Cardiolipin induces the formation of active oligomers of u-calpain. Implications for mitochondria functioning

INTRODUCTION: - and m-calpain are heterodimeric intracellular cysteine endopeptidases requiring micromolar and millimolar Ca2+ concentrations for their activation in vitro. In living cells they are activated by nanomolar Ca2+, and they are inhibited by same concentrations of their endogenous inhibitor, calpastatin. Among other mechanisms, the interaction of calpains with membrane phospholipids has been proposed to reduce in vivo Ca2+ requirement. However, to date the functional interaction of calpain with mitochondrial membranes has not been so far deeply investigated. Here we present in vitro data on the Ca2+ requirement for proteolytic activity of calpains in the presence of different lipids, demonstrating the structural and functional role of cardiolipin (CL), a specific anionic phospholipid of the inner mitochondrial membrane released during apoptosis.MATERIALS AND METHODS: -calpain was purified from human erythrocytes as described elsewhere. Small angle X-ray scattering (SAXS) experiments were carried out at LURE (Orsay-France). In vitro activities were assayed both using human denaturated globin and Suc-Leu-Tyr-AMC as calpain substrates. Mitochondria were isolated from control or apoptotic HaCaT cells using discontinuous Percoll density gradient.RESULTS: Among different lipids, here we report a functional role of CL, in reducing -calpain Ca2+ requirement. Furthermore, we characterized, using SAXS analysis, a CL-induced functional oligomerization of -calpain leading to a specific quaternary structure showing less sensitivity to the natural inhibitor, calpastatin. We also address some basic issues related to CL-induced -calpain activation in a simplified in vitro model using both liposomes with known CL concentration and mitochondria isolated from cultured cells. We found that -calpain is activated only at submicellar concentration of CL, while liposomes containing CL did not influence calpain activity. Finally we found increased calpain concentration and activity in mitochondria isolated from apoptotic cells with respect to the control. Taken together these results allow us to suggest a new pathway leading to the activation of calpain system in mitochondria.Dainese et al., (2002) JBC 277, 40296-40301.[...]