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Updated: Oct 13, 2026
Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry
Published on: March 24, 2023
Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and
H R Stennicke1, M Renatus, M Meldal
1Program for Apoptosis & Cell Death Research, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.
Abstract:
Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.
Insights
Caspase proteases exhibit high specificity, particularly at the S(1) subsite, preferring aspartic acid. Discrimination at the P(1)
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Protease specificity is crucial for biological processes like apoptosis.
- Caspases are key proteases in apoptosis, and their substrate recognition is vital.
- Previous studies highlighted the S(4) subsite's role in caspase specificity.
Purpose of the Study:
- To investigate the P(4), P(1), and P(1)' subsite preferences of human caspases.
- To elucidate the influence of S(1) and S(1)' subsites on caspase substrate recognition.
- To understand how subsite interactions dictate caspase specificity in apoptosis.
Main Methods:
- Utilized internally quenched fluorescent peptide substrates.
- Employed substrates containing o-aminobenzoyl and 3-nitro-tyrosine.
- Examined subsite preferences for human caspases 1, 3, 6, 7, and 8.
Main Results:
- The S(1) subsite shows extreme selectivity, preferring aspartic acid over glutamic acid by 20,000-fold.
- Caspases discriminate at the P(1)' position, favoring small residues like glycine, alanine, and serine.
- While general preferences exist, individual caspase profiles vary, with caspase-3 showing promiscuity.
Conclusions:
- Caspase subsite preferences, especially S(1) and P(1)', significantly define their stringent specificity.
- These preferences can predict natural substrates, though second-site interactions may override optimal cleavage sequences.
- Caspases are among the most selective endopeptidases known, with complex recognition mechanisms.
