Fibrils Colocalize Caspase-3 with Procaspase-3 to Foster Maturation

Most proteases are expressed as inactive precursors, or zymogens, that become activated by limited proteolysis. We previously identified a small molecule, termed 1541, that dramatically promotes the maturation of the zymogen, procaspase-3, to its mature form, caspase-3. Surprisingly, compound 1541 self-assembles into nanofibrils, and localization of procaspase-3 to the fibrils promotes activation. Here, we interrogate the biochemical mechanism of procaspase-3 activation on 1541 fibrils in addition to proteogenic amyloid-β(1–40) fibrils. In contrast to previous reports, we find no evidence that procaspase-3 alone is capable of self-activation, consistent with its fate-determining role in executing apoptosis. In fact, mature caspase-3 is >107-fold more active than procaspase-3, making this proenzyme a remarkably inactive zymogen. However, we also show that fibril-induced colocalization of trace amounts of caspase-3 or other initiator proteases with procaspase-3 dramatically stimulates maturation of the proenzyme in vitro. Thus, similar to known cellular signaling complexes, these synthetic or natural fibrils can serve as platforms to concentrate procaspase-3 for trans-activation by upstream proteases. Background: Procaspase-3 is a critical protease in apoptosis. Results: Procaspase-3 has less than 1/10,000,000 the activity of mature caspase-3 and does not detectably autoprocess. Small molecule and proteogenic fibrils promote procaspase-3 maturation through induced proximity to an active protease. Conclusion: Fibrils enhance procaspase-3 maturation in vitro through colocalization with upstream proteases. Significance: These studies demonstrate the importance of scaffolding and colocalization with active proteases for procaspase-3 processing and activation.