Calpastatin Subdomains A and C Are Activators of Calpain*

The inhibitory domains of calpastatin contain three highly conserved regions, A, B, and C, of which A and C bind calpain in a strictly Ca2+-dependent manner but have no inhibitory activity whereas region B inhibits calpain on its own. We synthesized the 19-mer oligopeptides corresponding to regions A and C of human calpastatin domain I and tested their effect on human erythrocyte μ-calpain and rat m-calpain. The two peptides significantly activate both calpains: the Ca2+ concentration required for half-maximal activity is lowered from 4.3 to 2.4 μm for μ-calpain and from 250 to 140 μm for m-calpain. The EC50concentration of the peptides is 7.5 μm for μ-calpain and 25 μm for m-calpain. It is noteworthy that at low Ca2+ concentrations (1–2 μm for μ-calpain and 70–110 μm for m-calpain) both enzymes are activated about 10-fold by the peptides. Based on these findings, it is suggested that calpastatin fragments may have a role in calpain activationin vivo. Furthermore, these activators open new avenues to cell biological studies of calpain function and eventually may alleviate pathological states caused by calpain malfunction.

[1]  R. Ellis.  Macromolecular crowding : obvious but underappreciated , 2022 .

[2]  K. Suzuki,et al.  The structure of calpain. , 2001, Journal of biochemistry.

[3]  Changlian Zhu,et al.  Synergistic Activation of Caspase-3 by m-Calpain after Neonatal Hypoxia-Ischemia , 2001, The Journal of Biological Chemistry.

[4]  K. Suzuki,et al.  Domain III of calpain is a ca2+-regulated phospholipid-binding domain. , 2001, Biochemical and biophysical research communications.

[5]  I. Donkor.  A survey of calpain inhibitors. , 2000, Current medicinal chemistry.

[6]  C. Bogardus,et al.  A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. , 2000, The Journal of clinical investigation.

[7]  Tom H. Lindner,et al.  Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus , 2000, Nature Genetics.

[8]  E. Melloni,et al.  Differential degradation of calpastatin by μ‐ and m‐calpain in Ca2+‐enriched human neuroblastoma LAN‐5 cells , 2000, FEBS letters.

[9]  P. Greer,et al.  Disruption of the Murine Calpain Small Subunit Gene, Capn4: Calpain Is Essential for Embryonic Development but Not for Cell Growth and Division , 2000, Molecular and Cellular Biology.

[10]  M. Maki,et al.  Caspases cleave the amino-terminal calpain inhibitory unit of calpastatin during apoptosis in human Jurkat T cells. , 2000, Journal of biochemistry.

[11]  D A Lauffenburger,et al.  Epidermal Growth Factor Receptor Activation of Calpain Is Required for Fibroblast Motility and Occurs via an ERK/MAP Kinase Signaling Pathway* , 2000, The Journal of Biological Chemistry.

[12]  R. Huber,et al.  The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Potter,et al.  Inhibition of Calpain Blocks Platelet Secretion, Aggregation, and Spreading* , 1999, The Journal of Biological Chemistry.

[14]  Z. Jia,et al.  Crystal structure of calpain reveals the structural basis for Ca2+‐dependent protease activity and a novel mode of enzyme activation , 1999, The EMBO journal.

[15]  E. Melloni,et al.  Changes in intracellular localization of calpastatin during calpain activation. , 1999, The Biochemical journal.

[16]  E. Melloni,et al.  Mechanism of action of a new component of the Ca(2+)-dependent proteolytic system in rat brain: the calpain activator. , 1998, Biochemical and biophysical research communications.

[17]  H. Sorimachi,et al.  A novel aspect of calpain activation , 1998, FEBS letters.

[18]  K Suzuki,et al.  Functional Defects of a Muscle-specific Calpain, p94, Caused by Mutations Associated with Limb-Girdle Muscular Dystrophy Type 2A* , 1998, The Journal of Biological Chemistry.

[19]  K. Suzuki,et al.  Structure and physiological function of calpains. , 1997, The Biochemical journal.

[20]  R. Mellgren,et al.  Evidence for participation of a calpain-like cysteine protease in cell cycle progression through late G1 phase. , 1997, Biochemical and biophysical research communications.

[21]  P. Tompa,et al.  Autolysis parallels activation of mu-calpain. , 1996, The Biochemical journal.

[22]  E. Melloni,et al.  Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization , 1996, FEBS letters.

[23]  E. Lunney,et al.  An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[24]  P. Davies,et al.  Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography. , 1995, Protein engineering.

[25]  Isabelle Richard,et al.  Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A , 1995, Cell.

[26]  M. Maki,et al.  Preference of calcium‐dependent interactions between calmodulin‐like domains of calpain and calpastatin subdomains , 1995, FEBS letters.

[27]  K. Wang,et al.  Calpain inhibition: an overview of its therapeutic potential. , 1994, Trends in pharmacological sciences.

[28]  M. Maki,et al.  Amino-terminal conserved region in proteinase inhibitor domain of calpastatin potentiates its calpain inhibitory activity by interacting with calmodulin-like domain of the proteinase. , 1994, The Journal of biological chemistry.

[29]  M. Maki,et al.  Analysis of calcium-dependent interaction between amino-terminal conserved region of calpastatin functional domain and calmodulin-like domain of mu-calpain large subunit. , 1994, The Journal of biological chemistry.

[30]  M. Maki,et al.  Requirement of different subdomains of calpastatin for calpain inhibition and for binding to calmodulin-like domains. , 1993, Journal of biochemistry.

[31]  R. Mellgren,et al.  m-Calpain requires DNA for activity on nuclear proteins at low calcium concentrations. , 1993, The Journal of biological chemistry.

[32]  K. Suzuki,et al.  Positive regulation of mu-calpain action by polyphosphoinositides. , 1992, The Journal of biological chemistry.

[33]  D. E. Goll,et al.  Binding of calpain fragments to calpastatin. , 1991, The Journal of biological chemistry.

[34]  M. Maki,et al.  Inhibition of calpain by a synthetic oligopeptide corresponding to an exon of the human calpastatin gene. , 1989, The Journal of biological chemistry.

[35]  D. E. Goll,et al.  Effect of Ca2+ on binding of the calpains to calpastatin. , 1989, The Journal of biological chemistry.

[36]  M. Maki,et al.  Analysis of structure-function relationship of pig calpastatin by expression of mutated cDNAs in Escherichia coli. , 1988, The Journal of biological chemistry.

[37]  K. Titani,et al.  Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis. , 1988, Biochemistry.

[38]  H. Kawasaki,et al.  All four repeating domains of the endogenous inhibitor for calcium-dependent protease independently retain inhibitory activity. Expression of the cDNA fragments in Escherichia coli. , 1988, The Journal of biological chemistry.

[39]  M. Maki,et al.  All four internally repetitive domains of pig calpastatin possess inhibitory activities against calpains I and II , 1987, FEBS letters.

[40]  D. E. Goll,et al.  Purification of the Ca2+-dependent proteinase inhibitor from bovine cardiac muscle and its interaction with the millimolar Ca2+-dependent proteinase. , 1987, The Journal of biological chemistry.

[41]  K. Suzuki,et al.  Reversible interaction between Ca2+‐activated neutral protease (CANP) and its endogenous inhibitor , 1985, FEBS letters.

[42]  K. Imahori,et al.  Autolysis of calcium-activated neutral protease of chicken skeletal muscle. , 1981, Journal of biochemistry.

[43]  K. Wang,et al.  Development and therapeutic potential of calpain inhibitors. , 1997, Advances in pharmacology.