carbobenzoxy-leucyl-leucyl-norvalinal and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Impairment in ubiquitin-proteasome system has been shown to be implicated in the pathogenesis in neurodegenerative disorders, such as Parkinson's disease. Flavonoid baicalein has demonstrated anti-oxidant and anti-inflammatory effects. However, the effect of baicalein on the neuronal cell death due to proteasome inhibition has not been studied. Thus, in the respect of the cell death process, we assessed the effect of baicalein on the proteasome inhibition-induced apoptosis using differentiated PC12 cells. The proteasome inhibitors MG132 and MG115 induced a decrease in Bid, Bcl-2, Bcl-xL and survivin protein levels, an increase in Bax levels, loss of the mitochondrial transmembrane potential, release of cytochrome c, activation of caspases (-8, -9 and -3), an increase in the tumor suppressor p53 levels and cleavage of PARP-1. Baicalein attenuated the proteasome inhibition-induced changes in the levels of apoptosis-related proteins, formation of reactive oxygen species, depletion of GSH, DNA damage and cell death. The results show that baicalein may attenuate the proteasome inhibition-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The preventive effect appears to be attributed to its inhibitory effect on the formation of reactive oxygen species and depletion of GSH.

Topics: Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Caspase 8; Cell Differentiation; Cell Line; Cell Nucleus; Flavanones; Glutathione; Leupeptins; Mitochondria; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Reactive Oxygen Species

Methionine adenosyltransferase (MAT) is an important enzyme for metabolic processes, to the extent that its product, S-adenosylmethionine (AdoMet), plays a key role in trans-methylation, trans-sulphuration and polyamine synthesis. Previous studies have shown that a MAT-overexpressing strain of Leishmania donovani controls AdoMet production, keeping the intracellular AdoMet concentration at levels that are compatible with cell survival. This unexpected result, together with the fact that MAT activity and abundance changed with time in culture, suggests that different regulatory mechanisms acting beyond the post-transcriptional level are controlling this protein. In order to gain an insight into these mechanisms, several experiments were carried out to explain the MAT abundance during promastigote cell growth. Determination of MAT turnover in cycloheximide (CHX)-treated cultures resulted in a surprising 5-fold increase in MAT turnover compared to CHX-untreated cultures. This increase agrees with a stabilization of the MAT protein, whose integrity was maintained during culture. The presence of proteasome inhibitors, namely MG-132, MG-115, epoxomycin and lactacystin in the culture medium prevented MAT degradation in both MAT-overexpressing and 'mock-transfected' leishmanial strains. The role of the ubiquitin (Ub) pathway in MAT down-regulation was supported using immunoprecipitation experiments. Immunoprecipitated MAT cross-reacted with anti-Ub antibodies, which provides evidence of a proteasome-mediated down-regulation of the leishmanial MAT abundance.

Topics: Acetylcysteine; Cell Culture Techniques; Cloning, Molecular; Cycloheximide; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Gene Expression; Immunoprecipitation; Kinetics; Leishmania donovani; Leishmaniasis, Visceral; Leupeptins; Methionine Adenosyltransferase; Oligopeptides; Plasmids; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Processing, Post-Translational; Recombinant Proteins; S-Adenosylmethionine; Transfection; Ubiquitin

Melanoma antigen (MAGE)-A4 is processed to generate a C-terminal fragment with proapoptotic activity. Here we demonstrate that Adriamycin promotes generation of the processed MAGE-A4 by activating the proteasome. The proteasome is known to prevent accumulation of toxic proteins to maintain cellular homeostasis.. Treatment of hepatoma cells expressing MAGE-A4 with a sublethal dose of Adriamycin increased the MAGE-A4 processing and sensitized the cells to Adriamycin-induced apoptosis. The processing of MAGE-A4 was inhibited by the proteasome inhibitors MG115, MG132, lactacystin and epoxamicin. MAGE-A4 was coimmunoprecipitated with the S6 proteasomal ATPase, and present in the fractions containing the proteasome during glycerol gradient centrifugation. Consistent with the notion that the proteasome cleaves MAGE-A4, the 26S proteasome, ubiquitin, and cell lysates were necessary for efficient in vitrocleavage of MAGE-A4.. The present study suggests that a low dose of Adriamycin increases the proteasome activity, which either maintains cellular homeostasis or leads to apoptosis depending, at least under the present conditions, on the expression of MAGE-A4.

Topics: Acetylcysteine; Animals; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Doxorubicin; HEK293 Cells; Humans; Leupeptins; Liver Neoplasms; Neoplasm Proteins; Proteasome Endopeptidase Complex; Ubiquitin

Inhibition of the proteasome proteolytic pathway occurs as the result of normal aging, as well as in a variety of neurodegenerative conditions, and is believed to promote cellular toxicity in each of these conditions through diverse mechanisms. In the present study, we examined whether proteasome inhibition alters the protein kinase receptor-like endoplasmic reticulum kinase (PERK). Our studies demonstrate that proteasome inhibitors induce the transient activation of PERK in both primary rat neurons as well as the N2a neural cell line. Experiments with siRNA to PERK demonstrated that the modulation of PERK was not significant involved in regulating toxicity, ubiquitinated protein levels, or ribosome perturbations in response to proteasome inhibitor treatment. Surprisingly, PERK was observed to be involved in the up-regulation of p38 kinase following proteasome inhibitor treatment. Taken together, these data demonstrate the ability of proteasome inhibition to activate PERK and demonstrate evidence for novel cross-talk between PERK and the activation of p38 kinase in neural cells following proteasome inhibition. Taken together, these data have implications for understanding the basis by which proteasome inhibition alters neural homeostasis, and the basis by which cell signaling cascades are regulated by proteasome inhibition.

