Abstract
Two fundamental questions with regard to proteolytic networks and pathways concern the structural repertoire and kinetic threshold that distinguish legitimate signaling substrates. We used N-terminal proteomics to address these issues by identifying cleavage sites within the Escherichia coli proteome that are driven by the apoptotic signaling protease caspase-3 and the bacterial protease glutamyl endopeptidase (GluC). Defying the dogma that proteases cleave primarily in natively unstructured loops, we found that both caspase-3 and GluC cleave in α-helices nearly as frequently as in extended loops. Notably, biochemical and kinetic characterization revealed that E. coli caspase-3 substrates are greatly inferior to natural substrates, suggesting protease and substrate coevolution. Engineering an E. coli substrate to match natural catalytic rates defined a kinetic threshold that depicts a signaling event. This unique combination of proteomics, biochemistry, kinetics and substrate engineering reveals new insights into the structure-function relationship of protease targets and their validation from large-scale approaches.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Puente, X.S., Sanchez, L.M., Overall, C.M. & Lopez-Otin, C. Human and mouse proteases: a comparative genomic approach. Nat. Rev. Genet. 4, 544–558 (2003).
Salvesen, G.S. & Abrams, J.M. Caspase activation—stepping on the gas or releasing the brakes? Lessons from humans and flies. Oncogene 23, 2774–2784 (2004).
Gevaert, K. et al. Applications of diagonal chromatography for proteome-wide characterization of protein modifications and activity-based analyses. FEBS J. 274, 6277–6289 (2007).
Timmer, J.C. & Salvesen, G.S. Caspase substrates. Cell Death Differ. 14, 66–72 (2007).
Ding, L. et al. Origins of the specificity of tissue-type plasminogen activator. Proc. Natl. Acad. Sci. USA 92, 7627–7631 (1995).
Smith, M.M., Shi, L. & Navre, M. Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage peptide display libraries. J. Biol. Chem. 270, 6440–6449 (1995).
Thornberry, N.A. et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272, 17907–17911 (1997).
Stennicke, H.R., Renatus, M., Meldal, M. & Salvesen, G.S. Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8. Biochem. J. 350, 563–568 (2000).
Deng, S.J. et al. Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library. J. Biol. Chem. 275, 31422–31427 (2000).
Harris, J.L. et al. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc. Natl. Acad. Sci. USA 97, 7754–7759 (2000).
Nazif, T. & Bogyo, M. Global analysis of proteasomal substrate specificity using positional-scanning libraries of covalent inhibitors. Proc. Natl. Acad. Sci. USA 98, 2967–2972 (2001).
Turk, B.E., Huang, L.L., Piro, E.T. & Cantley, L.C. Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat. Biotechnol. 19, 661–667 (2001).
Hubbard, S.J., Campbell, S.F. & Thornton, J.M. Molecular recognition. Conformational analysis of limited proteolytic sites and serine proteinase protein inhibitors. J. Mol. Biol. 220, 507–530 (1991).
Coombs, G.S., Bergstrom, R.C., Madison, E.L. & Corey, D.R. Directing sequence-specific proteolysis to new targets. The influence of loop size and target sequence on selective proteolysis by tissue-type plasminogen activator and urokinase-type plasminogen activator. J. Biol. Chem. 273, 4323–4328 (1998).
Gettins, P.G. Serpin structure, mechanism, and function. Chem. Rev. 102, 4751–4804 (2002).
Kelly, C.A., Laskowski, M., Jr. & Qasim, M.A. The role of scaffolding in standard mechanism serine proteinase inhibitors. Protein Pept. Lett. 12, 465–471 (2005).
Timmer, J.C. et al. Profiling constitutive proteolytic events in vivo. Biochem. J. 407, 41–48 (2007).
Elias, J.E., Haas, W., Faherty, B.K. & Gygi, S.P. Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations. Nat. Methods 2, 667–675 (2005).
Crooks, G.E., Hon, G., Chandonia, J.M. & Brenner, S.E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).
Vacic, V., Iakoucheva, L.M. & Radivojac, P. Two Sample Logo: a graphical representation of the differences between two sets of sequence alignments. Bioinformatics 22, 1536–1537 (2006).
Kabsch, W. & Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983).
Jones, D.T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195–202 (1999).
Mahrus, S. et al. Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134, 866–876 (2008).
Kitagawa, M. et al. Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res. 12, 291–299 (2006).
Fischer, U., Janicke, R.U. & Schulze-Osthoff, K. Many cuts to ruin: a comprehensive update of caspase substrates. Cell Death Differ. 10, 76–100 (2003).
