Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Protease inhibitor-induced apoptosis: accumulation of wt p53, p21WAF1/CIP1, and induction of apoptosis are independent markers of proteasome inhibition. 1091 53

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.
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PMID:Accumulation of phosphorylated neurofilaments and increase in apoptosis-specific protein and phosphorylated c-Jun induced by proteasome inhibitors. 1100 89

alpha-Catenin is an essential component of the cadherin-catenin cell-cell adhesion complex. An excess amount of alpha-catenin also affects the Wnt signaling pathway probably through its direct binding to beta-catenin. Here, we examined the molecular mechanisms of the posttranscriptional regulation of alpha-catenin expression. We constructed an expression vector with alpha-catenin cDNA lacking the 5'-untranslated sequence. In L cell transfectants stably expressing mRNA derived from this vector, the amount of exogenous alpha-catenin protein was about 10-fold higher than that of the endogenous protein. The expression level of the exogenously expressed alpha-catenin mRNA, however, was about 80% of that of endogenous molecule. Most of the endogenous and exogenous alpha-catenin protein in cadherin-negative cells was degraded 5 h after inhibition of protein synthesis. Although alpha-catenin contains the PEST sequence, various proteasome and calpain inhibitors did not affect the level of expression of endogenous alpha-catenin protein in L cells. Overexpressed alpha-catenin showed cytoplasmic localization, disturbed the nuclear localization of stabilized beta-catenin, and inhibited TCF-4-responsive transactivation after Wnt-3a treatment. These results suggested that the low-efficiency of translation and unidentified degradation mechanisms maintained the low levels of alpha-catenin expression in the cytoplasm as a necessary condition for the Wnt signaling pathway.
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PMID:Posttranscriptional regulation of alpha-catenin expression is required for Wnt signaling in L cells. 1106 15

The effect of indomethacin, a non-steroidal anti-inflammatory drug upon purified calpain has been studied. Also, its effects upon Ca2+-mediated degradation of cytoskeletal proteins (neurofilament) in spinal cord homogenate has been investigated. A dose-dependent inhibition of purified calpain activity was observed. A 50% inhibition of 14C-caseinolytic activity was obtained with less than 1.1 mM of indomethacin while the activity was completely inhibited at 3.3 mM concentration. The inhibitory effect of ketorlac, another non-steroidal anti-inflammatory drug, upon calpain was weaker than that of indomethacin. The degradation of myelin basic protein (MBP) by cathepsin B, a lysosomal cysteine protease, was significantly inhibited by indomethacin. It also inhibited the Ca2+-mediated degradation of neurofilament protein (NFP) in spinal cord homogenate. The extent of NFP degradation was analyzed by SDS-PAGE and the inhibition shown by indomethacin was weaker than that observed with leupeptin and the calpain inhibitor E64-d. The inhibitory effect of indomethacin on the activity of multicatalytic proteinase complex was negligible. These results suggest that indomethacin, a non-steroidal anti-inflammatory drug and cyclooxygenase inhibitor also inhibits proteinases, including cathepsin B and calpain.
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PMID:Inhibition of proteolysis by a cyclooxygenase inhibitor, indomethacin. 1107 71

A 22-year-old man developed unconsciousness, severe quadriplegia and muscle atrophy, and had markedly elevated serum creatine kinase levels after using the high-dose steroid and nondepolarizing neuromuscular blocking agents during the course of sepsis and DIC. On neurological examination, he was lethargic. The patient had generalized muscle weakness and wasting, and diminished deep tendon reflexes. He weakly responsed to painful stimuli on the legs. The motor nerve conduction study demonstrated decreased CMAP (compound muscle action potential) amplitudes. Motor and sensory nerve conduction velocities and their distal latencies were normal. Muscle biopsy revealed marked muscle fiber atrophy predominantly in type 2 fibers and numerous basophilic and a few necrotic fibers. Some atrophic fibers had decreased to absent myosin adenosine triphosphatase activity in their center. Accordingly, he was diagnosed as having acute quadriplegic myopathy (AQM), which has been reported mainly in Western countries. The mechanism of muscle fiber degradation in this myopathy is still unknown. On immunohistochemical analysis to our patient, enzyme activities of various proteases such as calpain, cathepsin B, and proteasomes were increased in the sarcoplasm, especially in the atrophic fibers. We suggest that lysosomal cathepsin, nonlysosomal calpain, and ATP-ubiquitin-proteasome proteolytic pathways participate in muscle fiber degradation in AQM.
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PMID:[A case of acute quadriplegic myopathy]. 1108 98