Topics: Animals; Cells, Cultured; eIF-2 Kinase; Enzyme Activation; Leupeptins; Neurons; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley

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-kappaB activation, IkappaBalpha 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 IkappaBalpha) underscores proteasome-mediated inhibition of osteolytic bone conditions.

Topics: Actins; Animals; Bone Resorption; Cell Line; Cysteine; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Deubiquitinating Enzyme CYLD; Erythropoietin; Humans; I-kappa B Proteins; Leupeptins; Mice; Mice, Inbred C57BL; Microtubules; NF-kappa B; NF-KappaB Inhibitor alpha; Oligopeptides; Osteoclasts; Proteasome Endopeptidase Complex; Proteasome Inhibitors; RANK Ligand; Signal Transduction; Synaptotagmin I; TNF Receptor-Associated Factor 6; Transcription Factor TFIIH; Transcription Factors

A method for studying 20S proteasome inhibitors by capillary electrophoresis (CE) has been developed. Proteasome plays a fundamental role in degrading key regulatory proteins. The 20S proteasome can degrade intrinsically disordered proteins in an ATP-independent manner without additional "helper" molecules. The discovery of new proteasome inhibitors with little or no toxicity is highly desirable in anticancer therapy. In this study, the inhibitory effects of MG132 and MG115 on the 20S proteasome were evaluated by CE for the first time. The optimized CE conditions were as follows: fused-silica capillary of 30 cm effective length and 75 microm internal diameter, pressure injection of 0.5 psi for 5 s, 50 mM Hepes buffer (pH 7.6) with 2% dimethyl sulfoxide, constant voltage of 20 kV, and detection wavelength at 340 nm. Also, the new method was used to study the inhibitory effects of 30 novel peptidyl vinyl ester derivatives of MG132. The 50% inhibition concentrations (IC(50) values) of MG132 and MG115 were 40.0 and 84.7 nM, respectively. Two new compounds, XP32 and XP35, showed considerable inhibitory effects on the 20S proteasome. When the concentrations of them were fixed at 172 nM, their inhibition rates were 36.2% and 29.1%, respectively. The results showed that the CE method was powerful, sensitive, and fast and required little sample. It could be employed as one of the reliable drug screening methods for 20S proteasome inhibitors.

Topics: Animals; Coumarins; Drug Evaluation, Preclinical; Electrophoresis, Capillary; Esters; Hydrolysis; Inhibitory Concentration 50; Leupeptins; Linear Models; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rabbits; Reproducibility of Results

Proteasomes are multisubunit enzymes responsible for the degradation of many cytosolic proteins. The inhibition of the proteasome has been the subject of intense interest in the development of drug therapies. We have previously demonstrated that simultaneous administration of a tripeptide aldehyde proteasome inhibitor (MG115 or MG132) with a peptide (Cys-Trp-Lys18) DNA condensate boosted gene expression by 30-fold in cell culture. In the present study, we have developed a convergent synthesis to allow the incorporation of a proteasome inhibitor tripeptide into the C-terminal end of a gene delivery peptide. The resulting peptides formed DNA condensates that mediated a 100-fold enhancement in gene expression over a control peptide lacking all or part of the tripeptide inhibitor. Gene transfer peptides possessing intrinsic proteasome inhibitors were also found to be nontoxic to cells in culture. These results suggest that intrinsic proteasome inhibition may also be used to boost the efficiency of peptide-mediated nonviral gene delivery systems in vivo.

Topics: Amino Acid Sequence; Cell Line; Cell Survival; DNA; Esters; Gene Transfer Techniques; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Up-Regulation

Previous studies have shown that proteasome inhibitors promote the accumulation of steroidogenic acute regulatory protein (StAR) in cultured rat adrenocortical cells. Unexpectedly, this response was associated with a moderate lowering in the corticosterone secretion and proliferation rate of cultured cells. Hence, we studied the effects of proteasome inhibitors MG115 and MG132 on the secretion and proliferative activity of the regenerating adrenal cortex in rats 5 days after surgery. Animals were given two subcutaneous injections of 0.15 or 1.5 nmol/100 g of inhibitors 24 and 12 h before decapitation. Real-time PCR and Western blotting showed that StAR expression, both mRNA and protein, was markedly lower in regenerating adrenals than in the intact gland of sham-operated rats. Neither MG115 nor MG132 affected StAR expression in regenerating gland. Inhibitors induced a slight decrease in the plasma concentrations of aldosterone and corticosterone, but did not significantly alter metaphase index of the regenerating adrenal cortex. Our findings provide the first evidence that down-regulation of StAR occurs during the early stages of adrenal regeneration. Moreover, this suggests that the steroidogenic pathway is more sensitive to proteasome inhibitors than that regulating proliferative activity of regenerating adrenal cortex in the rat.

Topics: Adrenal Cortex; Animals; Enzyme Inhibitors; Female; Gene Expression Regulation; Leupeptins; Phosphoproteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Regeneration; Steroids

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization at 100 microM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization.

Topics: Acrosome Reaction; Animals; Female; Fertilization; Leucine; Leupeptins; Male; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Sea Urchins; Sperm-Ovum Interactions; Spermatozoa; Ubiquitin; Vitelline Membrane

The sperm proteasome has been reported to be involved in sperm penetration through the proteinaceous egg-coat during fertilization in ascidians and mammals. However, such an extracellular role for the sperm proteasome in fertilization is not known in other deuterostomes. Here, we investigated the effects of two proteasome inhibitors on fertilization of the sea urchin Anthocidaris crassispina. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization, whereas E-64-d, chymostatin or leupeptin showed no inhibition at 100 microM. MG-132 inhibited the egg-jelly-induced acrosome reaction, but not the reaction induced by the Ca(2+) ionophore ionomycin. MG-132 and MG-115, but not E-64-d, inhibited the fertilization of dejellied eggs by acrosome-reacted sperm. Furthermore, MG-132-susceptible proteasome activity was detected in the acrosomal contents. These results suggest that the sperm proteasome plays a key role not only in the acrosome reaction, in particular, in a process before the increase in intracellular Ca(2+) concentration but also in the sperm penetration through the vitelline coat, most probably as a lysin.

