The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform
E Melloni - One of the best experts on this subject based on the ideXlab platform.
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Association of Calpastatin with Inactive Calpain
2020Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S PontremoliAbstract:ing the physiological conditions under which calpain retains its inactive conformational state. The Calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4 –7. This Calpastatin region recognizes a calpain sequence located near the end of the DIIdomain. Interaction of calpain with Calpastatins lacking these sequences becomes strictly Ca 2 -dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca 2 -free calpain and various recombinant Calpastatin forms has been demonstrated by the following experimental results. Addition of Calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when Calpastatin was immunoprecipitated. The Calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-Calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-Calpastatin interaction at a physiological concentration of Ca 2 represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca 2 influx.
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Differential regulation of the calpain-Calpastatin complex by the L-domain of Calpastatin.
Biochimica et Biophysica Acta, 2014Co-Authors: R De Tullio, M Averna, S Pontremoli, Marco Pedrazzi, Bianca Sparatore, Franca Salamino, E MelloniAbstract:Abstract Here we demonstrate that the presence of the L-domain in Calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of Calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind Calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain–Calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional Calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of Calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca2 +] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which Calpastatin levels are increased. Our findings show that the L-domain of Calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length Calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.
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adaptive modifications in the calpain Calpastatin system in brain cells after persistent alteration in ca2 homeostasis
Journal of Biological Chemistry, 2010Co-Authors: Roberto Stifanese, M Averna, R De Tullio, F Salamino, S Pontremoli, Marco Pedrazzi, Francesco Beccaria, Marco Milanese, Giambattista Bonanno, E MelloniAbstract:Abstract Persistent dysregulation in Ca2+ homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-Calpastatin system in −G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca2+]i elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca2+ levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca2+]i, 3) intracellular activation of calpain is always associated with diffusion of Calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-Calpastatin complex, and 4) a conservative fragmentation of Calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca2+]i. Thus, Calpastatin diffusion and formation of the calpain-Calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca2+ homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.
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Role of the calpain-Calpastatin system in the density-dependent growth arrest.
Archives of Biochemistry and Biophysics, 2008Co-Authors: Roberto Stifanese, M Averna, R De Tullio, S Pontremoli, Franca Salamino, Claudia Cantoni, Maria Cristina Mingari, Carola Prato, E MelloniAbstract:Abstract In dividing cells Calpastatin diffuses from aggregates into cytosol, indicating the requirement for a tight regulation of calpain. Accordingly, the involvement of the calpain–Calpastatin system in cell proliferation and in the density-dependent growth arrest was studied in JA3 cells stably transfected with a Calpastatin form permanently localized in cytosol. In Calpastatin overexpressing cells, cell cycle rate is 50% reduced, and cells enter the ungrowing, still fully reversible, stage at a 3-fold higher cell density. Furthermore, in cell density growth arrest phase, down regulation of α- and θ-PKC isoforms, as well as FAK and talin occurs. In Calpastatin overexpressing cells, degradation of these calpain substrate proteins is prevented and delayed. Thus, calpain activity plays a crucial role in inducing the cell entry into a functional quiescent phase.
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association of Calpastatin with inactive calpain a novel mechanism to control the activation of the protease
Journal of Biological Chemistry, 2006Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S PontremoliAbstract:Abstract It is generally accepted that the Ca2+-dependent interaction of calpain with Calpastatin is the most relevant mechanism involved in the regulation of Ca2+-induced proteolysis. We now report that a calpain-Calpastatin association can occur also in the absence of Ca2+ or at very low Ca2+ concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The Calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4-7. This Calpastatin region recognizes a calpain sequence located near the end of the DII-domain. Interaction of calpain with Calpastatins lacking these sequences becomes strictly Ca2+-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca2+-free calpain and various recombinant Calpastatin forms has been demonstrated by the following experimental results. Addition of Calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when Calpastatin was immunoprecipitated. The Calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-Calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-Calpastatin interaction at a physiological concentration of Ca2+ represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca2+ influx.
M Averna - One of the best experts on this subject based on the ideXlab platform.
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Association of Calpastatin with Inactive Calpain
2020Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S PontremoliAbstract:ing the physiological conditions under which calpain retains its inactive conformational state. The Calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4 –7. This Calpastatin region recognizes a calpain sequence located near the end of the DIIdomain. Interaction of calpain with Calpastatins lacking these sequences becomes strictly Ca 2 -dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca 2 -free calpain and various recombinant Calpastatin forms has been demonstrated by the following experimental results. Addition of Calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when Calpastatin was immunoprecipitated. The Calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-Calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-Calpastatin interaction at a physiological concentration of Ca 2 represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca 2 influx.
