Mechanistic insights into caspase-9 activation by the structure of the apoptosome holoenzyme

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Mammalian intrinsic apoptosis relies on caspase-9 activation to initiate programmed cell death.
• The Apaf-1 apoptosome is essential for forming a holoenzyme that activates caspase-9.
• The precise mechanism of caspase-9 activation by the apoptosome has remained unclear.

Purpose of the Study:

• To elucidate the molecular mechanism of caspase-9 activation within the apoptotic holoenzyme.
• To determine the structural basis of caspase-9 recruitment and activation by the Apaf-1 apoptosome.

Main Methods:

• Cryoelectron microscopy (cryo-EM) to determine the structure of the apoptotic holoenzyme.
• Structure-guided biochemical analyses to validate functional interactions.

Main Results:

• The cryo-EM structure revealed two distinct Apaf-1/caspase-9 CARD complex formations (4:4 and 2:1) interacting with the apoptosome.
• Specific interfaces between CARD complexes and the apoptosome's central hub are crucial for caspase-9 activation.
• Free caspase-9's CARD domain was found to inhibit its proteolytic activity.

Conclusions:

• The Apaf-1 apoptosome activates caspase-9 by sequestering its inhibitory CARD domain.
• Structural rearrangements of CARDs within the holoenzyme are key to releasing caspase-9 inhibition.
• This study provides critical insights into the regulation of apoptosis at the molecular level.