Topics: Acrosome; Animals; Cyclic AMP-Dependent Protein Kinases; Female; Fertilization; Leupeptins; Male; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Sea Urchins

The purpose of this study was to investigate whether proteasome inhibition acts as a thermal sensitizing agent to induce tumor cell death in a colon cancer cell line.. HT-29 colon cancer cells were exposed to hyperthermia (43 degrees C) in the presence of proteasome inhibition for 1 h. Viable cell mass and apoptosis were measured by MTT and annexin V staining, respectively. Protein levels were determined by Western blot analysis.. A significant synergistic effect on cell viability with proteasome inhibition was noted under hyperthermic conditions compared to hyperthermia alone (p < 0.05). Increases in phosphorylated ERK and decreases in HSP27 levels were observed in the cells exposed to proteasome inhibition at 43 degrees C. Pretreatment with an inhibitor of ERK yielded an additional increase in apoptosis when used in combination with proteasome inhibition and hyperthermia. Decreased expression of HSP27 by siRNA also resulted in increased thermally induced apoptotic cell death.. Thermal sensitization through proteasome inhibition may represent a novel approach to increase the efficacy of hyperthermia as an anticancer modality.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Butadienes; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Synergism; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Heat-Shock Proteins; HT29 Cells; Humans; Hyperthermia, Induced; Leupeptins; Nitriles; Protease Inhibitors; Proteasome Inhibitors; RNA, Small Interfering

Ku70 plays an important role in DNA damage repair and prevention of cell death. Previously, we reported that apoptosis caused a decrease in cellular Ku70 levels. In this study, we analyzed the mechanism of how Ku70 levels decrease during drug-induced apoptosis. In HeLa cells, staurosporin (STS) caused a decrease in Ku70 levels without significantly affecting Ku70 mRNA levels. We found that Ku70 protein was highly ubiquitinated in various cell types, such as HeLa, HEK293T, Dami (a megakaryocytic cell line), endothelial, and rat kidney cells. An increase in ubiquitinated Ku70 protein was observed in apoptotic cells, and proteasome inhibitors attenuated the decrease in Ku70 levels in apoptotic cells. These results suggest that the ubiquitin-proteasome proteolytic pathway plays a role in decreasing Ku70 levels in apoptotic cells. Ku70 forms a heterodimer with Ku80, which is required for the DNA repair activity of Ku proteins. We also found that Ku80 levels decreased in apoptotic cells and that Ku80 is a target of ubiquitin. Ubiquitinated Ku70 was not found in the Ku70-Ku80 heterodimer, suggesting that modification by ubiquitin inhibits Ku heterodimer formation. We propose that the ubiquitin-dependent modification of Ku70 plays an important role in the control of cellular levels of Ku70.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antigens, Nuclear; Apoptosis; Caspase Inhibitors; Cell Line; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Doxorubicin; Gene Expression; HeLa Cells; Humans; Ku Autoantigen; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Signal Transduction; Staurosporine; Ubiquitin; Ubiquitin-Protein Ligase Complexes

The ubiquitin-proteasome pathway is responsible for regulating cell cycle proteins, tumor-suppressor molecules, oncogenes, transcription factors, and pro- and anti-apoptotic proteins. The aim of this study is to evaluate the effects of proteasome inhibitors on human hepatocellular carcinoma (HCC) cells. HCC cells SK-Hep1, HLE and HepG2 were treated with the proteasome inhibitors MG132 and MG115. Our data showed that both inhibitors induce apoptosis in the three cell types tested in a dose-dependent manner. Moreover, subtoxic levels of MG132 and MG115 sensitized HCC cells to TRAIL-induced apoptosis. To investigate the mechanism of increased TRAIL sensitivity in HCC cells, we first examined surface expression of TRAIL and its receptors. MG132 upregulated TRAIL and its receptors (TRAIL-R1 and -R2) in SK-Hep1 and HLE, whereas MG115 upregulated them in SK-Hep1. MG132 downregulated expression of X-linked inhibitor of apoptosis protein (XIAP) in SK-Hep1 and HLE, and of survivin in all three cell-types. MG115 downregulated expression of XIAP in SK-Hep1, and survivin in SK-Hep1 and HepG2. Furthermore, MG132 downregulated phospho-AKT and its downstream target phospho-BAD, indicating that MG132 activated the mitochondrial apoptosis pathway by inhibiting phosphorylation of AKT and BAD. In conclusion, proteasome inhibitors induced apoptosis and augmented TRAIL sensitivity via both the IAP family and AKT pathways. Thus, combining proteasome inhibitors with a TRAIL agonist may provide a new therapeutic strategy for HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase Inhibitors; Caspases; Down-Regulation; Drug Synergism; Humans; Leupeptins; Mice; Oncogene Protein v-akt; Phosphorylation; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; TNF-Related Apoptosis-Inducing Ligand

