Fig. 3: Mpro cleaves NEMO.
a, SARS-CoV-2 Mpro in increasing concentrations (0, 5, 10 and 25 µM; 120 min) degraded full-length human NEMO (fused to GST) while several cleavage products emerged (representative of at least six experiments at different conditions). The full immunoblots are shown in Extended Data Fig. 6. b, Mouse NEMO in bEnd.3 cell extracts was cleaved to a short fragment after incubation with increasing concentrations of Mpro (0, 5 and 10 µM) for 120 min (representative of three experiments). c, In human brain endothelial hCMEC/D3 cells, Mpro-HA degraded NEMO-2A. After transfecting the cells with pCAG-NEMO-2A-GFP ± pCAG-Mpro-HA, immunoblots of cell lysates were performed (representative of at least nine experiments). d, Tryptic digestion and tandem mass spectrometry (MS/MS) analysis identified five Mpro cleavage sites in human NEMO as illustrated in the schematic. For the protein sequence, see Supplementary Fig. 2k. e, Mpro cleaved human NEMO at Q231. An extracted ion chromatogram of the tryptic peptide 227LAQLQ231 (m/z, 572.34142+; retention time (RT), 11.6 min) derived from NEMO after incubation with Mpro (5 µM, inset) and the MS/MS spectrum that was used for peptide identification are shown. The experiment was performed once. f, A synthetic peptide corresponding to the human NEMO sequence confirmed that Q231 is an Mpro cleavage site. Total ion chromatograms after incubation of the synthetic peptide h-NEMO_222-241 (EEKRKLAQLQVAYHQLFQEY) in the presence or absence of Mpro (2.5 µM, inset) are shown. In the presence of Mpro, the proteolysis product 222EEKRKLAQLQ231 (m/z, 414.91093+; RT, 5.1 min) was detected. The mutant peptide h-NEMO-Q231A_222-241 (EEKRKLAQLAVAYHQLFQEY) was not cleaved by Mpro (inset). The MS/MS spectrum of the peptide 222EEKRKLAQLQ231 is shown (representative of five experiments with the synthetic peptide h-NEMO_222-241 and four experiments with the mutant peptide h-NEMO-Q231A_222-241).
