calpain and lactacystin

The dysfunction of the proteasome system is implicated in the pathomechanism of several chronic neurodegenerative diseases. Lactacystin (LC), an irreversible proteasome inhibitor, induces cell death in primary cortical neurons, however, the molecular mechanisms of its neurotoxic action has been only partially unraveled. In this study we aimed to elucidate an involvement of the key enzymatic pathways responsible for LC-induced neuronal cell death. Incubation of primary cortical neurons with LC (0.25-50 μg/ml) evoked neuronal cell death in concentration- and time-dependent manner. Lactacystin (2.5 μg/ml; 6.6μM) enhanced caspase-3 activity, but caspase-3 inhibitor, Ac-DEVD-CHO did not attenuate the LC-evoked cell damage. Western blot analysis showed a time-dependent, prolonged activation of MAPK/ERK1/2 pathway after LC exposure. Moreover, inhibitors of MAPK/ERK1/2 signaling, U0126 and PD98052 attenuated the LC-evoked cell death. We also found that LC-treatment resulted in the induction of calpains and calpain inhibitors (MDL28170 and calpeptin) protected neurons against the LC-induced cell damage. Neuroprotective action of MAPK/ERK1/2 and calpain inhibitors were connected with attenuation of LC-induced DNA fragmentation measured by Hoechst 33342 staining and TUNEL assay. However, only MAPK/ERK1/2 but not calpain inhibitors, attenuated the LC-induced AIF (apoptosis inducing factor) release. Further studies showed no synergy between neuroprotective effects of MAPK/ERK1/2 and calpain inhibitors given in combination when compared to their effects alone. The obtained data provided evidence for neuroprotective potency of MAPK/ERK1/2 and calpain, but not caspase-3 inhibition against the neurotoxic effects of LC in primary cortical neurons and give rationale for using these inhibitors in the treatment of neurodegenerative diseases connected with proteasome dysfunction.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Butadienes; Calpain; Caspase 3; Caspase Inhibitors; Cells, Cultured; Cerebral Cortex; Cysteine Proteinase Inhibitors; Cytoprotection; Dipeptides; Dose-Response Relationship, Drug; Enzyme Activation; Gestational Age; In Situ Nick-End Labeling; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Neuroprotective Agents; Nitriles; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Kinase Inhibitors; Signal Transduction; Spectrin; Time Factors

Paclitaxel (Taxol) is one of the most effective treatment options for patients suffering from a variety of cancers. A major side effect seen in a high percentage of patients treated with paclitaxel is irreversible peripheral neuropathy. We previously reported that prolonged treatment with paclitaxel activates a calcium-dependent enzyme, calpain, which degrades neuronal calcium sensor 1 (NCS-1) and subsequent loss of intracellular calcium signaling. Because it appears that activation of calpain is an early step in this destructive cascade, we proposed that inhibition of calpain will protect against the unwanted side effects of paclitaxel treatment. First, NCS-1 levels and intracellular calcium signaling were found to be protected by the presence of lactacystin, a protesome inhibitor. To reinforce the role of calpain in this process, we showed that increased concentrations of calpastatin, a naturally occurring calpain inhibitor, were protective. Next, we tested two mutated versions of NCS-1 developed with point mutations at the P2 position of the calpain cleavage site of NCS-1 to decrease the likelihood of NCS-1 degradation. One mutant was cleaved more favorably by calpain compared with NCS-1 WT, whereas the other mutant was less favorably cleaved. Expression of either mutated version of NCS-1 in neuroblastoma cells protected intracellular calcium signals from paclitaxel-induced changes. These results support our hypothesis that it is possible to protect cells from paclitaxel-induced degradation of NCS-1 by inhibiting calpain activity.

Topics: Acetylcysteine; Calcium Signaling; Calpain; Cell Line, Tumor; Humans; Mutation; Neuroblastoma; Neuronal Calcium-Sensor Proteins; Neurons; Neuropeptides; Paclitaxel; Point Mutation; Protease Inhibitors; Protein Isoforms; Signal Transduction; Thermodynamics

p120-catenin contributes to the cadherin-mediated adhesion and aggregation of cells. mu-Calpain was activated and p120-catenin was degraded after 36 h of ischemia in differentiated SH-SY5Y cells. Calpain inhibitors Cbz-Val-Phe-H (MDL28170, 20 microM) and N-acetyl-leucyl-leucyl-norleucinal (ALLN, 20 microM) increased the levels of dephosphorylated p120-catenin, aggregation, and cell survival as detected by reduced LDH release in ischemic cells. However, a proteasome inhibitor lactacystin had no such effects. This is the first report of the calpain-mediated degradation of p120-catenin and an association between the level of dephosphorylated p120-catenin and cell aggregation in ischemic neuronal cells.

Topics: Acetylcysteine; Calpain; Catenins; Cell Adhesion Molecules; Cell Aggregation; Cell Death; Cell Line, Tumor; Delta Catenin; Dipeptides; Humans; Ischemia; Leupeptins; Neuroblastoma; Phosphoproteins

