Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures

Evidence has accumulated showing that pharmacological inhibition of proteasome activity can both induce and prevent neuronal apoptosis. We tested the hypothesis that these paradoxical effects of proteasome inhibitors depend on the degree of reduced proteasome activity and investigated underlying mechanisms. Murine cortical cell cultures exposed to 0.1 microM MG132 underwent widespread neuronal apoptosis and showed partial inhibition of proteasome activity down to 30-50%. Interestingly, administration of 1-10 microM MG132 almost completely blocked proteasome activity but resulted in reduced neuronal apoptosis. Similar results were produced in cortical cultures exposed to other proteasome inhibitors, proteasome inhibitor I and lactacystin. Administration of 0.1 microM MG132 led to activation of a mitochondria-dependent apoptotic signaling cascade involving cytochrome c, caspase-9, caspase-3 and degradation of tau protein; such activation was markedly reduced with 10 microM MG132. High doses of MG132 prevented the degradation of inhibitor of apoptosis proteins (IAPs) cIAP and X chromosome-linked IAP, suggesting that complete blockade of proteasome activity interferes with progression of apoptosis. In support of this, addition of high doses of proteasome inhibitors attenuated apoptosis of cortical neurons deprived of serum. Taken together, the present results indicate that inhibition of proteasome activity can induce or prevent neuronal cell apoptosis through regulation of mitochondria-mediated apoptotic pathways and IAPs.     

Lamotrigine Attenuates Proteasome Inhibition-Induced Apoptosis by Suppressing the Activation of the Mitochondrial Pathway and the Caspase-8- and Bid-Dependent Pathways

Proteasome impairment has been shown to be involved in neuronal degeneration. Antiepileptic lamotrigine has been demonstrated to have a neuroprotective effect. However, the effect of lamotrigine on the proteasome inhibition-induced neuronal cell death has not been studied. Therefore, we assessed the effect of lamotrigine on the proteasome inhibition-induced neuronal cell apoptosis in relation to cell death process using differentiated PC12 cells and SH-SY5Y cells. The proteasome inhibitors MG132 and MG115 induced a decrease in the levels of Bid and Bcl-2 proteins, an increase in the levels of Bax and p53, loss of the mitochondrial transmembrane potential, cytochrome c release and activation of caspases (-8, -9 and -3). The addition of lamotrigine reduced the proteasome inhibitor-induced changes in the apoptosis-related protein levels, production of reactive oxygen species, depletion and oxidation of glutathione (GSH), and cell death in both cell lines. Lamotrigine and N-acetylcysteine alone did not affect the levels of 26S proteasome and activity of 20S proteasome. MG132 did not alter the levels of 26S proteasome but decreased activity of 20S proteasome. Lamotrigine and N-acetylcysteine attenuated MG132-induced decrease in the activity of 20S proteasome. The results show that lamotrigine appears to suppress the proteasome inhibitor-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The suppressive effect of lamotrigine appears to be associated with its inhibitory effect on the production of reactive oxygen species, the depletion and oxidation of GSH and the activity reduction of 20S proteasome.     

Design,synthesis and biological evaluation of novel non-covalent piperidine-containing peptidyl proteasome inhibitors

A series of novel non-covalent piperidine-containing dipeptidyl derivatives were designed, synthesized and evaluated as proteasome inhibitors. All target compounds were tested for their proteasome chymotrypsin-like inhibitory activities, and selected derivatives were evaluated for the anti-proliferation activities against two multiple myeloma (MM) cell lines RPMI 8226 and MM-1S. Among all of these compounds, eight exhibited significant proteasome inhibitory activities with IC₅₀ less than 20 nM, and four are more potent than the positive control Carfilzomib. Compound 28 displayed the most potent proteasome inhibitory activity (IC₅₀: 1.4 ± 0.1 nM) and cytotoxicities with IC₅₀ values at 13.9 ± 1.8 nM and 9.5 ± 0.5 nM against RPMI 8226 and MM-1S, respectively. Additionally, the ex vivo blood cell proteasome inhibitory activities of compounds 24 and 27–29 demonstrated that the enzymatic metabolism in the whole blood could be well tolerated. All these experiments confirmed that the piperidine-containing non-covalent proteasome inhibitors are potential leads for exploring new anti-cancer drugs.     

