A nucleotide binding site in caspase-9 regulates apoptosome activation.

Area of Science:

• Molecular Biology
• Cell Death Pathways
• Biochemistry

Background:

• Apoptotic protease activating factor 1 (Apaf-1) and caspase-9 (Csp9) form the apoptosome, a key complex in initiating programmed cell death.
• Adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) are known activators of Apaf-1, leading to Csp9 activation.
• The precise regulatory mechanisms of Csp9 activation by nucleotides remain incompletely understood.

Purpose of the Study:

• To investigate the dual role of ATP and dATP in regulating Apaf-1-mediated Csp9 activation.
• To identify potential inhibitory mechanisms of ATP/dATP on Csp9 activity.
• To characterize the nucleotide-binding properties of Csp9.

Main Methods:

• Utilized affinity labeling with 3'-O-(5-fluoro-2,4-dinitrophenyl)-ATP (FDNP-ATP) to probe nucleotide binding to Csp9.
• Performed activity assays on full-length proCsp9, truncated variants (DeltaproCsp9, p18/p10), and processed Csp9 (p35/p10).
• Quantified nucleotide binding stoichiometry and inhibition constants (IC50).

Main Results:

• ATP and dATP activate Apaf-1 for apoptosome formation and Csp9 activation.
• At concentrations > 1 mM, ATP and dATP inhibit Csp9 activity, acting as negative regulators of apoptosis.
• FDNP-ATP binds potently to full-length proCsp9 (IC50 ~5-11 nM), covalently labeling it and preventing apoptosome formation.
• ATP and dATP bind to processed Csp9 (p35/p10), suggesting a role in regulating the active enzyme.

Conclusions:

• ATP and dATP exhibit a dual role in apoptosis: activation at low concentrations and inhibition at high concentrations.
• High-affinity binding of nucleotides to proCsp9 and processed Csp9 suggests a novel regulatory mechanism for apoptosis.
• The findings reveal previously unrecognized nucleotide-binding sites on Csp9 that modulate its activation and function.