Epidemiological studies have indicated that malnutrition during early life may programme chronic degenerative disease in adulthood. In an animal model of fetal malnutrition, rats received an isoenergetic, low-protein (LP) diet during gestation. This reduced fetal beta-cell proliferation and insulin secretion. Supplementation during gestation with taurine prevented these alterations. Since proteases are involved in secretion and proliferation, we investigated which proteases were associated with these alterations and their restoration in fetal LP islets. Insulin secretion and proliferation of fetal control and LP islets exposed to different protease modulators were measured. Lactacystin and calpain inhibitor I, but not isovaleryl-L-carnitine, raised insulin secretion in control islets, indicating that proteasome and cysteinyl cathepsin(s), but not mu-calpain, are involved in fetal insulin secretion. Insulin secretion from LP islets responded normally to lactacystin but was insensitive to calpain inhibitor I, indicating a loss of cysteinyl cathepsin activity. Taurine supplementation prevented this by restoring the response to calpain inhibitor I. Control islet cell proliferation was reduced by calpain inhibitor I and raised by isovaleryl-L-carnitine, indicating an involvement of calpain. Calpain activity appeared to be lost in LP islets and not restored by taurine. Most modifications in the mRNA expression of cysteinyl cathepsins, calpains and calpastatin due to maternal protein restriction were consistent with reduced protease activity and were restored by taurine. Thus, maternal protein restriction affected cysteinyl cathepsins and the calpain-calpastatin system. Taurine normalised fetal LP insulin secretion by protecting cysteinyl cathepsin(s), but the restoration of LP islet cell proliferation by taurine did not implicate calpains.

Topics: Acetylcysteine; Animals; Calcium-Binding Proteins; Calpain; Cell Proliferation; Cells, Cultured; Diet, Protein-Restricted; Disease Models, Animal; Female; Fetal Nutrition Disorders; Gene Expression Regulation, Developmental; Gene Expression Regulation, Enzymologic; Glycoproteins; Insulin; Insulin Secretion; Islets of Langerhans; Maternal-Fetal Exchange; Peptide Hydrolases; Pregnancy; Proteasome Endopeptidase Complex; Protein Array Analysis; Rats; Rats, Wistar; Taurine

Treatment of OM10.1 cells latently infected with human immunodeficiency virus type 1 (HIV-1) with phorbol ester and calcium ionophore (A23187) induced virus replication which was blocked by N-Ac-Leu-Leu-norleucinal (ALLnL), a calpain inhibitor I, and not by lactacystin, a specific proteasome inhibitor. When the purified NF-kappa B/I kappa B complex was treated with mu-calpain, the specific DNA-binding activity was demonstrated by using electrophoretic mobility shift assay in vitro. This effect of mu-calpain was inhibited by ALLnL and calpastatin and not by lactacystin. In fact, we found that mu-calpain efficiently degraded I kappa B alpha. Furthermore, our Western blotting analysis has revealed that mu-calpain cleaves I kappa B alpha at its N-terminal and C-terminal regions that were previously reported to be involved in the interaction with NF-kappa B p65. These observations indicate that in monocyte/macrophage cells calcium signaling is involved in NF-kappa B activation through activation of calpain and thus calpain inhibitors may be effective in inhibiting the activation of latently infected HIV.

Topics: Acetylcysteine; Binding Sites; Calcimycin; Calcium Signaling; Calcium-Binding Proteins; Calpain; Cell Line; Glycoproteins; HIV-1; Humans; I-kappa B Proteins; In Vitro Techniques; Ionophores; Leucine; NF-kappa B; NF-KappaB Inhibitor alpha; Recombinant Fusion Proteins; Tetradecanoylphorbol Acetate; Virus Replication

Cyclin-dependent kinase 5 (cdk5) is involved in the development of the nervous system and neuronal process outgrowth, and it regulates several intracellular processes including cytoskeletal dynamics. Dysregulation of cdk5 has been implicated in many disorders of the nervous system. The activity of the kinase is regulated by binding of cdk5 activators (p35, p39, p67). We examined the phosphorylation of p35, and the role of phosphorylation in regulating the proteolysis of the p35 protein. By detecting changes in electrophoretic mobility, we observed that a significant proportion of p35 is phosphorylated in rat brain tissue. In cultured neurons, the phosphorylation was prevented by roscovitine, an inhibitor of cdk5 and some other cdks. The phosphatase inhibitor okadaic acid induced p35 degradation in neuronal cultures which was sensitive to the proteasome inhibitor lactacystin. These latter results agree with some previous studies showing that phosphorylation regulates proteasomal degradation of p35. Treatment of brain homogenate with okadaic acid in the presence of ATP led to accumulation of p35 phosphorylated also by a kinase that was not inhibited by roscovitine. This implies that the effect of okadaic acid on p35 degradation could also be contributed by a non-cdk kinase. The calpain protease has been shown to cleave p35. Our results suggest that this process may also be modulated by p35 phosphorylation under some conditions. We conclude that p35 phosphorylation influences the proteasome-mediated degradation of p35 and calpain-mediated cleavage of p35 to p25.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Animals, Newborn; Brain Chemistry; Calpain; Cells, Cultured; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Embryo, Mammalian; Enzyme Inhibitors; Hippocampus; Multienzyme Complexes; Nerve Tissue Proteins; Neurons; Okadaic Acid; Phosphorylation; Proteasome Endopeptidase Complex; Purines; Rats; Rats, Wistar; Roscovitine

Within 24 h of hormonally stimulated 3T3-L1 adipocyte differentiation, there are dramatic changes in the protein levels of p130 and p107, two members of the retinoblastoma tumor suppressor gene family. Designated the "p103:p107" switch, this alteration is characterized by a rapid and transient drop in p130 protein levels accompanied by a transient increase in both p107 mRNA and protein levels. Using protease inhibitors, the specific proteolytic pathway involved in degradation of p130 was examined. Treatment of cells with N-acetyl-leu-leu-norleucinal, an inhibitor that blocks proteolytic activity of type I calpain and the 26S proteasome, resulted in a complete block in the degradation of p130 protein, as well as adipocyte differentiation, suggesting that one of these pathways is involved in regulating p130 protein levels. Similar analysis with lactacystin, a specific inhibitor of the 26S proteasome, also resulted in a complete block in both differentiation and p130 degradation. Furthermore, both inhibitors blocked the increase in p107 protein levels normally observed on Day 1, suggesting that the p130:p107 switch is required for adipocyte differentiation and one of the early molecular events involved in activating the p130:p107 switch is the specific degradation of p130 by the 26S proteasome.

