Author response: Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome (2015)

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans. https://doi.org/10.7554/eLife.08467.001 eLife digest Cells have numerous methods for removing proteins that have been damaged or are no longer needed. One of these methods is carried out by a large protein complex called the proteasome. Because of its central role in maintaining protein balance, drugs that stop the proteasome functioning often kill cancer cells grown in dishes. However, these proteasome inhibitors tend not to work against most tumors in patients. Moreover, tumors that do respond to these drugs ultimately become resistant to them. Tsvetkov et al. used a genetic screen to find the mutations that allowed cancer cells to withstand exposure to proteasome inhibitors. The proteasome complex contains two types of subunits: regulatory subunits that recognize the proteins that need to be degraded; and catalytic subunits that degrade the proteins. Surprisingly, individually inactivating the genes for many different regulatory subunits provided protection against proteasome inhibitors. When the regulatory subunits were reduced, the proteasomes shifted into a state that ultimately protected the cells. This mechanism was observed to protect both yeast and human cells and may be a widespread mechanism for establishing stress-resistant states. The next challenge will be to identify the vulnerabilities of cells that have reduced regulatory subunits. Research is also needed to find out if this reduction varies from cell to cell, making some cells more able to withstand treatment. https://doi.org/10.7554/eLife.08467.002 Introduction Maintaining the integrity of the proteome is vital for all cells. Protein chaperone systems and the ubiquitin-proteasome pathway are essential components of the global architecture that sustains protein homeostasis. The importance of these systems is underscored by the fact that perturbation of protein homeostasis is central to diverse human diseases (Balch et al., 2008; Labbadia and Morimoto, 2015). In neurodegenerative diseases, cells succumb to an overload of toxic protein aggregates. Whereas in cancer, malignant cells co-opt protein quality control systems to accommodate the severe proteotoxic stresses that arise from high mutation loads, relentless biomass accumulation and protein turnover (Mendillo et al., 2012; Deshaies, 2014; Scherz-Shouval et al., 2014). Because the ubiquitin-proteasome system is the major mechanism regulating protein turnover, cells are highly vulnerable to insults that impair the function of this system (Varshavsky, 2012; Petrocca et al., 2013). Proteasome function can be hampered in many ways. For example, a multitude of environmental stresses cause the toxic accumulation of misfolded and aggregated proteins (Parsell et al., 1994; Gidalevitz et al., 2011). Such proteins can bind the proteasome and stabilize an inactive closed conformation (Deriziotis et al., 2011; Ayyadevara et al., 2015). In addition, chemically diverse natural product inhibitors of the ubiquitin-proteasome pathway are elaborated by organisms as diverse as terrestrial and marine bacteria, fungi, and plants (Kisselev et al., 2006; Schneekloth and Crews, 2011; Kisselev et al., 2012). These proteasome inhibitors can thwart the proliferation of neighboring organisms and thereby provide a competitive growth advantage. Applying this lesson from nature toward the treatment of cancer has been an effective strategy. Malignant cells in which the rates of protein synthesis outstrip proteasome degradation capacity are exceedingly vulnerable to proteasome inhibition (Adams et al., 2000; Cenci et al., 2012; Deshaies, 2014). Targeting the 26S proteasome with the proteasome inhibitor bortezomib (Velcade, PS-341) is particularly effective in hematopoietic tumors. Indeed, it is broadly applied as a therapy for patients with myeloma or mantle cell lymphoma (Chen et al., 2011; Crawford and Irvine, 2013). The 26S proteasome is an elaborate multi-subunit protein complex that is present in all eukaryotes. This complex is comprised of a 20S catalytic core that orchestrates peptide bond cleavage and a 19S regulatory complex that can be attached to either or both ends of the 20S core (Besche et al., 2009; Matyskiela et al., 2013; Tomko and Hochstrasser, 2013). The 19S complex recognizes ubiquitin-tagged substrates, cleaves ubiquitin chains, unfolds substrates and translocates the unfolded proteins into the catalytic chamber of the 20S core (Hochstrasser, 1996; Finley, 2009; Matyskiela et al., 2013). Despite an exquisitely detailed understanding of proteasome function and of the mechanism of action of proteasome inhibitors, we have a limited understanding of the molecular mechanisms that cells deploy to resist the cytotoxic effects of reduced flux through the proteasome (Kale and Moore, 2012). Such an understanding is of great importance in the dynamics of natural ecosystems in the face of diverse proteotoxic stresses and in the clinic, where pre-existing intrinsic resistance and acquired resistance following drug