leupeptins and carfilzomib

Proteasome inhibitors are an important class of chemotherapeutic drugs. In this study, we performed a large-scale ubiquitylome analysis of the three proteasome inhibitors MG132, bortezomib and carfilzomib. Although carfilzomib is currently being used for the treatment of multiple myeloma, it has not yet been subjected to a global ubiquitylome analysis. In this study, we identified more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. We introduced stringent criteria to determine the correct ubiquitylation site ratios and used five biological replicates to achieve increased statistical power. With the vast amount of data acquired, we made proteome-wide comparisons between the proteasome inhibitors and indicate candidate proteins that will benefit from further study. We find that in addition to the expected increase in ubiquitylation in the majority of proteins, unexpectedly a select few are specifically and significantly decreased in ubiquitylation at specific sites after treatment with proteasome inhibitors. We chose to follow-up on Mortality factor 4-like 1 (MORF4L1), which was significantly decreased in ubiquitylation at lysine 187 and lysine 104 upon proteasome inhibition, but increased in protein abundance by approximately two-fold. We demonstrate that the endogenous protein level of MORF4L1 is highly regulated by the ubiquitin proteasome system. SIGNIFICANCE: This study provides a highly curated dataset of more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. For the proper quantification of ubiquitylation sites, we introduced a higher standard by quantifying only those ubiquitylation sites that are not flanked by neighboring ubiquitylation, thereby avoiding the report of incorrect ratios. The sites identified will serve to identify important targets of the ubiquitin proteasome system and aid to better understand the repertoire of proteins that are affected by inhibiting the proteasome with MG132, bortezomib, and carfilzomib. In addition, we investigated the unusual observation that ubiquitylation of the tumor suppressor Mortality factor 4-like (MORF4L1) protein decreases rather than increases upon proteasome inhibition, which may contribute to an additional anti-tumor effect of bortezomib and carfilzomib.

Topics: Antineoplastic Agents; Bortezomib; Humans; Leupeptins; Multiple Myeloma; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteomics; Ubiquitination

Organic anion transporter 1 (OAT1), expressed at the basolateral membrane of renal proximal tubule epithelial cells, mediates the renal excretion of many clinically important drugs. Previous study in our laboratory demonstrated that ubiquitin conjugation to OAT1 leads to OAT1 internalization from the cell surface and subsequent degradation. The current study showed that the ubiquitinated OAT1 accumulated in the presence of the proteasomal inhibitors MG132 and ALLN rather than the lysosomal inhibitors leupeptin and pepstatin A, suggesting that ubiquitinated OAT1 degrades through proteasomes. Anticancer drugs bortezomib and carfilzomib target the ubiquitin-proteasome pathway. We therefore investigate the roles of bortezomib and carfilzomib in reversing the ubiquitination-induced downregulation of OAT1 expression and transport activity. We showed that bortezomib and carfilzomib extremely increased the ubiquitinated OAT1, which correlated well with an enhanced OAT1-mediated transport of p-aminohippuric acid and an enhanced OAT1 surface expression. The augmented OAT1 expression and transport activity after the treatment with bortezomib and carfilzomib resulted from a reduced rate of OAT1 degradation. Consistent with this, we found decreased 20S proteasomal activity in cells that were exposed to bortezomib and carfilzomib. In conclusion, this study identified the pathway in which ubiquitinated OAT1 degrades and unveiled a novel role of anticancer drugs bortezomib and carfilzomib in their regulation of OAT1 expression and transport activity. SIGNIFICANCE STATEMENT: Bortezomib and carfilzomib are two Food and Drug Administration-approved anticancer drugs, and proteasome is the drug target. In this study, we unveiled a new role of bortezomib and carfilzomib in enhancing OAT1 expression and transport activity by preventing the degradation of ubiquitinated OAT1 in proteasomes. This finding provides a new strategy in regulating OAT1 function that can be used to accelerate the clearance of drugs, metabolites, or toxins and reverse the decreased expression under disease conditions.

Topics: Antineoplastic Agents; Biological Transport, Active; Bortezomib; HEK293 Cells; Humans; Leupeptins; Oligopeptides; Organic Anion Transport Protein 1; p-Aminohippuric Acid; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Ubiquitination

Proteases are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult

Topics: Animals; Antineoplastic Agents; Bortezomib; Caspases; Cysteine Proteinase Inhibitors; Drug Delivery Systems; Hep G2 Cells; Humans; Leupeptins; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Schistosoma mansoni

