title: The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism.
abstract: The major mechanism of cytotoxic lymphocyte killing involves the directed release of granules containing perforin and a number of proteases onto the target cell membrane. One of these proteases, granzyme B, has an unusual substrate site preference for Asp residues, a property that it shares with members of the emerging interleukin-1beta-converting enzyme (ICE)/CED-3 family of proteases. Here we show that granzyme B is sufficient to reproduce rapidly all of the key features of apoptosis, including the degradation of several protein substrates, when introduced into Jurkat cell-free extracts. Granzyme B-induced apoptosis was neutralized by a tetrapeptide inhibitor of the ICE/CED-3 family protease, CPP32, whereas a similar inhibitor of ICE had no effect. Granzyme B was found to convert CPP32, but not ICE, to its active form by cleaving between the large and small subunits of the CPP32 proenzyme, resulting in removal of the prodomain via an autocatalytic step. The cowpox virus protein CrmA, a known inhibitor of ICE family proteases as well as granzyme B, inhibited granzyme B-mediated CPP32 processing and apoptosis. These data demonstrate that CPP32 activation is a key event during apoptosis initiated by granzyme B.
authors.affiliation: La Jolla Institute for Allergy and Immunology, CA 92037, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Martin
authors.forename: G P
authors.initials: GP
authors.lastname: Amarante-Mendes
authors.forename: L
authors.initials: L
authors.lastname: Shi
authors.forename: T H
authors.initials: TH
authors.lastname: Chuang
authors.forename: C A
authors.initials: CA
authors.lastname: Casiano
authors.forename: G A
authors.initials: GA
authors.lastname: O'Brien
authors.forename: P
authors.initials: P
authors.lastname: Fitzgerald
authors.forename: E M
authors.initials: EM
authors.lastname: Tan
authors.forename: G M
authors.initials: GM
authors.lastname: Bokoch
authors.forename: A H
authors.initials: AH
authors.lastname: Greenberg
authors.forename: D R
authors.initials: DR
authors.lastname: Green
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D020170:Caspase 1; D053148:Caspase 3; D020169:Caspases; D002474:Cell-Free System; D003546:Cysteine Endopeptidases / Q000145:classification / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D053804:Granzymes; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000473:pathology; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000502:physiology*; D015843:Serpins / Q000494:pharmacology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014764:Viral Proteins
chemical_list: D009363:Neoplasm Proteins; D011480:Protease Inhibitors; D015843:Serpins; D014764:Viral Proteins; C074641:interleukin-1beta-converting enzyme inhibitor; D053804:Granzymes; D012697:Serine Endopeptidases; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1