exposure have limited the effectiveness of bortezomib as a therapeutic. To gain insights into the mechanisms that allow cells to withstand reduced flux through the proteasome, we took advantage of highly specific chemical inhibitors. While proteasome function can be impaired by many factors, none can be controlled with the dosage-dependent precision of proteasome inhibitors such as bortezomib and the peptide aldehyde MG132. These inhibit both 20S and 26S proteasomes by targeting the core proteolytic catalytic activity of the 20S subunits (Kisselev et al., 2006, 2012; Goldberg, 2012). We used these inhibitors at toxic levels in an unbiased, genome-wide screen. We selected for cells that were resistant to the inhibitors from a library of 100 million gene-trap insertions, using a human cell line that is haploid for all chromosomes except chromosome 8 (Carette et al., 2009, 2011a). This approach is analogous to screens so broadly and effectively used in haploid yeast and identifies loss-of-function events that allow human cells to survive diverse toxic insults (Guimaraes et al., 2011; Reiling et al., 2011; Carette et al., 2011a, 2011b; Winter et al., 2014). The results of our screen led us to the discovery of a surprising and highly conserved strategy by which organisms can protect themselves from the toxic effects of reduced flux through the proteasome. Results Unbiased mammalian screen identifies 19S subunits as key determinants of resistance to proteasome inhibitors For our screens, we used a library of near-haploid human chronic myeloid leukemia cells (KBM7) containing approximately 100 million retroviral gene-trap insertions that target over 98% of transcribed genes. To identify genes that increase resistance to proteasome inhibition, we exposed cells for 4 weeks to either MG132 or bortezomib. We then further expanded the pools of resistant cells to enable the amplification and sequencing of the insertion sites (Figure 1A). Figure 1 with 1 supplement see all Download asset Open asset The 19S regulatory subunits of the proteasome are the most significant mediators of resistance to proteasome inhibitor toxicity. (A) Schematic representation of the screen. One hundred million KBM7 cells subjected to random gene deletion using retroviral gene-trap insertions were exposed to either MG132 (700 nM) or bortezomib (18 nM) for 4 weeks. Surviving cells were expanded and insertions identified by sequencing. (B) The p-values of the recovered insertions from the MG132 screen are plotted (log2). Bubble sizes represent the number of insertions. (C) Compilation of the most significant gene deletions conferring resistance to MG132 with the gene name, number of inserts, and p-value. The subunits of the 19S regulatory complex are highlighted with orange. https://doi.org/10.7554/eLife.08467.003 For the MG132 resistance screen, we identified 992 independent insertion sites in the pool of surviving cells. Surprisingly, all insertions that reached a high level of statistical enrichment (p-value < 1 e−7) lay in genes encoding subunits of the proteasome 19S regulatory complex (Figure 1B,C; Supplementary file 1). These included both ATPase subunits (PSMC2, PSMC3, PSMC4, PSMC5, and PSMC6) as well as non-ATPase subunits (PSMD2, PSMD6, PSMD7, and PSMD12). No insertions were recovered in genes encoding subunits of the 20S catalytic core (Supplementary file 1). For the bortezomib resistance screen, we recovered 538 independent insertions. The results of the 2 screens were remarkably similar with seven of the ten most highly enriched genes encoding subunits of the 19S complex (Supplementary file 1). Such strikingly similar results from 2 unbiased screens with chemically distinct proteasome inhibitors strongly indicated that altering the 19S complex can protect cells against compounds that inhibit the 20S catalytic core. From the resistant pools of cells, we then attempted to isolate stable clones that contained 19S subunit gene insertions. We were unable to do so. Next, we attempted to delete PSMD12 in a near-haploid fibroblast cell line (HAP1) (Carette et al., 2010; Essletzbichler et al., 2014). With this targeted approach using CRISPR constructs, we were only able to recover diploid cell variants in which just one of the two PSMD12 alleles was disrupted. Finally, from a collection of haploid mouse embryonic stem cells that harbor reversible gene-trap cassettes (Elling et al., 2011), we identified two clones with cassettes located in the first intron of the PSMC2 or PSMD12 genes. In these cells, inversion of the cassettes would generally be expected to inactivate the targeted gene. We induced Cre-mediated inversion in over 3000 cells harboring each cassette, but less than 1% of the cells survived. We confirmed that inversion had occurred in the surviving cells. However, all of the stable clones that emerged retained expression of the targeted subunits (Figure 1—figure supplement 1). These findings confirm that, as others have found in yeast and Drosophila, the function of the 19S regulatory complex is essential for sustained proliferation of mammalian cells under basal