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In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipophilic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds 13, 20, and 21 showed the best proteasome inhibition activity with IC50 values of 1.42, 1.56, and 1.80 µM, respectively, which are three- to four-fold more potent than the proteasome inhibition controls LLM-F and lactacystin.

Background

Because of the vital importance of the proteasome pathway, chemicals affecting proteasome activity could disrupt essential cellular processes. Although the toxicity of organotins to both invertebrates and vertebrates is well known, the essential cellular target of organotins has not been well identified. We hypothesize that the proteasome is a molecular target of environmental toxic organotins.

Objectives

Our goal was to test the above hypothesis by investigating whether organotins could inhibit the activity of purified and cellular proteasomes and, if so, the involved molecular mechanisms and downstream events.

Results

We found that some toxic organotins [e.g., triphenyltin (TPT)] can potently and preferentially inhibit the chymotrypsin-like activity of purified 20S proteasomes and human breast cancer cellular 26S proteasomes. Direct binding of tin atoms to cellular proteasomes is responsible for the observed irreversible inhibition. Inhibition of cellular proteasomes by TPT in several human cell lines results in the accumulation of ubiquitinated proteins and natural proteasome target proteins, accompanied by induction of cell death.

Conclusions

The proteasome is one of the molecular targets of environmental toxic organotins in human cells, and proteasome inhibition by organotins contributes to their cellular toxicity.

Introduction

Proteasome inhibition is an attractive approach to anticancer therapy and may have relevancy in breast cancer treatment. Natural products, such as dietary flavonoids, have been suggested as natural proteasome inhibitors with potential use for cancer prevention and therapeutics. We previously reported that apigenin, a flavonoid widely distributed in many fruits and vegetables, can inhibit proteasome activity and can induce apoptosis in cultured leukemia Jurkat T cells. Whether apigenin has proteasome-inhibitory activity in the highly metastatic human breast MDA-MB-231 cells and xenografts, however, is unknown.

Methods

MDA-MB-231 breast cancer cell cultures and xenografts were treated with apigenin, followed by measurement of reduced cellular viability/proliferation, proteasome inhibition, and apoptosis induction. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity, by ubiquitinated proteins, and by accumulation of proteasome target proteins in extracts of the treated cells or tumors. Apoptotic cell death was measured by capase-3/caspase-7 activation, poly(ADP-ribose) polymerase cleavage, and immunohistochemistry for terminal nucleotidyl transferase-mediated nick end labeling positivity.

Results

We report for the first time that apigenin inhibits the proteasomal chymotrypsin-like activity and induces apoptosis not only in cultured MDA-MB-231 cells but also in MDA-MB-231 xenografts. Furthermore, while apigenin has antibreast tumor activity, no apparent toxicity to the tested animals was observed.

Conclusion

We have shown that apigenin is an effective proteasome inhibitor in cultured breast cancer cells and in breast cancer xenografts. Furthermore, apigenin induces apoptotic cell death in human breast cancer cells and exhibits anticancer activities in tumors. The results suggest its potential benefits in breast cancer prevention and treatment.

Betulinic acid, a triterpenoid isolated from the methyl alcohol extract of the leaves of Syzigium claviflorum, was found to have a potent inhibitory activity against human immunodeficiency virus type 1 (HIV-1). Betulinic acid derivatives were synthesized to enhance the anti-HIV activity. Among the derivatives, 3-O-(3′,3′-dimethylsuccinyl) betulinic acid, designated YK-FH312, showed the highest activity against HIV-induced cytopathic effects in HIV-1-infected MT-4 cells. To determine the step(s) of HIV replication affected by YK-FH312, a syncytium formation inhibition assay in MOLT-4/HIV-1IIIB and MOLT-4 coculture, a multinuclear-activation-of-galactosidase-indicator (MAGI) assay in MAGI-CCR5 cells, electron microscopic observation, and a time-of-addition assay were performed. In the syncytium formation inhibition assay or in the MAGI assay for de novo infection, the compound did not show inhibitory effects against HIV replication. Conversely, no virions were detected in HIV-1-infected cell cultures treated with YK-FH312 either by electron microscopic observation or by viral yield in the supernatant. In accordance with a p24 enzyme-linked immunosorbent assay of culture supernatant in the time-of-addition assay, YK-FH312 inhibited virus expression in the supernatant when it was added 18 h postinfection. However, Western blot analysis of the cells in the time-of-addition assay revealed that the production of viral proteins in the cells was not inhibited completely by YK-FH312. These results suggest that YK-FH312 might affect the step(s) of virion assembly and/or budding of virions, and this is a novel mechanism of action of an anti-HIV compound.

