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Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases

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Matrix Metalloproteinase Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 622))

Abstract

A continuous assay method, such as the one that utilizes an increase in fluorescence upon hydrolysis, allows for rapid and convenient kinetic evaluation of proteases. To better understand MMP behaviors and to aid in the design of MMP inhibitors, a variety of sequence specificity, phage display, and combinatorial chemistry studies have been performed. Results of these studies have been valuable for defining the differences in MMPs and for creating quenched fluorescent substrates that utilize fluorescence resonance energy transfer (FRET)/intramolecular fluorescence energy transfer (IFET). FRET triple-helical substrates have been constructed to examine the collagenolytic activity of MMP family members. The present chapter provides an overview of MMP and related FRET substrates and describes how to construct and utilize these substrates.

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References

  1. Overall, C. M. and Lopez-Otin, C. (2002) Strategies for MMP inhibition in cancer: innovations for the post-trial era. Nat Rev Cancer 2, 657–672.

    Article  PubMed  CAS  Google Scholar 

  2. Egeblad, M. and Werb, Z. (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2, 161–174.

    Article  PubMed  CAS  Google Scholar 

  3. Song, F., Wisithphrom, K., Zhou, J., and Windsor, L. J. (2006) Matrix metalloproteinase dependent and independent collagen degradation. Frontiers Biosci 11, 3100–3120.

    Article  CAS  Google Scholar 

  4. Fingleton, B. (2007) Matrix metalloproteinases as valid clinical targets. Curr Pharm Design 13, 333–346.

    Article  CAS  Google Scholar 

  5. Bodey, B., Bodey, J. B., Siegel, S. E., and Kaiser, H. F. (2001) Matrix metalloproteinase expression in malignant melanomas: tumor–extracellular matrix interactions in invasion and metastasis. In Vivo 15, 57–64.

    PubMed  CAS  Google Scholar 

  6. Woessner, J. F. and Nagase, H. (2000) Matrix metalloproteinases and TIMPs. Oxford: Oxford University Press.

    Google Scholar 

  7. Hofmann, U. B., Westphal, J. R., van Muijen, G. N. P., and Ruiter, D. J. (2000) Matrix metalloproteinases in human melanoma. J Invest Dermatol 115, 337–344.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  9. Nagase, H. and Fields, G. B. (1996) Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptides. Biopolymers 40, 399–416.

    Article  PubMed  CAS  Google Scholar 

  10. Fields, G. B. (2001) Using fluorogenic peptide substrates to assay matrix metalloproteinases. In Clark, I. M. (ed.), Methods in molecular biology 151: matrix metalloproteinase protocols. Totowa, NJ: Humana Press, pp. 495–518.

    Google Scholar 

  11. Ohkubo, S., Miyadera, K., Sugimoto, Y., Matsuo, K.-I., Wierzba, K., and Yamada, Y. (1999) Identification of substrate sequences for membrane type-1 matrix metalloproteinase using bacteriophage peptide display library. Biochem Biophys Res Commun 266, 308–313.

    Article  PubMed  CAS  Google Scholar 

  12. Deng, S.-J., Bickett, D. M., Mitchell, J. L., Lambert, M. H., Blackburn, R. K., Carter, H. L., III, Neugebauer, J., Pahel, G., Weiner, M. P., and Moss, M. L. (2000) Substrate specificity of human collagenase 3 assessed using a phage-displayed peptide library. J Biol Chem 275, 31422–31427.

    Article  PubMed  Google Scholar 

  13. Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C. (2001) Determination of protease cleavage site motifs using mixture-based oriented peptide libraries. Nat Biotech 19, 661–667.

    Article  CAS  Google Scholar 

  14. Kridel, S. J., Sawai, H., Ratnikov, B. I., Chen, E. I., Li, W., Godzik, A., Strongin, A. Y., and Smith, J. W. (2002) A unique substrate binding mode discriminates membrane type 1-matrix metalloproteinase (MT1-MMP) from other matrix metalloproteinases. J Biol Chem 277, 23788–23793.

    Article  PubMed  CAS  Google Scholar 

  15. Chen, E. I., Kridel, S. J., Howard, E. W., Li, W., Godzik, A., and Smith, J. W. (2002) A unique substrate recognition profile for matrix metalloproteinase-2. J Biol Chem 277, 4485–4491.

