Mitochondrial calpain-1 truncates ATP synthase beta subunit

Calpains cleave proteins in a calcium concentration-dependent manner, modulating their intracellular functions. Calpain-1, a member of the calpain family, is localized in the cytosol and mitochondria. Mitochondrial calpain-1 induces mitochondrial dysfunction and apoptosis by cleaving its substrate. Thus, identifying the substrate of calpain-1 is essential to understand its function. However, little is known about the substrates of mitochondrial calpain-1. To address this issue, we screened mitochondrial proteins using bioinformatics approaches and two-dimensional gel electrophoresis. We identified ATP5B as a potential substrate of mitochondrial calpain-1. Calpeptin, a pan-calpain inhibitor, and Tat-μCL, a mitochondrial calpain-1 specific inhibitor, prevented the truncation of ATP5B during in vitro Ca²⁺ incubation. Using recombinant human calpain-1 and ATP5B proteins, we demonstrated that calpain-1 directly cleaved ATP5B, generating a fragment of ATP5B. Based on the predicted cleavage sites in ATP5B, this cleavage may disrupt its interaction with ATP5A1, leading to mitochondrial dysfunction in ATP production. This study identified ATP5B as a novel substrate of mitochondrial calpain-1. The results provide new insights into mitochondrial dysfunction.