Technical note: a rapid method for quantification of calpain and calpastatin activities in muscle.
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Stepwise and continuous gradient ion-exchange chromatography were compared for yield of calpains and calpastatin from ovine muscle in a study designed to quantify their activities for comparative purposes. In Exp. 1, a continuous (25 to 400 mM NaCl) gradient and a two-step gradient method (200 mM NaCl to coelute mu-calpain and calpastatin together and then 400 mM NaCl to elute mu-calpain) were compared. For the two-step method, mu-calpain activities were determined by subtracting calpastatin activities before and after heat inactivation of mu-calpain. Both the two-step and the continuous gradient method yielded similar results over a broad range of activities. The stepwise gradient method does not require the use of fraction collectors and pumps, and it can be completed in a fraction of the time required for the continuous gradient method. In Exp. 2, the two-step method was compared with a three-step method (100 mM NaCl to elute calpastatin, then 200 mM NaCl to elute mu-calpain, and then 400 mM NaCl to elute m-calpain). Unlike the continuous gradient method, calpastatin and mu-calpain could not be completely separated using the three-step chromatography method. Thus, the three-step gradient method should not be used to quantify the components of the calpain proteolytic system. The present results indicate that the two-step gradient method is a fast and inexpensive method to determine calpain and calpastatin activities in studies designed to quantify the components of the calpain proteolytic system in skeletal muscle. (+info)
Epidermal growth factor receptor activation of calpain is required for fibroblast motility and occurs via an ERK/MAP kinase signaling pathway.
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To become migratory, cells must reorganize their connections to the substratum, and during locomotion they must break rear attachments. The molecular and biochemical mechanisms underlying these biophysical processes are unknown. Recent studies have implicated both extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase and calpain (EC 3.4.22.17) in these processes, but it is uncertain whether these are two distinct pathways acting on different modes of motility. We report that cell deadhesion involved in epidermal growth factor (EGF) receptor-mediated fibroblast motility requires activation of M-calpain downstream of ERK/MAP kinase signaling. NR6 fibroblasts expressing full-length wild type epidermal growth factor receptor required both calpain and ERK activation, as demonstrated by pharmacological inhibitors (calpeptin and calpain inhibitor I and PD98059, respectively) for EGF-induced deadhesion and motility. EGF induced rapid activation of calpain that was preventable by molecular inhibition of the Ras-Raf-MEK but not phospholipase Cgamma signaling pathway, and calpain was stimulated by transfection of constitutively active MEK. Enhanced calpain activity was not mirrored by increased calpain protein levels or decreased levels of its endogenous inhibitor calpastatin. The link between ERK/MAP kinase signaling and cell motility required the M-isoform of calpain (calpain II), as determined by specific antisense-mediated down-regulation. These data promote a previously undescribed signaling pathway of ERK/MAP kinases activating calpain to destabilize cell-substratum adhesions in response to EGF stimulation. (+info)
Association of L-arginine transporters with fodrin: implications for hypoxic inhibition of arginine uptake.
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In this study, we investigated the possible interaction between the cationic amino acid transporter (CAT)-1 arginine transporter and ankyrin or fodrin. Because ankyrin and fodrin are substrates for calpain and because hypoxia increases calpain expression and activity in pulmonary artery endothelial cells (PAEC), we also studied the effect of hypoxia on ankyrin, fodrin, and CAT-1 contents in PAEC. Exposure to long-term hypoxia (24 h) inhibited L-arginine uptake by PAEC, and this inhibition was prevented by calpain inhibitor 1. The effects of hypoxia and calpain inhibitor 1 were not associated with changes in CAT-1 transporter content in PAEC plasma membranes. However, hypoxia stimulated the hydrolysis of ankyrin and fodrin in PAEC, and this could be prevented by calpain inhibitor 1. Incubation of solubilized plasma membrane proteins with anti-fodrin antibodies resulted in a 70% depletion of CAT-1 immunoreactivity and in a 60% decrease in L-arginine transport activity in reconstituted proteoliposomes (3,291 +/- 117 vs. 8,101 +/- 481 pmol. mg protein(-1). 3 min(-1) in control). Incubation with anti-ankyrin antibodies had no effect on CAT-1 content or L-arginine transport in reconstituted proteoliposomes. These results demonstrate that CAT-1 arginine transporters in PAEC are associated with fodrin, but not with ankyrin, and that long-term hypoxia decreases L-arginine transport by a calpain-mediated mechanism that may involve fodrin proteolysis. (+info)
Direct cleavage by the calcium-activated protease calpain can lead to inactivation of caspases.
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Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases. (+info)
Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer.