    Article  PubMed  CAS  Google Scholar 

  16. Park, H. I., Turk, B. E., Gerkema, F. E., Cantley, L. C., and Sang, Q.-X. A. (2002) Peptide substrate specificities and protein cleavage sites of human endometase/matrilysin-2/matrix metalloproteinase-26. J Biol Chem 277, 35168–35175.

    Article  PubMed  CAS  Google Scholar 

  17. Pan, W., Arnone, M., Kendall, M., Grafstrom, R. H., Seitz, S. P., Wasserman, Z. R., and Albright, C. F. (2003) Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display. J Biol Chem 278, 27820–27827.

    Article  PubMed  CAS  Google Scholar 

  18. Knight, C. G. (1995) Fluorometric assays of proteolytic enzymes. Methods Enzymol 248, 18–34.

    Article  PubMed  CAS  Google Scholar 

  19. Gershkovich, A. A. and Kholodovych, V. V. (1996) Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS). J Biochem Biophys Methods 33, 135–162.

    Article  PubMed  CAS  Google Scholar 

  20. Stack, M. S. and Gray, R. D. (1989) Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide. J Biol Chem 264, 4277–4281.

    PubMed  CAS  Google Scholar 

  21. Niedzwiecki, L., Teahan, J., Harrison, R. K., and Stein, R. L. (1992) Substrate specificity of the human matrix metalloproteinase stromelysin and the development of continuous fluorometric assays. Biochemistry 31, 12618–12623.

    Article  PubMed  CAS  Google Scholar 

  22. Knight, C. G., Willenbrock, F., and Murphy, G. (1992) A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases. FEBS Lett 296, 263–266.

    Article  PubMed  CAS  Google Scholar 

  23. Bouvier, J., Schneider, P., and Malcolm, B. (1993) A fluorescent peptide substrate for the surface metalloprotease of Leishmania. Exp Parasitol 76, 146–155.

    Article  PubMed  CAS  Google Scholar 

  24. Nagase, H., Fields, C. G., and Fields, G. B. (1994) Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3). J Biol Chem 269, 20952–20957.

    PubMed  CAS  Google Scholar 

  25. Bickett, D. M., Green, M. D., Berman, J., Dezube, M., Howe, A. S., Brown, P. J., Roth, J. T., and McGeehan, G. M. (1993) A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9). Anal Biochem 212, 58–64.

    Article  PubMed  CAS  Google Scholar 

  26. Beekman, B., Wouter, J., Bloemhoff, W., Ronday, K., Tak, P. P., and te Koppele, J. M. (1996) Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media. FEBS Lett 390, 221–225.

    Article  PubMed  CAS  Google Scholar 

  27. Geoghegan, K. F., Emery, M. J., Martin, W. H., McColl, A. S., and Daumy, G. O. (1993) Site-directed double fluorescent tagging of human renin and collagenase (MMP-1) substrate peptides using the periodate oxidation of N-terminal serine. An apparently general strategy for provision of energy-transfer substrates for proteases. Bioconjugate Chem 4, 537–544.

    Article  CAS  Google Scholar 

  28. Geoghegan, K. F. (1996) Improved method for converting an unmodified peptide to an energy transfer substrate for proteinase. Bioconjugate Chem 7, 385–391.

    Article  CAS  Google Scholar 

  29. Rakhmanova, V., Meyer, R., and Tong, X. (2007) New substrates for FRET-based assays: use of a long-wavelength fluorophore to detect the activity of MMPs. Gen Eng Biotechnol News October 15, 40.

    Google Scholar 

  30. Moss, M. L. and Rasmussen, F. H. (2007) Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening. Anal Biochem 366, 144–148.

    Article  PubMed  CAS  Google Scholar 

  31. Buchardt, J., Ferreras, M., Krog-Jensen, C., Delaisse, J.-M., Foged, N. T., and Meldal, M. (1999) Phosphinic peptide matrix metalloproteinase-9 inhibitors by solid-phase synthesis using a building block approach. Chem Eur J 5, 2877–2884.

