Fig 2: AF potently enhances E3 activity in vitro and accelerates ERAD in cells.a Effects of AF on E3 activity in in vitro E3 autoubiquitination assay. RBX complex: Elongin B/Elongin C/VHL/CUL2/RBX1. b IB of UBA1a (UBA1), UBA1b, and UBA1c used in the E3 activity assay in (c). UBA1a, UBA1b, and UBA1c translation start at methionine 1, 41, and 67, respectively. c In vitro gp78c autoubiquitination assay in the presence of UBA1a, UBA1b, and UBA1c, respectively, and the effects of AF. d AF decreased NHK protein levels in a dose-dependent manner. HeLa cells stably expressing NHK-HA were treated with AF for 24 h and then subject to IB. AF versus Control, p = 0.1482, p = 0.0065 and p = 0.0018 at 25, 50 and 100 µM, respectively. e AF-induced NHK downregulation is blocked by proteasome inhibitor, bortezomib (BTZ). AF versus Control, p = 0.0018. f AF increases NHK-HA ubiquitination in HeLa cells. Anti-HA reIP was performed to determine NHK-HA ubiquitination. g AF increases NHK dislocation in a dose-dependent manner. drGFP intensity was monitored and quantified in IncuCyte S3 Live-Cell Analysis System and expressed as mean ± S.D., n = 4 wells/treatment. **p < 0.01. AF versus Control, p = 0.0076, p = 0.0086, p = 0.008, p = 0.007 and p = 0.0079 at 3.125, 6.25, 12.5, 25 and 50 µM, respectively. p-values were calculated by two-tailed paired t-test. h AF decreased CD3d protein levels in a dose-dependent manner. AF versus Control, p = 0.2442, p = 0.0238 and p = 0.0052 at 25, 50 and 100 µM, respectively The graphs in (d, e, and h) show % increase for each condition relative to DMSO-treated control. Data are presented as mean values ± S.D., n = 3 biologically independent experiments. *p < 0.05, **p < 0.01. p-values were calculated by two-tailed paired t-test. Source data are provided as a Source Data file.

Fig 3: AF binds to the UFD of UBA1 and conjugates to cysteine 1039.a Effect of Iodoacetamide or DTT on the interaction of UBE2G2 with the UFD fragment of UBA1. b C1039A but not C1040A mutation diminishes AF-enhanced UBA1-UBE2G2 interaction in cells. HeLa cells transfected with the indicated plasmids were treated with 100 nM AF for 2 h, followed by processing for IP and IB. c C1039A but not C1040A mutation diminishes AF-enhanced MBP-UFD and UBE2G2 interaction in vitro. Recombinant MBP-UFD and its mutants were incubated with UBE2G2 along with different doses of AF. UBE2G2 bound to MBP-UFD, or its mutants was detected by IB. d Predicted binding complex of UBA1 with auranofin and E2G2. The binding interaction of the portion of Au-PEt3 of auranofin at the UFD domain is shown in a close-up panel. The protein structure of UBA1 (PDB 6DC6) and E2G2 (PDB 4LAD) are represented in ribbons. The gold Au (I) is coordinated to Cys1039 and rendered as balls in blue, the triethylphosphine ligand of auranofin in the pocket is shown in sticks. The domain structure of UBA1 is shown on top of the model. e Mutation of two predicted AF binding residues, E1037 and E1049, respectively, abrogates AF-enhanced UBA1-UBE2G2 interaction by coIP. Source data are provided as a Source Data file.

Fig 4: AF facilitates ubiquitin charging to E2s.The effects of AF on ubiquitin charging in vitro to UBA1 (a), UBE2G2 (b, c), and UBE2D1 (d). The assays were performed using purified recombinant proteins. 250 nM UBA1 alone or in combination with 4 µM His-E2s (His-UBE2G2 or His-UBE2D1) were treated with increasing concentrations of AF in reaction buffer for 1 min. ATP (50 µM) was added to initiate the reaction. The reactions proceeded at 15 oC for 45 s and stopped by adding non-reducing loading buffer. 5 mM DTT was used to disrupt the thioester bond that links ubiquitin to the active cysteine of E2s. Source data are provided as a Source Data file.

Fig 5: Auranofin (AF) binds to UBA1 and enhances UBA1 interaction with UBE2G2.a Chemical structure of AF. b, c AF enhances UBA1 interaction with UBE2G2 in cells. Total lysates from 293 T cells treated with 100 nM AF for 3 h were used for anti-UBA1 (a) or anti-UBE2G2 (b) co-immunoprecipitation (coIP) assays. Lane 3 is anti-IgG control. d Effects of AF on the thermal stability of UBA1 and AUP1 in UBE2G2 knockout 293 T cells as revealed by CETSA. The indicated proteins were blotted in soluble fractions. e, f The intensities of the bands for UBA1 and AUP1 in (d) were quantified. Data are presented as mean values ± S.D., n = 3 biologically independent experiments. p-value was calculated by two-tailed paired t-test. *p < 0.05 and **p < 0.01 for UBA1. p = 0.008, p = 0.0421 and p = 0.0285 at 47, 52 and 57 oC, respectively. No difference for AUP1. Source data are provided as a Source Data file.