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Excitatory synaptic activity can evoke transient and substantial elevations of postsynaptic calcium. Downstream effects of elevated calcium include the activation of the calcium-dependent protease calpain. We have developed a reagent that identifies dendritic spines in which calpain has been activated. A fusion protein was expressed that contained enhanced yellow and enhanced cyan fluorescent protein (EYFP and ECFP, respectively) linked by a peptide that included the micro-calpain cleavage site from alpha-spectrin. A PDZ-binding site fused to ECFP anchored this protein to postsynaptic densities. The fusion protein exhibited fluorescence resonance energy transfer (FRET), and diminution of FRET by proteolysis was used to localize calpain activity in situ by fluorescence microscopy. Incubation of the fusion protein with calpain in the presence of calcium resulted in the separation of EYFP and ECFP into monomeric fluorophores. In transiently transfected cell lines and dissociated hippocampal neurons, FRET was diminished by raising intracellular calcium levels with an ionophore or with glutamatergic agonists. Calpain inhibitors blocked these changes. Under control conditions, FRET levels in different dendritic spines of cultured neurons and in hippocampal slices were heterogeneous but showed robust decreases upon treatment with glutamatergic agonists. Immunostaining of cultured neurons with antibodies to a spectrin epitope produced by calpain-mediated digestion revealed an inverse correlation between the amount of FRET present at postsynaptic elements and the concentration of spectrin breakdown products. These results suggest that the FRET methodology identifies sites of synaptically induced calpain activity and that it may be useful in analyzing synapses undergoing changes in efficacy. (+info)
Novel characteristics of glutamate-induced cell death in primary septohippocampal cultures: relationship to calpain and caspase-3 protease activation.
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Studies examined the phenotypic characteristics of glutamate-induced cell death and their relationship to calpain and caspase-3 activation. Cell viability was assessed by fluorescein diacetate and propidium iodide staining and lactate dehydrogenase release. Calpain and caspase-3 activity was inferred from signature proteolytic fragmentation of alpha-spectrin. Characterization of cell death phenotypes was assessed by Hoechst 33258 and DNA fragmentation assays. Exposure of septohippocampal cultures to 1.0, 2.0, and 4.0 mmol/L glutamate induced a dose-dependent cell death with an LD50 of 2.0 mmol/L glutamate after 24 hours of incubation. Glutamate treatment induced cell death in neurons and astroglia and produced morphological alterations that differed from necrotic or apoptotic changes observed after maitotoxin or staurosporine exposure, respectively. After glutamate treatment, cell nuclei were enlarged and eccentrically shaped, and aggregated chromatin appeared in a diffusely speckled pattern. Furthermore, no dose of glutamate produced evidence of internucleosomal DNA fragmentation. Incubation with varying doses of glutamate produced calpain and caspase-3 activation. Calpain inhibitor II (N-acetyl-Leu-Leu-methionyl) provided protection only with a narrow dose range, whereas carbobenzoxy-Asp-CH2-OC(O)-2,6-dichlorobenzene (Z-D-DCB; pan-caspase inhibitor) and MK-801 (N-methyl-D-aspartate receptor antagonist) were potently effective across a wider dose range. Cycloheximide did not reduce cell death or protease activation. (+info)
Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart.
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OBJECTIVE: The involvement of Calpain-I mediated proteolysis has been implicated in myofibrillar dysfunction of reperfused myocardium following ischemia (stunning). This study addresses the question whether ultrastructural alterations might be responsible for the depressed contractility. METHODS: Mechanical properties and protein composition of isolated myocytes after Calpain-I exposure (1.25 U/ml; 10 min; 15 degrees C; pCa 5.0) and of ischemic rat hearts following reperfusion were characterized. RESULTS: Maximal isometric force (44 +/- 5 kN/m2) at pCa 4.5 (pCa = -log[Ca2+]) decreased by 42.5% in Triton permeabilized myocytes (n = 11) after Calpain-I treatment. Force (and consequent myofilament disarrangement) during Calpain-I treatment was prevented by 40 mM BDM. The contractile force of Calpain-I exposed myocytes was significantly higher at submaximal levels of activation (pCa 5.5, 5.4 and 5.3) before maximal force development (pCa 4.5) than after maximal force development. The pCa50 value (5.40 +/- 0.02) determined from these initial test contractures did not differ significantly from that of untreated controls (5.44 +/- 0.03). However, after full activation Ca(2+)-sensitivity of force production in Calpain-I treated myocytes was significantly reduced (pCa50 5.34 +/- 0.02). This change in pCa50 was positively correlated with the reduction in maximal isometric force and was accompanied by sarcomere disorder. These findings imply that at least part of the Calpain-I induced mechanical alterations are dependent on force history. Measurements of the rate of force redevelopment after unloaded shortening suggested that Calpain-I did not affect cross-bridge kinetics. SDS gel electrophoresis and Western immunoblotting of Calpain-I treated myocytes revealed desmin degradation. The desmin content of postischemic myocardium was also reduced. CONCLUSION: Our results indicate that ultrastructural alterations may play an important role in the Calpain-I mediated cardiac dysfunction. (+info)
Caspases cleave the amino-terminal calpain inhibitory unit of calpastatin during apoptosis in human Jurkat T cells.
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We have previously reported the activation of procalpain mu (precursor for low-calcium-requiring calpain) in apoptotic cells using a cleavage-site-directed antibody specific to active calpain [Kikuchi, H. and Imajoh-Ohmi, S. (1995) Cell Death Differ. 2, 195-199]. In this study, calpastatin, the endogenous inhibitor protein for calpain, was cleaved to a 90-kDa polypeptide during apoptosis in human Jurkat T cells. The limited proteolysis of calpastatin preceded the autolytic activation of procalpain. Inhibitors for caspases rescued the cells from apoptosis and simultaneously inhibited the cleavage of calpastatin. The full-length recombinant calpastatin was also cleaved by caspase-3 or caspase-7 at Asp-233 into the same size fragment. Cys-241 was also targeted by these caspases in vitro but not in apoptotic cells. Caspase-digested calpastatin lost its amino-terminal inhibitory unit, and inhibited three moles of calpain per mole. Our findings suggest that caspases trigger the decontrol of calpain activity suppression by degrading calpastatin. (+info)