In order to test directly if

In order to test directly if changes in these proteasome modifications affect proteasome function, we first searched for commercial enzyme preparations that can digest the modifications of nuclear proteasomes. We discovered that a combination of venom phosphodiesterase-1 and S1 nuclease was efficient in trimming these modifications and collapsing modified Rpt2 read this post here into its correct subunit-size species (Fig. 3A). Next, we combined this enzyme protocol with an in-vitro proteasome-mediated proteolysis assay, which uses a ubiquitinated model protein as substrate (Matyskiela et al., 2013) (Figs. 3B, S4). We scaled up cell growth and affinity-purified proteasomes from total (i.e. including nuclear) lysate. Proteasomes bound to affinity-resin were mock-treated or treated with PDE1/S1 at a sub-optimal (20°C) temperature, after which resin was washed to remove enzymes. Next, proteasomes were eluted to generate a control and a PDE1/S1-treated proteasome preparation. Most subunits of PDE1/S1-treated versus untreated proteasomes were very similar, but for example Rpn12 showed pronounced differences (Fig. S3). We then incubated the proteasome preparations with a ubiquitinated model protein (Matyskiela et al., 2013) (Fig. S4) in order to assess the ability of these proteasome preparations to process a ubiquitinated substrate. At low proteasome/substrate ratio at 30°C, the PDE1/S1-treated proteasomes were impaired in degrading ubiquitinated substrate (Fig. 3C). However, increasing the ratio overcame this defect, with both preparations degrading substrate equivalently. At even higher proteasome/substrate ratio, and at 37°C, proteasomes processed the substrate towards deubiquitination rather than degradation, and again both preparations behaved equivalently (Fig. 3B). In addition, we found that changes in the redox state of the reaction conditions uncovered qualitative differences between enzyme-treated and untreated proteasomes (Fig. 3D): in the oxidizing conditions minus DTT, enzyme-treated proteasomes removed streptag epitope (i.e. substrate) more efficiently than untreated proteasomes but did not shift down the ubiquitin signal correspondingly, indicating that these enzyme-treated proteasomes only partially digested the substrate protein – starting from the tagged carboxyterminus – before premature release. In contrast, under reducing conditions, untreated proteasomes were more efficient in degrading substrate than enzyme-treated proteasomes (see also Fig. 3C). In sum, our data indicate that, under certain experimental conditions, changes in proteasome modifications affect proteasome function, thereby strengthening the case that Bortezomib-triggered changes in proteasome modifications within cells also affect proteasome function.
Discussion In summary, our data reveal a dramatic inhibition of proteasome activity in MM cells after a (low) IC50 Bortezomib challenge, and suggest that this inhibition is the compound result of, first, inhibition of a subset of active-sites, and, second, structural changes in the proteasome which further impair hydrolytic activity (Fig. 3E). Engagement of PIs with active-sites changes proteasome conformation and stabilizes the (distant) CP–RP (Kleijnen et al., 2007; Park et al., 2008) and RP-hPLIC/ubiquilin (Kleijnen et al., 2000) interactions, thus providing a possible signalling mechanism into the cell that may enable active-site inhibition to directly trigger activation of the cellular machinery that then changes the posttranslational modifications of the proteasomes. Whereas it has been very difficult to explain why MM cells die from a nanomolar IC50 Bortezomib challenge when assuming that the modest level of proteasome inhibition observed in-vitro holds true in-vivo (Bianchi et al., 2009; Kisselev et al., 2006; Shabaneh et al., 2013), it is not surprising that a myeloma cell with over 95% CT-inhibition and observable proteasome stress (i.e. accumulation of ubiquitin conjugates, Fig. 1a) will undergo apoptosis. In addition, our data show that Bortezomib, in cancer cells which are Bortezomib-resistant, does not achieve the same degree of proteasome inhibition as in (Bortezomib-sensitive) MM cells (Figs. 1F, S2D), thus providing a molecular mechanism explaining what differentiates MM from most other cancers which Bortezomib cannot treat. Please note that our data indicate that a high proteasome workload in MM cells (Bianchi et al., 2009; Meister et al., 2007; Shabaneh et al., 2013) cannot be the primary reason for MM cells\' sensitivity to Bortezomib: for this explanation to work, all proteasomes in a cell would need to be fully engaged – with no spare capacity left – in order for a minimal inhibition of proteasomes to produce proteasome stress; instead, we observed that MM cells have much spare proteasome capacity, and that reducing capacity even to 20% still did not yield proteasome stress (Fig. 1a). Understanding the cellular mechanism via which Bortezomib amplifies its effect on proteasome function may enable future intervention to re-sensitize Bortezomib-resistant cells to treatment.