Photorhabdus luminescens Tc toxin is inhibited by the protease inhibitor MG132 and activated by protease cleavage resulting in increased binding to target cells

Photorhabdus luminescens Tc toxins consist of the cell‐binding component TcA, the linker component TcB, and the enzyme component TcC. TccC3, a specific isoform of TcC, ADP‐ribosylates actin and causes redistribution of the actin cytoskeleton. TccC5, another isoform of TcC, ADP‐ribosylates and activates Rho proteins. Here, we report that the proteasome inhibitor MG132 blocks the intoxication of cells by Tc toxin. The inhibitory effect of MG132 was not observed, when the ADP‐ribosyltransferase domain of the TcC component was introduced into target cells by protective antigen, which is the binding and delivery component of anthrax toxin. Additionally, MG132 affected neither pore formation by TcA in artificial membranes nor binding of the toxin to cells. Furthermore, the in vitro ADP‐ribosylation of actin by the enzyme domain of TccC3 was not affected by MG132. Similar to MG132, several calpain inhibitors blocked the action of the Tc toxin. Proteolytic cleavage of the binding component TcA induced by P. luminescens protease PrtA1 or by collagenase largely increased the toxicity of the Tc toxin. MG132 exhibited no inhibitory effect on the cleaved TcA component. Moreover, binding of TcA to target cells was largely increased after cleavage. The data indicate that Tc toxin is activated by proteolytic processing of the TcA component, resulting in increased receptor binding. Toxin processing is probably inhibited by MG132.     

Activation of multiple caspases and modification of cell surface fas (CD95) in proteasome inhibitor-induced apoptosis of rat natural killer cells

The proteasome is a multi-subunit protease complex that is involved in intracellular protein degradation in eukaryotes. Previously, we have reported that selective, synthetic chymotryptic proteasome inhibitors inhibit A-NK cell-mediated cytotoxicity by approximately 50%; however, the exact role of the proteasome in NK cell-mediated cytotoxicity remains unknown. Herein, we report that proteasome inhibitors, MG115 and MG132, decreased the proteasome chymotrypsin-like activity in the rat natural killer cell line RNK16 by 85% at a concentration of 5 microM. The viability of RNK16 cells was also reduced in the presence of these inhibitors. Both inhibitors induced the apoptosis of RNK16 cells, as shown by DNA fragmentation, caspase-3 activation and the appearance of sub-G-cell populations. An increase in the fraction of apoptotic cells was observed in a dose- and time-dependent manner in our studies. In addition, the activity of caspase-1, -2, -6, -7, -8, and -9, was increased following the treatment of RNK16 cells with these inhibitors. Further investigation revealed that the expression of Fas (CD95) protein on the RNK16 cell surface was increased after the treatment by MG115 or MG132, indicating that apoptosis induced by proteasome inhibitors in RNK16 cells might be mediated through the Fas (CD95)-mediated death pathway as well. Our studies indicate, for the first time, that proteasomal chymotryptic inhibitors can reduce natural killer cell viability and therefore indirectly inhibit cell-mediated cytotoxicity via the apoptosis-inducing properties of these agents.     

Effects of proteasome inhibitor, MG132, on proteasome activity and oxidative status of rat liver

In vivo effects of N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal (MG132) on chymotryptic-like (ChT-L), tryptic-like, and post-glutamyl peptide hydrolytic-like proteasome activities, protein oxidation, lipid peroxidation (LP), glutathione (GSH) level, as well as on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione-reductase) in the rat liver were studied. The possibility of MG132 provoking the formation of free oxygen radicals was also assayed in primary hepatocytes. The following results were obtained: (1) In vivo, MG132 did not change the spontaneous LP, but increased Fe-induced LP and the amount of oxidized proteins; it decreased the GSH level in liver. From the proteasome activities studied in liver cytosol only ChT-L activity was significantly decreased after MG132 administration. Furthermore, MG132 increased antioxidant enzyme activities of SOD, CAT, and GSH-Px. (2) In vitro, MG132 increased free radical oxygen species in hepatocytes; this effect disappeared in the presence of CAT or mannitol. In conclusion, since nowadays proteasome inhibitors are entering into the swing of laboratory and clinical practice, the present data could provide useful information for MG132 action. Consequently, future in vivo experiments with MG132 could highlight the possibility of its use at different pathological conditions.     

