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Báo cáo khoa học: The calpain 1–a-actinin interaction Resting complex between the calcium-dependant protease and its target in cytoskeleton
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Calpain 1 behaviour toward cytoskeletal targets was inves-tigated using twoa-actinin isoforms from smooth and skel-etal muscles. These two isoforms which are, respectively, sensitiveandresistant tocalpaincleavage, interactwiththe protease when usinginvitro binding assays. The stability of the complexes in EGTA [Kd(–Ca2+)¼0.5 ± 0.1lM] was improved in the presence of 1 mMcalcium ions [Kd(+Ca2+)¼0.05 ± 0.01lM]. Location of the binding structures shows that the C-terminal domain ofa-actinin and each calpain subunit, 28 and 80 kDa, participates in the interaction | Eur. J. Biochem. 270 4662-4670 2003 FEBS 2003 doi 10.1046 j.1432-1033.2003.03859.x The calpain 1-a-actinin interaction Resting complex between the calcium-dependant protease and its target in cytoskeleton Fabrice Ravnaud1. Chantal Bonnal1 Eric Fernandez2 Laure Bremaud3 Martine Cerutti4 Marie-Christine Lebart1 Claude Roustan1 Ahmed Ouali2 and Yves Benyamin1 1UMR 5539 - CNRS laboratoire de Motilité Cellulaire - EPHE cc107 USTL Montpellier France 2Station de Recherches sur la Viande INRA-Theix St-Genee-Champanelle France 3Inetitut Sciences de la Vie et de la Sante Genetique Moleculaire Animale UR 1061 INRA-Univereite de Limogee Faculte dee Sciences Limogee France 4Laboratoire de Pathologie Comparee INRA-CNRS URA5087 Saint Chrietol Lee Alee France Calpain 1 behaviour toward cytoskeletal targets was investigated using two a-actinin isoforms from smooth and skeletal muscles. These two isoforms which are respectively sensitive and resistant to calpain cleavage interact with the protease when using in vtlro binding assays. The stability of the complexes in EGTA Kd -Ca2 0.5 0.1 im was improved in the presence of 1 mM calcium ions Kd Ca2 0.05 0.01 im . Location of the binding structures shows that the C-terminal domain of a-actinin and each calpain subunit 28 and 80 kDa participates in the interaction. In particular the autolysed calpain form 76 18 affords a similar binding compared to the 80 28 intact enzyme with an identified binding site in the catalytic subunit located in the C-terminal region of the chain domain III-IV . The in vivo colocalization of calpain 1 and a-actinin was shown to be likely in the presence of calcium when permeabilized muscle fibres were supplemented by exogenous calpain 1 and the presence of cal-pain 1 in Z-line cores was shown by gold-labelled antibodies. The demonstration of such a colocalization was brought by coimmunoprecipitation experiments of calpain 1 and a-actinin from C2.7 myogenic cells. We propose that calpain 1 interacts in a resting