Calpain 3 Is Activated through Autolysis within the Active Site and Lyses Sarcomeric and Sarcolemmal Components

Abstract

Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.

ACKNOWLEDGMENTS

We are grateful to the in vivo evaluation department of Genethon, especially to Philippe Rameau, Isabelle Adamski, Daniel Stockholm, and Françoise Fougerousse. We thank Noriyuki Kioka, Paul Mangeat, Carol Otey, and Siegfried Labeit for providing antibodies. We are grateful to Olivier Danos for constant support, Beatrice Benayoun and Suzanne Cure for critical reading of the manuscript, and Stephanie Penninck for technical help.

This work was funded by the Association Française contre les Myopathies. M.T. and G.S. are AFM and MRE fellows.