(A) Computational molecular docking of Au+ with UCHL5 of 19S
proteasomes. The hydrolysed form of chloro (triethylphosphine) gold or Aur,
(triethylphosphine) gold cation (L2, left), and its binding mode at the active
site of UCHL5 were shown (right). (B) Effect of Aur on DUB activities in cell
lysate. Cell lysate was treated with Aur (2μM) or NEM (N-ethylmaleimide,
2 mM), then the DUB activity at different times was recorded by using the
Ub-AMC substrate. The experiment was repeated three times, yielding the similar
results. (C) Inhibition of the DUB activity in 26S proteasomes. Purified 26S
proteasomes were treated with increasing doses of Aur, then DUB activity was
kinetically detected as in (B). (D) NAC rescues Aur-mediated DUB inhibition.
Purified 26S proteasomes were treated with Aur (2 μM), Aur+NAC (100
μM), or NEM (2 mM) for 15 min, then DUB activity was detected. (E)
Ubiquitin chain disassembly assay. K48-linked ubiquitin tetramers were
disassembled by the 26S proteasomes after treatment with Aur (2.0, 40
μM). (F) Active-site–directed labeling of proteasomal DUBs. Purified 26S
proteasomes were treated with Aur (2.0, 40 μM) for 10 min and then
labeled with HA-UbVS and fractionated via SDS-PAGE. The covalently bound
HA-UbVS was detected by western blot for the HA tag. (G) The effect of 26S
proteasome disassembly by siRNA-mediated knockdown of RPN11 on Aur induced
Ub-prs accumulation. HepG2 cells were transfected with specific siRNA against
RPN11 for 48 h, and then treated with Aur (0.5, 1.0, 2.0 μM) for 6 h.
Scrambled siRNA was used as control. K48-linked polyubiquitin and RPN11 protein
was detected by western blot analyses. GAPDH was used as a loading control. (H)
GFPu accumulation with RPN11 siRNA silencing and Aur treatment. GFPu-HEK293
cells were transfected with control siRNA or RPN11 siRNA for 48 h, and then
treated with 0.5 μM Aur for 6 h. GFPu and RPN11 protein was detected by
western blot analyses.