Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation.

Although several proteasome subunits have been shown to bind ubiquitin (Ub) chains, many ubiquitylated substrates also associate with 26S proteasomes via "shuttling factors." Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a Ub-associated domain; therefore, it is unclear how they bind Ub. Here, we show that deletion of Ddi2 leads to the accumulation of Ub conjugates with K11/K48 branched chains. We found using affinity copurifications that Ddi2 binds Ub conjugates through its Ub-like domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding Ub conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of Ub conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking the endoprotease activity of Ddi2 either genetically or with the HIV protease inhibitor nelfinavir increased its binding to Ub conjugates but decreased its binding to proteasomes and reduced subsequent protein degradation by proteasomes leading to further accumulation of Ub conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum-associated protein degradation and delivers K11/K48-ubiquitylated proteins to the proteasome. We conclude that the protease activity of Ddi2 influences this shuttling factor activity, promotes protein turnover, and helps prevent endoplasmic reticulum stress, which may explain nelfinavir's ability to enhance cell killing by proteasome inhibitors.

Multiple myeloma cells are exceptionally sensitive to heat shock, which overwhelms their proteostasis network and induces apoptosis.

Proteasome inhibitors, such as bortezomib (BTZ), are highly effective and widely used treatments for multiple myeloma. One proposed reason for myeloma cells' exceptional sensitivity to proteasome inhibition is that they produce and continually degrade unusually large amounts of abnormal immunoglobulins. We, therefore, hypothesized that, heat shock may also be especially toxic to myeloma cells by causing protein unfolding, increasing further the substrate load on proteasomes, and, thus, putting further stress on their capacity for protein homeostasis. After a shift from 37 to 43 °C, all four myeloma lines studied underwent extensive apoptosis in 4 h, unlike 13 nonmyeloma cell lines, even though the myeloma cells induced heat-shock proteins and increased protein degradation similar to other cells. Furthermore, two myeloma lines resistant to proteasome inhibitors were also more resistant to 43 °C. Shifting myeloma cells to 43, 41, or 39 °C (which was not cytotoxic) dramatically increased their killing by proteasome inhibitors and inhibitors of ubiquitination or p97/VCP. Combining increased temperature with BTZ increased the accumulation of misfolded proteins and substrate load on the 26S proteasome. The apoptosis seen at 43 °C and at 39 °C with BTZ was mediated by caspase-9 and was linked to an accumulation of the proapoptotic Bcl-2-family member Noxa. Thus, myeloma cells are exceptionally sensitive to increased temperatures, which greatly increase substrate load on the ubiquitin-proteasome system and eventually activate the intrinsic apoptotic pathway. Consequently, for myeloma, mild hyperthermia may be a beneficial approach to enhance the therapeutic efficacy of proteasome inhibitors.

Inhibiting ubiquitination causes an accumulation of SUMOylated newly synthesized nuclear proteins at PML bodies.

Protein ubiquitination and SUMOylation are required for the maintenance of cellular protein homeostasis, and both increase in proteotoxic conditions (e.g. heat shock or proteasome inhibition). However, we found that when ubiquitination was blocked in several human cell lines by inhibiting the ubiquitin-activating enzyme with TAK243, there was an unexpected, large accumulation of proteins modified by SUMO2/3 chains or SUMO1, but not by several other ubiquitin-like proteins. This buildup of SUMOylated proteins was evident within 3-4 h. It required the small ubiquitin-like modifier (SUMO)-conjugating enzyme, UBC9, and the promyelocytic leukemia protein (PML) and thus was not due to nonspecific SUMO conjugation by ubiquitination enzymes. The SUMOylated proteins accumulated predominantly bound to chromatin and were localized to PML nuclear bodies. Because blocking protein synthesis with cycloheximide prevented the buildup of SUMOylated proteins, they appeared to be newly-synthesized proteins. The proteins SUMOylated after inhibition of ubiquitination were purified and analyzed by MS. In HeLa and U2OS cells, there was a cycloheximide-sensitive increase in a similar set of SUMOylated proteins (including transcription factors and proteins involved in DNA damage repair). Surprisingly, the inhibition of ubiquitination also caused a cycloheximide-sensitive decrease in a distinct set of SUMOylated proteins (including proteins for chromosome modification and mRNA splicing). More than 80% of the SUMOylated proteins whose levels rose or fell upon inhibiting ubiquitination inhibition underwent similar cycloheximide-sensitive increases or decreases upon proteasome inhibition. Thus, when nuclear substrates of the ubiquitin-proteasome pathway are not efficiently degraded, many become SUMO-modified and accumulate in PML bodies.

