calcimycin and calpastatin

calcimycin has been researched along with calpastatin* in 13 studies

Other Studies

13 other study(ies) available for calcimycin and calpastatin

ArticleYear
mu-Calpain mediated cleavage of the Na+/Ca2+ exchanger in isolated mitochondria under A23187 induced Ca2+ stimulation.
    Archives of biochemistry and biophysics, 2009, Volume: 482, Issue:1-2

    Treatment of bovine pulmonary artery smooth muscle mitochondria with the calcium ionophore, A23187 (0.2 microM) stimulates mu-calpain activity and subsequently cleaves Na(+)/Ca(2+) exchanger (NCX). Pretreatment of the A23187 treated mitochondria with the calpain inhibitors, calpeptin or MDL28170 or with Ca(2+) chelator, EGTA does not cleave NCX. Treatment of the mitochondria with A23187 increases Ca(2+) level in the mitochondria, which subsequently dissociates mu-calpain-calpastatin association leading to the activation of mu-calpain. Immunoblot study of the A23187 treated mitochondria with the NCX polyclonal antibody indicates the degradation of mitochondrial inner membrane NCX (110kDa) resulting in the doublet of approximately 54-56kDa NCX fragments. Moreover, in vitro cleavage of mitochondrial purified NCX by mitochondrial purified mu-calpain supports our conclusion. This cleavage of NCX may be interpreted as the main cause of Ca(2+) overload and could lay a key role in the activation of apoptotic process in pulmonary smooth muscle.

    Topics: Animals; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cattle; Dipeptides; Intracellular Membranes; Mitochondria, Muscle; Mitochondrial Membranes; Muscle, Smooth, Vascular; Phosphatidylcholines; Phospholipid Ethers; Pulmonary Artery; Sodium-Calcium Exchanger

2009
Low activity by the calpain system in primate lenses causes resistance to calcium-induced proteolysis.
    Experimental eye research, 2006, Volume: 83, Issue:3

    The human genome contains 14 genes for 80 kDa catalytic subunit of the calcium-activated protease calpain (EC 34.22.17), yet no calpain-like cleavage sites have been detected on human lens crystallins in vivo. The purpose of the present study was to provide a comprehensive study of calpain activation in human and macaque lenses developing experimental cataract due to lens culture in ionophore A23187. Zymography was used to measure calpain activity; SDS-PAGE and immunoblotting were used to detect hydrolysis of potential lens protein substrates. Quantitative PCR was used to measure transcripts for calpains and the endogenous inhibitor calpastatin. We found that the lack of appreciable calpain-induced proteolysis in primate lenses is most likely due to relatively low levels of endogenous calpain activity compared to the high levels of endogenous calpain inhibitor, calpastatin.

    Topics: Aged; Aged, 80 and over; Animals; Apoptosis; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Caseins; Cataract; Eye Proteins; Humans; Immunoblotting; In Situ Nick-End Labeling; Ionophores; Lens, Crystalline; Macaca mulatta; Models, Animal; Reverse Transcriptase Polymerase Chain Reaction; Staining and Labeling

2006
Calpain system regulates the differentiation of adult primitive mesenchymal ST-13 adipocytes.
    Endocrinology, 2006, Volume: 147, Issue:10