Herpes simplex virus type 1 (HSV-1) is a potent inducer of nuclear factor-KB (NF-kappaB), a cellular transcription factor with a crucial role in promoting inflammation and controlling cell proliferation and survival.. On the basis of the recent demonstration that HSV-1-induced NF-kappaB is actively recruited to KB-binding sites in the HSV-1 infected-cell protein 0 (ICPO) promoter enhancing viral transcription and replication, we investigated the effect of proteasome inhibitors MG132, MG115 and epoxomicin, which block NF-kappaB function by preventing the degradation of the inhibitory proteins IkappaBalpha, on HSV-1-induced NF-kappaB activation and viral replication.. Antiviral activity of proteasome inhibitors was analysed in HSV-1-infected HEp2 cells by determining infective virus titres by CPE50%, viral RNA synthesis by RT-PCR, and viral protein synthesis by immunoblot analysis or immunofluorescence. ICPO transcription was studied in transient transfection experiments using the ICPO promoter-luciferase IE1-Luc construct. IkappaBalpha degradation and NF-kappaB activity were determined by immunoblot analysis and EMSA, respectively.. Proteasome inhibitors were found to prevent HSV-1-induced NF-kappaB activation in the early phase of infection. Block of virus-induced NF-kappaB activation resulted in inhibiting HSV-1 ICPO gene expression, in decreasing the level of immediate-early and late viral proteins, and ultimately in greatly suppressing viral replication. The antiviral effect was lost if treatment was started after NF-kappaB activation, and appeared to be independent of the HSV-1-induced activation of the JNK pathway.. Proteasome inhibitors possess NF-kappaB-dependent antiherpetic activity. The results described further identify the IKK/NF-kappaB pathway as a suitable target for novel antiherpetic drugs.

Topics: Animals; Antiviral Agents; Cell Line, Tumor; Chlorocebus aethiops; Down-Regulation; Herpesvirus 1, Human; Humans; Immediate-Early Proteins; Leupeptins; NF-kappa B; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Transcription Factor AP-1; Vero Cells

Proteasomes are multi-subunit proteases involved in several mechanisms and thought to contribute to the regulation of cellular homeostasis. Here, we report for the first time biochemical evidence for the existence of a ubiquitin-proteasome proteolytic pathway in this parasite. Proteasomes from both cercariae and adult worms exhibited a high preference for hydrolysis of the substrate Suc-LLVY-AMC, although in the cercariae extract the rate of hydrolysis was 50% lower when compared to adult worms extracts. The same difference in proteasome activities was observed when endogenous proteins were broken down in the presence of ATP and ubiquitin. Additionally, accumulation of high molecular weight conjugates was observed when cercariae were pre-incubated with proteasome inhibitors. Finally, we present evidence that during experimental schistosomiasis, proteasome inhibitors were able to reduce the number of lung stage schistosomula, reduce the worm burden and consequently decrease the egg output in infected mice.

Topics: Adenosine Triphosphate; Animals; Biomphalaria; Coumarins; Host-Parasite Interactions; Hydrolysis; Leupeptins; Lung; Mice; Mice, Inbred BALB C; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Schistosoma mansoni; Schistosomiasis mansoni; Ubiquitin

The proteasome is a multisubunit cytosolic protein complex responsible for degrading cytosolic proteins. Several studies have implicated its involvement in the processing of viral particles used for gene delivery, thereby limiting the efficiency of gene transfer. Peptide-based nonviral gene delivery systems are sufficiently similar to viral particles in their size and surface properties and thereby could also be recognized and metabolized by the proteasome. The present study utilized proteasome inhibitors (MG 115 and MG 132) to establish that peptide DNA condensates are metabolized by the proteasome, thereby limiting their gene transfer efficiency. Transfection of HepG2 or cystic fibrosis/T1 (CF/T1) cells with CWK18 DNA condensates in the presence of MG 115 or MG 132 resulted in significantly enhanced gene expression. MG 115 and MG 132 increased luciferase expression 30-fold in a dose-dependent manner in HepG2 and CF/T1. The enhanced gene expression correlated directly with proteasome inhibition, and was not the result of lysosomal enzyme inhibition. The enhanced transfection was specific for peptide DNA condensates, whereas Lipofectamine- and polyethylenimine-mediated gene transfer were significantly blocked. A series of novel gene transfer peptides containing intrinsic GA proteasome inhibitors (CWK18(GA)n, where n=4, 6, 8 and 10) were synthesized and found to inhibit the proteasome. The gene transfer efficiency mediated by these peptides in four different cell lines established that a GA repeat of four is sufficient to block the proteasome and significantly enhance the gene transfer. Together, these results implicate the proteasome as a previously undiscovered route of metabolism of peptide-based nonviral gene delivery systems and provide a rationale for the use of proteasome inhibition to increase gene transfer efficiency.

Topics: Cell Line; Cell Line, Tumor; Cystic Fibrosis; DNA; Gene Expression; Genetic Therapy; Humans; Leupeptins; Lipids; Luciferases; Lung; Oligopeptides; Peptides; Polyethyleneimine; Protease Inhibitors; Proteasome Endopeptidase Complex; Transfection

Elicitation of defense reactions in tobacco by cryptogein, triggered a production of active oxygen species (AOS) via the NADPH oxidase, NtrbohD, and an accumulation of beta1din, a defense induced beta-type subunit of 20S proteasome. The proteasome inhibitor, MG132, stimulated this AOS production. Tobacco cells transformed with sense constructs of beta1din showed an inhibition of the AOS production following elicitin treatment, whereas the antisense transformed cells showed a strongly enhanced AOS production. In cells transformed with sense construct of beta1din, the NtrbohD transcripts failed to be induced by cryptogein as observed in control and antisense transformed cells. Conversely, in tobacco cells transformed with antisense constructs for NtrbohD, beta1din transcripts remained at a low level after elicitation. These results constitute the first demonstration of proteasome comprising beta1din acting as a negative regulator of NtrbohD and contributes to the regulation of AOS generation during plant defense reactions.

Topics: Cysteine Proteinase Inhibitors; Leupeptins; NADPH Oxidases; Nicotiana; Oligonucleotides, Antisense; Plant Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Subunits; Reactive Oxygen Species

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.