Measuring the Overall Rate of Protein Breakdown in Cells and the Contributions of the Ubiquitin-Proteasome and Autophagy-Lysosomal Pathways.

In certain physiological or pathological states (e.g., starvation, heat shock, or muscle atrophy) and upon drug treatments, the overall rate of protein degradation in cells may increase or decrease. These adaptations and pathological responses can occur through alterations in substrate flux through the ubiquitin-proteasome pathway (UPP), the autophagy-lysosomal system, or both. Therefore, it is important to precisely measure the activities of these degradation pathways in degrading cell proteins under different physiological states or upon treatment with drugs. In particular, proteasome inhibitors have become very important agents for treating multiple myeloma and very useful tools in basic research. To evaluate rigorously their efficacy and the cellular responses to other inhibitors, it is essential to know the degree of inhibition of protein breakdown. Unfortunately, commonly used assays of the activities of the UPP or autophagy rely on qualitative, indirect approaches that do not directly reflect the actual rates of protein degradation by these pathways. In this chapter, we describe isotopic pulse-chase methods to directly measure overall rates of protein degradation in cells by radiolabeling cell proteins and following their subsequent degradation to radioactive amino acids, which diffuse from cells into the medium and can be easily quantitated. While pulse-chase methods have often been used to follow degradation of specific proteins, the methods described here allow quantification of the total cellular activity in degrading either long-lived proteins (the great bulk of cell constituents) or the fraction with short half-lives. Moreover, by use of specific inhibitors of proteasomes or lysosomes, it is also possible to measure precisely the total contributions of the UPP or lysosomal proteases. These approaches have already been proven very useful in defining the effects of inhibitors, growth factors, nutrients, ubiquitination, and different proteasome activators on overall proteolysis and on substrate flux through the proteasomal and lysosomal pathways.

Rapid induction of p62 and GABARAPL1 upon proteasome inhibition promotes survival before autophagy activation.

Proteasome inhibitors are used as research tools and to treat multiple myeloma, and proteasome activity is diminished in several neurodegenerative diseases. We therefore studied how cells compensate for proteasome inhibition. In 4 h, proteasome inhibitor treatment caused dramatic and selective induction of GABARAPL1 (but not other autophagy genes) and p62, which binds ubiquitinated proteins and GABARAPL1 on autophagosomes. Knockdown of p62 or GABARAPL1 reduced cell survival upon proteasome inhibition. p62 induction requires the transcription factor nuclear factor (erythroid-derived 2)-like 1 (Nrf1), which simultaneously induces proteasome genes. After 20-h exposure to proteasome inhibitors, cells activated autophagy and expression of most autophagy genes by an Nrf1-independent mechanism. Although p62 facilitates the association of ubiquitinated proteins with autophagosomes, its knockdown in neuroblastoma cells blocked the buildup of ubiquitin conjugates in perinuclear aggresomes and of sumoylated proteins in nuclear inclusions but did not reduce the degradation of ubiquitinated proteins. Thus, upon proteasome inhibition, cells rapidly induce p62 expression, which enhances survival primarily by sequestering ubiquitinated proteins in inclusions.

Inhibition of the Proteasome ß2 Site Sensitizes Triple-Negative Breast Cancer Cells to ß5 Inhibitors and Suppresses Nrf1 Activation.

The proteasome inhibitors carfilzomib (Cfz) and bortezomib (Btz) are used successfully to treat multiple myeloma, but have not shown clinical efficacy in solid tumors. Here we show that clinically achievable inhibition of the ß5 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 ß1 and ß2 to demonstrate that inhibiting a second site of the proteasome, particularly the ß2 site, sensitizes cell lines to Btz and Cfz in vitro and in vivo. Inhibiting both ß5 and ß2 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 ß5 and ß2 inhibitors for the treatment of solid tumors.

Reply to Vangala et al.: Complete inhibition of the proteasome reduces new proteasome production by causing Nrf1 aggregation.

