Extended Data Fig. 3: Dap-containing proteases selectively capture known substrates of their parent enzymes.
From: Mechanism-based traps enable protease and hydrolase substrate discovery
(a) The change of AMC fluorescence resulting from cleavage from the C-terminus of Ubiquitin (Ub) or ubiquitin-like molecules (SUMO, NEDD8, ISG15) was followed upon mixing with UL36USP. WT UL36USP from human herpesvirus 1 specifically hydrolyses Ub-AMC, but not other ubiquitin-like protein-AMC (UBL-AMC) molecules. The deubiquitination activity of UL36USP is lost when Cys65 is mutated to Ser. This data is consistent with the previously reported specificity of UL36USP58. (b) UL36USP(C65Dap) specifically reacts with Ub-AMC to form the UL36USP(Dap)-Ub conjugate. In contrast, no conjugates were observed between UL36USP(Dap) and SUMO1, NEDD8 or ISG15. (c) WT SCoV2-PLpro selectively hydrolyses ISG15-AMC in preference to other Ub/UBL-AMC molecules; this data is consistent with the previously reported specificity of SCoV2-PLpro59. The hydrolysis of ISG15-AMC is abrogated when the catalytic Cys111 of SCoV2-PLpro is mutated to Ala. (d) SCoV2-PLpro(C111Dap) specifically reacts with ISG15-AMC, generating the SCoV2-PLpro(Dap)-ISG15 conjugate. *: UBL-AMC independent higher MW bands resulting from PLpro(C111Dap) self-reaction. (a) and (c) were generated using n = 3 independent measurements. The line represents the means of three measurements. (b) was repeated twice and (d) was performed in three biological replicates with similar results. For gel source data, see Supplementary Fig. 1.
