OBJECTIVE: Proteasomes are conserved proteases crucial for proteostasis in eukaryotes and are promising drug targets for protozoan parasites. Yet, the proteasomes of Entamoeba histolytica remain understudied. The study\'s objective was to analyse the differences in the substrate binding pockets of amoeba proteasomes from those of host, and computational modelling of β5 catalytic subunit, with the goal of finding selective inhibitors.
RESULTS: Comparative sequence analysis revealed differences in substrate binding sites of E. histolytica proteasomes, especially in the S1 and S3 pockets of the catalytic beta subunits, implying differences in substrate preference and susceptibility to inhibitors from host proteasomes. This was strongly supported by significantly lower sensitivity to MG132 mediated inhibition of amoebic proteasome β5 subunit\'s chymotryptic activity compared to human proteasomes, also reflected in lower sensitivity of E. histolytica to MG132 for inhibition of proliferation. Computational models of β4 and β5 subunits, and a docked β4-β5 model revealed a binding pocket between β4-β5, similar to that of Leishmania tarentolae. Selective inhibitors for visceral leishmaniasis, LXE408 and compound 8, docked well to this pocket. This functional and sequence-based analysis predicts differences between amoebic and host proteasomes that can be utilized to develop rationally designed, selective inhibitors against E. histolytica.

Pyruvate dehydrogenase kinase (PDK), which phosphorylates the pyruvate dehydrogenase complex, regulates glucose metabolism in skeletal muscle. PDK1, an isozyme whose expression is controlled by hypoxia-inducible factor-1α (HIF-1α), is thought to play a role in muscle adaptation to hypoxia. While transcriptional upregulation of PDK1 by HIF-1α is well characterised, mechanisms controlling proteolysis of PDK1 in skeletal muscle have not been thoroughly investigated. Proteasome inhibitor MG132 paradoxically reduced the abundance of PDK1 in human cancer cells and rat L6 myotubes, suggesting that MG132 might direct PDK1 towards autophagic degradation. The objectives of our current study were to determine (1) whether MG132 suppresses PDK1 levels in primary human myotubes, (2) whether chloroquine, an inhibitor of autophagy, prevents MG132-induced suppression of PDK1 in L6 myotubes, and (3) whether PYR-41, an inhibitor of ubiquitination, suppresses PDK1 in L6 myotubes. Using qPCR and/or immunoblotting, we found that despite markedly upregulating HIF-1α protein, MG132 did not alter the PDK1 expression in cultured primary human myotubes, while it suppressed both PDK1 mRNA and protein in L6 myotubes. The PDK1 levels in L6 myotubes were suppressed also during co-treatment with chloroquine and MG132. PYR-41 markedly increased the abundance of HIF-1α in primary human and L6 myotubes, while reducing the abundance of PDK1. In L6 myotubes treated with PYR-41, chloroquine increased the abundance of the epidermal growth factor receptor, but did not prevent the suppression of PDK1. Collectively, our results suggest that cultured myotubes degrade PDK1 via a pathway that cannot be inhibited by MG132, PYR-41, and/or chloroquine.

BACKGROUND: MG132, a proteasome inhibitor, is widely used to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity by proteasome-mediated degradation of IκB. It has been marketed as a specific, reversible, cell-permeable and low-cost inhibitor. However, adverse effects of the compound have been reported in the literature. We recently discovered and characterised a point mutation in the acute phase protein serum amyloid A (SAA) in chickens, by overexpressing the protein in chicken hepatocellular carcinoma (LMH) cells. This serine to arginine exchange at amino acid position 90 (SAA.R90S) leads to intra- and extracellular accumulation of SAA, which is surprisingly counteracted by MG132 treatment, independent of SAA\'s intrinsic promoter.
RESULTS: To test, whether low proteasomal degradation of SAA.R90S is responsible for the observed intra- and extracellular SAA accumulation, we intended to inhibit the proteasome in SAA wild type (SAA.WT) overexpressing cells with MG132. However, we observed an unexpected drastic decrease in SAA protein expression at the transcript level. NF-κB gene expression was unchanged by MG132 at the measured time point.
CONCLUSIONS: The observed results demonstrate that MG132 inhibits SAA expression at the transcript level, independent of its endogenous promoter. Further, the data might indicate that NF-κB is not involved in the observed MG132-induced inhibition of SAA expression. We, consequently, question in this brief report whether MG132 should truly be categorised as a specific ubiquitin proteasome inhibitor and recommend the usage of alternative compounds.