Mitotane is the only drug approved for treatment of the orphan disease adrenocortical carcinoma (ACC) and was recently shown to be the first clinically used drug acting through endoplasmic reticulum (ER)-stress induced by toxic lipids. Since mitotane has limited clinical activity as monotherapy, we here study the potential of activating ER-stress through alternative pathways. The single reliable NCI-H295 cell culture model for ACC was used to study the impact MG132, bortezomib (BTZ) and carfilzomib (CFZ) on mRNA and protein expression of ER-stress markers, cell viability and steroid hormone secretion. We found all proteasome inhibitors alone to trigger expression of mRNA (spliced X-box protein 1, XBP1) and protein markers indicative of the inositol-requiring enzyme 1 (IRE1) dependent pathway of ER-stress but not phosphorylation of eukaryotic initiation factor 2α (eIF2α), a marker of the PRKR-like endoplasmic reticulum kinase (PERK)-dependent pathway. Whereas mitotane alone activated both pathways, combination of BTZ and CFZ with low-dose mitotane blocked mitotane-induced eIF2α phosphorylation but increased XBP1-mRNA splicing indicating that proteasome inhibitors can commit signalling towards a single ER-stress pathway in ACC cells. By applying the median effect model of drug combinations using cell viability as a read out, we determined significant drug synergism between mitotane and both BTZ and CFZ. In conclusion, combination of mitotane with activators of ER-stress through the unfolded protein response is synergistic in an ACC cell culture model. Since proteasome inhibitors are readily available clinically, they are attractive candidates to study for ACC treatment in clinical trials in combination with mitotane.

Topics: Adrenal Cortex Neoplasms; Adrenocortical Carcinoma; Antineoplastic Agents, Hormonal; Bortezomib; Cell Line, Tumor; Cell Survival; Drug Synergism; Drug Therapy, Combination; Endoplasmic Reticulum Stress; Endoribonucleases; Eukaryotic Initiation Factor-2; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Mitotane; Oligopeptides; Phosphorylation; Proteasome Inhibitors; Protein Serine-Threonine Kinases; X-Box Binding Protein 1

The ubiquitin-proteasome system (UPS) has been successfully targeted by both academia and the pharmaceutical industry for oncological and immunological applications. Typical proteasome inhibitors are based on a peptidic backbone endowed with an electrophilic C-terminus by which they react with the active proteolytic sites. Although the peptide moiety has attracted much attention in terms of subunit selectivity, the target specificity and biological stability of the compounds are largely determined by the reactive warheads. In this study, we have carried out a systematic investigation of described electrophiles by a combination of in vitro, in vivo, and structural methods in order to disclose the implications of altered functionality and chemical reactivity. Thereby, we were able to introduce and characterize the class of α-ketoamides as the most potent reversible inhibitors with possible applications for the therapy of solid tumors as well as autoimmune disorders.

Topics: Binding Sites; Boronic Acids; Bortezomib; Catalytic Domain; Crystallography, X-Ray; HeLa Cells; Humans; Leupeptins; Molecular Dynamics Simulation; Oligopeptides; Peptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Pyrazines

The ubiquitin-proteasome pathway degrades ubiquitinated proteins to remove damaged or misfolded protein and thus plays an important role in the maintenance of many important cellular processes. Because the pathway is also crucial for tumor cell growth and survival, proteasome inhibition by specific inhibitors exhibits potent antitumor effects in many cancer cells. xCT, a subunit of the cystine antiporter system xc (-), plays an important role in cellular cysteine and glutathione homeostasis. Several recent reports have revealed that xCT is involved in cancer cell survival; however, it was unknown whether xCT affects the cytotoxic effects of proteasome inhibitors. In this study, we found that two stress-inducible transcription factors, Nrf2 and ATF4, were upregulated by proteasome inhibition and cooperatively enhance human xCT gene expression upon proteasome inhibition. In addition, we demonstrated that the knockdown of xCT by small interfering RNA (siRNA) or pharmacological inhibition of xCT by sulfasalazine (SASP) or (S)-4-carboxyphenylglycine (CPG) significantly increased the sensitivity of T24 cells to proteasome inhibition. These results suggest that the simultaneous inhibition of both the proteasome and xCT could have therapeutic benefits in the treatment of bladder tumors.

Topics: Activating Transcription Factor 4; Amino Acid Transport System y+; Antineoplastic Agents; Boronic Acids; Bortezomib; Cell Line, Tumor; Cysteine; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glutathione; Glycine; HEK293 Cells; HeLa Cells; Humans; Leupeptins; NF-E2-Related Factor 2; Oligopeptides; Proteasome Endopeptidase Complex; Pyrazines; RNA, Small Interfering; Sulfasalazine; Urinary Bladder Neoplasms

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.

Topics: Antineoplastic Agents; Boronic Acids; Bortezomib; Cell Line, Tumor; HEK293 Cells; Humans; Inhibitory Concentration 50; Intracellular Space; Leupeptins; Oligopeptides; Peptides; Proteasome Inhibitors; Pyrazines