Topics: Animals; Apoptosis; Caspases; Cell Line; Cysteine Endopeptidases; Enzyme Activation; fas Receptor; Killer Cells, Natural; Leupeptins; Multienzyme Complexes; Protease Inhibitors; Proteasome Endopeptidase Complex; Rats

Chronic ethanol consumption causes increased oxidative damage in the liver. Induction of CYP2E1 is one pathway involved in how ethanol produces oxidative stress. Ethanol can cause protein accumulation, decreased proteolysis, and decreased proteasome activity. The objective of this study was to investigate the effect of inhibition of the proteasome activity on CYP2E1-dependent toxicity. HepG2 cells over-expressing CYP2E1 (E47 cells) were treated with arachidonic acid (AA) plus iron, agents important in development of alcoholic liver injury and which are toxic to E47 cells by a mechanism dependent on CYP2E1, oxidative stress, and lipid peroxidation. Addition of various proteasome inhibitors was associated with significant potentiation of the loss of cell viability caused by AA plus iron. Potentiation of toxicity was associated with increased oxidative damage as reflected by an increase in lipid peroxidation and accumulation of oxidized and nitrated proteins in E47 cells and an enhanced decline in mitochondrial membrane potential. Antioxidants prevented the loss of viability and the potentiation of this loss of viability by proteasome inhibition. CYP2E1 levels were elevated about 3-fold by the proteasome inhibitors. Inhibition of proteasome activity also potentiated toxicity of AA alone and toxicity after treatment to remove glutathione (GSH). Similar results were found in hepatocytes from pyrazole-treated rats with high levels of CYP2E1. In conclusion, proteasome activity plays an important role in modulating CYP2E1-mediated toxicity in HepG2 cells by regulating CYP2E1 levels and by removal of oxidized proteins. Such interactions may be important in CYP2E1-catalyzed toxicity of hepatotoxins and in alcohol-induced liver injury.

Topics: Antioxidants; Arachidonic Acid; Buthionine Sulfoximine; Carcinoma, Hepatocellular; Cysteine Endopeptidases; Cytochrome P-450 CYP2E1; Drug Synergism; Endopeptidases; Humans; Iron; Leupeptins; Lipid Peroxides; Liver Neoplasms; Lysosomes; Membrane Potentials; Mitochondria, Liver; Multienzyme Complexes; Proteasome Endopeptidase Complex; Tumor Cells, Cultured; Tyrosine

Growth related oncogene protein-alpha (GRO-alpha) is a member of C-X-C chemokine and plays an important role in inflammatory responses. Expression of GRO gene family is regulated by a number of factors at both transcriptional and posttranscriptional levels. In the present study, we have addressed the possible regulation of GRO-alpha expression by ubiquitin-proteasome system. Cultures of human umbilical vein endothelial cells were treated with a proteasome inhibitor, MG132, and the levels of GRO-alpha mRNA were analyzed by reverse transcription-polymerase chain reaction or northern blotting. Levels of GRO-alpha protein in the cell-conditioned medium were determined by enzyme-linked immunosorbent assay. MG132 alone increased the levels of GRO-alpha mRNA and protein; however, it did not affect the GRO-alpha mRNA induced by lipopolysaccharide (LPS) and inhibited the LPS-induced decrease in IkappaB levels. Other proteasome inhibitors, MG115 and lactacystin, also induced the expression of GRO-alpha mRNA. MG132 induced the phosphorylation of p38 MAPK, MEK and JNK. Pretreatment of the cells with SB203580, an inhibitor of p38 MAPK, suppressed the MG132-induced GRO-alpha expression, but pretreatment of the cells with U0126, PD98059 or SP600125, inhibitors of MEK1/2 or JNK, did not influence the effect of MG132. We conclude that MG132 upregulates GRO-alpha expression in vascular endothelial cells, at least in part, through the activation of p38 MAPK.

Topics: Acetylcysteine; Anthracenes; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Cysteine Proteinase Inhibitors; Endothelium, Vascular; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation; Humans; I-kappa B Proteins; Imidazoles; Intercellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Leupeptins; MAP Kinase Kinase Kinase 1; MAP Kinase Kinase Kinases; Mitogen-Activated Protein Kinases; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Umbilical Veins

We previously reported that sperm proteasome is responsible for degradation of the ubiquitinated vitelline-coat during fertilization in the ascidian Halocynthia roretzi. Here, we report the roles in fertilization and localization in the sperm cell surface of H. roretzi sperm proteasome. An anti-proteasome antibody, as well as the proteasome inhibitors MG115 and MG132, inhibited the fertilization, indicating that the sperm proteasome functions extracellularly in ascidian fertilization. In order to further assess this issue, the sperm surface proteasome activity was labeled with a cell-impermeable labeling reagent, NHS-LC-biotin, extracted with 0.1% CHAPS, and was subjected to a pull-down assay with avidin-agarose beads. It was found that a substantial amount of sperm proteasome is exposed to the cell surface. Partition analysis with Triton X-114 also revealed that a considerable amount of the sperm proteasome activity is partitioned into a lipid layer. Localization of the proteasome activity was investigated by fluorescence microscopy with succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide as a substrate. The sperm proteasome activity was specifically detected in the sperm head region, and it was markedly activated upon sperm activation. The membrane-associated proteasome was purified from the membrane fraction of H. roretzi sperm by affinity chromatography using an anti-20S proteasome antibody-immobilized Sepharose column. SDS-PAGE of the purified preparation showed a similar pattern of subunit composition to that of the 26S proteasome of mammalian origin. Taken together, these results indicate that H. roretzi sperm has the membrane-associated proteasome on its head, which is activated upon sperm activation, and that sperm proteasome plays an essential role in H. roretzi fertilization.