Stennicke, H.R. et al. Pro-caspase-3 is a major physiologic target of caspase-8. J. Biol. Chem. 273, 27084–27090 (1998).
Casciola-Rosen, L. et al. Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death. J. Exp. Med. 183, 1957–1964 (1996).
Ishihama, Y. et al. Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics 9, 102 (2008).
Impens, F. et al. Mechanistic insight into taxol-induced cell death. Oncogene 27, 4580–4591 (2008).
Gevaert, K. et al. Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat. Biotechnol. 21, 566–569 (2003).
Schilling, O. & Overall, C.M. Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites. Nat. Biotechnol. 26, 685–694 (2008).
Dean, R.A. & Overall, C.M. Proteomics discovery of metalloproteinase substrates in the cellular context by iTRAQ labeling reveals a diverse MMP-2 substrate degradome. Mol. Cell. Proteomics 6, 611–623 (2007).
Enoksson, M. et al. Identification of proteolytic cleavage sites by quantitative proteomics. J. Proteome Res. 6, 2850–2858 (2007).
Dix, M.M., Simon, G.M. & Cravatt, B.F. Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell 134, 679–691 (2008).
McDonald, L., Robertson, D.H., Hurst, J.L. & Beynon, R.J. Positional proteomics: selective recovery and analysis of N-terminal proteolytic peptides. Nat. Methods 2, 955–957 (2005).
Igarashi, Y. et al. CutDB: a proteolytic event database. Nucleic Acids Res. 35, D546–D549 (2007).
Stubbs, M.T. & Bode, W. The clot thickens: clues provided by thrombin structure. Trends Biochem. Sci. 20, 23–28 (1995).
Overall, C.M. Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites. Mol. Biotechnol. 22, 51–86 (2002).
Denault, J.B. & Salvesen, G.S. Apoptotic caspase activation and activity. Methods Mol. Biol. 414, 191–220 (2008).
Yates, J.R., III, Eng, J.K. & McCormack, A.L. Mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases. Anal. Chem. 67, 3202–3210 (1995).
Morrison, J.F. The slow-binding and slow, tight-binding inhibition of enzyme-catalysed reactions. Trends Biochem. Sci. 7, 102–105 (1982).
Acknowledgements
This work was supported by the US National Institutes of Health (NIH) Roadmap Initiative National Biotechnology Resource Center grant RR20843 for the Center on Proteolytic Pathways, CA69381 from the National Cancer Institute (NCI) and by Training Grant 5T32CA77109-9 from the NCI. We thank M. Enoksson for helpful discussions.
Author information
Authors and Affiliations
Contributions
J.C.T. designed and performed most experiments and interpreted results; W.Z. performed LC-MS/MS analysis and database searching; C.P. designed and performed Km determination experiments; T.R. prepared some N-terminomic samples; S.J.S. performed Edman degradation; A.M.E. provided advice and helped revise the manuscript; S.J.R. aided in structural interpretation; G.S.S. designed the scope of the study, interpreted results and, together with J.C.T. and S.J.R., wrote the manuscript.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–10 and Supplementary Table 5 (PDF 3156 kb)
Supplementary Table 1
N-terminome of caspase-3 treated E. coli lysate & controls (XLS 395 kb)
Supplementary Table 2
N-terminome of GluC treated E. coli lysate & controls (XLS 354 kb)
Supplementary Table 3
Caspase-3 cleavage-sites in the E. coli proteome determined by N-terminomics. (XLS 21 kb)
Supplementary Table 4
GluC cleavage-sites in the E. coli proteome determined by N-terminomics. (XLS 26 kb)
Rights and permissions
About this article
Cite this article
Timmer, J., Zhu, W., Pop, C. et al. Structural and kinetic determinants of protease substrates. Nat Struct Mol Biol 16, 1101–1108 (2009). https://doi.org/10.1038/nsmb.1668
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.1668
This article is cited by
-
Structural determinants of specificity and regulation of activity in the allosteric loop network of human KLK8/neuropsin
Scientific Reports (2018)
-
Cacidases: caspases can cleave after aspartate, glutamate and phosphoserine residues
Cell Death & Differentiation (2016)
-
A novel method to isolate protein N-terminal peptides from proteome samples using sulfydryl tagging and gold-nanoparticle-based depletion
Analytical and Bioanalytical Chemistry (2016)
-
Protease signalling: the cutting edge
The EMBO Journal (2012)
-
Metacaspases
Cell Death & Differentiation (2011)