Topics: Acetylcysteine; Adipocytes; Animals; Calpain; Cell Differentiation; Cell Line; Cysteine Proteinase Inhibitors; Kinetics; Leupeptins; Nuclear Proteins; Peptide Hydrolases; Phosphoproteins; Proteasome Endopeptidase Complex; Proteins; Retinoblastoma-Like Protein p130

Preproparathyroid hormone (prepro-PTH) is one of the proteins abundantly synthesized by parathyroid chief cells; yet under normal growth conditions, little or no prepro-PTH can be detected in these cells. Although this may be attributed to effective cotranslational translocation and proteolytic processing, proteasome-mediated degradation of PTH precursors may be important in the regulation of the levels of these precursors and hence PTH secretion. The effects of N-acetyl-Leu-Leu-norleucinal, N-acetyl-Leu-Leu-methional, carbobenzoxy-Leu-Leu-leucinal (MG132), benzyloxycarbonyl-Ile-Glu(t-butyl)-Ala-leucinal (proteasome inhibitor I), and lactacystin on the biosynthesis and secretion of PTH were examined in dispersed bovine parathyroid cells. We demonstrate that treatment of these cells with proteasome inhibitors caused the accumulation of prepro-PTH and pro-PTH. Compared with mock-treated cells, the processing of pro-PTH to PTH was delayed, and the secretion of intact PTH decreased in proteasome inhibitor-treated cells. Relieving the inhibition of the proteasome by chasing MG132-treated cells in medium without the inhibitor led to the rapid disappearance of the accumulated prepro-PTH, and the rate of PTH secretion was restored to levels comparable to those in mock-treated cells. Furthermore, overexpression of the Hsp70 family of molecular chaperones was observed in proteasome inhibitor-treated cells, and we show that PTH/PTH precursors interact with these molecular chaperones. These data suggest the involvement of parathyroid cell proteasomes in the quality control of PTH biosynthesis.

Topics: Acetylcysteine; Animals; Brefeldin A; Calpain; Cattle; Cells, Cultured; Chloroquine; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; HSP70 Heat-Shock Proteins; Multienzyme Complexes; Parathyroid Glands; Parathyroid Hormone; Proteasome Endopeptidase Complex; Protein Precursors

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.

Topics: Acetylcysteine; Alternative Splicing; Animals; Calpain; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytokines; Dimerization; Enzyme Inhibitors; Enzyme Stability; Humans; Lysosomes; Mice; Multienzyme Complexes; Mutation; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Serine Proteinase Inhibitors; Transcription, Genetic

Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin-proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin-proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT.

Topics: Acetylcysteine; Adenine; Alcohol Dehydrogenase; Animals; Calpain; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dihydrotestosterone; Electrophoresis, Polyacrylamide Gel; Hepatocytes; Leupeptins; Liver; Lysosomes; Male; Multienzyme Complexes; Precipitin Tests; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Ubiquitins

Previously, we have shown that translation eukaryotic initiation factor (eIF) 4GI is cleaved during anti-Fas-mediated apoptosis. Here, we have investigated the effects of the proteasome inhibitors, MG132 and lactacystin, and the immunosuppressants, 2-amino-2[2-(4-octylphenyl)ethyl]-1,3,propane diol (FTY720) and cyclosporin A, on the integrity of eIF4GI and eIF4GII in T cells. Using wild-type Jurkat T cells, we show that the proteasome inhibitors MG132 and lactacystin promote the cleavage of eIF4G, activate caspase-8 and caspase-3-like activities and decrease cell viability. Furthermore, MG132 also promotes the cleavage of eIF4G and the activation of caspase-3-like activity in a caspase-8-deficient Jurkat cell line which is resistant to anti-Fas-mediated apoptosis. Using specific anti-peptide antisera, we show that both eIF4GI and eIF4GII are cleaved in either cell line in response to MG132 and lactacystin. In response to such treatments, we demonstrate that the fragments of eIF4GI generated include those previously observed with anti-Fas antiserum together with a novel product which lacks the ability to interact with eIF4E. In contrast, cells treated with the immunosuppressants FTY720 and cyclosporin A appear to contain only the novel cleavage fragment of eIF4GI and to lack those characteristic of cells treated with anti-Fas antiserum. These data suggest that caspase-8 activation is not required for apoptosis and eIF4G cleavage mediated by proteasome inhibitors and immunosuppressants in human T cells.

