Figure 5
From: Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system
Binding of compounds to USP14. (a) In silico docking of different compounds to USP14 (left) and structure of the catalytic domain USP14 (PDB-id: 2AYO) in complex with ubiquitin aldehyde (right). Residues included in the catalytic triad are indicated in orange with the sulfur from Cys114 colored yellow, tryptophans in green and the blocking loops (BL1 and BL2) in purple. (b) Plot of Tm from thermal unfolding screen of full-length USP14 and its catalytic domain with 10 hit compounds analyzed with the barycentric mean and integrated intensity. (c) Dilution recovery of DUB activity. 19S proteasomes were exposed to DMSO, 1 μM or 20 μM for 20 min at 37 °C and followed by analysis of Ub-rhodamine cleavage activity. Samples were diluted 20-fold where indicated (20 μM - > 1 μM); *p < 0.05, ***p < 0.005. (d) MALDI-TOF analysis of incubation of USP14 with hit compounds. Recombinant USP14 (20 μg) was incubated with 20 μM compound for 1 hour at 37 °C and analysed by MALDI-TOF. Shifts in molecular masses were calculated (n = 3, mean + S.E.M.). Horisontal bars show the shifts in mass expected from binding of one molecule. (e) thermostability of UPS14 analysed by CETSA (cellular thermal shift assay)46. Statistic significant was calculated by matched pairs t-test (n = 6).