    Article  CAS  Google Scholar 

  32. Buchardt, J., Schiodt, C. B., Krog-Jensen, C., Delaissé, J.-M., Foged, N. T., and Meldal, M. (2000) Solid phase combinatorial library of phosphinic peptides for discovery of matrix metalloproteinase inhibitors. J Comb Chem 2, 624–638.

    Article  PubMed  CAS  Google Scholar 

  33. Ito, A. S., Turchiello, R. D. F., Hirata, I. Y., Cezari, M. H. S., Meldal, M., and Juliano, L. (1999) Fluorescent properties of amino acids labeled with ortho-aminobenzoic acid. Biospectroscopy 4, 395–402.

    Article  Google Scholar 

  34. de Souzaa, E. S., Hiratab, I. Y., Julianob, L., and Itoa, A. S. (2000) End-to-end distance distribution in bradykinin observed by Förster resonance energy transfer. Biochim Biophys Acta 1474, 251–261.

    Article  Google Scholar 

  35. Takara, M. and Ito, A. S. (2005) General and specific solvent effects in optical spectra of ortho-aminobenzoic acid. J Fluores 15, 171–177.

    Article  CAS  Google Scholar 

  36. George, J., Teear, M. L., Norey, C. G., and Burns, D. D. (2003) Evaluation of an imaging platform during the development of a FRET protease assay. J Biomol Screen 8, 72–80.

    Article  PubMed  CAS  Google Scholar 

  37. Knight, C. G. (1991) A quenched fluorescent substrate for thimet peptidase containing a new fluorescent amino acid, DL-2-amino-3-(7-methoxy-4-coumaryl)propionic acid. Biochem J 274, 45–48.

    PubMed  CAS  Google Scholar 

  38. Murphy, G., Allan, J. A., Willenbrock, F., Cockett, M. I., O’Connell, J. P., and Docherty, A. J. P. (1992) The role of the C-terminal domain in collagenase and stromelysin specificity. J Biol Chem 267, 9612–9618.

    PubMed  CAS  Google Scholar 

  39. Upadhye, S. and Ananthanarayanan, V. S. (1995) Interaction of peptide substrates of fibroblast collagenase with divalent cations: Ca++ binding by substrate as a suggested recognition signal for collagenase action. Biochem Biophys Res Commun 215, 474–482.

    Article  PubMed  CAS  Google Scholar 

  40. Liko, Z., Botyanszki, J., Bodi, J., Vass, E., Majer, Z., Hollosi, M., and Suli-Vargha, H. (1996) Effect of Ca2+ on the secondary structure of linear and cyclic collagen sequence analogs. Biochem Biophys Res Commun 227, 351–359.

    Article  PubMed  CAS  Google Scholar 

  41. Fields, G. B. (1988) The application of solid phase peptide synthesis to the study of structure–function relationships in the collagen–collagenase system, Ph.D. dissertation, Florida State University, Tallahassee, 213p.

    Google Scholar 

  42. Netzel-Arnett, S., Sang, Q. X., Moore, W. G. I., Navre, M., Birkedal-Hansen, B., and VanWart, H. E. (1993) Comparative sequence specificities of human 72- and 92-kDa gelatinases (type IV collagenases) and PUMP (matrilysin). Biochemistry 32, 6427–6432.

    Article  PubMed  CAS  Google Scholar 

  43. Netzel-Arnett, S., Mallya, S. K., Nagase, H., Birkedahl-Hansen, H., and Van Wart, H. E. (1991) Continuously recording fluorescent assays optimized for five human matrix metalloproteinases. Anal Biochem 195, 86–92.

    Article  PubMed  CAS  Google Scholar 

  44. Xia, T., Akers, K., Eisen, A. Z., and Seltzer, J. L. (1996) Comparison of cleavage site specificity of gelatinases A and B using collagenous peptides. Biochim Biophys Acta 1293, 259–266.

    Article  PubMed  Google Scholar 

  45. Knäuper, V., López-Otin, C., Smith, B., Knight, G., and Murphy, G. (1996) Biochemical characterization of human collagenase-3. J Biol Chem 271, 1544–1550.

