Fig 1: Characterization of 3CLpro cleavage specificity(A and B) MALDI-TOF-MS spectra of synthetic peptides spanning P4–P4’ of protein cleavage sites after incubation with 3CLpro (1:20 molar ratio, E:S). Product generation (red) and substrate consumption (black) were calculated as the peak area normalized to the total peak area in the spectrum. Apparent (app) kcat/KM values for 1 μM 3CLpro to convert 50% of substrate in 5, 15, 30, 60, 120 or 240 min are listed alongside bins of 4 peptides that share similar kinetic values arranged on a row-by-row basis. P4–P4’ sequence alignment using the Shapley color scale. Green protein names had cut sites identified by Edman sequencing of recombinant substrate digests. Boxed peptides, no cleavage.(C–I) Structures of the highest-ranked of 50,000 models of the active site of 3CLpro protomer 1 (PDB: 6XHM) docked with P4–P4’ peptides from six 3CLpro substrates exhibiting a range of appkcat/KM values (circled in B). (C) 3CLpro dimer. Protomer 1, gray surface or green ribbons with catalytic Cys145 shown. Protomer 2, orange surface with Ser1 shown. Docking models with P4–P4’ peptide of: (C and D) RPAP1 (I_sc = −31.7), (E) IMA4 (I_sc = −30.6), (F) PTBP1 (I_sc = −31.7), (G) RBM15 (I_sc = −39.65), (H) MAP4K5 (I_sc = −28.1), and (I) CREB1 (I_sc = −30.3). Blue and red sticks, P and P’ amino acid residues, respectively. Yellow dashed sticks, hydrogen-bonds.See also Figure S2.
Fig 2: Hippo pathway substrate validation(A) 3CLpro cleavage sites in MAP4K5 and CREB1 identified by TAILS neo-N-terminal peptides (red) and Edman sequencing (green). Representative MS/MS spectra of cleaved neo-N-terminal peptides.(B) MALDI-TOF-MS kinetic analyses of 3CLpro cleavage of P4–P4’ peptides of MAP4K5 and CREB1.(C) SDS-PAGE, Edman sequencing (green) and immunoblot validation of human MAP4K5 and CREB1 substrates incubated with 3CLpro+/− inhibitor GC376, or 3CLpro-C145A (1:5 mol/mol, E:S). ΔMAP4K5 or ΔCREB1, no sequence obtained.(D and H) (D) YAP1, MAP4K5, CREB1 and (H) FYCO1 and FAF1 immunoblots of lysates from primary HAECs incubated with 3CLpro or 3CLpro-C145A (1:200 w/w, E:S) for 18 h, 37°C.(E and F) (E) YAP1 and (F) MAP4K5 immunoblots of Vero E6 cells at 24 (n = 4) and 48 (n = 4) hpi (MOI 0.1) or mock (n = 3). MAP4K5 activity assay measured as ATP consumption using myelin basic protein as substrate. The area under the curve was calculated and compared by Student’s t test, (mean ± SD, n = 2, ∗∗p ≤ 0.01).(G, I, and J) (G) Lysates of human Calu-3 lung cells infected with SARS-CoV-2 (MOI 0.1 and 1.0, n = 4, mock n = 2) were immunoblotted for (G) CREB1 48 hpi, (I) FYCO1 24 hpi and (J) FAF1 48 hpi. Statistical analysis of the relative amount of full-length protein (E and F) or proteolytic bands (G, I, and J) identified by molecular weights relative to β-actin was assessed by one-way ANOVA and Dunnett’s multiple comparisons test. Box and whiskers (min to max) plots, ∗∗∗p ≤ 0.001, ∗∗p ≤ 0.01, ∗p ≤ 0.05, ns p > 0.05. β-actin and β-tubulin loading controls.See also Figures S3 and S4.
Supplier Page from Sino Biological, Inc. for Human MAP4K5 / MEKKK5 Protein (His & GST Tag)