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Published on: March 5, 2018
Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways
G P McStay1, G S Salvesen, D R Green
1Department of Immunology, St. Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105, USA.
Abstract:
Caspases orchestrate the controlled demise of a cell after an apoptotic signal through specific protease activity and cleavage of many substrates altering protein function and ensuring apoptosis proceeds efficiently. Comparing a variety of substrates of each apoptotic caspase (2, 3, 6, 7, 8, 9 and 10) showed that the cleavage sites had a general motif, sometimes specific for one caspase, but other times specific for several caspases. Using commercially available short peptide-based substrates and inhibitors the promiscuity for different cleavage motifs was indicated, with caspase-3 able to cleave most substrates more efficiently than those caspases to which the substrates are reportedly specific. In a cell-free system, immunodepletion of caspases before or after cytochrome c-dependent activation of the apoptosome indicated that the majority of activity on synthetic substrates was dependent on caspase-3, with minor roles played by caspases-6 and -7. Putative inhibitors of individual caspases were able to abolish all cytochrome c-induced caspase activity in a cell-free system and inhibit apoptosis in whole cells through the extrinsic and intrinsic pathways, raising issues regarding the use of such inhibitors to define relevant caspases and pathways. Finally, caspase activity in cells lacking caspase-9 displayed substrate cleavage activity of a putative caspase-9-specific substrate underlining the lack of selectivity of peptide-based substrates and inhibitors of caspases.
Insights
Caspase activity in apoptosis is complex, with caspase-3 showing broad substrate cleavage. Peptide-based substrates and inhibitors lack selectivity, challenging their use in defining specific caspase roles.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Caspases are key proteases regulating apoptosis.
- Their substrate specificity is crucial for controlled cell death.
Purpose of the Study:
- To investigate the substrate specificity of various caspases.
- To evaluate the utility of peptide-based substrates and inhibitors in studying caspase function.
Main Methods:
- Comparative analysis of caspase cleavage sites on various substrates.
- Assays using commercially available peptide-based substrates and inhibitors.
- Cell-free systems with immunodepletion of caspases.
- Apoptosis induction via extrinsic and intrinsic pathways in whole cells.
Main Results:
- Caspase cleavage sites exhibit general motifs, sometimes shared among caspases.
- Caspase-3 demonstrates promiscuous cleavage activity on multiple substrates.
- Caspase activity in cell-free systems is largely caspase-3 dependent.
- Inhibitors abolish caspase activity but lack specificity, impacting pathway definitions.
Conclusions:
- Peptide-based substrates and inhibitors show limited selectivity for individual caspases.
- Caspase-3 plays a dominant role in substrate cleavage during apoptosis.
- The specificity of current tools is insufficient for definitively assigning roles to specific caspases in apoptotic pathways.
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