    Article  PubMed  Google Scholar 

  46. Knäuper, V., Cowell, S., Smith, B., Lopez-Otin, C., O’Shea, M., Morris, H., Zardi, L., and Murphy, G. (1997) The role of the C-terminal domain of human collagenase-3 (MMP-13) in the activation of procollagenase-3, substrate specificity, and tissue inhibitor of metalloproteinase interaction. J Biol Chem 272, 7608–7616.

    Article  PubMed  Google Scholar 

  47. Wang, Y., Johnson, A. R., Ye, Q.-Z., and Dyer, R. D. (1999) Catalytic activities and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain. J Biol Chem 274, 33043–33049.

    Article  PubMed  CAS  Google Scholar 

  48. Park, H. I., Ni, J., Gerkema, F. E., Liu, D., Belozerov, V. E., and Sang, Q.-X. A. (2000) Identification and characterization of human endometase (matrix metalloproteinase-26) from endometrial tumor. J Biol Chem 275, 20540–20544.

    Article  PubMed  CAS  Google Scholar 

  49. English, W. R., Velasco, G., Stracke, J. O., Knauper, V., and Murphy, G. (2001) Catalytic activities of membrane-type 6 matrix metalloproteinase (MMP25). FEBS Lett 491, 137–142.

    Article  PubMed  CAS  Google Scholar 

  50. Hurst, D. R., Schwartz, M. A., Ghaffari, M. A., Jin, Y., Tschesche, H., Fields, G. B., and Sang, Q.-X. A. (2004) Catalytic- and ecto-domains of membrane type 1-matrix metalloproteinase have similar inhibition profiles but distinct endopeptidase activities. Biochem J 377, 775–779.

    Article  PubMed  CAS  Google Scholar 

  51. Neumann, U., Kubota, H., Frei, K., Ganu, V., and Leppert, D. (2004) Characterization of Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, a fluorogenic substrate with increased specificity constants for collagenases and tumor necrosis factor converting enzyme. Anal Biochem 328, 166–173.

    Article  PubMed  CAS  Google Scholar 

  52. Lauer-Fields, J. L., Tuzinski, K. A., Shimokawa, K., Nagase, H., and Fields, G. B. (2000) Hydrolysis of triple-helical collagen peptide models by matrix metalloproteinases. J Biol Chem 275, 13282–13290.

    Article  PubMed  CAS  Google Scholar 

  53. Lauer-Fields, J. L., Nagase, H., and Fields, G. B. (2000) Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases. J Chromatogr A 890, 117–125.

    Article  PubMed  CAS  Google Scholar 

  54. Ottl, J., Battistuta, R., Pieper, M., Tschesche, H., Bode, W., Kühn, K., and Moroder, L. (1996) Design and synthesis of heterotrimeric collagen peptides with a built-in cystine-knot. FEBS Lett 398, 31–36.

    Article  PubMed  CAS  Google Scholar 

  55. Ottl, J., Gabriel, D., Murphy, G., Knäuper, V., Tominaga, Y., Nagase, H., Kröger, M., Tschesche, H., Bode, W., and Moroder, L. (2000) Recognition and catabolism of synthetic heterotrimeric collagen peptides by matrix metalloproteinases. Chem Biol 7, 119–132.

    Article  PubMed  CAS  Google Scholar 

  56. Ottl, J. and Moroder, L. (1999) Disulfide-bridged heterotrimeric collagen peptides containing the collagenase cleavage site of collagen type I: synthesis and conformational properties. J Am Chem Soc 121, 653–661.

    Article  CAS  Google Scholar 

  57. Lauer-Fields, J. L., Broder, T., Sritharan, T., Nagase, H., and Fields, G. B. (2001) Kinetic analysis of matrix metalloproteinase triple-helicase activity using fluorogenic substrates. Biochemistry 40, 5795–5803.

    Article  PubMed  CAS  Google Scholar 

  58. Lauer-Fields, J. L. and Fields, G. B. (2002) Triple-helical peptide analysis of collagenolytic protease activity. Biol Chem 383, 1095–1105.

    Article  PubMed  CAS  Google Scholar 

  59. Lauer-Fields, J. L., Juska, D., and Fields, G. B. (2002) Matrix metalloproteinases and collagen catabolism. Biopolymers (Pept Sci) 66, 19–32.

