Actin cleavage by CPP-32/apopain during the development of apoptosis

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Interleukin-1beta-converting enzyme (ICE)/ced-3 family proteases are critical for apoptosis.
• The specific cellular substrates targeted by these proteases during apoptosis remain largely unidentified.
• Previous work indicated a distinct ICE family protease cleaves actin during VP-16-induced apoptosis.

Purpose of the Study:

• To identify the specific ICE family protease responsible for actin cleavage during apoptosis.
• To investigate the role of actin cleavage in the mechanism of apoptosis.
• To determine if inhibiting this protease affects cellular apoptosis.

Main Methods:

• In vitro cleavage assays using apoptotic cell extracts and purified actin.
• Western blot analysis using antibodies against specific actin fragments.
• In vivo studies using VP-16-treated U937 cells and a selective CPP-32(-like) protease inhibitor (Z-EVD-CH2-DCB).

Main Results:

• Activated CPP-32/apopain was identified as the protease cleaving actin in vitro, generating 15 kDa and 31 kDa fragments.
• In vivo cleavage of actin during apoptosis was confirmed using an antibody specific to the N-terminus of the 15 kDa fragment.
• Inhibition of CPP-32(-like) protease activity with Z-EVD-CH2-DCB effectively blocked actin cleavage and VP-16-induced apoptosis in U937 cells.

Conclusions:

• Actin serves as a direct substrate for CPP-32/apopain(-like) protease in both in vitro and in vivo settings.
• The cleavage of actin by CPP-32/apopain is a significant event during apoptosis.
• These findings suggest a role for actin in the regulation of cell growth and programmed cell death.