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Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugates

Abstract

Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis–Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD–thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.

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Figure 1: QD–peptide sensor architecture and optical characteristics of the fluorophores used.
Figure 2: FRET efficiency data from QD–peptide nanosensors.
Figure 3: Caspase-1, collagenase and chymotrypsin proteolytic assays.
Figure 4: Thrombin inhibition assay and screening for thrombin inhibitors.

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References

  1. Puente, X. S., Sanchez, L. M., Overall, C. M. & Lopez-Otin, C. Human and mouse proteases: A comparative genomic approach. Nature Rev. Genet. 4, 544–558 (2003).

    Article  Google Scholar 

  2. Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).

    Article  Google Scholar 

  3. Vihinen, P., Ala-Aho, R. & Kahari, V. M. Matrix metalloproteinases as therapeutic targets in cancer. Curr. Cancer Drug Targets 5, 203–220 (2005).

    Article  Google Scholar 

  4. Richard, I. The genetic and molecular bases of monogenic disorders affecting proteolytic systems. J. Med. Genet. 42, 529–539 (2005).

    Article  Google Scholar 

  5. Wu, Y. M., Wang, X. Y., Liu, X. & Wang, Y. F. Data-mining approaches reveal hidden families of proteases in the genome of malaria parasite. Genome Res. 13, 601–616 (2003).

    Article  Google Scholar 

  6. Shao, F., Merritt, P. M., Bao, Z. Q., Innes, R. W. & Dixon, J. E. A Yersinia effector and a pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis. Cell 109, 575–588 (2002).

    Article  Google Scholar 

  7. Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J. R. & Hilgenfeld, R. Coronavirus main proteinase (3CL(pro)) structure: Basis for design of anti-SARS drugs. Science 300, 1763–1767 (2003).

    Article  Google Scholar 

  8. Mahajan, N. P., Harrison-Shostak, D. C., Michaux, J. & Herman, B. Novel mutant green fluorescent protein protease substrates reveal the activation of specific caspases during apoptosis. Chem. Biol. 6, 401–409 (1999).

    Article  Google Scholar 

  9. Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. Creating new fluorescent probes for cell biology. Nature Rev. Mol. Cell Biol. 3, 906–918 (2002).

    Article  Google Scholar 

  10. Rodems, S. M. et al. A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay Drug Dev. Technol. 1, 9–19 (2002).

    Article  Google Scholar 

  11. Miyawaki, A., Sawano, A. & Kogure, T. Lighting up cells: labelling proteins with fluorophores. Nature Cell Biol. 5, S1–S7 (2003).

    Article  Google Scholar 

  12. Miyawaki, A. Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell 4, 295–305 (2003).

    Article  Google Scholar 

  13. Lakowicz, J. R. Principles of Fluorescence Spectroscopy 2nd edn (Kluwer Academic/Plenum, 1999).

    Book  Google Scholar 

  14. Michalet, X. et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307, 538–544 (2005).

    Article  Google Scholar 

  15. Medintz, I., Uyeda, H., Goldman, E. & Mattoussi, H. Quantum dot bioconjugates for imaging, labelling and sensing. Nature Mater. 4, 435–446 (2005).

    Article  Google Scholar 

  16. Bruchez, M. Jr, Moronne, M., Gin, P., Weiss, S. & Alivisatos, A. P. Semiconductor nanocrystals as fluorescent biological labels. Science 281, 2013–2016 (1998).

    Article  Google Scholar 

  17. Clapp, A. R. et al. Fluorescence resonance energy transfer between quantum dot donors and dye-labeled protein acceptors. J. Am. Chem. Soc. 126, 301–310 (2004).

    Article  Google Scholar 

  18. Goldman, E. et al. A hybrid quantum dot-antibody fragment fluorescence resonance energy transfer-based TNT sensor. J. Am. Chem. Soc. 127, 6744–6751 (2005).

    Article  Google Scholar 

  19. Medintz, I. L. et al. Self-assembled nanoscale biosensors based on quantum dot FRET donors. Nature Mater. 2, 630–638 (2003).

    Article  Google Scholar 

  20. Zhang, C. Y., Yeh, H. C., Kuroki, M. T. & Wang, T. H. Single-quantum-dot-based DNA nanosensor. Nature Mater. 4, 826–831 (2005).

    Article  Google Scholar 

  21. Patolsky, F. et al. Lighting-up the dynamics of telomerization and DNA replication by CdSe–ZnS quantum dots. J. Am. Chem. Soc. 125, 13918–13919 (2003).

    Article  Google Scholar 

  22. Levy, M., Cater, S. F. & Ellington, A. D. Quantum-dot aptamer beacons for the detection of proteins. Chem. Biochem. 6, 1–4 (2005).

    Google Scholar 

  23. Cohen, G. M. Caspaces: the executioners of apoptosis. Biochem. J. 326, 1–16 (1997).

    Article  Google Scholar 

  24. Thornberry, N. A. et al. A novel heterodimeric cysteine protease is required for interleukin-1-beta processing in monocytes. Nature 356, 768–774 (1992).

    Article  Google Scholar 

  25. 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).

    Article  Google Scholar 

  26. Linkins, L. A. & Weitz, J. I. New anticoagulant therapy. Ann. Rev. Med. 56, 63–77 (2005).

    Article  Google Scholar 

  27. Gurm, H. S. & Bhatt, D. L. Thrombin, an ideal target for pharmacological inhibition: A review of direct thrombin inhibitors. Am. Heart J. 149, S43–S53 (2005).

