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Analytical and Bioanalytical Chemistry

, Volume 409, Issue 5, pp 1173–1183 | Cite as

Interspecies comparison of peptide substrate reporter metabolism using compartment-based modeling

  • Allison J. Tierney
  • Nhat Pham
  • Kunwei Yang
  • Brooks K. Emerick
  • Michelle L. Kovarik
Paper in Forefront

Abstract

Peptide substrate reporters are fluorescently labeled peptides that can be acted upon by one or more enzymes of interest. Peptide substrates are readily synthesized and more easily separated than full-length protein substrates; however, they are often more rapidly degraded by peptidases. As a result, peptide reporters must be made resistant to proteolysis in order to study enzymes in intact cells and lysates. This is typically achieved by optimizing the reporter sequence in a single cell type or model organism, but studies of reporter stability in a variety of organisms are needed to establish the robustness and broader utility of these molecular tools. We measured peptidase activity toward a peptide substrate reporter for protein kinase B (Akt) in E. coli, D. discoideum, and S. cerevisiae using capillary electrophoresis with laser-induced fluorescence (CE-LIF). Using compartment-based modeling, we determined individual rate constants for all potential peptidase reactions and explored how these rate constants differed between species. We found the reporter to be stable in D. discoideum (t 1/2 = 82–103 min) and S. cerevisiae (t 1/2 = 279–314 min), but less stable in E. coli (t 1/2 = 21–44 min). These data suggest that the reporter is sufficiently stable to be used for kinase assays in eukaryotic cell types while also demonstrating the potential utility of compartment-based models in peptide substrate reporter design.

Graphical abstract

Cell lysates from several evolutionarily divergent species were incubated with a peptide substrate reporter, and compartment-based modeling was used to determine key steps in the metabolism of the reporter in each cell type

Keywords

Amino acids/peptides Capillary electrophoresis/electrophoresis Modeling Bioanalytical methods Biological samples Enzymes 

Notes

Acknowledgments

The authors thank the Allbritton Laboratory at the University of North Carolina for generously providing advice and peptide standards, particularly Angela Proctor and Emilie Mainz for helpful discussions and their collaborator Qunzhao Wang for synthesis of the peptides. We also thank Jeremiah Marden and the Graf Laboratory at the University of Connecticut for assistance with the S. cerevisiae and E. coli cultures and lysis. This work was supported by Trinity College.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2016_85_MOESM1_ESM.pdf (399 kb)
ESM 1 (PDF 399 kb)

