Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and is not required for activation

Citation
K. Nakagawa et al., Dissociation of m-calpain subunits occurs after autolysis of the N-terminus of the catalytic subunit, and is not required for activation, J BIOCHEM, 130(5), 2001, pp. 605-611
Citations number
43
Categorie Soggetti
Biochemistry & Biophysics
Journal title
JOURNAL OF BIOCHEMISTRY
ISSN journal
0021924X → ACNP
Volume
130
Issue
5
Year of publication
2001
Pages
605 - 611
Database
ISI
SICI code
0021-924X(200111)130:5<605:DOMSOA>2.0.ZU;2-U
Abstract
Calpain is a heterodimeric, intracellular Ca2+-dependent, "bio-modulator" t hat alters the properties of substrates through site-specific proteolysis. It has been proposed that calpains are activated by autolysis of the N-term inus of the large subunit and/or its dissociation into the subunits. It is, however, unclear whether the dissociation into subunits is required for th e expression of protease activity and/or for in vivo function. Recently, th e crystal structure of m-calpain in the absence of Ca2+ has been resolved. The 3D structure clearly shows that the N-terminus of the m-calpain large s ubunit (mCL) makes contact with the 30K subunit, suggesting that autolysis of the N-terminus of mCL changes the interaction of both subunits. To exami ne the relationship between autolysis, dissociation, and activation, we mad e and analysed a series of N-terminal mutants of mCL that mimic the autolys ed forms or have substituted amino acid residue(s) interacting with 30K. As a result, the mutant m-calpains, which are incapable of autolysis, did not dissociate into subunits, whereas those lacking the N-terminal 19 residues (Delta 19), but not those lacking only nine residues (Delta9), dissociated into subunits even in the absence of Ca2+. Moreover, both Delta9 and Delta 19 mutants showed an equivalent reduced Ca2+ requirement for protease acti vity. These results indicate that autolysis is necessary for the dissociati on of the m-calpain subunits, and that the dissociation occurs after, but i s not necessary for, activation.