Loss of WD2 subdomain of Apaf-1 forms an apoptosome structure which blocks activation of caspase-3 and caspase-9

Area of Science:

• Molecular Biology
• Cell Death Pathways
• Protein Interactions

Background:

• Apaf-1 is crucial for apoptosome assembly, a key step in the mitochondrial cell death pathway.
• Apaf-1's inactive conformation is maintained by intramolecular interactions between its nucleotide-binding domain and WD40 domains.
• Apaf-1 activation involves cytochrome c binding to WD1 and WD2, promoting oligomerization and caspase-9 activation.

Purpose of the Study:

• To investigate the impact of WD domain deletion on Apaf-1 oligomerization and function.
• To determine if Apaf-1 with a single WD domain can be activated by cytochrome c.
• To elucidate the role of individual WD domains in Apaf-1-mediated caspase activation.

Main Methods:

• Utilized a split luciferase complementary assay to assess Apaf-1 oligomerization.
• Studied a truncated Apaf-1 mutant (Apaf-1 1-921) lacking one WD domain.
• Examined caspase-9 activation in the presence and absence of cytochrome c.

Main Results:

• Apaf-1 1-921 failed to activate caspase-9, even when supplemented with cytochrome c.
• The presence of a single WD domain was sufficient to lock Apaf-1 in an inactive conformation.
• Cytochrome c could not overcome the inhibitory state induced by the single WD domain.

Conclusions:

• A single WD domain in Apaf-1 is sufficient to maintain an inactive state.
• Both WD1 and WD2 domains are essential for cytochrome c-induced apoptosome formation and caspase activation.
• This study refines the understanding of Apaf-1 regulation in the intrinsic apoptosis pathway.