Expression, refolding, and autocatalytic proteolytic processing of the interleukin-1 beta-converting enzyme precursor

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Interleukin-1 beta-converting enzyme (ICE) is a cysteine protease involved in apoptosis.
• ICE is synthesized as a 45-kDa inactive precursor.
• Understanding ICE processing is crucial for its role in biological pathways.

Purpose of the Study:

• To investigate the autoproteolytic processing and activation of the interleukin-1 beta-converting enzyme precursor.
• To identify factors influencing the conversion of the 45-kDa ICE precursor to its active heterodimeric form.
• To explore the potential regulatory role of the ICE precursor's N-terminal region.

Main Methods:

• Expression of full-length ICE precursor in Escherichia coli.
• Solubilization and refolding of the expressed protein.
• Analysis of proteolytic processing using inhibitors and varying refolding conditions.
• Time-course studies to determine subunit processing order.

Main Results:

• Autoproteolytic conversion of the 45-kDa ICE precursor to a 10- and 20-kDa heterodimeric form was observed upon refolding.
• Catalytic activity was confirmed against interleukin-1 beta precursor and synthetic substrates.
• Inclusion of a specific inhibitor (SDZ 223-941) or refolding under nonreducing conditions prevented processing to the active form.
• Time-course experiments indicated sequential release of the 10-kDa subunit before the 20-kDa subunit.

Conclusions:

• The interleukin-1 beta-converting enzyme precursor undergoes autoproteolytic processing to generate its active heterodimeric form.
• Specific inhibitors and nonreducing conditions can block this activation process.
• The sequential release of subunits suggests a regulatory role for the N-terminal portion of the precursor, which is released last.