NleC cleaves p65 by its zinc protease domain.

(A) Activity of purified GST-NleC protein. For lysates, 1×10⁶ of unstimulated HeLa cells were lysed by NET150 (10 mM Tris-Cl pH 8.0, 0.5% Triton-X100, 150 mM NaCl, 10% glycerol) without EDTA or protease inhibitors. GST protein or GST-NleC protein expressed in bacteria was purified and mixed with the HeLa lysates. The reaction mixtures were incubated at 25°C for 8 hours and then analyzed with antibodies against p65 (N-terminal or C-terminal specific) and α-tubulin (as a loading control). (B) Cleavage of p65 by NleC alone. p65, GST, and GST-NleC were expressed in bacteria and purified. The p65 was then mixed with either GST or GST-NleC in the reaction buffer. The mixtures were incubated at 25°C for 8 hours and analyzed with anti-p65 (N-term.) and anti-p65 (C-term.) antibodies. (C) Zinc protease motif in NleC. Schematic diagram showing the zinc protease domain in NleC. A Histidine (H) at 187 was replaced with Tyrosine (Y) by site-directed mutagenesis. (D) NleC's cleavage activity required the zinc protease motif and divalent metal ions. p65, GST, GST-NleC wild type (GST-NleC), and GST-NleC mutant (GST-NleCmut) were expressed in bacteria and purified. The p65 was mixed with GST, GST-NleC, or GST-NleCmut in the reaction buffer. The cleavage reaction was performed at 25°C for 8 hours. Samples were analyzed with anti-GST and anti-p65 antibodies against either the N- or C-terminal region. For the EDTA inhibition, EDTA was included in the reaction buffer at a final concentration of 10 mM. (E) NF-κB activity in cells infected with the nleCmut-expressing strain. Two days prior to infection, HeLa cells were transfected with NF-κB and luciferase reporter plasmids. Forty-eight hours post-transfection, cells were infected with TOB02/HA, TOB02/nleC-HA (nleC), or TOB02/nleCmut-HA (nleCmut) for 3 hours. Cells were rinsed and incubated in fresh DMEM containing 0.1 mg/ml gentamicin and HKE (10⁸/ml). Cells were stimulated for 8 hours, then the medium and cell lysates were analyzed for NF-κB SEAP and Luciferase reporter activities. Non-infected/non-stimulated (N.I/N.S) and non-infected/stimulated (N.S/S) HeLa cells served as negative and positive controls, respectively.