Summary Background Proteasome inhibitors are widely used in treatment of multiple myeloma and as research tools. Diminished proteasome function also may contribute to neuronal dysfunction. In response to these inhibitors, cells enhance the expression of proteasome subunits by the transcription factor Nrf1. Here we investigate the mechanisms by which decreased proteasome function triggers production of new proteasomes via Nrf1. Results Exposure of myeloma or neuronal cells to proteasome inhibitors (bortezomib, epoxomicin, MG132), but not to proteotoxic or ER stress, caused a 2–4-fold increase within 4h in mRNAs for all 26S subunits. In addition, p97 and its cofactors (Npl4, Ufd1, p47), PA200 and Usp14 were induced, but expression of immunoproteasome-specific subunits was suppressed. Nrf1 mediates this induction of proteasomes and p97, but only upon exposure to low concentrations of inhibitors that inhibit proteolysis partially. Surprisingly, high concentrations of these inhibitors prevent this compensatory response. Nrf1 is normally ER-bound, and its release requires its deglycosylation and ubiquitination. Normally ubiquitinated Nrf1 is rapidly degraded, but when partially inhibited, proteasomes carry out limited proteolysis and release the processed Nrf1 (lacking its N-terminal region) from the ER, which allows it to enter the nucleus and promote gene expression. Conclusions When fully active, proteasomes degrade Nrf1, but when partially inhibited, they perform limited proteolysis which generates the active form of Nrf1. This elegant mechanism allows cells to compensate for reduced proteasome function by enhancing production of 26S subunits and p97.

Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively polyubiquitinated by the proteasome-associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat-shock or arsenite treatment, when poly-ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin-conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients.

Summary eIF3 promotes translation initiation, but relatively little is known about its full range of activities in the cell. Here, we employed affinity purification and highly sensitive LC-MS/MS to decipher the fission yeast eIF3 interactome, which was found to contain 230 proteins. eIF3 assembles into a large supercomplex, the translasome, which contains elongation factors, tRNA-synthetases, 40S and 60S ribosomal proteins, chaperones, and the proteasome. eIF3 also associates with ribosome biogenesis factors and the importins-β Kap123p and Sal3p. Our genetic data indicated that the binding to both importins-β is essential for cell growth, and photobleaching experiments revealed a critical role for Sal3p in the nuclear import of one of the translasome constituents, the proteasome. Our data reveal the breadth of the eIF3 interactome and suggest that factors involved in translation initiation, ribosome biogenesis, translation elongation, quality control, and transport are physically linked to facilitate efficient protein synthesis.

Summary The proteasome inhibitors Carfilzomib (Cfz) and Bortezomib (Btz) are used successfully to treat MM, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the β5 site of the proteasome by Cfz and Btz does not result in loss of viability of triple-negative breast cancer cell lines. We use site-specific inhibitors and CRISPR-mediated genetic inactivation of β1 and β2 to demonstrate that inhibiting a second site of the proteasome, particularly the β2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both β5 and β2 suppresses production of the soluble, active form of the transcription factor Nrf1 and prevents the recovery of proteasome activity through induction of new proteasomes. These findings provide a strong rationale for the development of dual β5 and β2 inhibitors for the treatment of solid tumors.

Cells are thought to adapt to proteasome inhibition by using alternative pathways for degradation such as autophagy. Sha et al. now report that cells rapidly induce GABARAPL1 and p62 upon proteasome inhibition, but this promotes cell survival by sequestering ubiquitinated and sumoylated proteins long before the cells induce other Atg genes and activate autophagy.