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Production and Purification of Recombinant Calpastatin.
Methods of Molecular Biology, 2019Co-Authors: R De Tullio, M AvernaAbstract:The production of recombinant Calpastatin in E. coli has become an efficient tool to obtain discrete amounts of a specific Calpastatin species that can be present concomitantly with other Calpastatin fragments/forms in the same tissue or cell type in a given condition. Indeed, at present, it is still difficult to distinguish the various Calpastatin species for several reasons among which: Calpastatins differ only at the N-terminus, can undergo calpain-dependent cleavage generating discrete fragments, and show anomalous electrophoretic mobility. Another benefit of using recombinant Calpastatin is that, as the wild-type forms, it is heat resistant and thus can be efficiently isolated taking advantage of a simple quick purification step. Finally, the lack of posttranslational modifications makes recombinant Calpastatin species particularly suitable for studying in vitro the biochemical features of specific parts of the inhibitor that following controlled posttranslational modifications change their functional interaction with calpain. In this chapter, we describe, starting from the mRNA sequence, how to produce rat Calpastatin Type I in E. coli. We use routinely the same method, with minor modifications, for the production of other Calpastatin species deriving from different tissues or organisms and Calpastatin constructs having only specific domains. The possibility to obtain large amounts of a single calpain inhibitor form is a great advantage for studying the calpain/Calpastatin system in vitro.
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Immunoblotting for Calpastatin Expression.
Methods of Molecular Biology, 2019Co-Authors: M Averna, R De TullioAbstract:Immunoblotting is a procedure routinely used to analyze Calpastatin expression. However, immunoblotting alone may not be adequate for this task, since Calpastatin isoforms can vary by tissue, can be modified by partial digestion, and can undergo posttranslational modifications. Here we describe a method for more precise evaluation of Calpastatin expression by combining immunoblot analysis with an assay for the inhibitory activity of a single Calpastatin species isolated by SDS-PAGE and protein elution from the gel.
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Isolation of Endogenous Calpastatin.
Methods of Molecular Biology, 2019Co-Authors: R De Tullio, M AvernaAbstract:We here describe the purification of Calpastatin from human erythrocytes. When Calpastatin is purified from tissues, it is necessary to measure its inhibitory activity against calpain in the presence of Ca2+ to specifically identify the protein. Thus, the purification steps necessary to obtain the inhibitor protein were originally designed to obtain calpain from the same tissue. For this reason, in addition to Calpastatin purification, we also include a method for purifying human erythrocyte calpain and globin. We routinely use these two components for assaying Calpastatin inhibition.
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Differential regulation of the calpain-Calpastatin complex by the L-domain of Calpastatin.
Biochimica et Biophysica Acta, 2014Co-Authors: R De Tullio, M Averna, S Pontremoli, Marco Pedrazzi, Bianca Sparatore, Franca Salamino, E MelloniAbstract:Abstract Here we demonstrate that the presence of the L-domain in Calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of Calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind Calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain–Calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional Calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of Calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca2 +] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which Calpastatin levels are increased. Our findings show that the L-domain of Calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length Calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.
R De Tullio - One of the best experts on this subject based on the ideXlab platform.
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Association of Calpastatin with Inactive Calpain
2020Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S PontremoliAbstract:ing the physiological conditions under which calpain retains its inactive conformational state. The Calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4 –7. This Calpastatin region recognizes a calpain sequence located near the end of the DIIdomain. Interaction of calpain with Calpastatins lacking these sequences becomes strictly Ca 2 -dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca 2 -free calpain and various recombinant Calpastatin forms has been demonstrated by the following experimental results. Addition of Calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when Calpastatin was immunoprecipitated. The Calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-Calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-Calpastatin interaction at a physiological concentration of Ca 2 represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca 2 influx.
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Production and Purification of Recombinant Calpastatin.
