Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Caspases are key proteases in apoptosis.
• The precise order of caspase activation in response to different stimuli is not fully understood.
• Existing methods for detecting caspase activity have limitations in single-cell analysis.

Purpose of the Study:

• To develop and utilize novel cell-permeable fluorogenic substrates for detecting specific caspase activities in intact apoptotic cells.
• To elucidate the order of caspase activation in mouse thymocytes induced by dexamethasone and anti-Fas antibody.
• To investigate the effectiveness of carbobenzoxy-valyl-alanyl-aspartyl(beta-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK) in inhibiting activated caspases.

Main Methods:

• Synthesis of fluorogenic caspase substrates with recognition motifs for caspases 1, 3/7, 6, 8, and 9.
• Treatment of mouse thymocytes with dexamethasone or anti-Fas antibody.
• Assessment of caspase activities using flow cytometry and confocal microscopy.
• Evaluation of Z-VAD[OMe]-FMK inhibition of caspase activities.

Main Results:

• Dexamethasone treatment induced caspase activation in the order: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase.
• Confocal microscopy confirmed VEIDase (caspase-6) activity preceded DEVDase (caspase-3/7) activity with dexamethasone.
• Anti-Fas antibody stimulation resulted in a different activation sequence: IETDase (caspase-8) was first, followed by DEVDase, then VEIDase.
• Z-VAD[OMe]-FMK did not significantly inhibit previously activated intracellular caspases.

Conclusions:

• The developed fluorogenic substrates enable direct observation of caspase cascades in intact apoptotic cells.
• The order of downstream caspase activation is stimulus-dependent.
• This provides a powerful tool for dissecting apoptotic signaling pathways.