Topics: Animals; Cell Membrane; Cholic Acids; Cysteine Proteinase Inhibitors; Detergents; Enzyme Activation; Female; Fertilization; Leupeptins; Male; Peptide Hydrolases; Proteasome Endopeptidase Complex; Spermatozoa; Urochordata

Invasive Salmonella induces macrophage apoptosis via the activation of caspase-1 by the bacterial protein SipB. Here we show that infection of macrophages with Salmonella causes the activation and degradation of Raf-1, an important intermediate in macrophage proliferation and activation. Raf-1 degradation is SipB- and caspase-1-dependent, and is prevented by proteasome inhibitors. To study the functional significance of Raf-1 in this process, the c-raf-1 gene was inactivated by Cre-loxP-mediated recombination in vivo. Macrophages lacking c-raf-1 are hypersensitive towards pathogen-induced apoptosis. Surprisingly, activation of the antiapoptotic mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and nuclear factor kappaB pathways is normal in Raf-1-deficient macrophages, and mitochondrial fragility is not increased. Instead, pathogen-mediated activation of caspase-1 is enhanced selectively, implying that Raf-1 antagonizes stimulus-induced caspase-1 activation and apoptosis.

Topics: Animals; Apoptosis; Bacterial Proteins; Bone Marrow Cells; Caspase 1; Cells, Cultured; Enzyme Activation; Leupeptins; Macrophages; Membrane Proteins; Mice; Mice, Inbred C57BL; Mitochondria; Mitogen-Activated Protein Kinases; NF-kappa B; Protease Inhibitors; Proto-Oncogene Proteins c-raf; Salmonella typhimurium

It was investigated whether proteasome activity was implicated in susceptibility of human vascular smooth muscle cells (VSMCs) to Fas-mediated death. Human fetal aorta smooth muscle cells were treated with agonistic anti-Fas antibody (CH11) and proteasome inhibitors (MG115 or MG132) and then cell death was determined by morphology, viability, and DNA fragmentation. The present study reports that: (a) crosslinking of Fas receptor with anti-Fas antibody in the presence of proteasome inhibitor-induced death and DNA degradation in human VSMCs that were blocked by caspases inhibitor z-DEVD.fmk; (b) cotreatment with anti-Fas antibody and proteasome inhibitor activated caspase-3; (c) proteasome inhibitors did not influence expression of procaspase-8, procaspase-3, c-FLIP, and Bcl-2; and (d) proteasome inhibitors up-regulated Fas and FADD. The data indicate that proteasome activity is important in survival of VSMCs and provide the first evidence that proteasome is involved in Fas signal transduction. The present study proposes novel mechanism(s) by which VSMCs become susceptible to FasL.

Topics: Antibodies; Blotting, Western; Caspase 3; Caspases; Cell Adhesion; Cell Death; Cell Membrane; Cells, Cultured; Cross-Linking Reagents; Cysteine Endopeptidases; DNA; DNA Fragmentation; fas Receptor; Humans; Leupeptins; Multienzyme Complexes; Muscle, Smooth, Vascular; Protease Inhibitors; Proteasome Endopeptidase Complex; Signal Transduction

1DNA topoisomerase II (topo II) is a nuclear enzyme that modifies DNA topology and also serves as a target to mediate the cytotoxicity of several antineoplastic agents. Several reports have demonstrated that a reduction of topo II is associated with reduced sensitivity to these agents. Topo II exists as two isoforms in mammalian cells: topo IIalpha and topo IIbeta. In MCF-7 cells, the half-life (mean +/- SEM) values of topo IIalpha and topo IIbeta in situ were 6.6 +/- 0.3 and 17.6 +/- 2.3 hr, respectively, as determined by [(35)S]methionine/cysteine pulse-chase analysis. Degradation of topo IIalpha in situ was abrogated by the presence of proteasome inhibitors, and the relative activities were carbobenzoxy-leucyl-leucyl-leucinal (MG132) > carbobenzoxy-leucyl-leucyl-norvalinal (MG115) > ALLN congruent with lactacystin. ATP-dependent degradation of topo IIalpha, but not topo IIbeta, was observed in extracts of asynchronously dividing HeLa and MCF-7 cells. Furthermore, degradation of topo IIalpha was abrogated by the proteasome inhibitors MG132 and MG115, but not by lactacystin, in extracts of asynchronously dividing MCF-7 cells. Finally, degradation of topo IIalpha, but not topo IIbeta, was observed to occur in a cell cycle-dependent fashion, in extracts of synchronized HeLa cells, with maximal loss of the alpha isoform occurring 2 hr after release from mitotic arrest. This degradation of topo IIalpha appeared to be facilitated by an ATP-dependent activity. Furthermore, high molecular weight bands (>200 kDa), which may represent polyubiquitinated-topo IIalpha conjugates, were also detected in extracts of synchronized HeLa cells. This study provides evidence for a role of the ubiquitin-proteasome pathway in the cell cycle-dependent regulation of topo IIalpha expression.