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.
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PMID:Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors. 1115 77

The present study investigates the role of two major proteolytic systems in transforming rabbit and rat muscles. The fast-to-slow transformation of rabbit muscle by chronic low-frequency stimulation (CLFS) induces fast-to-slow transitions of intact, mature fibers and replacement of degenerating fibers by newly formed slow fibers. Ubiquitination, an indicator of the ATP-dependent proteasome system, and calpain activity were measured in homogenates of control and stimulated extensor digitorum longus muscles. Calpain activity increased similarly (approximately 2-fold) in stimulated rat and rabbit muscles. CLFS had no effect on protein ubiquitination in rat muscle but led to elevations in ubiquitin protein conjugates in rabbit muscle. Immunohistochemistry was used to study the distribution of micro-calpain and m-calpain and of ubiquitinated proteins in myosin heavy chain-based fiber types. The findings suggest that both proteolytic systems are involved in fiber transformation and replacement. Transforming mature fibers displayed increases in micro-calpain and accumulation of ubiquitin protein conjugates. The majority of these fibers were identified as type IIA. Enhanced ubiquitination was also observed in degenerating and necrotic fibers. Such fibers additionally displayed elevated m-calpain levels. Conversely, p94, the skeletal muscle-specific calpain, decayed rapidly after stimulation onset and was hardly detectable after 4 days of CLFS.
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PMID:Fiber type-specific expression of major proteolytic systems in fast- to slow-transforming rabbit muscle. 1120 17

We describe a method that has allowed us to measure the synthesis, turnover and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons. For these studies, rat hippocampal cultures containing 74.5-83.0% neurons were established. B-27 (Gibco) supplement has been used to obtain an excellent long-term viability (up to 5 weeks) of hippocampal neurons in culture. For the synthesis, turnover, and assembly experiments the neurons were labeled with [35S]methionine, and chased with 10-fold excess of cold methionine for the turnover experiments. The cells were then lysed and immunoprecipitated with alpha, beta-erythroid, alpha, and beta-nonerythroid spectrin antibodies. Immunoprecipitated [35S]methionine-labeled spectrins of hippocampal neurons grown in vitro produced bands in 5% polyacrylamide minigels strong enough to be detected by the high sensitivity screens of a phosphorimager to generate graphs from which the synthesis or half-lives of alpha, beta-erythroid, alpha, and beta-nonerythroid spectrins were calculated. This method can be used to study the role of calpain, caspase-3, and the ubiquitin-proteasome system on the synthesis and turnover of erythroid and nonerythroid spectrins in resting and depolarized rat hippocampal neurons in culture.
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PMID:Measurement of the synthesis, turnover, and assembly of alpha- and beta-erythroid and nonerythroid spectrins in cultured rat hippocampal neurons. 1122 13

