Caspase-7 uses RNA to enhance proteolysis of poly(ADP-ribose) polymerase 1 and other RNA-binding proteins

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Apoptosis involves caspases cleaving essential proteins for cell removal.
• Caspase-7 utilizes exosites for specific substrate recognition beyond its active site.
• The mechanism of caspase-7 exosite-mediated substrate selection, particularly for PARP-1, is largely unknown.

Purpose of the Study:

• To elucidate the mechanism by which caspase-7's N-terminal exosite recognizes and cleaves substrates.
• To investigate the role of RNA in caspase-7 substrate selection and proteolysis.
• To compare substrate preference and RNA-mediated regulation between caspase-7 and caspase-3.

Main Methods:

• Analysis of caspase-7 exosite charge and its role in substrate binding.
• RNA interference and addition experiments to assess RNA's influence on PARP-1 proteolysis.
• Affinity chromatography and gel shift assays to detect nucleic acid binding by caspases.
• Comparative proteolysis assays using RNA-binding proteins (RNA-BPs) as substrates.

Main Results:

• The positive charge of the caspase-7 exosite is critical for substrate recognition.
• PARP-1 interaction with the exosite involves its Zn3 and BRCT domains and is mediated by RNA.
• Caspase-7, unlike caspase-3, binds nucleic acids and preferentially cleaves RNA-BPs, with RNA enhancing this activity.

Conclusions:

• Caspase-7 employs an evolutionarily conserved, positively charged exosite for substrate selection.
• RNA plays a crucial role in mediating caspase-7's interaction with substrates like PARP-1 and RNA-BPs.
• This study reveals an unconventional RNA-dependent mechanism for substrate specificity in caspase-7 activity during apoptosis.