Topics: Acetylcysteine; Adenosine Triphosphate; Antigens, Neoplasm; Cell Cycle; Cell Extracts; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Topoisomerases, Type II; DNA-Binding Proteins; HeLa Cells; Humans; Isoenzymes; Leupeptins; Multienzyme Complexes; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

Previously we demonstrated that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by ATP-dependent proteolysis in serotonin producing mast cells [Hasegawa et al. (1995) FEBS Lett. 368, 151-154]. We searched for the major proteases involved in the rapid degradation of TPH in RBL2H3 cells. Among various protease inhibitors tested, proteasome inhibitors MG115, MG101, MG132, and lactacystin effectively inhibited the intracellular degradation of TPH. Administration of the proteasome inhibitors to cultured cells caused more than a 5-fold accumulation of TPH. Administration of the inhibitors together with cycloheximide stabilized the amount of TPH with no appreciable increase or decrease. Although MG-series proteasome inhibitors could inhibit calpain, the involvement of calpain was excluded since the cysteine protease inhibitor E-64-d, which acts on calpain, had no effect. Extracts of RBL2H3 cells were shown to contain a protease that digests TPH in an ATP-dependent manner and is sensitive to proteasome inhibitors. The ubiquitination of TPH could be visualized by Western blot analysis using both anti-TPH and anti-ubiquitin antibodies. Based on these results, we conclude that 26S proteasomes are mainly involved in the degradation of TPH. In the reported amino acid sequences of TPH from various sources including human, rabbit, rat, and mouse, a PEST sequence that is widely shared among short-lived proteins has been recognized. It was noted that the PEST sequence lies within the most conserved portion of the enzyme, the pteridine binding site.

Topics: Amino Acid Motifs; Animals; Cell-Free System; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Hydrolysis; Leucine; Leupeptins; Mast Cells; Multienzyme Complexes; Proteasome Endopeptidase Complex; Rats; Serotonin; Tryptophan Hydroxylase; Tumor Cells, Cultured; Ubiquitins

Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of nuclear retinoid receptors.

Topics: Amino Acid Motifs; Cells, Cultured; Cycloheximide; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Gene Expression Regulation; HeLa Cells; Humans; Keratinocytes; Kinetics; Leupeptins; Multienzyme Complexes; Proteasome Endopeptidase Complex; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; Retinoid X Receptors; RNA, Messenger; Sequence Deletion; Transcription Factors; Transfection; Ubiquitins; Ultraviolet Rays

Depolarization and subsequent calcium entry exert essential neuroprotective effects but the ultimate effector by which calcium blocks apoptosis is not known. Here we show that inhibition of calcium entry into cerebellar neurons by switching from high to low extracellular K+ concentrations (30-5 mM) induces apoptosis, that correlates with a rapid accumulation of cyclin D1 (CD1), an early marker of the G1/S transition of the cell cycle. These effects on apoptosis and cyclin D1 are mimicked either by blocking calcium entry into neurons (LaCl3, 100 microM or nifedipine, 10(-6) M) or by inhibiting the calcium/calmodulin pathway (calmidazolium, 10(-7) M). The increased CD1 protein levels do not result from a transcriptional upregulation of the CD1 gene by the Ca2+/calmodulin pathway but rather reflect an accumulation due to the lack of degradation by the proteasome-dependent pathway. Specific proteasome antagonists: carbobenzoxyl-leucinyl-leucinyl-norvalinal-H (MG-115), carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG-132) and clastolactacystin beta-lactone, induce neuronal apoptosis by themselves. Finally, this pathway is functional only at neuroprotective concentrations of K+ (30 mM), suggesting that calcium/CamK signalling pathway may regulate neuronal death by regulating the proteasome-mediated degradation activity of rapidly turning-over proteins (constitutively expressed genes or pre-existing pools of mRNA).

Topics: Animals; Apoptosis; Calcium; Calmodulin; Cerebellum; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Primers; Enzyme Inhibitors; Gene Expression; Imidazoles; In Situ Nick-End Labeling; Isoquinolines; Leupeptins; Membrane Potentials; Mice; Mice, Inbred Strains; Multienzyme Complexes; Neurons; Potassium Chloride; Protease Inhibitors; Proteasome Endopeptidase Complex; RNA, Messenger; Signal Transduction; Sulfonamides; Ubiquitins

Phenylmethylsulphonyl fluoride, lactacystin (a selective inhibitor of the proteasome) and the peptide aldehydes carbobenzoxyleucylleucylnorvalinal and carbobenzoxyleucylleucylleucinal amplify the production of prostacyclin in rat liver cells incubated for 6 h with the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the TPA-type tumour promoters teleocidin and aplysiatoxin. Such stimulation is not dependent upon the simultaneous presence of the inhibitor and TPA. Preincubation of the cells with TPA followed by addition of the inhibitor or preincubation with the inhibitor followed by addition of TPA results in amplified prostacyclin production. Phenylmethylsulphonyl fluoride, lactacystin, and carbobenzoxyleucylleucylnorvaline also enhance prostacyclin production after incubation with interleukin-1beta and transforming growth factor-alpha. The Ca2+ chelator ethyleneglycol-O,O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid inhibits the phenylmethylsulphonyl fluoride-TPA or lactacystin-TPA amplifications. Cells, treated with phenylmethylsulphonyl fluoride, TPA, interleukin-1beta, lactacystin or the peptide aldehydes exhibit increased prostaglandin endoperoxide G/H synthase activity. The increased activities as well as the constitutive prostaglandin endoperoxide G/H synthase activity are inhibited by a selective prostaglandin endoperoxide G/H synthase-2 inhibitor, 1-[2-(4-fluorophenyl)-cyclopenten-1-yl]-4-(methysulphonyl)-b enzene, with an IC50 of approximately 0.5 microM. These results demonstrate that the C-9 rat liver cells express prostaglandin endoperoxide G/H synthase-2 constitutively and express induced prostaglandin endoperoxide G/H synthase-2. Inhibition of proteolytic activity amplifies agonist-stimulated arachidonic acid metabolism in these cells.