Background

Despite the effectiveness of currently available antiretroviral therapies in the treatment of HIV-1 infection, a continuing need exists for novel compounds that can be used in combination with existing drugs to slow the emergence of drug-resistant viruses. We previously reported that the small molecule 3-O-{3',3'-dimethylsuccinyl}-betulinic acid (DSB) specifically inhibits HIV-1 replication by delaying the processing of the CA-SP1 junction in Pr55Gag. By contrast, SIVmac239 replicates efficiently in the presence of high concentrations of DSB. To determine whether sequence differences in the CA-SP1 junction can fully account for the differential sensitivity of HIV-1 and SIV to DSB, we engineered mutations in this region of two viruses and tested their sensitivity to DSB in replication assays using activated human primary CD4+ T cells.

Results

Substitution of the P2 and P1 residues of HIV-1 by the corresponding amino acids of SIV resulted in strong resistance to DSB, but the mutant virus replicated with reduced efficiency. Conversely, replication of an SIV mutant containing three amino acid substitutions in the CA-SP1 cleavage site was highly sensitive to DSB, and the mutations resulted in delayed cleavage of the CA-SP1 junction in the presence of the drug.

Conclusions

These results demonstrate that the CA-SP1 junction in Pr55Gag represents the primary viral target of DSB. They further suggest that the therapeutic application of DSB will be accompanied by emergence of mutant viruses that are highly resistant to the drug but which exhibit reduced fitness relative to wild type HIV-1.

Summary

Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis (Mtb). Given that proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-ones kill non-replicating Mtb and act as selective suicide-substrate inhibitors of the Mtb proteasome by cyclo-carbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose H-bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-ones to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homolog.

Thiazole antibiotic, thiostrepton was recently identified as proteasome inhibitor. We investigated the therapeutic potential of the combination of thiostrepton and proteasome inhibitor bortezomib (Velcade) on various human tumor cell lines. Combination of sub-lethal concentrations of thiostrepton and bortezomib induced potent apoptosis and inhibition of long-term colony formation in a wide variety of human cancer cell lines. The synergistic relationship between thiostrepton and bortezomib combination was also quantitatively demonstrated by calculating their combination index values that were much lower than 1 in all studied cell lines. The synergy between these drugs was based on their proteasome inhibitory activities, because thiostrepton modification, thiostrepton methyl ester, which did not have intact quinaldic acid ring and did not inhibit proteasome activity failed to demonstrate any synergy in combination with bortezomib.

