Engineering a dimeric caspase-9: a re-evaluation of the induced proximity model for caspase activation

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Caspases execute programmed cell death (apoptosis) and require proteolytic activation.
• The induced proximity model suggests dimerization drives initiator caspase activation.
• Caspase-9, an initiator caspase, primarily exists as a monomer.

Purpose of the Study:

• To investigate the induced proximity model of caspase activation.
• To engineer a constitutively dimeric caspase-9 and assess its activity.
• To compare dimerization-mediated activation with Apaf-1-dependent activation.

Main Methods:

• Engineered a constitutively dimeric caspase-9 by altering the dimer interface.
• Determined the crystal structure of the engineered caspase-9.
• Assessed in vitro catalytic activity and cell death induction.
• Evaluated Apaf-1-dependent activity enhancement.

Main Results:

• Engineered dimeric caspase-9 retained structural similarity to wild-type (WT) caspase-9.
• The engineered dimer exhibited higher catalytic activity and induced more efficient cell death than WT caspase-9.
• Dimeric caspase-9 activity was a fraction of Apaf-1-activated caspase-9 and could not be further enhanced by Apaf-1.
• WT caspase-9 activity was significantly enhanced by Apaf-1.

Conclusions:

• Dimerization of caspase-9 may be distinct from Apaf-1-mediated activation.
• Findings support an induced conformation model for initiator caspase activation.
• Apaf-1-dependent activation likely involves conformational changes beyond simple dimerization.