    The activity of calpain, a calcium-activated protease, is required during the mitotic clonal expansion phase of 3T3-L1 embryonic preadipocyte differentiation. Here we examined the role of calpain in the adipogenesis of ST-13 preadipocytes established from adult primitive mesenchymal cells, which do not require mitotic clonal expansion. After exposure to the calpain inhibitor, N-benzyloxycarbonyl-L-leucyl-L-leucinal or overexpression of calpastatin, a specific endogenous inhibitor of calpain, ST-13 preadipocytes acquired the adipocyte phenotype. Overexpression of calpastatin in ST-13 adipocytes stimulated the expression of adipocyte-specific CCAAT/enhancer-binding protein-alpha (C/EBPalpha), peroxisome proliferator-activated receptor (PPAR)-gamma, sterol regulatory element-binding protein 1, and the insulin signaling molecules, insulin receptor alpha, insulin-receptor substrates, and GLUT4. However, insulin-stimulated glucose uptake was reduced by approximately 52%. The addition of calpain to the nuclear fraction of ST-13 adipocytes resulted in the Ca(2+)-dependent degradation of PPARgamma and C/EBPalpha but not sterol regulatory element-binding protein 1. Exposing ST-13 adipocytes to A23187 also led to losses of endogenous PPARgamma and C/EBPalpha. Under both conditions, calpain inhibitors almost completely prevented C/EBPalpha cleavage but partially blocked the decrease of PPARgamma. Two ubiquitous forms of calpain, mu- and m-calpain, localized to the cytosol and the nucleus, whereas the activated form of mu- but not m-calpain was found in the nucleus. Finally, stable dominant-negative mu-calpain transfectants showed accelerated adipogenesis and increase in the levels of PPARgamma and C/EBPalpha during adipocyte program. These results support evidence that the calpain system is involved in regulating the differentiation of adult primitive mesenchymal ST-13 preadipocytes.

    Topics: Adipocytes; Adipose Tissue; Antimetabolites; Blotting, Northern; Calcimycin; Calcium-Binding Proteins; Calpain; CCAAT-Enhancer-Binding Protein-alpha; Cell Differentiation; Deoxyglucose; Dipeptides; DNA, Complementary; Humans; Stem Cells; Sterol Regulatory Element Binding Protein 1; Subcellular Fractions

2006
Calpain is involved in the HIV replication from the latently infected OM10.1 cells.
    Biochemical and biophysical research communications, 2003, Apr-11, Volume: 303, Issue:3

    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

2003
Participation of the conventional calpains in apoptosis.
    Biochimica et biophysica acta, 2002, Jun-12, Volume: 1590, Issue:1-3

    The conventional calpains, m- and micro-calpain, are suggested to be involved in apoptosis triggered by many different mechanisms. However, it has not been possible to definitively associate calpain function with apoptosis, largely because of the incomplete selectivity of the cell permeable calpain inhibitors used in previous studies. In the present study, Chinese hamster ovary (CHO) cell lines overexpressing micro-calpain or the highly specific calpain inhibitor protein, calpastatin, have been utilized to explore apoptosis signals that are influenced by calpain content. This approach allows unambiguous alteration of calpain activity in cells. Serum depletion, treatment with the endoplasmic reticulum (ER) calcium ATPase inhibitor thapsigargin, and treatment with calcium ionophore A23187 produced apoptosis in CHO cells, which was increased in calpain overexpressing cells and decreased by induced expression of calpastatin. Inhibition of calpain activity protected beta-spectrin, but not alpha-spectrin, from proteolysis. The calpains seemed not to be involved in apoptosis triggered by a number of other treatments. Calpain protected against TNF-alpha induced apoptosis. In contrast to previous studies, we found no evidence that calpains proteolyze I kappa B-alpha in TNF-alpha-stimulated cells. These studies indicate that the conventional calpains participate in some, but not all, apoptotic signaling mechanisms. In most cases, they contributed to apoptosis, but in at least one case, they were protective.

    Topics: Animals; Apoptosis; Calcimycin; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Cells, Cultured; CHO Cells; Cricetinae; Enzyme Inhibitors; Gene Expression; Humans; Ionophores; Mice; Rats; Recombinant Proteins; Signal Transduction; Thapsigargin; Tumor Necrosis Factor-alpha

2002
Heterogeneous increases of cytoplasmic calcium: distinct effects on down-regulation of cell surface sodium channels and sodium channel subunit mRNA levels.
    British journal of pharmacology, 2001, Volume: 132, Issue:7