Screening efforts led to the identification of PI-8182 (1), an inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. Compound 1 contains a hydronaphthoquinone pharmacophore with a thioglycolic acid side chain at position 2 and thiophene sulfonamide at position 4. An efficient synthetic route to the hydronaphthoquinone sulfonamide scaffold was developed and compound 1 was synthesized in-house to confirm the structure and activity (IC50 = 3.0 ± 1.6 μM [n=25]). Novel hydronaphthoquinone derivatives of the hit 1 were designed, synthesized and evaluated as proteasome inhibitors. The structure activity relationship (SAR) guided synthesis of more than 170 derivatives revealed that the thioglycolic acid side chain is required and the carboxylic acid group of this side chain is critical to the CT-L inhibitory activity of compound 1. Furthermore, replacement of the carboxylic acid with carboxylic acid isosteres such as tetrazole or triazole greatly improves potency. Compounds with a thio-tetrazole or thio-triazole side chain in position 2, where the thiophene was replaced by hydrophobic aryl moieties were the most active compounds with up to 20-fold greater CT-L inhibitory than compound 1 (compounds 15e, 15f, 15h 15j, IC50 values around 200 nM and compound 29, IC50 = 150 nM). The synthetic iterations described here not only led to improving potency in vitro but also resulted in the identification of compounds that are more active such as 39 (IC50 = 0.44 to 1.01 μM) than 1 (IC50 = 3.54 to 7.22 μM) at inhibiting the proteasome CT-L activity in intact breast cancer cells. Treatment with 39 also resulted in the accumulation of ubiquitinated cellular proteins and inhibition of tumor cell proliferation of breast cancer cells. The hit 1 and its analog 39 inhibited proteasome CT-L activity irreversibly.

The compound 3-O-(3′,3′-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.

The nuclear proteasome activator REGγ/PA28γ is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REGγ-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.

Tea is a widely consumed beverage and its constituent polyphenols have been associated with potential health benefits. Although black tea polyphenols have been reported to possess potent anticancer activities, the effect of its polyphenols, theaflavins on the tumor’s cellular proteasome function, an important biological target in cancer prevention, has not been carefully studied. Here black tea extract (T5550) enriched in theaflavins inhibited the chymotrypsin-like (CT) activity of the proteasome and proliferation of human multiple myeloma cells in a dose-dependent manner. Also an isolated theaflavin (TF-1) can bind to, and inhibit the purified 20S proteasome, accompanied by suppression of tumor cell proliferation, suggesting that the tumor proteasome is an important target whose inhibition is at least partially responsible for the anti-cancer effects of black tea.

STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulates the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid -induced down regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP-1 and silencing of the SHP-1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescues betulinic acid-induced cell death. Betulinic acid also downregulated the expression of STAT3-regulated gene products such as bcl-xL, bcl-2, cyclin D1, and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub-G1 cell population and an increase in caspase-3–induced PARP cleavage. Consistent with these results, over expression of constitutive active STAT3 significantly reduced the betulinic acid-induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10% to 55%) and bortezomib (from 5% to 70%) in MM cells. Overall, our results suggest that betulinic acid down regulates STAT3 activation through upregulation of SHP-1 and this may have potential in sensitization of STAT3 over expressing tumors to chemotherapeutic agents.

SUMMARY

In Saccharomyces cerevisiae, chemical or genetic inhibition of proteasome activity induces new proteasome synthesis promoted by the transcription factor RPN4. This ensures that proteasome activity is matched to demand. This transcriptional feedback loop is conserved in mammals, but its molecular basis is not understood. Here we report that Nuclear factor erythroid derived 2-related factor 1 (Nrf1), a transcription factor of the cap ‘n’ collar basic leucine zipper family, but not the related Nrf2, is necessary for induced proteasome gene transcription in mouse embryonic fibroblasts (MEFs). Promoter-reporter assays revealed the importance of antioxidant response elements in Nrf1-mediated upregulation of proteasome subunit genes. Nrf1-/- MEFs were impaired in the recovery of proteasome activity after transient treatment with the covalent proteasome inhibitor YU101 and knockdown of Nrf1 in human cancer cells enhanced cell killing by YU101. Taken together, our results suggest that Nrf1-mediated proteasome homeostasis could be an attractive target for therapeutic intervention in cancer.

