title: Differential regulation of the calpain-calpastatin complex by the L-domain of calpastatin.
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 [Ca(2+)] 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.
authors.affiliation: Department of Experimental Medicine (DIMES), Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 7-16132 Genova, Italy. Electronic address: detullio@unige.it.
authors.forename: Roberta
authors.initials: R
authors.lastname: De Tullio
authors.affiliation: Department of Experimental Medicine (DIMES), Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 7-16132 Genova, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Averna
authors.affiliation: Department of Experimental Medicine (DIMES), Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 7-16132 Genova, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Pedrazzi
authors.affiliation: Department of Experimental Medicine (DIMES), Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 7-16132 Genova, Italy.
authors.forename: Bianca
authors.initials: B
authors.lastname: Sparatore
authors.affiliation: Department of Experimental Medicine (DIMES), Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 7-16132 Genova, Italy.
authors.forename: Franca
authors.initials: F
authors.lastname: Salamino
authors.affiliation: Department of Experimental Medicine (DIMES), Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 7-16132 Genova, Italy.
authors.forename: Sandro
authors.initials: S
authors.lastname: Pontremoli
authors.affiliation: Department of Experimental Medicine (DIMES), Biochemistry Section, University of Genova, Viale Benedetto XV, 1-16132 Genova, Italy; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV, 7-16132 Genova, Italy.
authors.forename: Edon
authors.initials: E
authors.lastname: Melloni
keywords: Calpain/calpastatin complex; Calpastatin L-domain; Inhibitory efficiency