Identification of TRAF6 as a ubiquitin ligase engaged in the ubiquitination of SopB, a virulence effector protein secreted by Salmonella typhimurium.

Abstract

The phosphoinositide phosphatase SopB is one of the effectors injected by Salmonella typhimurium (S.typhimurium) that diversifies its function through a ubiquitin-dependent differential localization. However, it is unclear which E3 ubiquitin ligase is responsible for ubiquitination of SopB. Based on the E1-E2-E3 trio of enzymes responsible for the ubiquitin activation and translocation to substrate proteins, we constructed an in vitro assay of SopB ubiquitination. Using this assay, we purified an E3 ubiquitin ligase, TRAF6, from the Henle-407 S100 extraction that may be responsible for the ubiquitination of SopB. To investigate the functional correlation of TRAF6, we showed that recombinant TRAF6 specifically ubiquitinates SopB in a dose-dependent manner in vitro. Upon infection, the ubiquitination of SopB was absolutely blocked by TRAF6 deletion, as shown in Traf6(-/-) mouse embryonic fibroblasts (MEFs) compared with Traf6(+/+) MEFs. However, the ectopic expression of TRAF6 in Traf6(-/-) MEFs rescued the two species of ubiquitin-conjugated SopB, which strengthens the role of TRAF6 in SopB ubiquitination. The analysis of E2 revealed that UbcH5c and not other E2 conjugating enzymes are required for TRAF6-mediated SopB ubiquitination both in vitro and in vivo. In summary, these results suggest the relevance of UbcH5c/TRAF6 in SopB during S.typhimurium infection and thereby imply that S.typhimurium has evolved a mechanism of utilizing the host's E3 ubiquitin ligase to modify and modulate the function of its effector protein in order to ensure pathogen and host cell survival.

• Authors: Hai-Hua Ruan, Ye Li, Xi-Xuan Zhang, Qiang Liu, He Ren, Kun-Sheng Zhang, Hui Zhao
• Journal: Biochemical and biophysical research communications (Biochem Biophys Res Commun) Vol. 447 Issue 1 Pg. 172-7 (Apr 25 2014) ISSN: 1090-2104 [Electronic] United States
• PMID: 24704445 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier Inc. All rights reserved.

Chemical References

• Bacterial Proteins
• TNF Receptor-Associated Factor 6
• UBE2D3 protein, mouse
• Ubiquitin-Conjugating Enzymes
• Ubiquitin-Protein Ligases
• SopB protein, Salmonella

Topics

• Animals
• Bacterial Proteins (metabolism)
• Cells, Cultured
• Fibroblasts
• Mice
• Salmonella typhimurium (genetics)
• TNF Receptor-Associated Factor 6 (genetics, metabolism)
• Ubiquitin-Conjugating Enzymes (metabolism)
• Ubiquitin-Protein Ligases (genetics, metabolism)
• Ubiquitination
• Virulence (genetics)