Topics: Acetylcysteine; Apoptosis; Calpain; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cyclosporine; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Eukaryotic Initiation Factor-4G; fas Receptor; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Jurkat Cells; Leupeptins; Multienzyme Complexes; Peptide Fragments; Peptide Initiation Factors; Propylene Glycols; Proteasome Endopeptidase Complex; Sphingosine; T-Lymphocytes

Prolonged treatment of rat adipocytes with TNF alpha increases lipolysis through a mechanism mediated, in part, by down-regulation of inhibitory G proteins (G(i)). Separately, down-regulation of G(i) by prolonged treatment with an A(1)-adenosine receptor agonist, N(6)-phenylisopropyl adenosine (PIA) increases lipolysis. To investigate the role of proteolysis in TNF alpha and PIA-mediated G(i) down-regulation and stimulation of lipolysis, we used the protease inhibitors lactacystin (proteasome inhibitor) and calpeptin (calpain inhibitor). Rat adipocytes were preincubated for 1 h with lactacystin (10 microM) or calpeptin (50 microM), before 30-h treatment with either TNF alpha (50 ng/ml) or PIA (300 nM). We then measured lipolysis (glycerol release), abundance of alpha-subunits of G(i)1 and G(i)2 in plasma membranes (Western blotting) and protease activities (in specific fluorogenic assays). TNF alpha and PIA stimulated lipolysis approximately 2-fold and caused G(i) down-regulation. Although neither lactacystin nor calpeptin affected basal lipolysis, lactacystin completely inhibited both TNF alpha and PIA-stimulated lipolysis (the 50% inhibitory concentration was approximately 2 microM), whereas calpeptin had no effect. Similarly, lactacystin but not calpeptin blocked both PIA and TNF alpha-induced G(i) down-regulation. These findings provide further evidence that the chronic lipolytic effect of TNF alpha and PIA is secondary to G(i) down-regulation and suggest that the mechanism involves proteolytic degradation mediated through the proteasome pathway.

Topics: Acetylcysteine; Adipocytes; Animals; Calpain; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptides; Down-Regulation; GTP-Binding Protein alpha Subunits, Gi-Go; Lipolysis; Male; Multienzyme Complexes; Phenylisopropyladenosine; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

The purpose of the present investigation was to develop a system for continuous evaluation of extralysosomal proteolytic activity and its regulation in polarized epithelial cells. Filter inserts containing a tight monolayer of primary cultured pig thyrocytes were placed in a thermostated aluminium block. The cell-permeable, fluorogenic calpain and proteasome substrate succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was added to the apical buffer and fluorescence changes were continuously measured via the fibre optics of a luminometer held at a fixed distance from the cell layer. Basal proteolytic activity was reduced by 60-70% by the proteasome inhibitor lactacystin. Proteolysis was increased within a few minutes after application of Ca(2+)-mobilizing agents (ionomycin, 4-bromo-A23187, thapsigargin and maitotoxin). Forskolin and staurosporine also enhanced the proteolytic activity. We conclude that Ca(2+)mobilization, and possibly also changes of protein kinase activity, rapidly increase non-lysosomal proteolysis in the intact thyroid epithelium.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Calcium; Calpain; Cell Polarity; Cells, Cultured; Colforsin; Coumarins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epithelial Cells; Fluorescence; L-Lactate Dehydrogenase; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; Swine; Thyroid Gland

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.

Topics: Acetylcysteine; Acrylates; Amino Acid Chloromethyl Ketones; Apoptosis; Boronic Acids; Bortezomib; Calpain; Cathepsins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Drug Synergism; Genes, p53; Humans; Jurkat Cells; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Neoplasms; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Tumor Cells, Cultured; Tumor Suppressor Protein p53; U937 Cells

The ubiquitin-proteasome system has been regarded as being important in the progression of neurodegenerative diseases, although its exact role remains uncertain. This in vitro study using PC12h cell cultures examined whether interference with the ubiquitin-proteasome system by proteasome inhibitors induces the neuropathological features of neurodegenerative diseases. Perikaryal accumulation of phosphorylated neurofilaments and an increase in c-Jun as well as phosphorylated form of c-Jun and apoptosis-specific protein were induced by the proteasome inhibitors lactacystin and N-carbobenzoxy-leucyl-leucyl-leucinal. These changes were not observed when only calpain was inhibited. The present study therefore suggests the possibility that a perturbation of the ubiquitin-proteasome system may be one of the causes that result in the development of neuropathological features. Additionally, activity assays showed that the proteasome inhibitor caused an increase in the activity of c-Jun N-terminal kinase (JNK/SAPK), which can phosphorylate neurofilaments and c-Jun, suggesting the possible involvement of JNK in phosphorylation of these proteins.

Topics: Acetylcysteine; Alkaline Phosphatase; Animals; Calpain; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Immunoblotting; JNK Mitogen-Activated Protein Kinases; Leupeptins; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Neurites; Neurofilament Proteins; PC12 Cells; Phosphorylation; Precipitin Tests; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-jun; Rats; Ubiquitins

To develop a system for continuous evaluation of proteolytic activity in human lens epithelium and to characterize factors of importance for the regulation of proteolytic activity in lens epithelial cells.. Human lens epithelial cells were obtained during cataract surgery. Capsule epithelium specimens consisted of the central parts of the anterior capsule and the underlying lens epithelium. The sample, with the cell-permeable substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was placed in a chamber, which was placed in a thermostat-controlled aluminum block. Fluorescence changes were continuously measured by the fiber optics of the luminometer, which was placed 5 mm above the buffer surface.. After administration of substrate to the medium overlying the cells, the substrate was degraded at a relatively slow rate. Approximately 10 picomoles of amino-4-methylcoumarin were formed per minute. A significant increase of proteolytic activity could be observed after application of 1 microM ionomycin or 2 microM thapsigargin. No leakage of lactate dehydrogenase from the cells was observed during these procedures. Basal proteolytic activity was totally inhibited by the proteasome inhibitor lactacystin. Lactacystin also attenuated the response to ionomycin and thapsigargin.. Human lens epithelium responds to increased Ca levels from external or internal stores with an increased proteolytic activity that may be mediated by calpain, by the proteasome, or by both. This calcium-dependent change in proteolytic activity may be of importance in the development of cataract.