    1. Long-term (> or = 12 h) treatment of cultured bovine adrenal chromaffin cells with A23187 (a Ca(2+) ionophore) or thapsigargin (TG) [an inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)] caused a time- and concentration-dependent reduction of cell surface [(3)H]-saxitoxin (STX) binding capacity, but did not change the K:(D:) value. In A23187- or TG-treated cells, veratridine-induced (22)Na(+) influx was reduced (with no change in veratridine EC(50) value) while it was enhanced by alpha-scorpion venom, beta-scorpion venom, or Ptychodiscus brevis toxin-3, like in nontreated cells. 2. The A23187- or TG-induced decrease of [(3)H]-STX binding was diminished by BAPTA-AM. EGTA also inhibited the decreasing effect of A23187. A23187 caused a rapid, monophasic and persistent increase in intracellular concentration of Ca(2+) ([Ca(2+)](i)) to a greater extent than that observed with TG. 2,5-Di-(t-butyl)-1,4-benzohydroquinone (DBHQ) (an inhibitor of SERCA) produced only a rapid monophasic increase in [Ca(2+)](i), without any effect on [(3)H]-STX binding. 3. Reduction in [(3)H]-STX binding capacity induced by A23187 or TG was attenuated by Gö6976 (an inhibitor of conventional protein kinase C) or calpastatin peptide (an inhibitor of calpain). When the internalization rate of cell surface Na(+) channels was measured in the presence of brefeldin A (an inhibitor of vesicular exit from the trans-Golgi network), A23187 or TG accelerated the reduction of [(3)H]-STX binding capacity. 4. Six hours treatment with A23187 lowered Na(+) channel alpha- and beta(1)-subunit mRNA levels, whereas TG had no effect. 5. These results suggest that elevation of [Ca(2+)](i) caused by A23187, TG or DBHQ exerted differential effects on down-regulation of cell surface functional Na(+) channels and Na(+) channel subunit mRNA levels.

    Topics: Animals; Binding, Competitive; Brefeldin A; Calcimycin; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Carbazoles; Cattle; Cells, Cultured; Chromaffin Cells; Dose-Response Relationship, Drug; Down-Regulation; Egtazic Acid; Enzyme Inhibitors; Hydroquinones; Indoles; Ionophores; Marine Toxins; Oxocins; Protein Subunits; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Saxitoxin; Scorpion Venoms; Sodium; Sodium Channels; Thapsigargin; Time Factors; Tritium; Veratridine

2001
Strategies for regulating calpain activities in living cells.
    Methods in molecular biology (Clifton, N.J.), 2000, Volume: 144

    Topics: Animals; Base Sequence; Blotting, Western; Calcimycin; Calcium-Binding Proteins; Calpain; Carrier Proteins; Cell Line; Cysteine Proteinase Inhibitors; DNA Primers; DNA, Complementary; Gene Expression; Ionophores; Microfilament Proteins; Molecular Sequence Data; Muscle Proteins; Muscle, Skeletal; Mutation; Protein Structure, Tertiary; Rats; Reverse Transcriptase Polymerase Chain Reaction; Transfection

2000
Possible role of calpain in normal processing of beta-amyloid precursor protein in human platelets.
    Biochemical and biophysical research communications, 2000, Jun-24, Volume: 273, Issue:1

    Abnormal proteolytic processing of beta-amyloid precursor protein (APP) underlies the formation of amyloid plaques in aging and Alzheimer's disease. The proteases involved in the process have not been identified. Here we found that spontaneous proteolysis of intact APP in detergent-lysed human platelets generated a N-terminal fragment that was immunologically indistinguishable from secreted APP, reminiscent of the action of a putative alpha-secretase. This proteolysis of APP was inhibited by EDTA, suggesting that a metal-dependent protease was involved. Among the several metals tested, calcium was the only one that enhanced APP proteolysis and the reaction was blocked by EGTA as well as by several calpain inhibitors. The APP fragments generated by spontaneous proteolysis in platelet lysates were identical to those produced by exposure of partially purified APP to exogenous calpain. Finally, the secretion of APP from intact platelets was inhibited by cell-permeable calpain inhibitors. Taken together, these results suggest that normal processing of APP in human platelets is mediated by a calcium-dependent protease that exhibits calpain-like properties.

    Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Blood Platelets; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cations, Divalent; Cell Extracts; Edetic Acid; Egtazic Acid; Endopeptidase K; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Molecular Weight; Peptide Fragments; Protease Inhibitors; Protein Processing, Post-Translational; Trypsin

2000
Changes in intracellular localization of calpastatin during calpain activation.
    The Biochemical journal, 1999, Oct-15, Volume: 343 Pt 2

    Localization of the two main components of the Ca(2+)-dependent proteolytic system has been investigated in human neuroblastoma LAN-5 cells. Using a monoclonal antibody which recognizes the N-terminal calpastatin domain, it has been shown that this inhibitory protein is almost completely confined in two granule-like structures not surrounded by membranes. Similar calpastatin distribution has been found in other human and in murine cell types, indicating that aggregation of calpastatin is a general property and not an exclusive characteristic of neuronal-like cells. The existence of such calpastatin aggregates is confirmed by the kinetics of calpastatin-activity release during rat liver homogenization, which does not correspond to the rate of appearance of cytosolic proteins or to the disruption of membrane-surrounded organelles. Calpastatin distribution is affected by the intracellular increase in free Ca(2+), which results in calpastatin progressively becoming a soluble protein. However, calpain is distributed in the soluble cell fraction and, in activating conditions, partially accumulates on the plasma membrane. Similar behaviour has been observed in calpastatin localization in LAN-5 cells induced with retinoic acid, suggesting that the proteolytic system is activated during the differentiation process of these cells. The involvement of calpastatin in controlling calpain activity, rather than its activation process, and the utilization of changes in calpastatin localization as a marker of activation of the system is discussed.

    Topics: Animals; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Membrane; Cell Membrane Permeability; Cytosol; Diffusion; Endopeptidases; Enzyme Activation; Humans; Liver; Mice; Neurons; Rats; Solubility; Tretinoin; Tumor Cells, Cultured

1999
Proteolysis by calpains: a possible contribution to degradation of p53.
    Molecular and cellular biology, 1997, Volume: 17, Issue:5

    p53 is a short-lived transcription factor that is frequently mutated in tumor cells. Work by several laboratories has already shown that the ubiquitin-proteasome pathway can largely account for p53 destruction, at least under specific experimental conditions. We report here that, in vitro, wild-type p53 is a sensitive substrate for milli- and microcalpain, which are abundant and ubiquitous cytoplasmic proteases. Degradation was dependent on p53 protein conformation. Mutants of p53 with altered tertiary structure displayed a wide range of susceptibility to calpains, some of them being largely resistant to degradation and others being more sensitive. This result suggests that the different mutants tested here adopt slightly different conformations to which calpains are sensitive but that cannot be discriminated by using monoclonal antibodies such as PAb1620 and PAb240. Inhibition of calpains by using the physiological inhibitor calpastatin leads to an elevation of p53 steady-state levels in cells expressing wild-type p53. Conversely, activation of calpains by calcium ionophore led to a reduction of p53 in mammalian cells, and the effect was blocked by cell-permeant calpain inhibitors. Cotransfection of p53-null cell lines with p53 and calpastatin expression vectors resulted in an increase in p53-dependent transcription activity. Taken together, these data support the idea that calpains may also contribute to the regulation of wild-type p53 protein levels in vivo.

