Cysteine Methylation Disrupts Ubiquitin-chain Sensing In NF-κB Activation

NF-κB pathway is crucial for innate immune defense against bacterial infection and many pathogens attenuate host NF-κB pathway to benefit their survival. When host cells are chanllenged by bacterial infection, Toll-like receptors would recongnize pathogen associated molecular patterns (PAMPs) from bacteria and lead to downstream NF-κB activation, which would initiate the transcription of genes responsible for anti-bacteria denfense. Meanwhile, many pathogens secret toxins or effectors into host cells to manipulate host cellular functions for their own goods. It was reported a type III secretion system-delivered effector called NleE from enteropathogenic Escherichia coli (EPEC) was mainly responsible for NF-κB inhibition during infection through an unknown mechanism. NleE is highly conserved in EPEC, enterohemorrhage Escherichia coli (EHEC), Shigella and some Salmonella species. Therefore the studies into the mechanism of NleE inhibiting host NF-κB pathway might shed light on the general mechanism adopted by enterobacteria to counteract host immune defense.According to our biochemical experiments, we found that NleE inhibited TAK1kinase activation to block NF-kB pathway. NleE showed specific interaction with TAB3in yeast two hybrid system and could be co-immunoprecipitated by both TAB2and TAB3(TAB2/3), suggesting that NleE targeted two homologous proteins TAB2and TAB3in TAK1complex. The major function of TAB2/3is to form complex with TAK1and TAB1and then recruit TAKl complex to ubiquitin chains through their C-terminal Np14-like zinc finger (NZF) domains, thus TAKl would be activated through oligmerization-mediated autophosphorylation. We found that NleE diminished ubiquitin-chain binding of full length TAB2/3as well as TAB2/3-NZF domains (truncated proteins only comprising the NZF domain) alone, indicating NleE targets NZF domains of TAB2/3. Further biochemical studies revealed that NleE was an unprecedented S-adenosyl-L-methionine-dependent methyltransferase that specifically methylates one of the four zinc-coordinating cysteines in the NZF domains of TAB2/3, namely Cys673in TAB2and Cys692in TBA3. Cysteine-methylated TAB2/3-NZF lost the zinc ion as well as the ubiquitin-chain binding activity. Ectopically expressed or type Ⅲ secretion system-delivered NleE methylated TAB2/3in host cells and diminished their ubiquitin-chain binding activity. Replacement of the NZF domain of TAB3with the NleE methylation-insensitive Np14-NZF domain resulted in NleE-resistant NF-κB activation. Therefore, our work not only elucidated the NF-κB inhibiting mechanism of NleE by cysteine methylation of TAB2/3, but also identified enzyme-catalyzed cysteine methylation as a mechanism of regulating signal transduction for the first time and further indicated the possibility of modulation of zinc finger motif-containing proteins by methylation in eukaryotic cells. In addition to TAB2/3, NleE also targets ZRANB3, a protein recently reported to be involved in maintaining genome integrity, for cysteine methylation in the NZF domain both in vivo and in vitro. NleE-catalyzed methylation of ZRANB3also disrupts its ubiquitin-chain binding as it does to TAB2/3. Given the prevalence of zinc-finger motifs and activation of cysteine thiol by zinc binding, our findings strongly suggests that methylation of zinc-finger cysteine might regulate other eukaryotic pathways in addition to NF-κB signalling.Our lab also solved the crystal structure of NleE in complex with SAM. The structure of NleE represents a novel class of methyltransferase which differs greatly from the known SAM-dependent methyltransferases. We also identified critical residues in NleE that contribute to the methyltransferase acitivity and their mutation would lead to loss of cysteine methyltransferase activity.