Topics: Acetylcysteine; Calcium; Calpain; Cell Membrane Permeability; Cell Survival; Coumarins; Cysteine Endopeptidases; Enzyme Inhibitors; Epithelium; Humans; Ionomycin; L-Lactate Dehydrogenase; Lens, Crystalline; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; Substrate Specificity; Thapsigargin

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Bucladesine; Calpain; Cyclin B; Cyclin B1; Cysteine Proteinase Inhibitors; Dipeptides; Fluorouracil; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ketones; Leupeptins; Lovastatin; Nuclear Proteins; Protein Processing, Post-Translational; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Tumor Cells, Cultured

We demonstrate here that both procaspase-3 (32 kDa) and PARP are calpain substrates. In calcium-channel opener maitotoxin-treated cells, a 30 kDa caspase-3 fragment is produced in a time and concentration-dependent manner. Formation of this fragment is prevented by calpain inhibitors but not by the pancaspase inhibitor, carbobenzoxy-Asp-CH(2)OC(O)-2,6-dichlorobenzene (Z-D-DCB) nor the selective proteasome inhibitor lactacystin. In maitotoxin-treated cells, PARP (113 kDa) is also cleaved into a 40 kDa immunoreactive fragment, in a calpain-inhibitor-sensitive manner. Both procaspase-3 and PARP are also cleaved in vitro by purified micro-calpain to a 30 kDa fragment and a 40 kDa fragment, respectively. Finally, we show that staurosporine-mediated caspase-3 activation is interrupted by maitotoxin pretreatment.

Topics: Acetylcysteine; Aspartic Acid; Calpain; Caspase 3; Caspase Inhibitors; Caspases; Cell Line; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Precursors; Humans; Marine Toxins; Oxocins; Poly(ADP-ribose) Polymerases; Protein Processing, Post-Translational; Staurosporine; Substrate Specificity

P-glycoprotein (Pgp) is a plasma-membrane glycoprotein that confers multi-drug resistance (MDR) on cells and displays ATP-driven drug pumping. The possible contribution of calpain-mediated proteolytic pathways to the functional regulation of the Pgp molecule was evaluated using K562/DXR, MDR cells. N-Acetyl-L-leucyl-L-leucyl-norleucinal was effluxed by Pgp, but N-benzyloxycarbonyl-L-leucyl-L-leucinal (zLLal), an inhibitor of calpain, retarded the degradation of Pgp leading to accumulation of the molecule largely at the cell surface membrane. Treatment with brefeldin A did not obstruct the zLLal-induced Pgp accumulation. NH4Cl increased the cytoplasmic Pgp level, with a slight to significant decrease at the cell surface membrane. Ubiquitin-ELISA and western blot analysis confirmed that the Pgp molecule, which accumulated mainly at the cell surface, was ubiquitinated. However, lactacystin did not show any accumulation of Pgp in either the cytoplasm or the cell surface membrane, suggesting that the proteasome did not participate in the phenomenon. Additionally, the Pgp was limitedly proteolyzed by calpain into two 98 kDa and 69 kDa, fragments within one minute. Despite the increased accumulation of Pgp at the cell surface after treatment with calpain inhibitor, the cytoplasmic doxorubicin level of the cells treated with a calpain inhibitor was higher than that of non-treated cells and approached that of parental cells. These results indicated that calpain involved Pgp turnover and that calpain inhibition induced ubiquitinated Pgp-accumulation mainly at the cell surface membrane with a reduction in its own functions suggesting that the modulation of Pgp-turnover involves MDR-reversal by another approach.

Topics: Acetylcysteine; Ammonium Chloride; ATP Binding Cassette Transporter, Subfamily B; Blotting, Western; Brefeldin A; Calpain; Cell Membrane; Cysteine Proteinase Inhibitors; Dipeptides; Doxorubicin; Enzyme-Linked Immunosorbent Assay; Glycoproteins; Humans; K562 Cells; Leucine; Leupeptins; Membrane Proteins; Protein Synthesis Inhibitors; Time Factors; Ubiquitins

p107 protein, a member of the retinoblastoma family protein, suppresses growth promotion in cancer cells. We have already reported evidence that calpain, a calcium dependent protease is involved in the cleavage of p107 protein. We show here that p107 protein can also be a substrate for ubiquitination. A negative growth regulator, the HMG-CoA reductase inhibitor lovastatin was found to induce loss of p107 protein which was reversible by a specific protease inhibitor lactacystin as well as calpain inhibitor. Following treatment with lovastatin higher molecular weight ubiquitinated forms of p107 were detected by anti-p107 immunoprecipitation and anti-ubiquitin Western blotting. These forms further increased when lactacystin was added to culture medium. These results indicate that ubiquitin-proteasome pathway plays a potential role in the degradation as well as calpain. The data presented here suggest a model in which calpain and ubiquitin-proteasome system possibly play a cooperative role in targeting the protein under certain conditions.

Topics: Acetylcysteine; Antineoplastic Agents; Blotting, Western; Calcium-Binding Proteins; Calpain; CDC2-CDC28 Kinases; Cell Cycle; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cysteine Proteinase Inhibitors; Humans; Lovastatin; Male; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Sarcoma, Ewing; Transfection; Tumor Cells, Cultured; Ubiquitins

N-myc is a short-lived transcription factor, frequently amplified in human neuroblastomas. The ubiquitin-proteasome system is involved in the degradation of many short-lived cellular proteins and previous studies have shown that ubiquitin-dependent proteolysis is implicated in the turn-over of N-myc in vitro. However, calpain has also been implicated in N-myc degradation in vitro. Here we report that, in vivo, N-myc is a sensitive substrate for the 26S proteasome in N-myc amplified neuroblastoma cells. We observed that inhibition of the 26S proteasome with two inhibitors, ALLnL and lactacystin, led to an elevation of the N-myc protein steady-state and increased N-myc protein polyubiquitination, as revealed by ubiquitin Western blotting. Pulse-chase experiments have shown that the increased N-myc levels resulted from stabilization of the protein. In contrast treatment with several calpain and cathepsin inhibitors failed to block N-myc degradation in vivo. Furthermore, fluorescence microscopy of ALLnL-treated cells localized N-myc exclusively to the nuclear compartment, suggesting the absence of a requirement for transport to the cytoplasm prior to degradation.

