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.     

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.     

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.     

Dysfunction of mitochondrial complex I and the proteasome: interactions between two biochemical deficits in a cellular model of Parkinson's disease

Two biochemical deficits have been described in the substantia nigra in Parkinson's disease, decreased activity of mitochondrial complex I and reduced proteasomal activity. We analysed interactions between these deficits in primary mesencephalic cultures. Proteasome inhibitors (epoxomicin, MG132) exacerbated the toxicity of complex I inhibitors [rotenone, 1-methyl-4-phenylpyridinium (MPP+)] and of the toxic dopamine analogue 6-hydroxydopamine, but not of inhibitors of mitochondrial complex II-V or excitotoxins [N-methyl-d-aspartate (NMDA), kainate]. Rotenone and MPP+ increased free radicals and reduced proteasomal activity via adenosine triphosphate (ATP) depletion. 6-hydroxydopamine also increased free radicals, but did not affect ATP levels and increased proteasomal activity, presumably in response to oxidative damage. Proteasome inhibition potentiated the toxicity of rotenone, MPP+ and 6-hydroxydopamine at concentrations at which they increased free radical levels >/= 40% above baseline, exceeding the cellular capacity to detoxify oxidized proteins reduced by proteasome inhibition, and also exacerbated ATP depletion caused by complex I inhibition. Consistently, both free radical scavenging and stimulation of ATP production by glucose supplementation protected against the synergistic toxicity. In summary, proteasome inhibition increases neuronal vulnerability to normally subtoxic levels of free radicals and amplifies energy depletion following complex I inhibition.     

Proteomic expression analysis of cardiomyocytes subjected to proteasome inhibition

We hypothesized that impaired proteasomal function affects gene expression in cardiomyocytes. To identify those genes, a proteomics-based analysis of neonatal rat cardiac myocytes treated with the proteasome inhibitor MG132 in comparison to vehicle treated control cells was performed. MG132 treatment induced reproducible changes in the protein expression profile, which was analyzed by two-dimensional difference gel electrophoresis followed by tryptic peptide mass fingerprinting for spot identification by MALDI-TOF mass spectrometry. The identified protein alterations could be grouped into three major categories: (1) induction of small heat shock proteins (HSPs) with chaperonic function, such as HSP27, alphaB-crystallin, and cardiovascular HSP, (2) altered expression of actin associated proteins, such as cofilin-1 and transgelin, and (3) induction of antioxidant proteins, such as peroxiredoxin-1, superoxide dismutase-1, and hemeoxygenase-1. Northern blotting revealed that expression was regulated at the mRNA level. Given that proteasomal activity is decreased in cardiovascular diseases, alterations in proteasome-dependent control of mRNA expression could provide a novel mechanism by which disease progression is modulated.     

Sulforaphane attenuates postnatal proteasome inhibition and improves spatial learning in adult mice

Proteasomes are known to degrade proteins involved in various processes like metabolism, signal transduction, cell-cycle regulation, inflammation, and apoptosis. Evidence showed that protein degradation has a strong influence on developing neurons as well as synaptic plasticity. Here, we have shown that sulforaphane (SFN) could prevent the deleterious effects of postnatal proteasomal inhibition on spatial reference and working memory of adult mice. One day old Balb/c mice received intracerebroventricular injections of MG132 and SFN. Sham received an equal volume of aCSF. We observed that SFN pre-administration could attenuate MG132 mediated decrease in proteasome and calpain activities. In vitro findings revealed that SFN could induce proteasomal activity by enhancing the expression of catalytic subunit-β5. SFN pre-administration prevented the hippocampus based spatial memory impairments during adulthood, mediated by postnatal MG132 exposure. Histological examination showed deleterious effects of MG132 on pyramidal neurons and granule cell neurons in DG and CA3 sub-regions respectively. Furthermore, SFN pre-administration has shown to attenuate the effect of MG132 on proteasome subunit-β5 expression and also induce the Nrf2 nuclear translocation. In addition, SFN pre-administered mice have also shown to induce expression of pCaMKII, pCreb, and mature/pro-Bdnf, molecules which play a crucial role in spatial learning and memory consolidation. Our findings have shown that proteasomes play an important role in hippocampal synaptic plasticity during the early postnatal period and SFN pre-administration could enhance the proteasomal activity as well as improve spatial learning and memory consolidation.     

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.     