Our recent studies with lactacystin, a prototype proteasome inhibitor, have suggested that the proteasome is a key regulator of LPS-induced signaling pathways contributing to the inflammatory process. Moreover, lactacystin protects animals from LPS-induced shock. Therefore, we sought to identify other less toxic compounds that would block the chymotrypsin-like activity of the proteasome or LPS-induced nitric oxide (NO). After screening over 100 natural compounds (based on chemistry and inhibition of LPS-induced biological activities), we now report for the first time that quercetin, like lactacystin (the prototype proteasome inhibitor), and mevinolin are also inhibitors of the chymotrypsin-like activity of the cellular proteasome within living cells. In addition, this study also suggests that mevinolin and quercetin both have relatively potent anti-inflammatory effects on LPS-treated macrophages in vitro. Interestingly, both of these compounds behave like lactacystin in that they block LPS-induced NO to a greater extent than TNF-α. The results of our experiments clearly suggest that mevinolin, in combination with the antibiotic imipenem, can provide protection against polymicrobial septic lethality induced by cecal-ligation and puncture in mice. Collectively, these studies strongly support the conclusion that therapeutic targeting of cellular proteasomes, in conjunction with standard antimicrobial therapy, may be of considerable survival benefit in the treatment of septic shock.

Betulinic acid (BA) derivatives can inhibit human immunodeficiency virus type 1 (HIV-1) entry or maturation depending on side chain modifications. While BA derivatives with antimaturation activity have attracted considerable interest, the anti-HIV-1 profile and molecular mechanism of BA derivatives with anti-HIV-1 entry activity (termed BA entry inhibitors) have not been well defined. In this study, we have found that two BA entry inhibitors, IC9564 and A43D, exhibited a broad spectrum of anti-HIV-1 activity. Both compounds inhibited multiple strains of HIV-1 from clades A, B, and C at submicromolar concentrations. Clade C viruses were more sensitive to the compounds than clade A and B viruses. Interestingly, IC9564 at subinhibitory concentrations could alter the antifusion activities of other entry inhibitors. IC9564 was especially potent in increasing the sensitivity of HIV-1YU2 Env-mediated membrane fusion to the CCR5 inhibitor TAK-779. Results from this study suggest that the V3 loop of gp120 is a critical determinant for the anti-HIV-1 activity of IC9564. IC9564 escape viruses contained mutations near the tip of the V3 loop. Moreover, IC9564 could compete with the binding of V3 monoclonal antibodies 447-52D and 39F. IC9564 also competed with the binding of gp120/CD4 complexes to chemokine receptors. In summary, these results suggest that BA entry inhibitors can potently inhibit a broad spectrum of primary HIV-1 isolates by targeting the V3 loop of gp120.

Curcumin (diferuloylmethane) is the major active ingredient of turmeric (curcuma longa) used in South Asian cuisine for centuries. Curcumin has been shown to inhibit the growth of transformed cells and to have a number of potential molecular targets. However, the essential molecular targets of curcumin under physiological conditions have not been completely defined. Herein, we report that the tumor cellular proteasome is most likely an important target of curcumin. Nucleophilic susceptibility and in silico docking studies show that both carbonyl carbons of the curcumin molecule are highly susceptible to a nucleophilic attack by the hydroxyl group of the N-terminal threonine of the proteasomal chymotrypsin-like subunit. Consistently, curcumin potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC50=1.85 µM) and cellular 26S proteasome. Furthermore, inhibition of proteasome activity by curcumin in human colon cancer HCT-116 and SW480 cell lines leads to accumulation of ubiquitinated proteins and several proteasome target proteins, and subsequent induction of apoptosis. Furthermore, treatment of HCT-116 colon tumor–bearing ICR SCID mice with curcumin resulted in decreased tumor growth, associated with proteasome inhibition, proliferation suppression and apoptosis induction in tumor tissues. Our study demonstrates that proteasome inhibition could be one of the mechanisms for the chemopreventive and/or therapaeutic roles of curcumin in human colon cancer. Based on its ability to inhibit the proteasome and induce apoptosis in both HCT-116 and metastatic SW480 colon cancer cell lines, our study suggests that curcumin could potentially be used for treatment of both early stage and late stage/refractory colon cancer.

A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of potency on proteasome inhibition. Among them, the phenylene diacetic acid hemiester derivative (13) displayed the most potent proteasome inhibition with IC50 = 1.9 μM. Enzyme kinetic analysis indicates that these lithocholic acid derivatives are non-competitive inhibitors of the proteasome.