Topics: Acetylcysteine; Calpain; Cysteine Proteinase Inhibitors; Humans; Lysosomes; Neuroblastoma; Peptide Hydrolases; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-myc; Substrate Specificity; Tumor Cells, Cultured; Ubiquitins

Objectives were to investigate the role of the proteasome and m-calpain to muscle cell differentiation. Accordingly, we investigated the effects of lactacystin, a proteasome inhibitor, and calpain inhibitor-II (CI-II) on L8 muscle cell differentiation and assessed concentrations of proteasomal and calpain subunit mRNAs during differentiation. L8 myoblasts were induced to differentiate by culturing in mitogen-depleted medium. To assess the importance of the proteasome and calpain to differentiation, we examined effects of lactacystin and CI-II on creatine kinase (CK) activity. In the absence of inhibitor, CK activity was detectable within 48 h of mitogen depletion and myotubes were formed. Addition of lactacystin or CI-II to cultures drastically reduced CK activity and prevented formation of myotubes. Hence, proteasome and calpain are both necessary for differentiation. In order to identify which proteasomal subunits were regulated during differentiation, we examined the concentrations of two 20S core subunits (C8 and C9) and three 22S ATPases (MSS1, S4 and TBP1) during differentiation. Concentrations of m-calpain and beta-tubulin mRNAs were also assessed. Differentiation was associated with slight increases (ca. 30%) in concentrations of mRNAs encoding the proteasomal 20S core subunits (C8 and C9) and with large increases (approximately 2-fold) in mRNAs encoding the regulatory subunit ATPases. m-calpain mRNA concentration also increased two-fold following mitogen depletion. beta-Tubulin mRNA concentration remained unchanged early in the differentiation process and thereafter declined. Of interest, changes in proteasomal and m-calpain mRNAs occurred within 6-24 h of mitogen depletion (i.e., at least 24-36 h prior to detectable changes in creatine kinase activity). These results indicate that changes in expression of proteasome and calpains subunits occur early in the differentiation process. These changes may be required for the normal course of differentiation to proceed. Differentiation is associated with larger changes in proteasomal ATPase mRNAs than in 20S core particle mRNAs indicating that either turnover rates of the 22S ATPase subunits are more rapid in differentiating cells than of the 20S core particles or that functions of the regulatory subunits become more important during muscle cell differentiation.

Topics: Acetylcysteine; Adenosine Triphosphatases; Animals; Calpain; Carrier Proteins; Cell Differentiation; Cell Line; Creatine Kinase; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Iron-Binding Proteins; Multienzyme Complexes; Muscles; Myogenin; Oligopeptides; Proteasome Endopeptidase Complex; Rats; Transferrin-Binding Proteins

A major problem in assessing the role of calpains in apoptosis induction concerns the fact that calpain inhibitors can also impair the activity of the proteasome, also reported to be involved in apoptosis. Herein we showed that apoptosis induced by calphostin C in U937 human promonocytic leukemia cells was associated, at its onset, with enhanced protein (poly)ubiquitination. This observation prompted us to study whether protein degradation through the ubiquitin/proteasome pathway was involved in apoptosis induction. We found that N-acetyl-Leu-Leu-norleucinal (50 microM), a proteasome as well as a calpain inhibitor, was able to reduce calphostin C-induced apoptosis by approximately 60%, whereas lactacystin (10 microM), a specific proteasome inhibitor, was ineffective. These results suggest that calphostin C-induced apoptosis is partly calpain-mediated, but does not require protein degradation through the ubiquitin/proteasome pathway.

Topics: Acetylcysteine; Antibiotics, Antineoplastic; Apoptosis; Calpain; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Leupeptins; Multienzyme Complexes; Naphthalenes; Neoplasm Proteins; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; U937 Cells; Ubiquitins

Calpain activity is thought to be essential for the execution of apoptotic cell death in certain experimental models. In the present study, the physiological inhibitor of calpain, calpastatin, was found to be cleaved in three different apoptotic systems. The 110-120 kDa calpastatin protein of Jurkat T-lymphocytes and U937 monocytic leukemia cells was cleaved to a 65-70 kDa form after the induction of apoptosis with anti-CD95 monoclonal antibody, staurosporine or TNF. Cleavage of calpastatin in apoptotic cells occurred simultaneously with the cleavage of the DNA repair enzyme, poly(ADP-ribose) polymerase. The caspase inhibitors VAD-cmk and IETD-fmk prevented calpastatin cleavage in all three systems. Calpain inhibitor I, however, suppressed calpastatin cleavage only during TNF-induced apoptosis. Other protease inhibitors, such as lactacystin and pepstatin A, did not confer any significant protection against apoptotic calpastatin cleavage. The results from in vitro incubations with cell lysates and purified enzymes showed that calpain I, calpain II and recombinant caspase-3, all cleaved calpastatin, with varying efficiency. In conclusion, the results of the present study suggest that caspases may cleave calpastatin and thus, regulate calpain activity during apoptotic cell death.