Inducible nitric-oxide synthase is regulated by the proteasome degradation pathway

Inducible nitric-oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. To determine the degradation pathway of iNOS, human epithelial kidney HEK293 cells with stable expression of human iNOS were incubated in the presence of various degradation pathway inhibitors. Treatment with the proteasomal inhibitors lactacystin, MG132, and N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal resulted in the accumulation of iNOS, indicating that these inhibitors blocked its degradation. Moreover, proteasomal inhibition blocked iNOS degradation in a dose- and time-dependent manner as well as when NO synthesis was inhibited by N(omega)-nitro-l-arginine methyl ester. Furthermore, proteasomal inhibition blocked the degradation of an iNOS splice variant that lacked the capacity to dimerize and of an iNOS mutant that lacks l-arginine binding ability, suggesting that iNOS is targeted by proteasomes, notwithstanding its capacity to produce NO, dimerize, or bind the substrate. In contrast to proteasomal inhibitors, the calpain inhibitor calpastatin and the lysosomal inhibitors trans-epoxysuccinyl-l-leucylamido-4-guanidino butane, leupeptin, pepstatin-A, chloroquine, and NH(4)Cl did not lead to significant accumulation of iNOS. Interestingly, when cytokines were used to induce iNOS in RT4 human epithelial cells, the effect of proteasomal inhibition was dichotomous. Lactacystin added prior to cytokine stimulation prevented iNOS induction by blocking the degradation of the NF-kappaB inhibitor IkappaB-alpha, thus preventing activation of NF-kappaB. In contrast, lactacystin added 48 h after iNOS induction led to the accumulation of iNOS. Similarly, in murine macrophage cell line RAW 264.7, lactacystin blocked iNOS degradation when added 48 h after iNOS induction by lipopolysaccharide. These data identify the proteasome as the primary degradation pathway for iNOS.     

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.     

Proteasome inhibition in oxidative stress neurotoxicity: implications for heat shock proteins

Recent studies have demonstrated that inhibition of the proteasome, an enzyme responsible for the majority of intracellular proteolysis, may contribute to the toxicity associated with oxidative stress. In the present study we demonstrate that exposure to oxidative injury (paraquat, H(2)O(2), FeSO(4)) induces a rapid increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential, inhibition of proteasome activity, and induction of cell death in neural SH-SY5Y cells. Application of proteasome inhibitors (MG115, epoxomycin) mimicked the effects of oxidative stressors on mitochondrial membrane potential and cell viability, and increased vulnerability to oxidative injury. Neural SH-SY5Y cells stably transfected with human HDJ-1, a member of the heat shock protein family, were more resistant to the cytotoxicity associated with oxidative stressors. Cells expressing increased levels of HDJ-1 displayed similar degrees of ROS formation following oxidative stressors, but demonstrated a greater preservation of mitochondrial function and proteasomal activity following oxidative injury. Cells transfected with HDJ-1 were also more resistant to the toxicity associated with proteasome inhibitor application. These data support a possible role for proteasome inhibition in the toxicity of oxidative stress, and suggest heat shock proteins may confer resistance to oxidative stress, by preserving proteasome function and attenuating the toxicity of proteasome inhibition.     

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.     

Proteasomal degradation of Kir6.2 channel protein and its inhibition by a Na+ channel blocker aprindine

ATP-sensitive K+ channels (K(ATP):SUR2A+Kir6.2) play a pivotal role in cardiac protection against ischemia and reperfusion injury. When expressed in COS cells, Kir6.2 was short-lived with a half-life time of 1.9 h. The half-life time of Kir6.2 was prolonged by proteasome inhibitors MG132, ALLN, proteasome inhibitor 1, and lactacystine, but not at all by a lysosomal inhibitor chloroquine. MG132 also increased the level of ubiquitinated Kir6.2 without affecting its localization in the endoplasmic reticulum and Golgi apparatus. In electrophysiological recordings, MG132 augmented nicorandil-activated K(ATP) currents in COS cells expressing SUR2A and Kir6.2 as well as the same currents in neonatal rat cardiomyocytes. Like MG132, a Na+ channel blocker aprindine prolonged the half-life time of Kir6.2 and augmented K(ATP). Finally, both aprindine and MG132 inhibited the 20S proteasome activity in vitro. These results suggest a novel activity of aprindine to enhance K(ATP) currents by inhibiting proteasomal degradation of Kir 6.2 channels, which may be beneficial in the setting of cardiac ischemia.