Proteasomes, the primary mediators of ubiquitin-protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that Usp14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin-protein conjugates, in vivo and in vitro. A catalytically inactive variant of Usp14 has reduced inhibitory activity, suggesting that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human Usp14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. Usp14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of Usp14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.

Despite the effectiveness of currently available human immunodeficiency virus type 1 (HIV-1) therapies, a continuing need exists for new drugs to treat HIV-1 infection. We investigated the mechanism by which 3-O-{3′,3′-dimethylsuccinyl}-betulinic acid (DSB) inhibits HIV-1 replication. DSB functions at a late stage of the virus life cycle but does not inhibit the HIV-1 protease in vitro or interfere with virus assembly or release. DSB specifically delays the cleavage of Gag between the capsid (CA) and p2, resulting in delayed formation of the mature viral core and reduced HIV-1 infectivity. Replication of simian immunodeficiency virus (SIV) was resistant to DSB; however, a chimeric SIV carrying CA-p2 sequences from HIV-1 was inhibited by the drug, indicating that susceptibility to DSB maps to the CA-p2 region of the HIV-1 Gag protein. A single point mutation at the CA-p2 cleavage site of HIV-1 conferred strong resistance to DSB, confirming the target of the drug. HIV-1 strains that are resistant to a variety of protease inhibitors were sensitive to DSB. These findings indicate that DSB specifically protects the CA-p2 cleavage site from processing by the viral protease during virion maturation, thereby revealing a novel mechanism for pharmacologic inhibition of HIV-1 replication.

Nuclear receptors (NRs) represent a class of transcription factors that associate with both positive and negative chromatin modifying complexes to activate or repress gene transcription. The 26S proteasome plays a major role in NR-regulated gene transcription by tightly regulating the levels of the receptor and coregulator complexes. Recent evidence suggests a robust nonproteolytic role for specific proteasome subunits in gene transcription mediated via alterations in specific histone modifications. The involvement of nuclear receptors and the proteasome with chromatin modifying complexes or proteins, particularly those that modify DNA and histone proteins, provides an opportunity to review two critical epigenetic mechanisms that control gene expression and heritable biological processes. Both nuclear receptors and the proteasome are targets of environmental factors including some which lead to epigenetic changes that can influence human diseases such as cancer. In this review, we will explore molecular mechanisms by which NR-mediated gene expression, under the control of the proteasome, can result in altered epigenetic landscapes.

Aims

The molecular mechanisms of triptolide responsible for its antitumor properties are not yet fully understood. The ubiquitin/proteasome system is an important pathway of protein degradation in cells. This study investigated whether triptolide may inhibit proteasomal activity and induce apoptosis in human cancer cells.

Materials and Methods

In vitro proteasome inhibition was measured by incubation of a purified 20S proteasome with triptolide. Human breast and prostate cancer cell lines were also treated with different doses of triptolide for different times, followed by measurement of proteasome inhibition (levels of the chymotrypsin-like activity, ubiquitinated proteins and three well-known proteasome target proteins, p27, IκB-α and Bax) and apoptosis induction (caspase-3 activity and PARP cleavage).

Results

Triptolide did not inhibit the chymotrypsin-like activity of purified 20S proteasome. However, treatment of triptolide was able to cause decreased levels of cellular proteasomal chymotrypsin-like activity and accumulation of ubiquitinated proteins and three well-known proteasome target proteins in human breast and prostate cancer cells, associated with apoptosis induction.

Conclusion

It is possible that at least one of metabolites of triptolide has proteasome-inhibitory activity.

