Abstract
Due to their altered metabolism cancer cells are more sensitive to proteasome inhibition or changes of copper levels than normal cells. Thus, the development of copper complexes endowed with proteasome inhibition features has emerged as a promising anticancer strategy. However, limited information is available about the exact mechanism by which copper inhibits proteasome. Here we show that Cu(II) ions simultaneously inhibit the three peptidase activities of isolated 20S proteasomes with potencies (IC50) in the micromolar range. Cu(II) ions, in cell-free conditions, neither catalyze red-ox reactions nor disrupt the assembly of the 20S proteasome but, rather, promote conformational changes associated to impaired channel gating. Notably, HeLa cells grown in a Cu(II)-supplemented medium exhibit decreased proteasome activity. This effect, however, was attenuated in the presence of an antioxidant. Our results suggest that if, on one hand, Cu(II)-inhibited 20S activities may be associated to conformational changes that favor the closed state of the core particle, on the other hand the complex effect induced by Cu(II) ions in cancer cells is the result of several concurring events including ROS-mediated proteasome flooding, and disassembly of the 26S proteasome into its 20S and 19S components.
Highlights
To address these issues, here we investigate the ability of Cu(II) ions to inhibit the peptidase activities, assembly and gating mechanisms of the 20S proteasome in cell-free conditions
Cu(II) ions inhibited all proteolytic activities with similar potencies in the micromolar range consistent with previous reports[27]
Reactive oxygen species (ROS) are present in HeLa cells grown in Cu(II)-supplemented media and concur
Summary
Here we investigate the ability of Cu(II) ions to inhibit the peptidase activities, assembly and gating mechanisms of the 20S proteasome in cell-free conditions. To further validate this hypothesis, we assayed the effect of Cu(II) ions on the ChT-L peptidase activity of a mutant proteasome (α3ΔN) where deletion of the first nine N terminal residues of the α3 subunit results in permanently open gate conformation[6]. To address this question we determined proteasome ChT-L activity in HeLa cells incubated for 24 h with different amounts of Cu(II) ions (ranging from 10 to 80 μM) (see Fig. 5 panel A, blue bars).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