Topics: Acetylcysteine; Apoptosis; Calcium-Binding Proteins; Calpain; Caspase 3; Caspases; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; fas Receptor; Flow Cytometry; Humans; Jurkat Cells; Pepstatins; Protease Inhibitors; Staurosporine; Tumor Necrosis Factor-alpha; U937 Cells

The accumulation of misfolded proteins in the cytosol leads to increased expression of heat-shock proteins, while accumulation of such proteins in the endoplasmic reticulum (ER) stimulates the expression of many ER resident proteins, most of which function as molecular chaperones. Recently, inhibitors of the proteasome have been identified that can block the rapid degradation of abnormal cytosolic and ER-associated proteins. We therefore tested whether these agents, by causing the accumulation of abnormal proteins, might stimulate the expression of cytosolic heat-shock proteins and/or ER molecular chaperones and thereby induce thermotolerance. Exposure of Madin-Darby canine kidney cells to various proteasome inhibitors, including the peptide aldehydes (MG132, MG115, N-acetyl-leucyl-leucyl-norleucinal) and lactacystin, inhibited the degradation of short-lived proteins and increased markedly the levels of mRNAs encoding cytosolic heat-shock proteins (Hsp70, polyubiquitin) and ER chaperones (BiP, Grp94, ERp72), as shown by Northern blot analysis. However, inhibitors of cysteine proteases (E64), serine proteases (leupeptin), or metalloproteases (1, 10-phenanthroline) had no effect on the levels of these mRNAs. The relative efficacies of the peptide aldehyde inhibitors in inducing these mRNAs correlated with their potencies against the proteasome. Furthermore, reduction of the aldehyde group of MG132 decreased its inhibitory effect on proteolysis and largely prevented the induction of these mRNAs. Although treatment with the proteasome inhibitors caused rapid increases in mRNA levels (as early as 2 h after treatment with MG132), the inhibitors did not detectably affect total protein synthesis, total protein secretion, ER morphology, or the retention of ER-lumenal proteins, even after 18 h of treatment. Together, the findings suggest that inhibition of proteasome function induces heat-shock proteins and ER chaperones due to the accumulation of sufficient amounts of abnormal proteins and/or the inhibition of degradation of a key regulatory factor (e.g. heat-shock factor). Since expression of heat-shock proteins can protect cells from thermal injury, we tested whether the proteasome inhibitors might also confer thermotolerance. Treatment of cells with MG132 for as little as 2 h, markedly increased the survival of cells subjected to high temperatures (up to 46 degrees C). Thus, these agents may have applications in protecting against cell injury.

Topics: Acetylcysteine; Animals; Calpain; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dogs; Endoplasmic Reticulum; Fungal Proteins; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Leupeptins; Membrane Glycoproteins; Membrane Proteins; Molecular Chaperones; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Conformation; RNA, Messenger

The crystal structure of the 20S proteasome from the yeast Saccharomyces cerevisiae shows that its 28 protein subunits are arranged as an (alpha1...alpha7, beta1...beta7)2 complex in four stacked rings and occupy unique locations. The interior of the particle, which harbours the active sites, is only accessible by some very narrow side entrances. The beta-type subunits are synthesized as proproteins before being proteolytically processed for assembly into the particle. The proforms of three of the seven different beta-type subunits, beta1/PRE3, beta2/PUP1 and beta5/PRE2, are cleaved between the threonine at position 1 and the last glycine of the pro-sequence, with release of the active-site residue Thr 1. These three beta-type subunits have inhibitor-binding sites, indicating that PRE2 has a chymotrypsin-like and a trypsin-like activity and that PRE3 has peptidylglutamyl peptide hydrolytic specificity. Other beta-type subunits are processed to an intermediate form, indicating that an additional nonspecific endopeptidase activity may exist which is important for peptide hydrolysis and for the generation of ligands for class I molecules of the major histocompatibility complex.

Topics: Acetylcysteine; Calpain; Crystallography, X-Ray; Cysteine Endopeptidases; Endopeptidases; Enzyme Inhibitors; Enzyme Precursors; Glycoproteins; Histocompatibility Antigens Class I; Models, Molecular; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Conformation; Saccharomyces cerevisiae; Thermoplasma; Threonine

Cerebral deposition of amyloid beta-protein (Abeta) as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Abeta falls into two major subspecies defined by their C-termini, Abeta40 and Abeta42, ending in Val-40 and Ala-42, respectively. Although Abeta42 accounts for only approximately 10% of secreted Abeta, Abeta42 is the predominant species accumulated in senile plaques in AD brain and appears to be the initially deposited species. Its secretion level has recently been reported to be increased in the plasma or culture media of fibroblasts from patients affected by any of early-onset familial AD (FAD). Thus, inhibition of Abeta42 production would be one of the therapeutic targets for AD. However, there is little information about the cleavage mechanism via which Abeta40 and Abeta42 are generated and its relationship to intracellular protease activity. Here, we examined by well-characterized enzyme immunoassay the effects of calpain and proteasome inhibitors on the levels of Abeta40 and Abeta42 secretion by cultured cells. A calpastatin peptide homologous to the inhibitory domain of calpastatin, an endogenous calpain specific inhibitor, induced a specific increase in secreted Abeta42 relative to the total secreted Abeta level, a characteristic of the cultured cells transfected with FAD-linked mutated genes, while a proteasome specific inhibitor, lactacystin, showed no such effect. These findings suggest that the Abeta42 secretion ratio is modulated by the calpain-calpastatin system and may point to the possibility of exploring particular compounds that inhibit Abeta42 secretion through this pathway.

