Copper(II) ions affect the gating dynamics of the 20S proteasome: a molecular and in cell study

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.

spectroscopic measurements were carried out by a UV Jasco J-670 spectrophotometer. UV spectra were recorded in a 350 -600 nm range with a 1nm step. Optical path was 1 cm.
Due to the presence of oxygen in the test tubes, Cu(II) ions may also catalyze the oxidation of cysteinyl thiols to produce S-S disulfide linkages with a resulting reduction of Cu(II) to Cu(I) 1 and formation of Cu(I) complexes. Hereby Cu(II) may induce the generation of reactive oxygen species (ROS) and convert the Cys into cystine. In a previous report the generation of Cu(I) in Cu(II)loaded CP samples was investigated by using bathocuproinedisulfonic acid sodium salt (BCS) according to a previously described method. (see Linder, et al Am. J. Clin. Nutr. 1998, 67, 965S;Xiao, et al. Int. J. Oncol. 2010, 37, 81). Indeed, it is known that Cu(I)-BCS complexes exhibit a specific absorption band at 480 nm which may be used to monitor the reduction of Cu(II) to Cu(I). Isolated 20S proteasome may promote the reduction of Cu(II) to Cu(I) species thus suggesting that the CP has an intrinsic reducing capacity. However, most of the commercially available 20S proteasome preparations, including those used in the reference study are delivered in a DTTenriched buffer which, due to its known reducing potential, may interfere with the assay. In order to ascertain if DTT may by itself promote the reduction of Cu(II) to Cu(I) we prepared a CP-free aqueous mimicking the DTT-enriched 20S proteasome solutions used in the UV studies ( Fig S5).
Our analysis on one hand confirmed the previously obtained results, but also evidenced that the UV band observed at 480 nm may be merely ascribed to the reducing effect of DTT. These data suggest that the hypothesized 20S-induced reduction of Cu(II) to Cu(I) is not confirmed in the experimental conditions here adopted. Furthermore, we have observed that BCS is also able to catalyze the formation of BCS-Cu(I) species even in the absence of any reducing agent. Therefore this assay cannot confirm the existence of Cu(I) species in CP assays.  Table 1 in the main main text. IC 50 values for the distinct peptidase activities and the related fitting parameters are reported in Table S1.

Table S1
Data fitting (see Figure S2) relative to the evaluation of the IC 50 values of Cu(II) ions for ChT-L, T-L and C-L peptidase activity of the CP measured in the presence and in the absence of 0.018% SDS. Curve fitting was performed by using equation 1.

20S peptidase activity
ChT-L T-L C-L  The solution containing the free peptide was filtered off from the resin and concentrated in vacuo at 30 °C. The peptide was precipitated with cold freshly distilled diethyl ether, then filtered and dried under vacuum. The resulting crude peptide was purified by RP-HPLC and characterized by MALDI-TOF MS. Analytical Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) analyses were performed using a Zorbax Eclipse XDB-C184.6x150 mm (5µm particle size) column at a flow rate of 1 ml/min. While, preparative RP-HPLC was carried out by means of Varian PrepStar 200 model SD-1 chromatography system equipped with a Prostar photodiode array detector on a Vydac C18 250x22 mm (300 Å pore size, 10-15 µm particle size) column at flow rate of 10 mL/min. Detection in both cases was at 222 nm. HPLC eluents were A: 0.1% TFA/water and B: 0.1% TFA/acetonitrile. The peptide sample was analysed using the following gradient elution with solvents A and B: Minutes 0,5,15,20,30,35 with % CH 3 CN 5,5,30,30,5,5,respectively. The molecular weight of peptide TED is 3084 Da. The yield of the synthesis was 60 %. The obtained crude TED peptide was purified by RP-HPLC and characterized by MALDI-TOF MS.
Figures S1 and S2 show respectively the chromatograms relatives to the crude and purified peptide.