In vitro Activation of CPP32 and Mch3 by Mch4, a Novel Human Apoptotic Cysteine Protease Containing Two FADD-Like Domains

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 15; pp. 7464 - 7469
• Main Authors: Fernandes-Alnemri, Teresa, Armstrong, Robert C., Krebs, Joseph, Srinivasula, Srinivasa M., Wang, Lijuan, Bullrich, Florencia, Fritz, Lawrence C., Trapani, Joseph A., Tomaselli, Kevin J., Litwack, Gerald, Alnemri, Emad S.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 23.07.1996; National Acad Sciences; National Academy of Sciences
• Subjects: Active sites; Amino Acid Sequence; Animals; Apoptosis; Base Sequence; Biochemistry; Caspase 10; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line; Chromosome Mapping; Chromosomes, Artificial, Yeast; Chromosomes, Human, Pair 2; Cloning, Molecular; Complementary DNA; Cysteine Endopeptidases - chemistry; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; DNA Primers; Enzyme Activation; Enzymes; Family members; Genes; Humans; Hybrid Cells; Ice; Kinetics; Molecular Sequence Data; Polymerase Chain Reaction; Proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Rodentia; Sequence Homology, Amino Acid; T lymphocytes; Tumor Cells, Cultured
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.15.7464

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartatespecific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in all known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Ki = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathway.