Granzyme B Mimics Apical Caspases

Abstract

Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the require-ments for these interactions remain unclarified. We ex-amined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is re-moved and cleavage occurs between the subunits. Strik-ingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propep-tide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a conse-quence, two proteases, caspase-3 and GrB, are required to activate procaspase-7. A family of cytosolic cysteine proteases, the caspases, stored in most cells as zymogens play an essential role in the execu-tion of apoptosis. The caspases involved in cell death are di-vided into apical (-2, -8, -9, and -10) and executioner subsets (-3, -6, and -7) (1, 2). The proteolytic signal initiated by the apical caspases is transmitted to the executioners (caspases-3, -6, and -7) (1, 2) whose action on cellular proteins defines apoptosis. The caspases are processed to form active heterodimeric en-zymes by cleavage at specific Asp residues. Activation is not thought to require removal of the propeptide; cleavage between the large and small subunits is the activating event. The zy-mogens of apical caspases are recruited by specific adapter molecules, either at the cytosolic face of death receptors (in the case of caspase-8 and -10) (3), or via a post-mitochondrial route