Several flavonoids have been reported to be proteasome inhibitors, but whether prenylated flavonoids are able to inhibit proteasome function remains unknown. We report for the first time that Sanggenon C, a natural prenylated flavonoid, inhibits tumor cellular proteasomal activity and cell viability. We found that (1) Sanggenon C inhibited tumor cell viability and induced cell cycle arrest at G0/G1 phase; (2) Sanggenon C inhibited the chymotrypsin-like activity of purified human 20S proteasome and 26S proteasome in H22 cell lysate, and Sanggenon C was able to dose-dependently accumulate ubiquitinated proteins and proteasome substrate protein p27; (3) Sanggenon C-induced proteasome inhibition occurred prior to cell death in murine H22 and P388 cell lines; (4) Sanggenon C induced death of human K562 cancer cells and primary cells isolated from leukemic patients. We conclude that Sanggenon C inhibits tumor cell viability via induction of cell cycle arrest and cell death, which is associated with its ability to inhibit the proteasome function and that proteasome inhibition by Sanggenon C at least partially contributes to the observed tumor cell growth-inhibitory activity.

Summary

The ubiquitin-proteasome system targets many cellular proteins for degradation and thereby controls most cellular processes. Although it is well established that proteasome inhibition is lethal, the underlying mechanism is unknown. Here, we show that proteasome inhibition results in a lethal amino acid shortage. In yeast, mammalian cells, and flies, the deleterious consequences of proteasome inhibition are rescued by amino acid supplementation. In all three systems, this rescuing effect occurs without noticeable changes in the levels of proteasome substrates. In mammalian cells, the amino acid scarcity resulting from proteasome inhibition is the signal that causes induction of both the integrated stress response and autophagy, in an unsuccessful attempt to replenish the pool of intracellular amino acids. These results reveal that cells can tolerate protein waste, but not the amino acid scarcity resulting from proteasome inhibition.

Graphical Abstract

Highlights

► Proteasome inhibition results in a lethal amino acid scarcity ► Amino acid supplementation rescues death caused by proteasome inhibition ► Proteasome-inhibited cells die by failure to maintain amino acid homeostasis ► Proteasome-inhibited cells can survive with their protein waste

Dedicated chaperones facilitate eukaryotic proteasome assembly, yet how they function remains largely unknown. Here we demonstrate that a yeast 20S proteasome assembly factor, Pba1–Pba2, requires a previously overlooked C-terminal HbYX (hydrophobic-tyrosine-X) motif for function. HbYX motifs in proteasome activators open the 20S proteasome entry pore, but Pba1–Pba2 instead binds inactive proteasomal precursors. We discovered an archaeal ortholog of this factor, here named PbaA, that also binds preferentially to proteasomal precursors in a HbYX-dependent fashion using the same proteasomal α-ring surface pockets bound by activators. Remarkably, PbaA and the related PbaB protein can be induced to bind mature 20S proteasomes if the active sites in the central chamber are occupied by inhibitors. Our data suggest an allosteric mechanism in which proteasome active-site maturation determines assembly chaperone binding, potentially shielding assembly intermediates or misassembled complexes from non-productive associations until assembly is complete.

Accumulating evidence has implicated the proteasome in the processing of protein along the major histocompatibility complex (MHC) class I presentation pathway. The availability of potent proteasome inhibitors provides an opportunity to examine the role of proteasome function in antigen presentation by MHC class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). We have investigated the processing and presenting of antigenic epitopes from influenza hemagglutinin in target cells treated with the inhibitor of proteasome activity MG132. In the absence of proteasome activity, the processing and presentation of the full- length hemagglutinin was abolished, suggesting the requirement for proteasome function in the processing and presentation of the hemagglutinin glycoprotein. Epitope-containing translation products as short as 21 amino acids when expressed in target cells required proteasome activity for processing and presentation of the hemagglutin epitope to CTLs. However, when endogenous peptides of 17 amino acids or shorter were expressed in target cells, the processing and presentation of epitopes contained in these peptides were insensitive to the proteasome inhibitor. Our results support the hypothesis that proteasome activity is required for the generation of peptides presented by MHC class I molecules and that the requirement for proteasome activity is dependent on the size of the translation product expressed in the target cell. The implications of these findings are discussed.