Topics: Acetylcysteine; Alzheimer Disease; Amyloid beta-Peptides; Calcimycin; Calcium-Binding Proteins; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Embryo, Mammalian; Humans; Kidney; Peptide Fragments; Transfection

Recent studies have demonstrated that cell-permeant protease inhibitors arrest human fibroblasts in late G1. The target for the inhibitors has been claimed to be either the proteasome, or a calpain-like cysteine protease activity. In the present investigation, the progression of serum-stimulated WI-38 fibroblasts into S-phase was partially inhibited by the cell-permeant general inhibitor of cysteine proteases, E64d, but not by its non-permeant anolog, E64c. Exposure of fibroblasts in late G1 to the proteasome inhibitor, lactacystin, produced only a modest inhibition of progression into S-phase, and did not influence the extensive inhibition produced by the calpain-selective inhibitor, ZLLY-DMK. ZLLnV-CHO and ZLLL-CHO, which are reportedly selective for the proteasome, were less potent than ZLLY-DMK as inhibitors of S-phase progression. These results argue for the involvement of a calpain-like protease acting in late G1 to allow transit into S-phase.

Topics: Acetylcysteine; Calpain; Cell Cycle; Cell Line; Cysteine Proteinase Inhibitors; Diazomethane; Fibroblasts; G1 Phase; Humans; Leupeptins; Oligopeptides; S Phase

Cyclin D1, a critical positive regulator of G1 progression, has been implicated in the pathogenesis of certain cancers. Regulation of cyclin D1 occurs at the transcriptional and posttranscriptional level. Here we present evidence that cyclin D1 levels are regulated at the posttranscriptional level by the Ca2+-activated protease calpain. Serum starvation of NIH 3T3 cells resulted in rapid loss of cyclin D1 protein that was completely reversible by calpain inhibitors. Actinomycin D and lovastatin induced rapid loss of cyclin D1 in prostate and breast cancer cells that was reversible by calpain inhibitors and not by phenylmethylsulfonyl fluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26 S proteasome. Treatment of intact NIH 3T3, prostate, and breast cancer cells with a calpain inhibitor dramatically increased the half-life of cyclin D1 protein. Addition of purified calpain to PC-3-M lysates resulted in Ca2+-dependent cyclin D1 degradation. Transient expression of the calpain inhibitor calpastatin increased cyclin D1 protein in serum-starved NIH 3T3 cells. Cyclins A, E, and B1 have been reported to be regulated by proteasome-associated proteolysis. The data presented here implicate calpain in cyclin D1 posttranslational regulation.

Topics: 3T3 Cells; Acetylcysteine; Animals; Calpain; Cyclin B; Cyclin B1; Cyclin D1; Cysteine Proteinase Inhibitors; Dactinomycin; Half-Life; Humans; Male; Mice; Prostatic Neoplasms; Protein Synthesis Inhibitors; Tumor Cells, Cultured

delta 9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt-washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.

Topics: Acetylcysteine; Animals; Calpain; Cell Fractionation; Cysteine Proteinase Inhibitors; Hydrogen-Ion Concentration; Liver; Lysosomes; Male; Microsomes; Molecular Weight; Rats; Rats, Sprague-Dawley; Sequence Analysis; Serine Proteinase Inhibitors; Stearoyl-CoA Desaturase; Temperature

Cell-permeant peptidyl aldehydes and diazomethylketones are frequently utilized as inhibitors of regulatory intracellular proteases. In the present study the specificities of several peptidyl inhibitors for purified human mu-calpain and 20 S proteasome were investigated. Acetyl-LLnL aldehyde, acetyl-LLM aldehyde, carbobenzyloxy-LLnV aldehyde (ZLLnVal), and carbobenzyloxy-LLY-diazomethyl ketone produced half-maximum inhibition of the caseinolytic activity of mu-calpain at concentrations of 1-5 x 10(-7) M. In contrast, only ZLLnVal was a reasonably potent inhibitor of the caseinolytic activity of 20 S proteasome, producing 50% inhibition at 10(-5) M. The other inhibitors were at least 10-fold less potent, producing substantial inhibition only at near saturating concentrations in the assay buffer. Further studies with ZLLnVal demonstrated that its inhibition of the proteasome was independent of casein concentration over a 25-fold range. Proteolysis of calpastatin or lysozyme by the proteasome was half-maximally inhibited by 4 and 22 microM ZLLnVal, respectively. Thus, while other studies have shown that ZLLnVal is a potent inhibitor of the hydrophobic peptidase activity of the proteasome, it appears to be a much weaker inhibitor of its proteinase activity. The ability of the cell permeant peptidyl inhibitors to inhibit growth of the yeast Saccharomyces cerevisiae was studied because this organism expresses proteasome but not calpains. Concentrations of ZLLnVal as high as 200 microM had no detectable effect on growth rates of overnight cultures. However, yeast cell lysates prepared from these cultures contained 2 microM ZLLnVal, an amount which should have been sufficient to fully inhibit hydrophobic peptidase activity of yeast proteasome. Degradation of ubiquitinylated proteins in yeast extracts by endogenous proteasome was likewise sensitive only to high concentrations of ZLLnVal. The higher sensitivity of the proteinase activity of calpains to inhibition by the cell permeant inhibitors suggests that calpain-like activities may be targets of these inhibitors in animal cells.

Topics: Acetylcysteine; Calcium-Binding Proteins; Calpain; Catalysis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Diazomethane; Enzyme Precursors; Humans; Kinetics; Leupeptins; Multienzyme Complexes; Muramidase; Oligopeptides; Plant Proteins; Protease Inhibitors; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Serine Proteinase Inhibitors; Ubiquitins