Methods of Molecular Biology, 2019Co-Authors: R De Tullio, M AvernaAbstract:The production of recombinant Calpastatin in E. coli has become an efficient tool to obtain discrete amounts of a specific Calpastatin species that can be present concomitantly with other Calpastatin fragments/forms in the same tissue or cell type in a given condition. Indeed, at present, it is still difficult to distinguish the various Calpastatin species for several reasons among which: Calpastatins differ only at the N-terminus, can undergo calpain-dependent cleavage generating discrete fragments, and show anomalous electrophoretic mobility. Another benefit of using recombinant Calpastatin is that, as the wild-type forms, it is heat resistant and thus can be efficiently isolated taking advantage of a simple quick purification step. Finally, the lack of posttranslational modifications makes recombinant Calpastatin species particularly suitable for studying in vitro the biochemical features of specific parts of the inhibitor that following controlled posttranslational modifications change their functional interaction with calpain. In this chapter, we describe, starting from the mRNA sequence, how to produce rat Calpastatin Type I in E. coli. We use routinely the same method, with minor modifications, for the production of other Calpastatin species deriving from different tissues or organisms and Calpastatin constructs having only specific domains. The possibility to obtain large amounts of a single calpain inhibitor form is a great advantage for studying the calpain/Calpastatin system in vitro.
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Immunoblotting for Calpastatin Expression.
Methods of Molecular Biology, 2019Co-Authors: M Averna, R De TullioAbstract:Immunoblotting is a procedure routinely used to analyze Calpastatin expression. However, immunoblotting alone may not be adequate for this task, since Calpastatin isoforms can vary by tissue, can be modified by partial digestion, and can undergo posttranslational modifications. Here we describe a method for more precise evaluation of Calpastatin expression by combining immunoblot analysis with an assay for the inhibitory activity of a single Calpastatin species isolated by SDS-PAGE and protein elution from the gel.
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Isolation of Endogenous Calpastatin.
Methods of Molecular Biology, 2019Co-Authors: R De Tullio, M AvernaAbstract:We here describe the purification of Calpastatin from human erythrocytes. When Calpastatin is purified from tissues, it is necessary to measure its inhibitory activity against calpain in the presence of Ca2+ to specifically identify the protein. Thus, the purification steps necessary to obtain the inhibitor protein were originally designed to obtain calpain from the same tissue. For this reason, in addition to Calpastatin purification, we also include a method for purifying human erythrocyte calpain and globin. We routinely use these two components for assaying Calpastatin inhibition.
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Differential regulation of the calpain-Calpastatin complex by the L-domain of Calpastatin.
Biochimica et Biophysica Acta, 2014Co-Authors: R De Tullio, M Averna, S Pontremoli, Marco Pedrazzi, Bianca Sparatore, Franca Salamino, E MelloniAbstract:Abstract Here we demonstrate that the presence of the L-domain in Calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of Calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind Calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain–Calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional Calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of Calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca2 +] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which Calpastatin levels are increased. Our findings show that the L-domain of Calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length Calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.
S Pontremoli - One of the best experts on this subject based on the ideXlab platform.
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Association of Calpastatin with Inactive Calpain
2020Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S PontremoliAbstract:ing the physiological conditions under which calpain retains its inactive conformational state. The Calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4 –7. This Calpastatin region recognizes a calpain sequence located near the end of the DIIdomain. Interaction of calpain with Calpastatins lacking these sequences becomes strictly Ca 2 -dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca 2 -free calpain and various recombinant Calpastatin forms has been demonstrated by the following experimental results. Addition of Calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when Calpastatin was immunoprecipitated. The Calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-Calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-Calpastatin interaction at a physiological concentration of Ca 2 represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca 2 influx.
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Differential regulation of the calpain-Calpastatin complex by the L-domain of Calpastatin.
Biochimica et Biophysica Acta, 2014Co-Authors: R De Tullio, M Averna, S Pontremoli, Marco Pedrazzi, Bianca Sparatore, Franca Salamino, E MelloniAbstract:Abstract Here we demonstrate that the presence of the L-domain in Calpastatins induces biphasic interaction with calpain. Competition experiments revealed that the L-domain is involved in positioning the first inhibitory unit in close and correct proximity to the calpain active site cleft, both in the closed and in the open conformation. At high concentrations of Calpastatin, the multiple EF-hand structures in domains IV and VI of calpain can bind Calpastatin, maintaining the active site accessible to substrate. Based on these observations, we hypothesize that two distinct calpain–Calpastatin complexes may occur in which calpain can be either fully inhibited (I) or fully active (II). In complex II the accessible calpain active site can be occupied by an additional Calpastatin molecule, now a cleavable substrate. The consequent proteolysis promotes the accumulation of Calpastatin free inhibitory units which are able of improving the capacity of the cell to inhibit calpain. This process operates under conditions of prolonged [Ca2 +] alteration, as seen for instance in Familial Amyotrophic Lateral Sclerosis (FALS) in which Calpastatin levels are increased. Our findings show that the L-domain of Calpastatin plays a crucial role in determining the formation of complexes with calpain in which calpain can be either inhibited or still active. Moreover, the presence of multiple inhibitory domains in native full-length Calpastatin molecules provides a reservoir of potential inhibitory units to be used to counteract aberrant calpain activity.