蛋白酶体抑制剂MG132诱导K562和HeLa细胞凋亡程度存在明显差异

蛋白酶体抑制剂MG132诱导人白血病细胞K562和宫颈癌细胞HeLa凋亡,用3个不同浓度的蛋白酶体抑制剂MG132处理人白血病细胞K562和宫颈癌细胞HeLa,通过MTT检测、annexin Ⅴ/ PI 双染法、流式细胞术、酶标仪和Western 印迹分别检测MG132对K562细胞和HeLa细胞的生长效应、细胞凋亡率、细胞内活性氧(ROS)水平和caspase-3活性变化的影响.蛋白酶体抑制剂MG132诱导K562细胞凋亡明显,对HeLa细胞诱导凋亡不明显.结果表明,蛋白酶体抑制剂MG132特异性诱导不同肿瘤细胞凋亡的程度存在明显差异.     

Two different stages of epidermal growth factor (EGF) receptor endocytosis are sensitive to free ubiquitin depletion produced by proteasome inhibitor MG132

Numerous studies implicate proteasomes in the regulation of EGF receptor (EGFR) endocytosis on the basis of the ability of inhibitors to decrease EGFR degradation, but the exact mechanisms remain obscure. We demonstrated that EGFR itself is not a direct target for proteasome, since it is delivered to lysosomes intact. Evidence is presented that the inhibitory effect of MG132 on EGF degradation is due mostly to free ubiquitin depletion resultant from the suppression of proteasomal functioning by MG132. By subcellular fractionation, we show two MG132-sensitive steps in the EGFR degradation pathway: sorting from early (EE) to late (LE) endosomes, and late stage of LE maturation. MG132 treatment resulted in stabilization of EGFR tyrosine phosphorylation and its association with c-Cbl. Nevertheless, ubiquitination of EGFR at late stages of endocytosis was significantly lower than that in control cells. Highly ubiquitinated forms of EGFR demonstrated more sensitivity to MG132 treatment.     

Inhibition of the Host Proteasome Facilitates Papaya Ringspot Virus Accumulation and Proteosomal Catalytic Activity Is Modulated by Viral Factor HcPro

The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant–pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro – PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation.     

Inhibition versus induction of apoptosis by proteasome inhibitors depends on concentration

We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.     

Design,synthesis and biological evaluation of tripeptide boronic acid proteasome inhibitors

A series of tripeptide boronate proteasome inhibitors were designed and synthesized on the basis of our previously built tripeptide aldehyde 3D-QSAR models. All the synthesized compounds were evaluated for their proteasome-inhibitory activities in an isolated 20S rabbit proteasome, and selected compounds were evaluated for their antitumor activities in vitro against four human cancer cell lines. Biological results showed bulky and negative substituents at P² position improved the proteasome-inhibitory potency obviously, which completely conformed to the theoretical models, while those at P³ position thoroughly deviated from the 3D-QSAR model. Most of the screened compounds showed less than 1 nM inhibitory potency and high selectivity against 20S proteasome, of which 7f is the most potent (IC₅₀ = 0.079 nM) and twofold more active than bortezomib (IC₅₀ = 0.161 nM). Cell viability indicated hydrophilic 4-hydroxyphenyl substituent at P² or P³ position was not favorable to the cellular activities. Especially for the two hematologic cancer cell lines, HL-60 and U266, 7f inhibited them at the level of less than 10 nM and was more potent than the control bortezomib. It is being considered a promising new lead to be developed for the treatment of various cancers.     

Proteasome inhibitor MG132 enhances TRAIL-induced apoptosis and inhibits invasion of human osteosarcoma OS732 cells

