Caspase-3 cleaves Apaf-1 into an approximately 30 kDa fragment that associates with an inappropriately oligomerized...

Area of Science:

• Cell biology
• Molecular biology
• Biochemistry

Background:

• Apoptosome complexes, formed by Apaf-1 oligomerization, are crucial for programmed cell death (apoptosis).
• Two distinct apoptosome complexes exist: a ~700 kDa active form and a ~1.4 MDa inactive form.
• The inactive ~1.4 MDa complex's inability to activate effector caspases remains poorly understood.

Purpose of the Study:

• To investigate the molecular basis for the inactivity of the ~1.4 MDa apoptosome complex.
• To identify factors contributing to the lack of effector caspase activation by the larger apoptosome.

Main Methods:

• Analysis of dATP-activated cell lysates and apoptotic cells.
• Identification and characterization of Apaf-1 fragments using immunoreactivity.
• In vitro assays with purified Apaf-1 fragments and recombinant proteins.
• Immunodepletion of XIAP (inhibitor of apoptosis protein).
• Proteolytic digestion of apoptosome complexes.

Main Results:

• A ~30 kDa Apaf-1 N-terminal fragment (p30) was exclusively found in the inactive ~1.4 MDa apoptosome complex.
• Caspase-3 cleaves Apaf-1 within its CED-4 domain to generate the p30 fragment.
• The p30 fragment showed weak inhibition of caspase-3 activation in vitro.
• Preventing p30 formation did not restore the large complex's activity.
• XIAP depletion did not relieve the inhibition of the large complex.
• Proteolytic digestion revealed conformationally distinct Apaf-1 forms in the active and inactive complexes.

Conclusions:

• The formation of the Apaf-1 p30 fragment is not the primary reason for the ~1.4 MDa apoptosome complex's inactivity.
• The inability of the ~1.4 MDa apoptosome complex to activate effector caspases likely stems from conformational alterations in Apaf-1, leading to improper folding or oligomerization.