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Role of the calpain-Calpastatin system in the density-dependent growth arrest.
Archives of Biochemistry and Biophysics, 2008Co-Authors: Roberto Stifanese, M Averna, R De Tullio, S Pontremoli, Franca Salamino, Claudia Cantoni, Maria Cristina Mingari, Carola Prato, E MelloniAbstract:Abstract In dividing cells Calpastatin diffuses from aggregates into cytosol, indicating the requirement for a tight regulation of calpain. Accordingly, the involvement of the calpain–Calpastatin system in cell proliferation and in the density-dependent growth arrest was studied in JA3 cells stably transfected with a Calpastatin form permanently localized in cytosol. In Calpastatin overexpressing cells, cell cycle rate is 50% reduced, and cells enter the ungrowing, still fully reversible, stage at a 3-fold higher cell density. Furthermore, in cell density growth arrest phase, down regulation of α- and θ-PKC isoforms, as well as FAK and talin occurs. In Calpastatin overexpressing cells, degradation of these calpain substrate proteins is prevented and delayed. Thus, calpain activity plays a crucial role in inducing the cell entry into a functional quiescent phase.
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Interaction between catalytically inactive calpain and Calpastatin
FEBS Journal, 2006Co-Authors: M Averna, R De Tullio, E Melloni, Roberto Stifanese, Enrico Defranchi, Franca Salamino, S PontremoliAbstract:Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca2+, substrate or Calpastatin, the affinity increases up to 10-fold, with Calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca2+] or extensive diffusion of Calpastatin into the cytosol without affecting Ca2+ homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that Calpastatin availability in the cytosol is the triggering event for calpain–Calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.
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changes in intracellular Calpastatin localization are mediated by reversible phosphorylation
Biochemical Journal, 2001Co-Authors: M Averna, R De Tullio, S Pontremoli, Franca Salamino, Mario Passalacqua, E MelloniAbstract:We have previously reported that, in neuroblastoma LAN-5 cells, Calpastatin is in an aggregated state, close to the cell nucleus [De Tullio, Passalacqua, Averna, Salamino, Melloni and Pontremoli (1999) Biochem. J. 343 , 467–472]. In the present paper, we demonstrate that aggregated Calpastatin is predominantly in a phosphorylated state. An increase in intracellular free [Ca 2+ ] induces both dephosphorylation of Calpastatin, through the action of a phosphoprotein phosphatase, and its redistribution as a soluble inhibitor species. cAMP, but not PMA-induced phosphorylation, reverses Calpastatin distribution favouring its aggregation. This intracellular reversible mechanism, regulating the level of cytosolic Calpastatin, could be considered a strategy through which calpain can escape Calpastatin inhibition, especially during earlier steps of its activation process.
F Salamino - One of the best experts on this subject based on the ideXlab platform.
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Association of Calpastatin with Inactive Calpain
2020Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S PontremoliAbstract:ing the physiological conditions under which calpain retains its inactive conformational state. The Calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4 –7. This Calpastatin region recognizes a calpain sequence located near the end of the DIIdomain. Interaction of calpain with Calpastatins lacking these sequences becomes strictly Ca 2 -dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca 2 -free calpain and various recombinant Calpastatin forms has been demonstrated by the following experimental results. Addition of Calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when Calpastatin was immunoprecipitated. The Calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-Calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-Calpastatin interaction at a physiological concentration of Ca 2 represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca 2 influx.