MG132 as a proteasome inhibitor could induce apoptosis in various cancer cells. This study aimed to discuss the effect of proteasome inhibitor MG132 on the TRAIL-induced apoptosis of human osteosarcoma OS732 cells. MG132 and TRAIL were applied on OS732 cells respectively or jointly. Cell survival rates, changes of cellular shape, cell apoptosis and cell invasion were analyzed, respectively, by 3-(4,5)-dimethylthiahiazo(-z-y1)-2,5-di-phenytetrazoliumromide (MTT) assay, inverted phase contrast microscope, flow cytometry, and transwell invasion chamber methods. The protein levels of DR5, caspase-3, caspase-8, p27^kip1 and MMP-9 were measured by Western blot analysis. The results indicated that combination of MG132 and TRAIL had the effect of up-regulating expression of DR5, caspase-3, caspase-8 and p27^kip1, down-regulating expression of MMP-9 and inducing apoptosis as well as suppressing the ability of invasion of OS732 cells. The survival rate of combined application of 10 μM MG132 and 100 ng/ml TRAIL on OS732 cells was significantly lower than that of the individual application (p < 0.01). Changes of cellular shape and apoptotic rates also indicated the apoptosis-inducing effect of combined application was much stronger than that of individual application. Cell cycle analysis showed combination of MG132 and TRAIL mostly caused OS732 cells arrested at G2–M-phase. The invasion ability of OS732 cells was restrained significantly in the combined group compared with the individual group and control group.     

Proteasome inhibitors improve the function of mutant lysosomal α-glucosidase in fibroblasts from Pompe disease patient carrying c.546G>T mutation

Pompe disease (glycogen storage disease type II) is an autosomal recessive myopathic disorder arising from the deficiency of lysosomal acid α-glucosidase (GAA). Recently, we found that mutant GAA in patient fibroblasts carrying c.546G>T mutation is stabilized by treatment with proteasome inhibitor as well as pharmacological chaperon N-butyl-deoxynojirimycin. In this study, we characterized the effect of two proteasome inhibitors, bortezomib and MG132, on maturation, subcellular localization and residual activity of mutant GAA in the patient fibroblasts carrying c.546G>T mutation. Each proteasome inhibitor promoted the stabilization of patient GAA and processing of them to mature forms without cytotoxic effect. Immunocytochemical analysis showed increased colocalization of GAA with the lysosomal marker LAMP2 in patient fibroblasts treated with proteasome inhibitors. Furthermore, bortezomib and MG132 also increased enzyme activity in the patient fibroblasts (about 4-fold and 2-fold, respectively). These findings indicate that proteasome inhibitor may be a novel drug as potential pharmacological chaperone therapy for Pompe disease patient carrying chaperon-responsive mutation.     

The Ubiquitin–Proteasome System and Cellular Proliferation and Regulation in Osteoblastic Cells

The 26S proteasome is the macromolecular assembly that mediates ATP- and ubiquitin-dependent extralysosomal intracellular protein degradation in eukaryotes. However, its contribution to the regulation of osteoblast proliferation and hormonal regulation remains poorly defined. Treating osteoblasts with MG-132 or lactacystin (membrane-permeable proteasome inhibitors) attenuates proliferation. Three proteasome activities (peptidylglutamyl–peptide bond hydrolase-, chymotrypsin-, and trypsin-like) were detected in osteoblasts. Catabolic doses of PTH stim-ulated these activities, and cotreatment with PTH and MG-132 blocked stimulation. The proteasome α- and β-subunits, polyubiquitins, and large ubiquitin–protein conjugates were detected by Western blotting. A 90-min treatment with 10 nM PTH had no effect on the amount of proteasome α or β subunit protein, but increased the relative amount of large ubiquitin-protein conjugates by 200%. MG-132 inhibited deubiquitination of large ubiquitin–protein conjugates. The protein kinase A inhibitor SQ22536 blocked much of the PTH-induced stimulation of MCP activities, while dibutyryl cAMP stimulated it, suggesting that protein kinase A-dependent phosphorylation is important in PTH stimulation of proteasome activities. In conclusion, the ubiquitin–proteasome system is essential for osteoblast proliferation under control and PTH-treated conditions. PTH mediates its metabolic effects on the osteoblast, in part, by enhancing ubiquitinylation of protein substrates and stimulating three major proteasome activities by a cAMP-dependent mechanism.     

