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  • Disclosure br Introduction The proteasome inhibitor PI Borte

    2018-11-13

    Disclosure
    Introduction The proteasome inhibitor (PI) Bortezomib is used as a first- and second-line treatment of multiple myeloma (MM) (Anderson et al., 2011). Proteasomes\' (Finley, 2009; Lander et al., 2012) main function is to degrade ubiquitinated proteins in a controlled manner (Bedford et al., 2011; Finley, 2009; Glickman and Ciechanover, 2002). Proteasomes are comprised of a cylindrical core particle (CP) capped at each end by a regulatory particle (RP) (Lander et al., 2012; He et al., 2012). The RP captures and denatures ubiquitin-marked protein substrates, and translocates their unfolded polypeptide chains towards proteolytic active-sites in the CP\'s lumen (Finley, 2009). CP contains three types of active-sites, each of which comprises a peptide-docking area and an exposed catalytic threonine. PIs including Bortezomib prevent protein hydrolysis by forming covalent adducts with the catalytic threonines of the active-sites (Groll et al., 2009; Beck et al., 2012). Some proteasome activity is necessary for any cell to live (Heinemeyer et al., 1997), not just MM herpes simplex virus 1 (Craxton et al., 2012; Suraweera et al., 2012). Although the Bortezomib concentrations at which cells of different cancers die differ widely, from low nanomolar to high micromolar IC50 concentrations, cells of the (incurable) B-cell malignancy MM are exquisitely sensitive (Shabaneh et al., 2013), hence Bortezomib\'s success in treatment of MM (Anderson et al., 2011). Intriguingly, Bortezomib at its low IC50 concentration causes only a small reduction in proteasomes\' ability to degrade proteins (Kisselev et al., 2006; Shabaneh et al., 2013). The reduction is small because Bortezomib preferentially inhibits the chymotrypsin-like (CT-Like) active-site, but – at IC50 – does not inhibit the caspase-like and trypsin-like active-sites (Fig. S2A) (Kisselev et al., 2006); however, protein substrates can be hydrolysed by any of the three types of active-sites (Kessler et al., 2001; Kisselev et al., 2006). Thus, the question arises why minimal inhibition of proteasome function suffices to induce apoptosis in MM but not in Bortezomib-insensitive cells. Several explanations have been proposed, including high proteasome workload in MM cells (Bianchi et al., 2009; Meister et al., 2007; Shabaneh et al., 2013). We report that, surprisingly, a (low) IC50 Bortezomib challenge – which in-vitro minimally inhibits proteasomes – in living MM cells severely inhibits proteasomes\' hydrolytic activity. Our data suggest that, in living MM cells, a Bortezomib-induced structural change in the proteasome (Pitcher et al., 2014) is responsible for this severe degree of proteasome inhibition.
    Experimental procedures Antibodies from Enzo Life Sciences: α-ubiquitin (FK2, PW8810), α-cleaved caspase 3 (PAb, ADI-AAS-103), α-20S α7 (MoAb LN43, BML-PW8110), α-Rpn12/S14 (PAb, BML-PW8815), α-Rpn10/S5a (MoAb S5a-18, BML-PW9250), α-Rpt5/S6a (MoAb TBP1-19, PW8770), α-Rpt4/S10b (MoAb p42-23, PW8830), α-Rpt2/S4 (PAb, BML-PW8305), and α-β5i/LMP7 (PAb, PW8355). Antibodies from other sources: α-streptag (Qiagen, MoAb 34850), α-PARP (CellSignalling, PAb, #9542) and α-procaspase3 (CellSignalling, PAb, #9661). CTAB-PAGE as described (Pitcher et al., 2014; Simpson, 2010). Additional Reagents: Ada-K(Biotin)-Ahx3-L3-VS, epoxomicin (Enzo LifeSciences), Bortezomib (Millennium/Takeda), Streptactin resin (Qiagen, 30004), Ni++NTA resin (Sigma-Aldrich, His Select HF Nickel Affinity Gel, H0537 (Fig. 2c), or His Select agarose, P2266). AnnexinV/7AAD apoptosis staining (EBioscience 88-8007-74) was used to assess cell viability. Enzymes: DNase1, micrococcal nuclease, RNaseA/T1, RNaseH, S1 nuclease and RNAse1 (Fermentas/Thermo), PDE1 from Crotalus adamanteus venom (Sigma, P3243-1VL). Caspase Inhibitor Set III (Enzo Life Sciences, ALX-850-227-KI01), used at 1:500 dilution=4μM. Antibodies were diluted in PBS+0.5% Tween20 for Western blotting, PVDF membranes were re-probed multiple times (Yeung and Stanley, 2009). Myeloma cell lines NCI-H929, KMS12-BM, RPMI-8226, OPM2, and the T-lymphocyte Jurkat cell line, were grown in RPMI (Sigma), supplemented with 10% v/v FBS, and 1% v/v Pen/strep. Cell fractionation procedure as described (Pitcher et al., 2014). For fractionation, a cytosol extraction (CE) buffer (25mM Tris–HCl [pH7.8], 5mM MgCl2/EDTA, 1mM ATP/ADP, 2mM DTT, 150mM NaCl, 0.1 or 0.5% NP-40/IGEPAL) and nuclear extraction (NE) buffer (Bakondi et al., 2011) (20mM HEPES [pH7.4], 420mM NaCl, 0.5mM EDTA, 0.5mM EGTA, 1mM DTT, 1 tablet of inhibitor cocktail (Roche, 11-873-580-001) per 50ml) were used. When using the NE for subsequent affinity-purification, 2 volumes of H2O were added to 1 volume NE to reduce the NaCl concentration to physiological levels. For non-fractionated lysate, cell pellets snap-frozen in LN and stored at −80°C were resuspended and combined in 10× volume of CE buffer and passed through a NanoDeBEE (BEE international) homogeniser at 19,000 PSI. Proteasome activity was measured basically as described (Kisselev and Goldberg, 2005; Vilchez et al., 2012), using fluorogenic proteasome substrates Suc-LLVY-AMC, Ac-RLR-AMC, Ac-GPLD-AMC (Enzo Life Sciences) for the three types of active-site. Cells were lysed in proteasome activity assay buffer (50mM Tris–HCl, pH7.5, 250mM sucrose, 5mM MgCl2, 0.5mM EDTA, 2mM ATP and 1mM DTT) by passing cells through a 29G needle ten times. Fluorescence (380nm excitation, 460nm emission) was monitored continuously on a microplate fluorometer for 1h at 37°C. To measure for the presence of free 20S proteasomes, 0.015% SDS was added to the proteasome activity assay buffer (Fig. S2C). For each well, a second was set up with the addition of 40mM Bortezomib; any activity in the Bortezomib wells was subtracted from corresponding wells to compensate for unspecific protease activity. The ubiquitinated substrate (G3P) (Matyskiela et al., 2013) was kindly provided by Dr Andreas Martin, and used as described. In-vitro degradation with purified yeast (Fig. S4) and human (Fig. 3C,D) proteasomes was done in PBS supplemented with 2.5mM ATP, 2.5mM MgCl2, 2.5mM DTT, and 10% DMSO in a total volume of 20μl. Reaction was performed for 1h either at 30°C or 37°C (Fig. 3C,D), after which the reaction was stopped by addition of 20μl 2× SDS sample buffer and boiled.