Topics: Acetylcysteine; Animals; Arachidonic Acid; Carcinogens; Cell Line; Endopeptidases; Epoprostenol; Leupeptins; Lyngbya Toxins; Phenylmethylsulfonyl Fluoride; Prostaglandin-Endoperoxide Synthases; Protease Inhibitors; Rats; Signal Transduction

To examine the cellular basis for secretion defect-type antithrombin deficiency, we expressed two mutants, P --> stop (Pro429 to stop codon) and deltaGlu (deletion of Glu313). Pulse-chase experiments using stably transfected BHK cells showed that little (< 5%) of P --> stop mutant as well as deltaGlu mutant was secreted and the total amount of radioactivity was significantly reduced, suggesting an intracellular degradation. The degradation was not inhibited by brefeldin A, indicating it occurring in a preGolgi apparatus. However, the degradation was strongly inhibited by proteasomal inhibitors, such as carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL), carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal (LLnV) and lactacystin. By endoglycosidase H digestion and immunofluorescence staining, these mutants were shown to localize in the endoplasmic reticulum (ER). These results suggest that the secretion defect-type mutants of antithrombin are degraded by proteasome through the ER-associated quality control mechanism in the cells.

Topics: Acetylcysteine; Animals; Antithrombin III; Brefeldin A; Cell Line; Cricetinae; Cyclopentanes; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Enzyme Inhibitors; Fluorescent Antibody Technique; Golgi Apparatus; Humans; Kidney; Leupeptins; Multienzyme Complexes; Mutagenesis, Site-Directed; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Transfection

A method was investigated for monitoring the activity of protease(s) in cytosol of a single starfish oocyte using succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid as the substrate, which was injected into the cell. After preincubation of immature oocytes with a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norvalinal, the initial hydrolysis of the substrate was remarkably inhibited. The inhibitor blocked 1-methyladenine-triggered cyclin degradation, which is known to be mediated by proteasome. However, calpain inhibitor E-64 did not inhibit the hydrolysis of the substrate. These results suggested that the protease activity measured by this method is mainly attributable to cytoplasmic proteasome. The hydrolysis of the substrate was partially inhibited by bestatin, suggesting that the substrate was cleaved by aminopeptidase. Thus, the initial velocity of hydrolysis of the substrate (V0) by proteasome was assayed in a living oocyte after preinjection of bestatin. The values of V0 increased gradually after 1-methyladenine addition and reached the maximum level at the time corresponding to cyclin degradation. The calculated maximum velocity of hydrolysis by a mature oocyte was approximately three times higher than that by an immature oocyte. The Michaelis-Menten constant value was also higher in mature than immature oocytes. These results suggest that proteasome-dependent proteolysis is regulated not only by ubiquitination of substrates, as is generally believed, but also by the proteasome activity itself.

Topics: Adenine; Aminopeptidases; Animals; Cell Membrane Permeability; Coumarins; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytosol; Fluorescent Dyes; Hydrolysis; Kinetics; Leucine; Leupeptins; Meiosis; Mesylates; Multienzyme Complexes; Oligopeptides; Oocytes; Proteasome Endopeptidase Complex; Spectrometry, Fluorescence; Starfish

We have studied whether various agents that inhibit purified yeast and mammalian 26 S proteasome can suppress the breakdown of different classes of proteins in Saccharomyces cerevisiae. The degradation of short-lived proteins was inhibited reversibly by peptide aldehyde inhibitors of proteasomes, carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132) and carbobenzoxyl-leucinyl-leucinyl-norvalinal (MG115), in a yeast mutant with enhanced permeability, but not in wild-type strains. Lactacystin, an irreversible proteasome inhibitor, had no effect, but the beta-lactone derivative of lactacystin, which directly reacts with proteasomes, inhibited the degradation of short-lived proteins. These inhibitors also blocked the rapid ubiquitin-dependent breakdown of a beta-galactosidase fusion protein and caused accumulation of enzymatically active molecules in cells. The degradation of the bulk of cell proteins, which are long-lived molecules, was not blocked by proteasome inhibitors, but could be blocked by phenylmethylsulfonyl fluoride. This agent, which inhibits multiple vacuolar proteases, did not affect the proteasome or breakdown of short-lived proteins. These two classes of inhibitors can thus be used to distinguish the cytosolic and vacuolar proteolytic pathways and to increase the cellular content of short-lived proteins.

Topics: Acetylcysteine; Animals; Kinetics; Leupeptins; Mammals; Muramidase; Peptide Hydrolases; Protease Inhibitors; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Substrate Specificity; Ubiquitins; Vacuoles

Multiple cell adhesion proteins are up-regulated in vascular endothelial cells in response to TNF alpha and other inflammatory cytokines. This increase in cell adhesion gene expression is thought to require the transcription factor NF-kappa B. Here, we show that peptide aldehyde inhibitors of the proteasome, a multicatalytic protease recently shown to be required for the activation of NF-kappa B, block TNF alpha induction of the leukocyte adhesion molecules E-selectin, VCAM-1, and ICAM-1. Striking functional consequences of this inhibition were observed in analyses of leukocyte-endothelial interactions under defined flow conditions. Lymphocyte attachment to TNF alpha-treated endothelial monolayers was totally blocked, while neutrophil attachment was partially reduced but transmigration was essentially prevented.

Topics: Base Sequence; Calpain; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Cysteine Endopeptidases; E-Selectin; Endothelium, Vascular; Gene Expression; Humans; In Vitro Techniques; Intercellular Adhesion Molecule-1; Interleukin-8; Leukocytes; Leupeptins; Molecular Sequence Data; Multienzyme Complexes; NF-kappa B; Oligodeoxyribonucleotides; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; RNA, Messenger; Time Factors; Transcription Factor RelB; Transcription Factors; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded, leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus, the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides, but also in the regulated processing of precursors into active proteins.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Cells, Cultured; Cysteine Endopeptidases; DNA-Binding Proteins; Humans; I-kappa B Proteins; Leupeptins; Models, Biological; Molecular Sequence Data; Multienzyme Complexes; NF-kappa B; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Precursors; Protein Processing, Post-Translational; Ubiquitins