Proteasome inhibitors impair RANKL‐induced NF‐κB activity in osteoclast‐like cells via disruption of p62, TRAF6, CYLD,and IκBα signaling cascades

Proteasome inhibitors represent a promising therapy for the treatment of relapsed and/or refractory multiple myeloma, a disease that is concomitant with osteolysis and enhanced osteoclast formation. While blockade of the proteosome pathway has been recently shown to influence osteoclast formation and function, the precise molecular cascade underlying these effects is presently unclear. Here, we provide evidence that proteasome inhibitors directly impair osteoclast formation and function via the disruption of key RANK‐mediated signaling cascades. Disruption of the proteosome pathway using selective inhibitors (MG‐132, MG‐115, and epoxomicin) resulted in the accumulation of p62 and CYLD, and altered the subcellular targeting and distribution of p62 and TRAF6 in osteoclast‐like cells. Proteosome inhibition also blocked RANKL‐induced NF‐κB activation, IκBα degradation and nuclear translocation of p65. The disruption in RANK‐signaling correlated dose‐dependently with an impairment in osteoclastogenesis, with relative potency epoxomicin > MG‐132 > MG‐115 based on equimolar concentrations. In addition, these inhibitors were found to impact osteoclastic microtubule organization and attenuate bone resorption. Based on these data we propose that deregulation of key RANK‐mediated signaling cascades (p62, TRAF6, CYLD, and IκBα) underscores proteasome‐mediated inhibition of osteolytic bone conditions. J. Cell. Physiol. 220: 450–459, 2009. © 2009 Wiley‐Liss, Inc.     

Proteasome Inhibitors Cause Induction of Heat Shock Proteins and Trehalose, Which Together Confer Thermotolerance in Saccharomyces cerevisiae

An accumulation in cells of unfolded proteins is believed to be the common signal triggering the induction of heat shock proteins (hsps). Accordingly, in Saccharomyces cerevisiae, inhibition of protein breakdown at 30°C with the proteasome inhibitor MG132 caused a coordinate induction of many heat shock proteins within 1 to 2 h. Concomitantly, MG132, at concentrations that had little or no effect on growth rate, caused a dramatic increase in the cells’ resistance to very high temperature. The magnitude of this effect depended on the extent and duration of the inhibition of proteolysis. A similar induction of hsps and thermotolerance was seen with another proteasome inhibitor, clasto-lactacystin β-lactone, but not with an inhibitor of vacuolar proteases. Surprisingly, when the reversible inhibitor MG132 was removed, thermotolerance decreased rapidly, while synthesis of hsps continued to increase. In addition, exposure to MG132 and 37°C together had synergistic effects in promoting thermotolerance but did not increase hsp expression beyond that seen with either stimulus alone. Although thermotolerance did not correlate with hsp content, another thermoprotectant trehalose accumulated upon exposure of cells to MG132, and the cellular content of this disaccharide, unlike that of hsps, quickly decreased upon removal of MG132. Also, MG132 and 37°C had additive effects in causing trehalose accumulation. Thus, the resistance to heat induced by proteasome inhibitors is not just due to induction of hsps but also requires a short-lived metabolite, probably trehalose, which accumulates when proteolysis is reduced.     

Evidence for proteasomal degradation of Kv1.5 channel protein

BACKGROUND: The voltage-gated potassium channel Kv1.5 plays a critical role in the maintenance of the membrane potential. While protein degradation is one of the major mechanisms for the regulation of channel functions, little is known on the degradation mechanism of Kv1.5. METHODS AND RESULTS: Kv1.5 was expressed in COS cells and its degradation, intracellular localization, and channel activities were assessed by pulse-chase analysis, immunofluorescence, and patch clamp techniques, respectively. Expressed Kv1.5 had a half-life time of approximately 6.7 h, which was prolonged by the proteasome inhibitors of MG132, ALLN, proteasomal inhibitor 1, or lactacystine, but not by a lysosomal inhibitor chloroquine. MG132 increased the protein level of Kv1.5, as well as the level of its ubiquitinated form in a dose-dependent manner. Similar effects of MG132 on endogenous Kv1.5 were seen in cultured rat atrial cells. Within a cell, Kv1.5 was mainly localized in both the endoplasmic reticulum and Golgi apparatus. MG132 increased the immunoreactivity of Kv1.5 in these compartments and also increased Ik(ur) currents through the cell-surface Kv1.5. Pretreatment with either brefeldin A or colchicine abolished MG132-induced increase in Ik(ur) currents. CONCLUSION: Kv1.5 is degraded by the proteasome. The inhibition of the proteasome increased Ik(ur) currents secondary to stabilization of the channel protein in the endoplasmic reticulum/Golgi apparatus.