| Procaspase-3 is the inactive zymogen precursor of caspase-3, an executioner caspase that exists in cells as a constitutive dimer built from an N-terminal prodomain connected to a large subunit and a smaller C-terminal subunit joined by an intersubunit linker, and activation requires proteolytic cleavage of this linker rather than any conformational change occurring in the absence of cutting. This cleavage is carried out by upstream initiator caspases, primarily caspase-9 acting within the apoptosome, a heptameric platform assembled when cytochrome c and dATP bind Apaf-1 in the cytosol, with each Apaf-1 subunit noncovalently coupled to a procaspase-9 subunit through their respective CARD domains; caspase-9 recruited to this platform cleaves procaspase-3 specifically at the site 172IETD-S, and this ISL cleavage event is required to generate catalytically active caspase-3. Full maturation of caspase-3 additionally requires removal of its N-terminal prodomain, a process occurring through two sequential cleavage events, an initial rapid cut at Asp9 followed by a slower cleavage at Asp28, and mutating the Asp9 site alone is sufficient to block prodomain removal and downstream caspase activation, while cells expressing caspase-3 engineered to lack its prodomain entirely become more susceptible to death signals without becoming constitutively active, indicating the prodomain restrains activation without being strictly required to sustain it once removed. Once activated, caspase-3 exerts a direct feedback effect on its own activator: caspase-3 cleaves procaspase-9 at a distinct site, Asp330, generating a smaller p10 subunit that removes the fifteen N-terminal amino acids of the p12 fragment, including the XIAP BIR3-binding motif, and this Asp330 cleavage increases apoptosome-driven caspase-3 activation up to eightfold compared with cleavage at the primary caspase-9 processing site alone, while a point mutant unable to be cleaved at Asp330 shows near-complete loss of apoptosome-mediated caspase-3 activity, establishing a caspase-3-to-caspase-9 amplification loop as a defined component of the apoptosome's overall catalytic output rather than a passive downstream consequence of caspase-9 activation. This same Asp330 cleavage event exposes a new N-terminal peptide sequence on the resulting p10 fragment that retains the capacity to be inhibited by XIAP, linking the caspase-3 feedback cleavage step directly to a defined apoptosis brake mechanism operating at the level of caspase-9 rather than of caspase-3 itself. |
