Complementary proteomic tools for the dissection of apoptotic proteolysis events

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Proteolysis regulates cell signaling via irreversible protein structure changes.
• Caspase-mediated proteolysis is crucial in apoptosis and other cellular processes.
• Accurate identification of caspase substrates is vital for understanding these pathways.

Purpose of the Study:

• To develop and validate a novel proteomic platform for comprehensive profiling of caspase substrates.
• To identify and quantify apoptotic-mediated proteolytic events across a wide dynamic range.
• To investigate caspase substrate cleavage events in both intrinsic and extrinsic apoptosis pathways.

Main Methods:

• Integrated proteomic workflow combining Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and C-terminal aspartic acid peptide enrichment.
• Application of the platform to etoposide-induced intrinsic apoptosis in Jurkat cells.
• Targeted proteomic analysis using BAX HCT116 isogenic cell lines and pro-apoptotic receptor agonists (PARA).

Main Results:

• Quantified 3346 proteins and identified 360 etoposide-induced proteolytic substrates, including 160 known caspase substrates.
• Dissected pre- and post-mitochondrial extrinsic apoptotic cleavage events.
• Identified novel caspase substrates such as Basic Transcription Factor 3, TRK-fused gene protein (TFG), and p62/Sequestosome, alongside known substrates BID and PARP-1.

Conclusions:

• The developed proteomic platform enables large-scale, dynamic range profiling of caspase substrates.
• This approach significantly expands the known repertoire of apoptotic proteolytic events.
• Provides insights into caspase substrate specificity and cleavage timing in different apoptotic contexts.