The Ced-3/interleukin 1 beta converting enzyme-like homolog Mch6 and the lamin-cleaving enzyme Mch2 alpha are substrates for the apoptotic mediator CPP32

Recent evidence suggests that CPP32 is an essential component of an aspartate-specific cysteine protease (ASCP) cascade responsible for apoptosis execution in mammalian cells, Activation of CPP32 could lead to activation of other downstream ASCPs, resulting in late morphological changes such as lamin cleavage and DNA fragmentation, observed in cells undergoing apoptosis, Here we describe the identification and cloning of a novel human ASCP named Mch6 from Jurkat T lymphocytes. We demonstrate that the pro-enzymes of Mch6 and the lamin-cleaving enzyme Mch2 alpha are substrates for mature CPP32, Site-directed mutagenesis revealed that CPP32 processes pro-Mch6 preferentially at Asp(330) to generate two subunits of molecular masses 37 kDa (p37) and 10 kDa (p10), However, CPP32 processes pro-Mch2 alpha at three aspartate processing sites (Asp(23), Asp(179), and Asp(193)) to produce the large (p18) and small (p11) subunits of the mature Mch2 alpha enzyme. The CPP32-processed Mch2 alpha is capable of cleaving the VEIDN lamin cleavage site, indicating that CPP32 can, in fact, activate pro-Mch2 alpha. Granzyme B at a concentration that allows processing and activation of CPP32 failed to process pro-Mch2 alpha. However, incubation of pro-Mch2 alpha with granzyme B in the presence of a cellular extract containing pro-CPP32 resulted in activation of pro CPP32 and subsequent processing of pro-Mch2 alpha. Interestingly, granzyme B can also process pro-Mch6 but at a site N-terminal to that cleaved by CPP32, These data suggest that Mch2 alpha and Mch6 are downstream proteases activated in CPP32- and granzyme B-mediated apoptosis, This is the first demonstration of a protease cascade involving granzyme B, CPP32, Mch2 alpha, and Mch6 and evidence that the lamin-cleaving enzyme Mch2 is a target of mature CPP32.