conditions. Presumably, cells carrying 19S mutations had become enriched in our initial MG132 and bortezomib screens because they provided cells with a short-term advantage for growth over several generations. Reducing 19S subunits protects human cancer cells from proteasome inhibitors Next, we asked if a simple reduction in the expression of 19S subunits could protect against the toxicity of proteasome inhibitors. We assembled a panel of shRNA-expressing lentiviruses targeting seventeen 19S subunits and three 20S subunits (PSMB5, PSMB7, and PSMA3). Each gene was targeted separately using four different shRNAs (Figure 2A, Supplementary file 2) and we averaged the effects on viability from these four hairpins. Figure 2 with 1 supplement see all Download asset Open asset Reducing expression of 19S subunits increases the levels of active 20S proteasomes and protects cancer cells from proteasome inhibition. (A) HepG2 cells were infected with 80 shRNAs targeting 20 different subunits of the proteasome and 10 control shRNAs. Infected cells were then exposed to 12 nM bortezomib and cell number was examined 4 days later. The plot represents the average (± SEM) of four different hairpins targeting the indicated proteasome subunits and their relative cell number following bortezomib treatment. 19S subunits are depicted with orange bars, the control with a blue bar, and 20S subunits with green bars. (B) HepG2 cells harboring either a control shGFP or shRNAs targeting two proteasome subunits (shPSMC5, shPSMD2) displayed significant growth differences in the presence of 12 nM bortezomib. (C) Proteasome complex content in the shGFP-, shPSMC5-, and shPSMD2-expressing HepG2 cells was analyzed by native gel electrophoresis after 24 hr of treatment with or without 12 nM bortezomib revealing an increase in 20S proteasome levels and activity in cells knocked down for PSMC5 or PSMD2. (D) The relative cell number of cells harboring a control shLacZ or each of 4 individual shRNAs targeting shPSMD2 was analyzed 4 days after addition of the indicated concentrations of bortezomib. (E) HepG2 cells stably expressing four different shRNAs targeting the PSMD2 subunit and a control shRNA (lacZ) were analyzed by Western blot for the indicated proteins 24 hr with or without bortezomib treatment. (F) Proteasome complex levels and activity in a control HepG2 cells and 4 cell lines with reduced PSMD2 levels with and without a 24 hr incubation with bortezomib (12 nM). Proteasome complex levels were detected by immunoblot analysis and 20S proteasome activity by measuring the hydrolysis of Suc-LLVY-AMC by substrate overlay assays. In B and C, the graph represents the average of four replicas and their SEM. The p-values were obtained by conducting a two-tailed unpaired t-test. *p < 0.05 **p < 0.01 Bortz- Bortezomib (E). https://doi.org/10.7554/eLife.08467.005 With or without bortezomib, knockdown of any of the subunits of the 20S catalytic core reduced viability (Figure 2A, Figure 2—figure supplement 1A–D). In contrast, the effects of knocking down several different 19S subunits had opposing effects, depending on the absence or presence of the inhibitor. In its absence, 19S subunit reduction had a fitness cost and decreased cell viability; in its presence, 19S subunit reduction provided a survival advantage and increased viability (Figure 2A, Figure 2—figure supplement 1A–D). To investigate further, we sought to recover stable clones with reduced levels of 19S subunits. Long-term 19S subunit reduction impeded the growth of most cells, but we were able to propagate two lines that proliferated normally (Figure 2—figure supplement 1E). These lines stably expressed shRNA targeting either PSMC5 or PSMD2. In both cases, the lines had only a modest reduction in protein levels (Figure 2—figure supplement 1F). At a concentration of bortezomib that completely inhibited the proliferation of control cells (12 nM), these cells continued proliferating (Figure 2B, figure 2—figure supplement 1G). We next used native gels to assess the levels and ratios of 20S and 26S proteasome complexes following a 24-hr exposure to bortezomib (Figure 2C). Bortezomib-induced cytotoxicity was not observed in control cells at this time point (Figure 2B, star). In cells with reduced levels of either PSMC5 or PSMD2, 26S proteasome complexes were reduced and 20S proteasome complexes were increased (Figure 2C, upper panel). These changes persisted in the presence of bortezomib (Figure 2C). Next, we assayed 20S proteasome complex activity with a fluorescently labeled substrate on native gels. Exposing control cells to 12 nM bortezomib for 24 hr led to a nearly complete inactivation of 20S proteasome function (Figure 2C, lower panel). Cells with reduced levels of either PSMC5 or PSMD2 maintained a significant level of 20S proteasome activity (Figure 2C). To validate our findings, we selected four additional cell lines each with a different shRNA driving modest reduction in PSMD2 protein levels. Again, all of these lines were significantly more resistant to bortezomib than the parental line (EC50 values increased by three to sixfold, Figure 2D). In these cells, we also observed that 26S proteasome complexes were reduced and that both the levels and the activity of 20S proteasome complexes were sharply increased (Figure 2E). Notably, in the presence of bortezomib, reducing 19S subunits enabled the relative preservation of the levels and activity of 20S proteasome complexes (Figure 2E). 