title: Identification of a Distinct Substrate-binding Domain in the Bacterial Cysteine Methyltransferase Effectors NleE and OspZ.
abstract: The type III secretion system effector protein NleE from enteropathogenic Escherichia coli plays a key role in the inhibition of NF-κB activation during infection. NleE inactivates the ubiquitin chain binding activity of host proteins TAK1-binding proteins 2 and 3 (TAB2 and TAB3) by modifying the Npl4 zinc finger domain through S-adenosyl methionine-dependent cysteine methylation. Using yeast two-hybrid protein interaction studies, we found that a conserved region between amino acids 34 and 52 of NleE, in particular the motif (49)GITR(52), was critical for TAB2 and TAB3 binding. NleE mutants lacking (49)GITR(52) were unable to methylate TAB3, and wild type NleE but not NleE(49AAAA52) where each of GITR was replaced with alanine restored the ability of an nleE mutant to inhibit IL-8 production during infection. Another NleE target, ZRANB3, also associated with NleE through the (49)GITR(52) motif. Ectopic expression of an N-terminal fragment of NleE (NleE(34-52)) in HeLa cells showed competitive inhibition of wild type NleE in the suppression of IL-8 secretion during enteropathogenic E. coli infection. Similar results were observed for the NleE homologue OspZ from Shigella flexneri 6 that also bound TAB3 through the (49)GITR(52) motif and decreased IL-8 transcription through modification of TAB3. In summary, we have identified a unique substrate-binding motif in NleE and OspZ that is required for the ability to inhibit the host inflammatory response.
authors.affiliation: From the Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: From the Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Mühlen
authors.affiliation: From the Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.
authors.forename: Clare V
authors.initials: CV
authors.lastname: Oates
authors.affiliation: From the Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia.
authors.forename: Jaclyn S
authors.initials: JS
authors.lastname: Pearson
authors.affiliation: From the Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria 3000, Australia hartland@unimelb.edu.au.
authors.forename: Elizabeth L
authors.identifier: 0000-0003-4254-2863
authors.initials: EL
authors.lastname: Hartland
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004405:Dysentery, Bacillary / Q000235:genetics / Q000378:metabolism*; D054308:Enteropathogenic Escherichia coli / Q000235:genetics / Q000378:metabolism*; D004927:Escherichia coli Infections / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012763:Shigella flexneri / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D029968:Escherichia coli Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526043:NleE protein, E coli; C410585:TAB2 protein, human; C481053:TAB3 protein, human; D037521:Virulence Factors; D004265:DNA Helicases; C555409:ZRANB3 protein, human
keywords: Escherichia coli (E. coli); NF-κB (NF-κB); infection; inflammation; substrate specificity