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)

The latent form of multicatalytic proteinase complex (MCP) was purified to homogeneity from ovine skeletal muscle. The MCP ran as a single band (M(r) 600,000) on nondenaturing polyacrylamide gel (PAGE) and dissociated to a number of subunits (M(r) 21,000 to 31,000) under denaturing and reducing conditions (SDS-PAGE). The proteinase complex was activated reversibly by heating at 60 degrees C and in the presence of SDS. Maximum activation (18-fold) was observed after 2 min at 60 degrees C and there was rapid inactivation beyond 2 min. Maximum proteolytic activity (12.8-fold) occurred in the presence of .25 mM SDS and diminished rapidly at higher SDS concentrations. The MCP was maximally active at pH 7.5 to 8.0 and 45 degrees C using radiolabeled alpha-casein. These and other results (e.g., proteinase inhibitor profiling) indicate that ovine skeletal muscle does indeed contain MCP and that its biochemical properties are the same as MCP isolated from other sources. By using [14C]-casein as a substrate, the specific activities (milligrams of protein degraded/milligrams of proteinase) for mu-, m-calpain, and MCP were 44.0, 59.7, and 2.0, respectively. Purified ovine myofibrils were incubated with mu-calpain or MCP. Classical effects of calpains, which include degradation of Z-disks, titin, desmin, troponin-T, and troponin-I and removal of alpha-actinin, were observed. However, only troponin-C and myosin light chains-2 and -3 were degraded by MCP. Morphologically, MCP had no detectable effect on myofibrils. Results suggest that MCP is not involved in the initial steps of myofibril disassembly. However, its involvement in the degradation of myofilaments remains to be determined.
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PMID:Ovine skeletal muscle multicatalytic proteinase complex (proteasome): purification, characterization, and comparison of its effects on myofibrils with mu-calpains. 147 9

This experiment was conducted to determine the relationship between 3-methylhistidine (3MH) production and proteinase activity in skeletal muscles of growing barrows. Barrows at 13 wk of age were randomly assigned to either control diet available on an ad libitum basis (21% of ME consisted of protein; control group), control diet fed restricted (pair-fed with barrows in protein-free group; intake-restricted group), or protein-free diet available on an ad libitum basis (protein-free group) for 14 d. During the last 3 d, blood samples were collected for determination of 3MH production rate, which is a measure of myofibrillar protein breakdown. At slaughter, two muscles were taken: masseter (M) and longissimus (L) muscles. The muscle samples were analyzed for calpastatin, mu-calpain, m-calpain, multicatalytic proteinase (MCP), cathepsin B, cathepsins B+L, and cystatins activities. Both muscles were also analyzed for amounts of DNA, RNA, total protein, and myofibrillar and sarcoplasmic proteins. Growth rate (kilograms/day) was influenced by dietary treatments (P < .05). Fractional breakdown rate (FBR, percentage/day) of skeletal muscle, as calculated from 3MH production rate (micromoles.kilogram-1.day-1), was 27% higher for the protein-free group than for the control group. However, no differences in proteinase activities were observed, except for lower MCP activity in the M muscle of the protein-free group than in that of the other groups (P < .05). In the present study, no direct relation was observed between myofibrillar protein degradation rate and proteinase activities in skeletal muscle during a protein-free feeding strategy.
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PMID:Comparison between 3-methylhistidine production and proteinase activity as measures of skeletal muscle breakdown in protein-deficient growing barrows. 856 63

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.
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PMID:Specificities of cell permeant peptidyl inhibitors for the proteinase activities of mu-calpain and the 20 S proteasome. 936 65

