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Cysteine methylation disrupts ubiquitin-chain sensing in NF-κB activation

Abstract

NF-κB is crucial for innate immune defence against microbial infection1,2. Inhibition of NF-κB signalling has been observed with various bacterial infections3,4. The NF-κB pathway critically requires multiple ubiquitin-chain signals of different natures5,6. The question of whether ubiquitin-chain signalling and its specificity in NF-κB activation are regulated during infection, and how this regulation takes place, has not been explored. Here we show that human TAB2 and TAB3, ubiquitin-chain sensory proteins involved in NF-κB signalling, are directly inactivated by enteropathogenic Escherichia coli NleE, a conserved bacterial type-III-secreted effector responsible for blocking host NF-κB signalling. NleE harboured an unprecedented S-adenosyl-l-methionine-dependent methyltransferase activity that specifically modified a zinc-coordinating cysteine in the Npl4 zinc finger (NZF) domains in TAB2 and TAB3. Cysteine-methylated TAB2-NZF and TAB3-NZF (truncated proteins only comprising the NZF domain) lost the zinc ion as well as the ubiquitin-chain binding activity. Ectopically expressed or type-III-secretion-system-delivered NleE methylated TAB2 and TAB3 in host cells and diminished their ubiquitin-chain binding activity. Replacement of the NZF domain of TAB3 with the NleE methylation-insensitive Npl4 NZF domain resulted in NleE-resistant NF-κB activation. Given the prevalence of zinc-finger motifs and activation of cysteine thiol by zinc binding, methylation of zinc-finger cysteine might regulate other eukaryotic pathways in addition to NF-κB signalling.

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Figure 1: NleE blocks NF-κB signalling downstream of TRAFs and upstream of TAK1.
Figure 2: NleE directly targets TAB2 and TAB3 and impairs their ubiquitin-chain binding activity.
Figure 3: NleE is a SAM-dependent methyltransferase that specifically modifies Cys 673/692 in TAB2/3-NZF domains.
Figure 4: Cysteine methylation-induced loss of ubiquitin-chain binding of TAB2/3 contributes to NleE inhibition of host NF-κB signalling.

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Acknowledgements

We thank I. Rosenshine for providing NleE deletion strains, K. Iwai for the HOIL-1L and HOIP expression plasmids, H. Sakurai for the phospho-TAK1 antibody, Z. Chen for TAB2/3 and Npl4-NZF chimera constructs, and S. Fukai for the NZF expression plasmid. We also thank members of the Shao laboratory for helpful discussions and technical assistance. This work was supported by the National Basic Research Program of China (973 Programs, 2010CB835400 and 2012CB518700).

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L.Z. performed the majority of the experiments, assisted by J.C., H.X. and Y.-N.G.; X.D. and S.C. performed mass spectrometry analysis and analysed the data. J.C., L.H., Y. Z., J.G., Q.L. and L.L. generated reagents. L.Z. and F.S. analysed the data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Feng Shao.

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The authors declare no competing financial interests.

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Zhang, L., Ding, X., Cui, J. et al. Cysteine methylation disrupts ubiquitin-chain sensing in NF-κB activation. Nature 481, 204–208 (2012). https://doi.org/10.1038/nature10690

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