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Calpastatin

The Experts below are selected from a list of 315 Experts worldwide ranked by ideXlab platform

E Melloni – 1st expert on this subject based on the ideXlab platform

  • Association of Calpastatin with Inactive Calpain
    , 2020
    Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S Pontremoli

    Abstract:

    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.

  • Differential regulation of the calpain-Calpastatin complex by the L-domain of Calpastatin.
    Biochimica et Biophysica Acta, 2014
    Co-Authors: R De Tullio, M Averna, S Pontremoli, Marco Pedrazzi, Bianca Sparatore, Franca Salamino, E Melloni

    Abstract:

    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.

  • adaptive modifications in the calpain Calpastatin system in brain cells after persistent alteration in ca2 homeostasis
    Journal of Biological Chemistry, 2010
    Co-Authors: Roberto Stifanese, M Averna, R De Tullio, F Salamino, S Pontremoli, Marco Pedrazzi, Francesco Beccaria, Marco Milanese, Giambattista Bonanno, E Melloni

    Abstract:

    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.

M Averna – 2nd expert on this subject based on the ideXlab platform

  • Association of Calpastatin with Inactive Calpain
    , 2020
    Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S Pontremoli

    Abstract:

    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.

  • Production and Purification of Recombinant Calpastatin.
    Methods of Molecular Biology, 2019
    Co-Authors: R De Tullio, M Averna

    Abstract:

    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.

  • Immunoblotting for Calpastatin Expression.
    Methods of Molecular Biology, 2019
    Co-Authors: M Averna, R De Tullio

    Abstract:

    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.

R De Tullio – 3rd expert on this subject based on the ideXlab platform

  • Association of Calpastatin with Inactive Calpain
    , 2020
    Co-Authors: E Melloni, M Averna, R De Tullio, F Salamino, Roberto Stifanese, Enrico Defranchi, S Pontremoli

    Abstract:

    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.

  • Production and Purification of Recombinant Calpastatin.
    Methods of Molecular Biology, 2019
    Co-Authors: R De Tullio, M Averna

    Abstract:

    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.

  • Immunoblotting for Calpastatin Expression.
    Methods of Molecular Biology, 2019
    Co-Authors: M Averna, R De Tullio

    Abstract:

    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.