References

  1. 1.
    Hardie DG. Peptide assay of protein kinases and use of variant peptides to determine recognition motifs. In: Walker J, Keyse S (eds) Stress response. Totowa: Humana Press; 2000. p. 191–201.Google Scholar
  2. 2.
    Wu D, Sylvester JE, Parker LL, Zhou G, Kron SJ. Peptide reporters of kinase activity in whole cell lysates. Biopolymers. 2010;94:475–86. doi: 10.1002/bip.21401.CrossRefGoogle Scholar
  3. 3.
    Yaron A, Carmel A, Katchalski-Katzir E. Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. Anal Biochem. 1979;95:228–35. doi: 10.1016/0003-2697(79)90210-0.CrossRefGoogle Scholar
  4. 4.
    Shults MD, Imperiali B. Versatile fluorescence probes of protein kinase activity. J Am Chem Soc. 2003;125:14248–9. doi: 10.1021/ja0380502.CrossRefGoogle Scholar
  5. 5.
    Kraft M, Radke D, Wieland GD, Zipfel PF, Horn U. A fluorogenic substrate as quantitative in vivo reporter to determine protein expression and folding of tobacco etch virus protease in Escherichia coli. Protein Expr Purif. 2007;52:478–84. doi: 10.1016/j.pep.2006.10.019.
  6. 6.
    Chen C-A, Yeh R-H, Lawrence DS. Design and synthesis of a fluorescent reporter of protein kinase activity. J Am Chem Soc. 2002;124:3840–1. doi: 10.1021/ja017530v.CrossRefGoogle Scholar
  7. 7.
    Arkhipov SN, Berezovski M, Jitkova J, Krylov SN. Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells. Cytometry. 2005;63A:41–7. doi: 10.1002/cyto.a.20100.CrossRefGoogle Scholar
  8. 8.
    Wang Q, Cahill SM, Blumenstein M, Lawrence DS. Self-reporting fluorescent substrates of protein tyrosine kinases. J Am Chem Soc. 2006;128:1808–9. doi: 10.1021/ja0577692.CrossRefGoogle Scholar
  9. 9.
    Phillips RM, Bair E, Lawrence DS, Sims CE, Allbritton NL. Measurement of protein tyrosine phosphatase activity in single cells by capillary electrophoresis. Anal Chem. 2013;85:6136–42. doi: 10.1021/ac401106e.CrossRefGoogle Scholar
  10. 10.
    Turner AH, Lebhar MS, Proctor A, Wang Q, Lawrence DS, Allbritton NL. Rational design of a dephosphorylation-resistant reporter enables single-cell measurement of tyrosine kinase activity. ACS Chem Biol. 2016;11:355–62. doi: 10.1021/acschembio.5b00667.CrossRefGoogle Scholar
  11. 11.
    Rehm M, Dussmann H, Janicke RU, Tavare JM, Kogel D, Prehn JHM. Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process. Role of caspase-3. J Biol Chem. 2002;277:24506–14. doi: 10.1074/jbc.M110789200.CrossRefGoogle Scholar
  12. 12.
    Ni Q, Titov DV, Zhang J. Analyzing protein kinase dynamics in living cells with FRET reporters. Methods. 2006;40:279–86. doi: 10.1016/j.ymeth.2006.06.013.CrossRefGoogle Scholar
  13. 13.
    Ng EX, Miller MA, Jing T, Chen C-H. Single cell multiplexed assay for proteolytic activity using droplet microfluidics. Biosens Bioelectron. 2016;81:408–14. doi: 10.1016/j.bios.2016.03.002.CrossRefGoogle Scholar
  14. 14.
    Lee CL, Linton J, Soughayer JS, Sims CE, Allbritton NL. Localized measurement of kinase activation in oocytes of Xenopus laevis. Nat Biotechnol. 1999;17:759–62. doi: 10.1038/11691.
  15. 15.
    Bozinovski S, Cristiano BE, Marmy-Conus N, Pearson RB. The synthetic peptide RPRAATF allows specific assay of Akt activity in cell lysates. Anal Biochem. 2002;305:32–9. doi: 10.1006/abio.2002.5659.CrossRefGoogle Scholar
  16. 16.
    Proctor A, Wang Q, Lawrence DS, Allbritton NL. Development of a peptidase-resistant substrate for single-cell measurement of protein kinase B activation. Anal Chem. 2012;84:7195–202. doi: 10.1021/ac301489d.CrossRefGoogle Scholar
  17. 17.
    Tung CH, Mahmood U, Bredow S, Weissleder R. In vivo imaging of proteolytic enzyme activity using a novel molecular reporter. Cancer Res. 2000;60:4953–8.Google Scholar
  18. 18.
    Dragulescu-Andrasi A, Liang G, Rao J. In vivo bioluminescence imaging of furin activity in breast cancer cells using bioluminogenic substrates. Bioconjug Chem. 2009;20:1660–6. doi: 10.1021/bc9002508.CrossRefGoogle Scholar
  19. 19.
    Brown RB, Hewel JA, Emili A, Audet J. Single amino acid resolution of proteolytic fragments generated in individual cells. Cytometry A. 2010;77:347–55. doi: 10.1002/cyto.a.20880.CrossRefGoogle Scholar
  20. 20.
    Yewdell JW, Reits E, Neefjes J. Making sense of mass destruction: quantitating MHC class I antigen presentation. Nat Rev Immunol. 2003;3:952–61. doi: 10.1038/nri1250.CrossRefGoogle Scholar
  21. 21.
    Proctor A, Wang Q, Lawrence DS, Allbritton NL. Metabolism of peptide reporters in cell lysates and single cells. Analyst. 2012;137:3028–38. doi: 10.1039/c2an16162a.CrossRefGoogle Scholar
  22. 22.
    Reits E, Griekspoor A, Neijssen J, Groothuis T, Jalink K, van Veelen P, et al. Peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by MHC class I. Immunity. 2003;18:97–108.CrossRefGoogle Scholar
  23. 23.