    Topics: Animals; Calcimycin; Calcium-Binding Proteins; Calpain; Cattle; Cricetinae; Cysteine Endopeptidases; Humans; Jurkat Cells; Mice; Multienzyme Complexes; Mutagenesis; Proteasome Endopeptidase Complex; Protein Conformation; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53; Ubiquitins

1997
Specific increase in amyloid beta-protein 42 secretion ratio by calpain inhibition.
    Biochemistry, 1997, Jul-08, Volume: 36, Issue:27

    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

1997
Identification of mu-, m-calpains and calpastatin and capture of mu-calpain activation in endothelial cells.
    Journal of cellular biochemistry, 1997, Aug-01, Volume: 66, Issue:2

    The presence of the calpain-calpastatin system in human umbilical vein endothelial cells (HUVEC) was investigated by means of ion exchange chromatography, Western blot analysis, and Northern blot analysis. On DEAE anion exchange chromatography, calpain and calpastatin activities were eluted at approximately 0.30 M and 0.15-0.25 M NaCl, respectively. For half-maximal activity, the protease required 800 microM Ca2+, comparable to the Ca2+ requirement of m-calpain. By Western blot analysis, the large subunit of mu-calpain (80 kDa) was found to be eluted with calpastatin (110 kDa). Both the large subunit of m-calpain (80 kDa) and calpastatin were detected in the respective active fractions. By Northern blot analysis, mRNAs for large subunits of mu- and m-calpains were detected in single bands, each corresponding to approximately 3.5 Kb. Calpastatin mRNA was observed in two bands corresponding to approximately 3.8 and 2.6 Kb. Furthermore, the activation of mu-calpain in HUVEC by a calcium ionophore was examined, using an antibody specifically recognizing an autolytic intermediate form of mu-calpain large subunit (78 kDa). Both talin and filamin of HUVEC were proteolyzed in a calcium-dependent manner, and the reactions were inhibited by calpeptin, a cell-permeable calpain specific inhibitor. Proteolysis of the cytoskeleton was preceded by the appearance of the autolytic intermediate form of mu-calpain, while the fully autolyzed postautolysis form of mu-calpain (76 kDa) remained below detectable levels at all time points examined. These results indicate that the calpain-calpastatin system is present in human endothelial cells and that mu-calpain may be involved in endothelial cell function mediated by Ca2+ via the limited proteolysis of various proteins.

    Topics: Antibody Specificity; Blotting, Western; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Cells, Cultured; Chromatography, DEAE-Cellulose; Cytoskeletal Proteins; Dipeptides; Endothelium, Vascular; Enzyme Activation; Enzyme Precursors; Extracellular Space; Humans; Hydrolysis; RNA, Messenger; Umbilical Veins

1997
Calpain secreted by activated human lymphoid cells degrades myelin.
    Journal of neuroscience research, 1995, Oct-01, Volume: 42, Issue:2

    Calpain secreted by lymphoid (MOLT-3, M.R.) or monocytic (U-937, THP-1) cell lines activated with PMA and A23187 degraded myelin antigens. The degradative effect of enzymes released in the extracellular medium was tested on purified myelin basic protein and rat central nervous system myelin in vitro. The extent of protein degradation was determined by SDS-PAGE and densitometric analysis. Various proteinase inhibitors were used to determine to what extent protein degradation was mediated by calpain and/or other enzymes. Lysosomal and serine proteinase inhibitors inhibited 20-40% of the myelin-degradative activity found in the incubation media of cell lines, whereas the calcium chelator (EGTA), the calpain-specific inhibitor (calpastatin), and a monoclonal antibody to m calpain blocked myelin degradation by 60-80%. Since breakdown products of MBP generated by calpain may include fragments with antigenic epitopes, this enzyme may play an important role in the initiation of immune-mediated demyelination.

    Topics: Animals; Antibodies, Monoclonal; Calcimycin; Calcium; Calcium-Binding Proteins; Calpain; Chelating Agents; Culture Media, Conditioned; Demyelinating Diseases; Egtazic Acid; Humans; Leukemia-Lymphoma, Adult T-Cell; Lymphoma, Large B-Cell, Diffuse; Monocytes; Myelin Basic Protein; Myelin Sheath; Neoplasm Proteins; Protease Inhibitors; Rabbits; Rats; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

1995