An important adaptation of cells to proteasome inhibition is the induction of new proteasomes via the transcription factor Nrf1 [1,2], which is produced as a precursor bound to the endoplasmic reticulum (ER) through its amino terminus. Nrf1 was reported to require proteolytic processing to enter the nucleus [3]. Increased proteasome production is induced by low concentrations of proteasome inhibitors that reduce proteolysis by <50%. Surprisingly, in earlier studies we found that proteasome induction and Nrf1 processing to its shorter form (which we estimated to be 75 kDa [2]) were suppressed by high concentrations of inhibitors that markedly reduce proteasome activity [4]. This unusual bimodal concentration dependence implied that some proteasome function was necessary for Nrf1 processing. Because we found that Nrf1 processing also required ubiquitin conjugation [2], we previously proposed that Nrf1 processing is catalyzed by partially inhibited proteasomes [2]. However, Vangala et al.[5] present compelling evidence that conversion of the ER-bound Nrf1 to the shorter form, which they describe as 110 kDa, is independent of proteasomes and is not blocked by high concentrations of proteasome inhibitors. Therefore, we investigated the basis for these differing results. Here we report that we and Vangala et al. have studied the same processed form of Nrf1, the actual molecular weight of which appears to be 90-95 kDa. We confirm our earlier finding [2] that high concentrations of proteasome inhibitors suppress proteasome induction and accumulation of processed Nrf1 in soluble lysates. However, we now show that the inhibitors do so not by blocking Nrf1 processing, but instead by causing the processed Nrf1 to aggregate. Therefore, Nrf1 must be cleaved by a non-proteasomal endoprotease that we show requires ubiquitination. Finally, we provide evidence supporting the recent report that Ddi1/Ddi2 is the critical protease [6,7].

Proteasome-mediated processing of Nrf1 is essential for coordinate induction of all proteasome subunits and p97.

BACKGROUND: Proteasome inhibitors are widely used in the treatment of multiple myeloma and as research tools. Additionally, diminished proteasome function 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, and MG132), but not to proteotoxic or ER stress, caused a 2- to 4-fold increase within 4 hr in mRNAs for all 26S subunits. In addition, p97 and its cofactors (Npl4, Ufd1, and 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 partially inhibit proteolysis. 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 that 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.

Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.

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 poly-ubiquitinated 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.

Proteasome nuclear import mediated by Arc3 can influence efficient DNA damage repair and mitosis in Schizosaccharomyces pombe.

Proteasomes must remove regulatory molecules and abnormal proteins throughout the cell, but how proteasomes can do so efficiently remains unclear. We have isolated a subunit of the Arp2/3 complex, Arc3, which binds proteasomes. When overexpressed, Arc3 rescues phenotypes associated with proteasome deficiencies; when its expression is repressed, proteasome deficiencies intensify. Arp2/3 is best known for regulating membrane dynamics and vesicular transport; thus, we performed photobleaching experiments and showed that proteasomes are readily imported into the nucleus but exit the nucleus slowly. Proteasome nuclear import is reduced when Arc3 is inactivated, leading to hypersensitivity to DNA damage and inefficient cyclin-B degradation, two events occurring in the nucleus. These data suggest that proteasomes display Arc3-dependent mobility in the cell, and mobile proteasomes can efficiently access substrates throughout the cell, allowing them to effectively regulate cell-compartment-specific activities.

The eIF3 interactome reveals the translasome, a supercomplex linking protein synthesis and degradation machineries.

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-beta Kap123p and Sal3p. Our genetic data indicated that the binding to both importins-beta 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.

Isolation of the Schizosaccharomyces pombe proteasome subunit Rpn7 and a structure-function study of the proteasome-COP9-initiation factor domain.

Proper assembly of the 26 S proteasome is required to efficiently degrade polyubiquitinated proteins. Many proteasome subunits contain the proteasome-COP9-initiation factor (PCI) domain, thus raising the possibility that the PCI domain may play a role in mediating proteasome assembly. We have previously characterized the PCI protein Yin6, a fission yeast ortholog of the mammalian Int6 that has been implicated in breast oncogenesis, and demonstrated that it binds and regulates the assembly of the proteasome. In this study, we isolated another PCI proteasome subunit, Rpn7, as a high copy suppressor that rescued the proteasome defects in yin6 null cells. To better define the function of the PCI domain, we aligned protein sequences to identify a conserved leucine residue that is present in nearly all known PCI domains. Replacing it with aspartate in yeast Rpn7, Yin6, and Rpn5 inactivated these proteins, and mutant human Int6 mislocalized in HeLa cells. Rpn7 and Rpn5 bind Rpn9 with high affinity, but their mutant versions do not. Our data suggest that this leucine may interact with several hydrophobic amino acid residues to influence the spatial arrangement either within the N-terminal tandem alpha-helical repeats or between these repeats and the more C-terminal winged helix subdomain. Disruption of such an arrangement in the PCI domain may substantially inactivate many PCI proteins and block their binding to other proteins.