Ferroptosis is involved in the pathogenesis of osteoarthritis (OA) while suppression of chondrocyte ferroptosis has a beneficial effect on OA. However, the molecular mechanism of ferroptosis in OA remains to be elucidated. P21, an indicator of aging, has been reported to inhibit ferroptosis, but the relationship between P21 and ferroptosis in OA remains unclear. Here, we aimed to investigate the expression and function of P21 in OA chondrocytes, and the involvement of P21 in the regulation of ferroptosis in chondrocytes. First, we demonstrated that high P21 expression was observed in the cartilage from OA patients and destabilized medial meniscus (DMM) mice, and in osteoarthritic chondrocytes induced by IL-1β, FAC and erastin. P21 knockdown exacerbated the reduction of Col2a1 and promoted the upregulation of MMP13 in osteoarthritic chondrocytes. Meanwhile, P21 knockdown exacerbated cartilage degradation in DMM-induced OA mouse models and decreased GPX4 expression in vivo. Furthermore, P21 knockdown sensitized chondrocytes to ferroptosis induced by erastin, which was closely associated with the accumulation of lipid peroxides. In mechanism, we demonstrated that P21 regulated the stability of GPX4 protein, and the regulation was independent of NRF2. Meanwhile, we found that P21 significantly affected the recruitment of GPX4 to linear ubiquitin chain assembly complex (LUBAC) and regulated the level of M1-linked ubiquitination of GPX4. Overall, our results suggest that P21 plays an essential anti-ferroptosis role in OA by regulating the stability of GPX4.

Purpose: This study clarifies the beneficial effects of MG132, a proteasomal inhibitor, on retinal vascular injury mediated by diabetes-induced oxidative stress through nuclear factor erythroid 2-related factor 2 (Nrf2). Methods: Diabetic rats and control animals were randomly assigned to receive MG132 or vehicle for 24 weeks, and human retinal endothelial cells (HRECs) were incubated with normal or high glucose with or without MG132. 26S proteasome activity in the rat retinas or cultured HRECs was measured using Suc-LLVY-7-amido-4-methylcoumarin. NADPH-quinone oxidoreduc-tase (NQO1), heme oxygenase (HO)-1, kelch-like ECH-associated protein 1 (Keap1) and Nrf2 were examined by Western blotting and real-time reverse transcription polymerase chain reaction. Cell apoptosis is measured through flow cytometry assay, mitochondrial reactive oxygen species (ROS) production, and retinal vascular leakage were assayed using CM-H2DCFDA fluorescent probes and Evans blue, respectively. Results: MG132 significantly inhibited the activation of 26S proteasome induced by diabetes or elevated glucose, and subsequently increased the expression of Nrf2, NQO1, and HO-1, and further reduced ROS accumulation. These changes were associated with a decrease of diabetes-induced retinal vascular leakage and retinal capillary cell apoptosis. Conclusions: MG132 decreases diabetes-induced 26S proteasome activation and exerts protective effects against retinal microvascular dysfunction in diabetic rats in association with the alleviation of retinal oxidative stress mediated by Nrf2.

Trichomonas gallinae is a protozoan parasite that is the causative agent of trichomoniasis, and infects captive and wild bird species throughout the world. Although metronidazole has been the drug of choice against trichomoniasis for decades, most Trichomonas gallinae strains have developed resistance. Therefore, drugs with new modes of action or targets are urgently needed. Here, we report the development and application of a cell-based CCK-8 method for the high-throughput screening and identification of new inhibitors of Trichomonas gallinae as a beginning point for the development of new treatments for trichomoniasis. We performed the high-throughput screening of 173 anti-parasitic compounds, and found 16 compounds that were potentially effective against Trichomonas gallinae. By measuring the median inhibitory concentration (IC50) and median cytotoxic concentration (CC50), we identified 3 potentially safe and effective compounds against Trichomonas gallinae: anisomycin, fumagillin, and MG132. In conclusion, this research successfully established a high-throughput screening method for compounds and identified 3 new safe and effective compounds against Trichomonas gallinae, providing a new treatment scheme for trichomoniasis.

Methylmercury (MeHg), an environmental pollutant, disrupts and impairs cellular function. MeHg binds to various cellular proteins, causing dysfunction and misfolding, which are considered underlying causes of MeHg toxicity. The p62 protein, also termed SQSTM1, is a ubiquitin-binding protein that targets ubiquitinated substrates to undergo autophagy and plays a key role in ameliorating MeHg toxicity. p62 also delivers ubiquitinated substrates to proteasomes. However, the role of these degradation systems in mitigating MeHg toxicity remains unknown. Herein, we explored the impact of the proteasome inhibitor MG132 on MeHg toxicity and examined the toxicity of co-treatment with MG132 and MeHg in p62KO mouse embryonic fibroblasts (MEFs) by analyzing cell viability, immunoblotting, mRNA levels, immunofluorescence, and the mercury content. The proteasome inhibitor MG132 enhanced MeHg-induced cytotoxicity while reducing intracellular mercury levels in MEFs. Co-treatment with MG132 and MeHg markedly increased levels of p62 and ubiquitinated proteins. Furthermore, co-treatment with MG132 and MeHg reduced p62KO MEF viability compared to that of wild-type MEFs. Our findings suggest that the proteasome participates in mitigating MeHg cytotoxicity, while p62 may play an important role in transporting MeHg-induced ubiquitinated proteins to the proteasome, as well as in autophagy. Collectively, these results imply that p62, and proteasome, and autophagy are vital for cytoprotection against MeHg toxicity.