    Article  Google Scholar 

  28. Steinberg, S. F. The cardiovascular actions of protease-activated receptors. Mol. Pharmacol. 67, 2–11 (2005).

    Article  Google Scholar 

  29. Lombard, C., Saulnier, J. & Wallach, J. Assays of matrix metalloproteinases (MMPs) activities: a review. Biochimie 87, 265–272 (2005).

    Article  Google Scholar 

  30. Ala-Aho, R. & Kahari, V. M. Collagenases in cancer. Biochimie 87, 273–286 (2005).

    Article  Google Scholar 

  31. Matter, H. & Schudok, M. Recent advances in the design of matrix metalloprotease inhibitors. Curr. Opin. Drug Discovery Devel. 7, 513–535 (2004).

    Google Scholar 

  32. Schnolzer, M., Jedrzejewski, P. & Lehmann, W. D. Protease-catalyzed incorporation of O-18 into peptide fragments and its application for protein sequencing by electrospray and matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 17, 945–953 (1996).

    Article  Google Scholar 

  33. Medintz, I. L. et al. A fluorescence resonance energy transfer derived structure of a quantum dot-protein bioconjugate nanoassembly. Proc. Natl Acad. Sci. USA 101, 9612–9617 (2004).

    Article  Google Scholar 

  34. Hainfeld, J. F., Liu, W., Halsey, C., Freimuth, P, M. R. & Powell, R., D. Ni-NTA-gold clusters target His-tagged proteins. J. Struct. Biol. 127, 185–198 (1999).

    Article  Google Scholar 

  35. Sandros, M. G., Gao, D., Gokdemir, C. & Benson, D. E. General, high-affinity approach for the synthesis of fluorophore appended protein nanoparticle assemblies. Chem. Commun. 22, 2832–2834 (2005).

    Article  Google Scholar 

  36. Grimmett, G. & Stirzaker, D. Probability and Random Processes 2nd edn (Oxford Univ. Press, Oxford, 1992).

    Google Scholar 

  37. Sapsford, K. E. et al. Surface-immobilized self-assembled protein-based quantum dot nanoassemblies. Langmuir 20, 7720–7728 (2004).

    Article  Google Scholar 

  38. Dabbousi, B. O. et al. (CdSe)ZnS core-shell quantum dots: synthesis and optical and structural characterization of a size series of highly luminescent materials. J. Phys. Chem. B 101, 9463–9475 (1997).

    Article  Google Scholar 

  39. Vencill, C. F., Rasnick, D., Crumley, K. V., Nishino, N. & Powers, J. C. Clostridium-Histolyticum Collagenase–development of new thio ester, fluorogenic, and depsipeptide substrates and new inhibitors. Biochemistry 24, 3149–3157 (1985).

    Article  Google Scholar 

  40. Simon, L. M., Kotorman, M., Szabo, A., Garab, G. & Laczko, I. Effects of polyethylene glycol on stability of alpha-chymotrypsin in aqueous ethanol solvent. Biochem. Biophys. Res. Commun. 317, 610–613 (2004).

    Article  Google Scholar 

  41. Bowden, A. C. Fundamentals of Enzyme Kinetics Revised edn (Portland Press, London, 1995).

    Google Scholar 

  42. Giegel, D. A. ICE processing and kinetic mechanism. J. Cellular Biochem. 64, 11–18 (1997).

    Article  Google Scholar 

  43. Szabelski, M., Rogiewicz, M. & Wiczk, W. Fluorogenic peptide substrates containing benzoxazol-5-yl-alanine derivatives for kinetic assay of cysteine proteases. Anal. Biochem. 342, 20–27 (2005).

    Article  Google Scholar 

  44. Santoro, M. F. et al. Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis. J. Biol. Chem. 273, 13119–13128 (1998).

    Article  Google Scholar 

  45. Pinaud, F., King, D., Moore, H.-P. & Weiss, S. Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. J. Am. Chem. Soc. 126, 6115–6123 (2004).

    Article  Google Scholar 

  46. Chen, L. M. et al. Design and validation of a bifunctional ligand display system for receptor targeting. Chem. Biol. 11, 1081–1091 (2004).

    Article  Google Scholar 

  47. Clapp, A. R. et al. Quantum dot-based multiplexed fluorescence resonance energy transfer. J. Am. Chem. Soc. 127, 18212–18221 (2005).

    Article  Google Scholar 

  48. Mattoussi, H. et al. Self-assembly of CdSe–ZnS quantum dot bioconjugates using an engineered recombinant protein. J. Am. Chem. Soc. 122, 12142–12150 (2000).

    Article  Google Scholar 

  49. Peng, Z. A. & Peng, X. Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor. J. Am. Chem. Soc. 123, 183–184 (2001).

    Article  Google Scholar 

  50. Gaffney, P. J. & Edgell, T. A. The International and NIH units for thrombin – How do they compare. Thrombosis Haemostasis 74, 900–903 (1995).

    Article  Google Scholar 

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Acknowledgements

The authors acknowledge NRL and L. Chrisey at the Office of Naval Research (ONR grant No N001404WX20270) and A. Krishnan at DARPA for support. A.R.C. is supported by a National Research Council Fellowship through NRL. P.E.D. acknowledges the Skaggs Institute for Chemical Biology. The authors thank E. Alnemri of the Kimmel Cancer Institute, Thomas Jefferson University for advice about caspase recognition sequences. The authors also thank J.M. Mauro (Invitrogen) for useful discussions.

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Correspondence to Igor L. Medintz or Hedi Mattoussi.

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Medintz, I., Clapp, A., Brunel, F. et al. Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugates. Nature Mater 5, 581–589 (2006). https://doi.org/10.1038/nmat1676

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