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adaptive modifications in the calpain Calpastatin system in brain cells after persistent alteration in ca2 homeostasis
Journal of Biological Chemistry, 2010Co-Authors: Roberto Stifanese, M Averna, R De Tullio, F Salamino, S Pontremoli, Marco Pedrazzi, Francesco Beccaria, Marco Milanese, Giambattista Bonanno, E MelloniAbstract:Abstract Persistent dysregulation in Ca2+ homeostasis is a pervasive pathogenic mechanism in most neurodegenerative diseases, and accordingly, calpain activation has been implicated in neuronal cells dysfunction and death. In this study we examined the intracellular functional state of the calpain-Calpastatin system in −G93A(+) SOD1 transgenic mice to establish if and how uncontrolled activation of calpain can be prevented in vivo during the course of prolonged [Ca2+]i elevation. The presented data indicate that 1) calpain activation is more extensive in motor cortex, in lumbar, and sacral spinal cord segments compared with the lower or almost undetectable activation of the protease in other brain areas, 2) direct measurements of the variations of Ca2+ levels established that the degree of the protease activation is correlated to the extent of elevation of [Ca2+]i, 3) intracellular activation of calpain is always associated with diffusion of Calpastatin from perinuclear aggregated forms into the cytosol and the formation of a calpain-Calpastatin complex, and 4) a conservative fragmentation of Calpastatin is accompanied by its increased expression and inhibitory capacity in conditions of prolonged increase in [Ca2+]i. Thus, Calpastatin diffusion and formation of the calpain-Calpastatin complex together with an increased synthesis of the inhibitor protein represent a cellular defense response to conditions of prolonged dysregulation in intracellular Ca2+ homeostasis. Altogether these findings provide a new understanding of the in vivo molecular mechanisms governing calpain activation that can be extended to many neurodegenerative diseases, potentially useful for the development of new therapeutic approaches.
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association of Calpastatin with inactive calpain a novel mechanism to control the activation of the protease
Journal of Biological Chemistry, 2006Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S PontremoliAbstract:Abstract It is generally accepted that the Ca2+-dependent interaction of calpain with Calpastatin is the most relevant mechanism involved in the regulation of Ca2+-induced proteolysis. We now report that a calpain-Calpastatin association can occur also in the absence of Ca2+ or at very low Ca2+ concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The Calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4-7. This Calpastatin region recognizes a calpain sequence located near the end of the DII-domain. Interaction of calpain with Calpastatins lacking these sequences becomes strictly Ca2+-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca2+-free calpain and various recombinant Calpastatin forms has been demonstrated by the following experimental results. Addition of Calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when Calpastatin was immunoprecipitated. The Calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-Calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-Calpastatin interaction at a physiological concentration of Ca2+ represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca2+ influx.
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age dependent degradation of Calpastatin in kidney of hypertensive rats
Journal of Biological Chemistry, 2001Co-Authors: M Averna, R De Tullio, F Salamino, S Pontremoli, Roberto Minafra, E MelloniAbstract:Abstract Hypertensive rats from the Milan strain show a significant decrease in Calpastatin activity as compared with normotensive control animals. Calpastatin deficiency is age-related and highly relevant in kidney, heart, and erythrocytes and of minor entity in brain tissue. In normotensives the changes during aging in the levels of Calpastatin activity and mRNA are consistent with an increase of Calpastatin protein. In hypertensive rats such a relationship during aging is not observed, because a progressive accumulation of mRNA is accompanied by a lower amount of Calpastatin protein as compared with control rats. Together with the low level of Calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa Calpastatin fragment, previously shown to represent a typical product of calpain-mediated Calpastatin degradation, is also observed. Evidence for such intracellular proteolysis by Ca2+-activated calpain is provided by the normalization of the Calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca2+ influx. Further evidence is provided by the disappearance, in these conditions, of the 15-kDa Calpastatin fragment. These data allow the conclusion that Calpastatin degradation is a relevant part of the overall mechanism for regulating calpain activity.
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Phosphorylation of rat brain Calpastatins by protein kinase C.
FEBS letters, 1999Co-Authors: M Averna, R De Tullio, F Salamino, E Melloni, S PontremoliAbstract:Calpastatin, the natural inhibitor of calpain, is present in rat brain in multiple forms, having different molecular masses, due to the presence of one (low Mr form) or four (high Mr form) repetitive inhibitory domains. Recombinant and native Calpastatin forms are substrates of protein kinase C, which phosphorylates a single serine residue at their N-terminus. Furthermore, both low and high Mr Calpastatins are phosphorylated by protein kinase C at the same site. These Calpastatin forms are phosphorylated also by protein kinase A, although with a lower efficiency. The incorporation of a phosphate group determines an increase in the concentration of Ca2+ required to induce the formation of the calpain-Calpastatin complex. This effect results in a large decrease of the inhibitory efficiency of Calpastatins. We suggest that phosphorylation of Calpastatin represents a mechanism capable to balance the actual amount of active Calpastatin to the level of calpain to be activated.