A negative correlation exists between calpastatin activity and meat tenderness. Therefore, it is important to determine the mechanism of calpastatin inactivation in postmortem skeletal muscle. Western immunoblot analysis was performed to determine the protease(s) responsible for degradation of muscle calpastatin during postmortem storage. To accomplish this, purified calpastatin was digested with different proteases in vitro, and their pattern of calpastatin degradation was compared with that of calpastatin degradation in postmortem muscle. Polyclonal antibodies raised in mice against recombinant bovine skeletal muscle calpastatin were used to monitor calpastatin degradation. Lamb longissimus was stored at 4 degrees C and sampled at 0, 6, 12, 24, 72, 168, and 336 h postmortem. Postmortem storage produced a discrete pattern of calpastatin degradation products that included immunoreactive bands at approximately 100, 80, 65, 54, 32, and 29 kDa. Undegraded calpastatin (130 kDa) was barely detectable after 72 h of postmortem storage at 4 degrees C, and no immunoreactive calpastatin was observed by 336 h postmortem. For in vitro proteolysis, lamb longissimus calpastatin (0 h postmortem) was purified using Affi-Gel Blue chromatography. Calpastatin was digested with m-calpain, mu-calpain, cathepsin B, proteasome, trypsin, or chymotrypsin. Each of these enzymes degraded calpastatin. Immunoreactive fragments resulting from digestion of calpastatin with m- and mu-calpain were similar to each other and closely resembled those observed during postmortem aging of lamb longissimus at 4 degrees C. Digestion of calpastatin with mu-calpain reduced calpastatin activity. Degradation of calpastatin by other proteases resulted in unique patterns of immunoreactive fragments, distinct from that observed in longissimus. Thus, m- and(or) mu-calpain seem to be responsible for calpastatin degradation during postmortem storage of meat.
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PMID:Immunoblot analysis of calpastatin degradation: evidence for cleavage by calpain in postmortem muscle. 1037 23

Polypeptide sequences enriched in proline (P), glutamate (E), serine (S), and threonine (T), dubbed PEST domains, are proposed to expedite the degradation of proteins. The proteolysis of one PEST-containing protein, IkappaBalpha, is prerequisite to the activation of the transcription factor NF-kappaB. Two mechanisms of IkappaBalpha degradation in vivo have been described, one well characterized through the ubiquitin-proteasome pathway, and another less characterized through calpain. In this report, a mutational analysis was done to identify any regions of IkappaBalpha that facilitate its recognition and proteolysis by calpain in vitro. These studies revealed that the PEST sequence of IkappaBalpha is critical for its calpain-dependent degradation. Furthermore, the IkappaBalpha-PEST domain binds to the calmodulin-like domain of the large subunit of mu-calpain (muCaMLD). Transfer of the IkappaBalpha-PEST domain to a protein incapable of either binding to or being degraded by mu-calpain allowed for the interaction of the chimeric protein with muCaMLD and resulted in its susceptibility to calpain proteolysis. Moreover, the muCaMLD of calpain acts as a competitive inhibitor of calpain-dependent IkappaBalpha degradation. Our data demonstrate that the IkappaBalpha-PEST sequence acts as a modular domain to promote the physical association with and subsequent degradation by mu-calpain and suggest a functional role for PEST sequences in other proteins as potential calpain-targeting units.
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PMID:The PEST domain of IkappaBalpha is necessary and sufficient for in vitro degradation by mu-calpain. 1052 80

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

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.
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PMID:Nutritional regulation of proteases involved in fetal rat insulin secretion and islet cell proliferation. 1587 69

Bfl-1/A1 is generally recognized as a Bcl-2-related inhibitor of apoptosis. We show that Bfl-1 undergoes constitutive ubiquitin/proteasome-mediated turnover. Moreover, while Bfl-1 suppresses apoptosis induced by staurosporine or cytokine withdrawal, it is proapoptotic in response to tumor necrosis factor (TNF) receptor activation in FL5.12 pro-B cells. Its anti- versus proapoptotic effect is regulated by two proteolytic events: (1) its constitutive proteasome-mediated turnover and (2) its TNF/cycloheximide (CHX)-induced cleavage by mu-calpain, or a calpain-like activity, coincident with acquisition of a proapoptotic phenotype. In vitro studies suggest that calpain-mediated cleavage of Bfl-1 occurs between its Bcl-2 homology (BH)4 and BH3 domains. This would be consistent with the generation of a proapoptotic Bax-like BH1-3 molecule. Overall, our studies uncovered two new regulatory mechanisms that play a decisive role in determining Bfl-1's prosurvival versus prodeath activities. These findings might provide important clues to counteract chemoresistance in tumor cells that highly express Bfl-1.
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PMID:Constitutive proteasome-mediated turnover of Bfl-1/A1 and its processing in response to TNF receptor activation in FL5.12 pro-B cells convert it into a prodeath factor. 1609 3