    Yang S, Proctor A, Cline LL, Houston KM, Waters ML, Allbritton NL. β-Turn sequences promote stability of peptide substrates for kinases within the cytosolic environment. Analyst. 2013;138:4305. doi: 10.1039/c3an00874f.CrossRefGoogle Scholar
  24. 24.
    Gladfelter AS. How nontraditional model systems can save us. Mol Biol Cell. 2015;26:3687–9. doi: 10.1091/mbc.E15-06-0429.CrossRefGoogle Scholar
  25. 25.
    Page MJ, Di Cera E. Evolution of peptidase diversity. J Biol Chem. 2008;283:30010–4. doi: 10.1074/jbc.M804650200.CrossRefGoogle Scholar
  26. 26.
    Tamura Y, Niinobe M, Arima T, Okuda H, Fujii S. Aminopeptidases and arylamidases in normal and cancer tissues in humans. Cancer Res. 1975;35:1030–4.Google Scholar
  27. 27.
    Mainz ER, Serafin DS, Nguyen TT, Tarrant TK, Sims CE, Allbritton NL. Single cell chemical cytometry of Akt activity in rheumatoid arthritis and normal fibroblast-like synoviocytes in response to tumor necrosis factor α. Anal Chem. 2016;88:7786–92. doi: 10.1021/acs.analchem.6b01801.
  28. 28.
    Fey P, Dodson RJ, Basu S, Chisholm RL. One stop shop for everything Dictyostelium: dictyBase and the Dicty stock center in 2012. In: Eichinger L, Rivero F, editors. Dictyostelium discoideum protocols. Totowa: Humana Press; 2013. p. 59–92.Google Scholar
  29. 29.
    Fey P, Kowal AS, Gaudet P, Pilcher KE, Chisholm RL. Protocols for growth and development of Dictyostelium discoideum. Nat Protoc. 2007;2:1307–16. doi: 10.1038/nprot.2007.178.
  30. 30.
    De Bernardo S, Weigele M, Toome V, Manhart K, Leimgruber W, Böhlen P, et al. Studies on the reaction of fluorescamine with primary amines. Arch Biochem Biophys. 1974;163:390–9.CrossRefGoogle Scholar
  31. 31.
    Kelley CT. Iterative methods for optimization. Philadelphia: SIAM; 1999.CrossRefGoogle Scholar
  32. 32.
    Alessi DR, Cohen P. Mechanism of activation and function of protein kinase B. Curr Opin Genet Dev. 1998;8:55–62. doi: 10.1016/S0959-437X(98)80062-2.CrossRefGoogle Scholar
  33. 33.
    Proctor A, Herrera-Loeza SG, Wang Q, Lawrence DS, Yeh JJ, Allbritton NL. Measurement of protein kinase B activity in single primary human pancreatic cancer cells. Anal Chem. 2014;86:4573–80. doi: 10.1021/ac500616q.CrossRefGoogle Scholar
  34. 34.
    Fabrizio P, Pozza F, Pletcher SD, Gendron CM, Longo VD. Regulation of longevity and stress resistance by Sch9 in yeast. Science. 2001;292:288–90. doi: 10.1126/science.1059497.CrossRefGoogle Scholar
  35. 35.
    Meili R, Ellsworth C, Lee S, Reddy TB, Ma H, Firtel RA. Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium. EMBO J. 1999;18:2092–105. doi: 10.1093/emboj/18.8.2092.
  36. 36.
    Anderson DH. Compartmental modeling and tracer kinetics. New York: Springer Science & Business Media; 2013.Google Scholar
  37. 37.
    Guan S, Price JC, Ghaemmaghami S, Prusiner SB, Burlingame AL. Compartment modeling for mammalian protein turnover studies by stable isotope metabolic labeling. Anal Chem. 2012;84:4014–21. doi: 10.1021/ac203330z.CrossRefGoogle Scholar
  38. 38.
    Kuzmic P. Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem. 1996;237:260–73. doi: 10.1006/abio.1996.0238.CrossRefGoogle Scholar
  39. 39.
    Petrera A, Lai ZW, Schilling O. Carboxyterminal protein processing in health and disease: key actors and emerging technologies. J Proteome Res. 2014;13:4497–504. doi: 10.1021/pr5005746.CrossRefGoogle Scholar
  40. 40.
    Weimershaus M, Evnouchidou I, Saveanu L, van Endert P. Peptidases trimming MHC class I ligands. Curr Opin Immunol. 2013;25:90–6. doi: 10.1016/j.coi.2012.10.001.CrossRefGoogle Scholar
  41. 41.
    Milo R, Jorgensen P, Moran U, Weber G, Springer M. BioNumbers—the database of key numbers in molecular and cell biology. Nucleic Acids Res. 2010;38:D750–3. doi: 10.1093/nar/gkp889.CrossRefGoogle Scholar
  42. 42.
    Rawlings ND. A large and accurate collection of peptidase cleavages in the MEROPS database. Database (Oxford). 2009;2009:bap015. doi:  10.1093/database/bap015.
  43. 43.
    Rawlings ND, Barrett AJ, Finn R. Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2016;44:D343–50. doi: 10.1093/nar/gkv1118.CrossRefGoogle Scholar
  44. 44.
    Cousin C, Derouiche A, Shi L, Pagot Y, Poncet S, Mijakovic I. Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation. FEMS Microbiol Lett. 2013;346:11–9. doi: 10.1111/1574-6968.12189.CrossRefGoogle Scholar
  45. 45.
    Krachler AM, Woolery AR, Orth K. Manipulation of kinase signaling by bacterial pathogens. J Cell Biol. 2011;195:1083–92. doi: 10.1083/jcb.201107132.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Allison J. Tierney
    • 1
  • Nhat Pham
    • 2
  • Kunwei Yang
    • 1
  • Brooks K. Emerick
    • 2
  • Michelle L. Kovarik
    • 1
  1. 1.Department of ChemistryTrinity CollegeHartfordUSA
  2. 2.Department of MathematicsTrinity CollegeHartfordUSA

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