Human cytomegalovirus (HCMV) stimulates arrested cells to enter the cell cycle by activating cyclin-dependent kinases (Cdks), notably Cdk2. Several mechanisms are involved in the activation of Cdk2. HCMV causes a substantial increase in the abundance of cyclin E and stimulates translocation of Cdk2 from the cytoplasm to the nucleus. Further, the abundance of the Cdk inhibitors (CKIs) p21cip1/waf1 (p21cip1) and p27kip1 is substantially reduced. The activity of cyclin E/Cdk2 increases as levels of CKIs, particularly p21cip1, fall. We have previously shown that these phenomena contribute to priming the cell for efficient replication of HCMV. In this study, the mechanisms responsible for the decrease in p21cip1 levels after HCMV infection were investigated by measuring p21cip1 RNA and protein levels in permissive human lung (LU) fibroblasts after HCMV infection. Northern blot analysis revealed that p21cip1 RNA levels increased briefly at 3 h after HCMV infection and then decreased to their nadir at 24 h; thereafter, RNA levels increased to about 60% of the preinfection level. Western blot analysis demonstrated that the relative abundance of p21cip1 protein roughly paralleled the observed changes in initial RNA levels; however, the final levels of protein were much lower than preinfection levels. After a transient increase at 3 h postinfection, p21cip1 abundance declined sharply over the next 24 h and remained at a very low level through 96 h postinfection. The disparity between p21cip1 RNA and protein levels suggested that the degradation of p21cip1 might be affected in HCMV-infected cells. Treatment of HCMV-infected cells with MG132, an inhibitor of proteasome-mediated proteolysis, provided substantial protection of p21cip1 in mock-infected cells, but MG132 was much less effective in protecting p21cip1 in HCMV-infected cells. The addition of E64d or Z-Leu-Leu-H, each an inhibitor of calpain activity, to HCMV-infected cells substantially increased the abundance of p21cip1 in a concentration-dependent manner. To verify that p21cip1 was a substrate for calpain, purified recombinant p21cip1 was incubated with either m-calpain or mu-calpain, which resulted in rapid proteolysis of p21cip1. E64d inhibited the proteolysis of p21cip1 catalyzed by either m-calpain or mu-calpain. Direct measurement of calpain activity in HCMV-infected LU cells indicated that HCMV infection induced a substantial and sustained increase in calpain activity, although there was no change in the abundance of either m- or mu-calpain or the endogenous calpain inhibitor calpastatin. The observed increase of calpain activity was consistent with the increases in intracellular free Ca2+ and phospholipid degradation in HCMV-infected LU cells reported previously from our laboratory. Considered together, these results suggest that the increase in calpain activity observed following HCMV infection contributes significantly to the reduction of p21cip1 levels and the resultant cell cycle progression.
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PMID:Degradation of p21cip1 in cells productively infected with human cytomegalovirus. 1126 51

Cachexia is a syndrome characterized by profound tissue wasting that frequently complicates malignancies. In a cancer cachexia model we have shown that protein depletion in the skeletal muscle, which is a prominent feature of the syndrome, is mostly due to enhanced proteolysis. There is consensus on the views that the ubiquitin/proteasome pathway plays an important role in such metabolic response and that cytotoxic cytokines such as TNFalpha are involved in its triggering (Costelli and Baccino, 2000), yet the mechanisms by which the relevant extracellular signals are transduced into protein hypercatabolism are largely unknown. Moreover, little information is presently available as to the possible involvement in muscle protein waste of the Ca(2+)-dependent proteolysis, which may provide a rapidly activated system in response to the extracellular signals. In the present work we have evaluated the status of the Ca(2+)-dependent proteolytic system in the gastrocnemius muscle of AH-130 tumour-bearing rats by assaying the activity of calpain as well as the levels of calpastatin, the natural calpain inhibitor, and of the 130 kDa Ca(2+)-ATPase, both of which are known calpain substrates. After tumour transplantation, total calpastatin activity progressively declined, while total calpain activity remained unchanged, resulting in a progressively increasing unbalance in the calpain/calpastatin ratio. A decrease was also observed for the 130 kDa plasma membrane form of Ca(2+)-ATPase, while there was no change in the level of the 90 kDa sarcoplasmic Ca(2+)-ATPase, which is resistant to the action of calpain. Decreased levels of both calpastatin and 130 kDa Ca(2+)-ATPase have been also detected in the heart of the tumour-bearers. These observations strongly suggest that Ca(2+)-dependent proteolysis was activated in the skeletal muscle and heart of tumour-bearing animals and raise the possibility that such activation may play a role in sparking off the muscle protein hypercatabolic response that characterizes cancer cachexia.
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PMID:Activation of Ca(2+)-dependent proteolysis in skeletal muscle and heart in cancer cachexia. 1128 75


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