TRAF2 and a mixture of E1, UbcH5b, ATP, and ubiquitin in the presence or absence of IpaH0722 or IpaH0722CA. (B) 293T cells were transfected with FLAG-TRAF2 with and without Myc-IpaH0722 or Myc-IpaH0722CA. After 24 h, the cells were treated with CHX (50 mg/ml) and cell lysates were prepared at the indicated time points. (C) 293T cells were transfected with FLAG-TRAF2 with and without FLAG-IpaH0722 or FLAG-IpaH0722CA. After 24 h, the cells were treated with DMSO or CHX (50 mg/ml) plus MG132 or E64D+pepstatin A. Cell lysates were prepared after 3 h and samples were subjected to immunoblotting. (D) Traf2/Traf5−/− MEFs and Traf2/Traf5−/− MEFs stably expressing Traf2 were infected with Shigella WT or ΔipaH0722. Cell lysates were harvested at the indicated time point and subjected to immunoblotting with anti- IκBα Anti-actin antibody was used as a loading control. (E) A model of this study. Shigella invasion induced phagosome membrane rupture of epithelial cells, which stimulates DAG–PKC–NF-κB activation. However, Shigella deliver IpaH0722 to inhibit PKC–NF-κB activation by ubiquitinating TRAF2 and promoting its proteasome degradation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675035&req=5

ppat-1003409-g006: IpaH0722 targets TRAF2 for ubiquitination.(A) In vitro ubiquitination assay using TRAF2 and a mixture of E1, UbcH5b, ATP, and ubiquitin in the presence or absence of IpaH0722 or IpaH0722CA. (B) 293T cells were transfected with FLAG-TRAF2 with and without Myc-IpaH0722 or Myc-IpaH0722CA. After 24 h, the cells were treated with CHX (50 mg/ml) and cell lysates were prepared at the indicated time points. (C) 293T cells were transfected with FLAG-TRAF2 with and without FLAG-IpaH0722 or FLAG-IpaH0722CA. After 24 h, the cells were treated with DMSO or CHX (50 mg/ml) plus MG132 or E64D+pepstatin A. Cell lysates were prepared after 3 h and samples were subjected to immunoblotting. (D) Traf2/Traf5−/− MEFs and Traf2/Traf5−/− MEFs stably expressing Traf2 were infected with Shigella WT or ΔipaH0722. Cell lysates were harvested at the indicated time point and subjected to immunoblotting with anti- IκBα Anti-actin antibody was used as a loading control. (E) A model of this study. Shigella invasion induced phagosome membrane rupture of epithelial cells, which stimulates DAG–PKC–NF-κB activation. However, Shigella deliver IpaH0722 to inhibit PKC–NF-κB activation by ubiquitinating TRAF2 and promoting its proteasome degradation.
Mentions: Since IpaH0722 inhibited NF-κB activation in an E3 ubiquitin ligase-dependent manner, we tested whether IpaH0722 ubiquitinated TRAF2 using an in vitro ubiquitination assay. TRAF2, IpaH0722, or IpaH0722CA (purified from E. coli) were combined with E1, ATP, UbcH5b (an E2 ubiqitin conjugating enzyme) in assay medium and subjected to immunoblotting. TRAF2 was ubiquitinated by IpaH0722 but not IpaH0722CA (Fig. 6A). To confirm the fate of ubiquitinated TRAF2, we measured the stability of TRAF2 in 293T cells ectopically expressing IpaH0722 or IpaH0722CA and treated with cycloheximide (CHX; a protein synthesis inhibitor) for 2, 4, and 6 h. TRAF2 degradation was faster in IpaH0722 expressing cells compared to IpaH0722CA expressing cells, which confirmed that IpaH0722 targeted TRAF2 for ubiquitin-mediated protein degradation (Fig. 6B). We also measured TRAF2 degradation in 293T cells that were co-transfected with TRAF2 plus IpaH0722 or IpaH0722CA, and treated with MG132 (a proteasome inhibitor) or E64D plus pepstain A (lysosome inhibitors). As shown in Fig. 6C, MG132 treatment prevented TRAF2 degradation in the presence of IpaH0722, whereas E64D plus pepstatin A treatment did not prevent TRAF2 degradation. These data confirm that IpaH0722 targets TRAF2 for ubiquitination and proteasomal degradation.

Bottom Line: Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation.These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway.IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

View Article: PubMed Central - PubMed

Affiliation: Division of Bacterial Infection Biology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

ABSTRACT
NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

Show MeSH
Related in: MedlinePlus