We report that protein adducts of iso[4]levuglandin E2 (iso[4]LGE2), a highly reactive product of free radical-induced lipid oxidation, accumulate in human glaucomatous trabecular meshwork (TM) but not in controls. Reactive oxygen species play a pathogenic role in primary open angle glaucoma by fostering changes that reduce permeability of the TM tissue and consequently impede aqueous humor outflow resulting in elevated intraocular pressure. IsoLGs covalently modify proteins and are especially effective in causing protein-protein cross-linking. We found elevated levels of calpain-1 in glaucomatous TM. However, calpain activity in glaucomatous TM is only about 50% of that in controls. This paradox is explicable by the fact that modification by isoLGs renders calpain-1 inactive. Thus, treatment of calpain-1 with iso[4]LGE2 in vitro results in covalent modification, inactivation, the formation of high molecular weight aggregates (as determined by Western and dynamic light scattering analyses), and resistance to proteasomal digestion. Iso[4]LGE2-modified calpain-1 undergoes ubiquitination, and its loading impairs the cellular proteasome activity, consistent with competitive inhibition and formation of suicidal high molecular weight aggregates. These data suggest that interference with proteasomal activity, owing to protein modification by isoLGs, could contribute to glaucoma pathophysiology by decreasing the ability of the TM to modulate outflow resistance.
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PMID:Isolevuglandin-modified proteins, including elevated levels of inactive calpain-1, accumulate in glaucomatous trabecular meshwork. 1808 99

Increasingly, published evidence links glutamate with the pathogenesis of Alzheimer's disease. We investigated the molecular mechanism underlying glutamate-induced neurotoxicity in hippocampus, which is primarily linked to cognitive dysfunction in Alzheimer's disease. Acute exposure of rat hippocampal slices to glutamate significantly induced cell death, as determined by media lactate dehydrogenase levels and PI staining. Moreover, this was accompanied by Ca2+ influx and calpain-1 activation, as confirmed by the proteolytic pattern of spectrin. Notably, glutamate-induced calpain-1 activation decreased the level of beta-catenin, and this process appeared to be independent of glycogen synthase kinase 3beta (GSK-3beta), since glutamate also led to loss of GSK-3beta. Calpeptin, a calpain inhibitor, attenuated the glutamate-mediated degradations of spectrin, synaptophysin, and beta-catenin except GSK-3beta and modestly increased cell survival. In contrast, the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) effectively reduced all glutamate-evoked responses, i.e., the breakdowns of spectrin, synaptophysin, beta-catenin and GSK-3beta, and cell death. Pharmacological studies and in vitro calpain-1 proteolysis confirmed that in the glutamate-treated hippocampus, calpain-1-mediated decrease of beta-catenin could occur independently of GSK-3beta and of proteasome, and that GSK-3beta degradation is independent of calpain-1. These findings together provide the first direct evidence that glutamate promotes the down-regulations of beta-catenin and GSK-3beta, which potently contribute to neurotoxicity in hippocampus during excitotoxic cell death, and a molecular basis for the protection afforded by calpeptin and APV from the neurotoxic effect of glutamate.
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PMID:Concomitant degradation of beta-catenin and GSK-3 beta potently contributes to glutamate-induced neurotoxicity in rat hippocampal slice cultures. 1844 33


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