19S subunit reduction does not activate classic cytoprotective stress responses and blunts bortezomib-mediated stress responses A likely mechanism by which 19S subunit reduction might promote resistance is by induction of the cytoprotective stress responses that allow cells to cope with the increase in proteotoxic stress caused by the proteasome inhibitor. One major response to such inhibition is the activation of NRF1, a transcriptional regulator of proteasome gene expression that increases the expression of proteasome subunits, elevates proteasome content, and promotes resistance (Radhakrishnan et al., 2010; Steffen et al., 2010; Radhakrishnan et al., 2014; Sha and Goldberg, 2014). A second major response to proteasome inhibitors is activation of heat-shock factor 1 (HSF1), the master regulator of the heat-shock response, which increases levels of HSP70 and other protein chaperones. Surprisingly, in the four cell lines with modest reductions of PSMD2, we did not detect constitutive activation of NRF1, and correspondingly, the expression of 20S subunits was unaltered (Figure 2F). Moreover, HSF1 was not activated as reflected by the stable expression of HSP70, the protein that is most highly responsive to proteotoxic stress (Figure 2F). Consistent with these findings, polyubiquitinated proteins did not accumulate in the PSMD2 knockdown cells (Figure 2F) suggesting that modestly reducing 19S subunit levels did not itself induce a cytoprotective stress response. Not only were stress response pathways not activated, but the response to bortezomib was blunted in cells with reduced PSMD2 levels. The accumulation of polyubiquitinated proteins was reduced relative to control cells treated with the inhibitor. The activation of NRF1 was also reduced (Figure 2F). We obtained very similar results in cells with PSMC5 knockdown except in this case HSP70 levels were also reduced (Figure 2—figure supplement 1H). Notably, the protective effect of 19S subunit reduction was specific to the toxicity caused by proteasome inhibitors. Cells with reduced PSMD2 levels remained fully sensitive to small molecule mediators of ER stress, HSP90 inhibition, thiol adduct formation or blockade of translation initiation or translation elongation among other stresses (Figure 2—figure supplement 1I–N). Thus, modest 19S subunit reduction protected cells by selectively lowering the proteotoxic stress that is generated by proteasome inhibition. Compromising the 19S regulatory complex suppresses bortezomib-induced stress responses To examine in detail the transcriptional changes that characterize cells with increased resistance to proteasome inhibition, we performed whole-genome RNA-sequencing in two lines with modest reductions in PSMD2. Sequence data confirmed that PSMD2 mRNA levels were reduced by ∼50% in both lines. We examined basal gene expression and the effects of bortezomib treatment on these cells, comparing them to cells carrying a control lacZ shRNA construct. Under basal conditions (in the absence of bortezomib), cells with reduction in PSMD2 showed a strong induction of components of the ribosome (gene set enrichment analysis false discovery rate [FDR] q-value = 4.0 e−22) (Supplementary file 3). Genes encoding the 20S subunits of the proteasome were not induced, consistent with our earlier observation that NRF1 is not activated and 20S subunit levels are unchanged (Figure 2F). The most strongly downregulated gene category involved components of diverse proteotoxic stress responses (FDR q-value = 2.31 e−9) (Supplementary file 3). These included genes for sentinel proteins that respond to heat-shock (e.g., HSPA1A, HSPA1B, HSPA8, HSPB1, and HSP90AA1), oxidative stress (e.g., HMOX1), and ER stress (e.g., CHOP/DDIT3, TRIB3, and HERPUD1) (Figure 3A). Genes previously identified as an HSF1-regulated cancer-specific transcriptional program were also downregulated (Mendillo et al., 2012; Santagata et al., 2013) (FDR q-value = 0.042; normalized enrichment score (NES) 1.57) (Figure 3—figure supplement 1A). Therefore, 19S subunit reduction not only fails to induce classic adaptive stress responses, but actually lowers basal levels of proteotoxic stress. Figure 3 with 1 supplement see all Download asset Open asset Inhibition of bortezomib-mediated transcriptional responses in PSMD2 knockdown cells. RNA-seq gene expression profiling was conducted on HepG2 cells that harbor two different shRNAs targeting PSMD2 cells used in Figure 2F) and on control cells The effects of reducing PSMD2 levels on gene expression were highly in both basal conditions = and following bortezomib treatment = oxidative and ER gene expression were all lower in the PSMD2 knockdown cells control cells under both basal conditions (A) and of 12 nM bortezomib for 24 hr (C) set enrichment analysis of genes in control but not in PSMD2 shRNA cells following bortezomib treatment. was for the indicated gene and is as a normalized enrichment score significant enrichment discovery rate [FDR] q-value < is in enrichment is in (D) levels of genes previously as of bortezomib-induced toxicity (Chen et al., are in the PSMD2 knockdown cells following the addition of bortezomib. (E) in mRNA levels of genes expressed in cells harboring control shRNA or PSMD2 shRNAs in the presence or absence of 12 nM bortezomib. enrichment is to the of the in control cells, bortezomib treatment a powerful transcriptional response by a increase in (FDR q-value = (Supplementary file 3). This bortezomib-induced stress response was in cells with reduced PSMD2 levels (Figure The genes involved in oxidative stress and ER stress responses, as well as genes in the heat-shock response (FDR q-value < (Figure and Figure 3—figure supplement For example, induction of HSP70 heat-shock genes and was reduced by was (Figure work has identified mediators of bortezomib many of which are bortezomib exposure (Chen et al., When we treated control cells with bortezomib, the mRNA of many of these genes increased significantly (e.g., and (Supplementary file 3). This response was also strongly in cells with reduced PSMD2 levels (Figure and Figure 3—figure supplement selectively 19S subunit expression broadly the diverse transcriptional responses that normally when flux through the proteasome is Compromising 19S function a cell response to bortezomib To further characterize the transcriptional effects of 19S subunit we performed a analysis on the genes the expression in our RNA-seq (Figure This analysis confirmed that PSMD2 reduction strongly blunted the bortezomib-mediated induction of stress response genes (FDR to also changes in genes involved in small molecule which to be One of genes highlighted by this analysis a the of the cell and increased resistance to bortezomib. In cells with reduced levels of PSMD2, bortezomib treatment strongly genes involved in (FDR q-value = and cell control (FDR q-value = These genes factors, and This response that cells with reduced 19S subunits are to a state when flux through the proteasome is reducing 19S subunits the effects of stable 19S subunit reduction To the effects of reduction of 19S subunits, we a cell line in which a shRNA is expressed from a (Figure The effects of reduction of PSMD2 the effects of stable PSMD2 it significantly increased resistance to both bortezomib (Figure Figure supplement and MG132 (Figure supplement Again, this resistance was and not accompanied by increased resistance to other small molecule (Figure supplement In the absence of bortezomib, 19S reduction did not activate NRF1 or HSF1 (Figure and reduced activation of NRF1 and HSF1 following bortezomib exposure (Figure Figure 4 with 1 supplement see all Download asset Open asset induction of PSMD2 shRNA is to promote resistance to proteasome inhibition. (A) Schematic representation of the (B) cells harboring a PSMD2 shRNA were grown in the presence or absence of 1 for Cells were then and in the absence of for 24 hr to exposure to concentrations of bortezomib. cell were 3 days later. (C) Protein content analysis by immunoblot for the indicated proteins on from control or cells for hr with by a of 24 hr and then incubation with 10 nM bortezomib for an additional 24 (D) gel analysis of proteasome complexes in cells as in The proteasome complex levels and activity of the 20S proteasome were by native gel were analyzed by immunoblot for PSMD2 and following (E) was conducted on cells for hr with by a of 24 hr and then incubation with nM bortezomib for an additional 24 Proteasome activity in the was with and proteasome content was analyzed by with and 20S subunits specific (F) The rate of degradation was analyzed in cells with reduced levels of PSMD2 control in the presence or absence of 10 nM bortezomib for 20 by the of in cells. of protein synthesis was in cells with reduced levels of PSMD2 control in the presence or absence of 10 nM bortezomib for 20 by measuring the rate of of for 1 The p-values were obtained by conducting a two-tailed unpaired t-test. **p < < 19S subunit reduction the effects of bortezomib on proteasome degradation capacity and protein translation We used native gels and to the levels and activity of proteasome complexes following reduction of PSMD2 (Figure Figure supplement reduction of this 19S subunit increased the ratio of proteasomes and levels of 20S proteasome activity (Figure without levels of 20S subunits (Figure Figure supplement to the results we obtained in stable knockdown cells (Figure 19S subunit reduction a of the 20S proteasome complexes following bortezomib treatment and a of their activity remained (Figure Figure supplement Next, we examined the of reducing PSMD2 levels on protein degradation and protein synthesis in cells labeled with for 24 We then separately the rates at which labeled proteins were through the proteasome and through the In the absence of bortezomib, reducing PSMD2 rates of by the (Figure supplement but it