Medullary thyroid cancer (MTC) is a rare thyroid tumour whose management in advanced stages is challenging, despite effective therapeutic options having expanded in recent years. Proteasome inhibitors (PrIn) have shown the ability to improve patient outcomes, including survival and quality of life, in several malignancies, due to their ability to impair cell proliferation and cause apoptosis through the inhibition of the proteasome activity. Consequently, these drugs could represent a useful tool, alone or in combination with other treatments, in MTC patients.
This review aims to summarize the available in vitro and in vivo data about the role of PrIn in MTC.
We performed an extensive search for relevant data sources, including full-published articles in international online databases (PubMed, Web of Science, Scopus), preliminary reports in selected international meeting abstract repositories, and short articles published as supplements of international meetings, by using the following terms: medullary thyroid carcinoma, proteasome inhibitors, bortezomib, carfilzomib, ixazomib, delanzomib, marizomib, oprozomib, and MG132. Additionally, we conducted with the same keywords, an in-depth search in registered clinical trials repositories.
Our search revealed in vitro studies in human and murine MTC cell lines, based on the use of PrIns, both alone and in combination with other anticancer drugs, and two pertinent clinical trials.
We found a strong discrepancy between the evidence of PrIns effects in preclinical studies, and the scarcity or early interruption of clinical trials. We might speculate that difficulties in enrolling patients, as happens in other rare diseases, may have discouraged trials\' implementation in favor of drugs already approved for MTC. However, given the concrete improvement in the comprehension of the molecular basis of PrIn effects in MTC, new clinical trials with accurate inclusion criteria of enrollment might be warranted, in order to ascertain whether this treatment, alone or in combination with other drugs, could indeed represent an option to enhance the therapeutic response, and to ultimately improve patients\' outcome and survival.

Cis-diamine-dichloroplatinum II (cisplatin, CDDP) is a key chemotherapeutic regimen in the treatment of oral squamous cell carcinoma (OSCC). However, the therapeutic efficacy of cisplatin in OSCC may be hampered by chemoresistance. Therefore, the development of novel combination therapy strategies to overcome the limitations of CDDP is of great importance. The proteasome inhibitor MG132 exhibits anti-cancer properties against various types of cancer. However, our knowledge of its anti-cancer effects in combination with CDDP in OSCC cells remains limited. In the current study, the synergetic effects of MG132 and CDDP were evaluated in the human CAL27 OSCC cell line. CAL27 cells were treated with CDDP alone or in combination with MG132. The results showed that MG132 significantly reduced cell viability in a dose-dependent manner. Additionally, cell viability was significantly reduced in CAL27 cells treated with 0.2 µM MG132 and 2 µM CDDP compared with cells treated with MG132 or CDDP alone. In addition, MG132 significantly enhanced the CDDP-induced generation of intracellular reactive oxygen species and DNA damage in OSCC cells. Furthermore, treatment with CDDP or MG132 alone notably inhibited colony formation and proliferation of OSCC cells. However, co-treatment of OSCC cells with MG132 and CDDP further hampered colony formation and proliferation compared with cells treated with either MG132 or CDDP alone. Finally, in cells co-treated with MG132 and CDDP, the expression of p53 was markedly elevated and the p53-mediated apoptotic pathway was further activated compared with cells treated with MG132 or CDDP alone, as shown by the enhanced cell apoptosis, Bax upregulation, and Bcl-2 downregulation. Overall, the results of the current study support the synergistic anti-cancer effects of a combination of MG132 and CDDP against OSCC, thus suggesting that the combination of MG132 and CDDP may be a promising therapeutic strategy for the management of OSCC.

We previously developed a stress-induced premature senescence (SIPS) model in which normal human fibroblast MRC-5 cells were treated with either the proteasome inhibitor MG132 or the vacuolar-type ATPase inhibitor bafilomycin A1 (BAFA1). To clarify the involvement of mitochondrial function in our SIPS model, MRC-5 cells were treated with MG132 or BAFA1 along with an inhibitor targeting either the electron transport chain complex I or complex III, or with a mitochondrial uncoupler. SIPS induced by MG132 or BAFA1 was significantly attenuated by short-term co-treatment with the complex III inhibitor, antimycin A (AA), but not the complex I inhibitor, rotenone or the mitochondrial uncoupler, carbonyl cyanide 3-chlorophenylhydrazone. By co-treatment with AA, mitochondrial and intracellular reactive oxygen species levels, accumulation of protein aggregates and mitochondrial unfolded protein responses (UPRmt ) were remarkably suppressed. Furthermore, AA co-treatment suppressed the hyperpolarization of the mitochondrial membrane and the induction of mitophagy observed in MG132-treated cells and enhanced mitochondrial biogenesis. These findings provide evidence that the temporal inhibition of mitochondrial respiration exerts protective effects against the progression of premature senescence caused by impaired proteostasis.