    Article  CAS  Google Scholar 

  60. Lauer-Fields, J. L., Kele, P., Sui, G., Nagase, H., Leblanc, R. M., and Fields, G. B. (2003) Analysis of matrix metalloproteinase activity using triple-helical substrates incorporating fluorogenic L- or D-amino acids. Anal Biochem 321, 105–115.

    Article  PubMed  CAS  Google Scholar 

  61. Lauer-Fields, J. L., Sritharan, T., Stack, M. S., Nagase, H., and Fields, G. B. (2003) Selective hydrolysis of triple-helical substrates by matrix metalloproteinase-2 and -9. J Biol Chem 278, 18140–18145.

    Article  PubMed  CAS  Google Scholar 

  62. Lauer-Fields, J. L., Nagase, H., and Fields, G. B. (2004) Development of a solid-phase assay for analysis of matrix metalloproteinase activity. J Biomol Tech 15, 305–316.

    PubMed  Google Scholar 

  63. Minond, D., Lauer-Fields, J. L., Nagase, H., and Fields, G. B. (2004) Matrix metalloproteinase triple-helical peptidase activities are differentially regulated by substrate stability. Biochemistry 43, 11474–11481.

    Article  PubMed  CAS  Google Scholar 

  64. Minond, D., Lauer-Fields, J. L., Cudic, M., Overall, C. M., Pei, D., Brew, K., Visse, R., Nagase, H., and Fields, G. B. (2006) The roles of substrate thermal stability and P2 and P1' subsite identity on matrix metalloproteinase triple-helical peptidase activity and collagen specificity. J Biol Chem 281, 38302–38313.

    Article  PubMed  CAS  Google Scholar 

  65. Minond, D., Lauer-Fields, J. L., Cudic, M., Overall, C. M., Pei, D., Brew, K., Moss, M. L., and Fields, G. B. (2007) Differentiation of secreted and membrane-type matrix metalloproteinase activities based on substitutions and interruptions of triple-helical sequences. Biochemistry 46, 3724–3733.

    Article  PubMed  CAS  Google Scholar 

  66. Lauer-Fields, J. L., Sritharan, T., Kashiwagi, M., Nagase, H., and Fields, G. B. (2007) Substrate conformation modulates aggrecanase (ADAMTS-4) affinity and sequence specificity: suggestion of a common topological specificity of functionally diverse proteases. J Biol Chem 282, 142–150.

    Article  PubMed  CAS  Google Scholar 

  67. Berndt, P., Fields, G. B., and Tirrell, M. (1995) Synthetic lipidation of peptides and amino acids: monolayer structure and properties. J Am Chem Soc 117, 9515–9522.

    Article  CAS  Google Scholar 

  68. Yu, Y.-C., Berndt, P., Tirrell, M., and Fields, G. B. (1996) Self-assembling amphiphiles for construction of protein molecular architecture. J Am Chem Soc 118, 12515–12520.

    Article  CAS  Google Scholar 

  69. Yu, Y.-C., Tirrell, M., and Fields, G. B. (1998) Minimal lipidation stabilizes protein-like molecular architecture. J Am Chem Soc 120, 9979–9987.

    Article  CAS  Google Scholar 

  70. Yu, Y.-C., Roontga, V., Daragan, V. A., Mayo, K. H., Tirrell, M., and Fields, G. B. (1999) Structure and dynamics of peptide-amphiphiles incorporating triple-helical proteinlike molecular architecture. Biochemistry 38, 1659–1668.

    Article  PubMed  CAS  Google Scholar 

  71. Fields, G. B., Lauer, J. L., Dori, Y., Forns, P., Yu, Y.-C., and Tirrell, M. (1998) Proteinlike molecular architecture: biomaterial applications for inducing cellular receptor binding and signal transduction. Biopolymers 47, 143–151.

    Article  PubMed  CAS  Google Scholar 

  72. Malkar, N. B., Lauer-Fields, J. L., Borgia, J. A., and Fields, G. B. (2002) Modulation of triple-helical stability and subsequent melanoma cellular responses by single-site substitution of fluoroproline derivatives. Biochemistry 41, 6054–6064.

    Article  PubMed  CAS  Google Scholar 

  73. Malkar, N. B., Lauer-Fields, J. L., Juska, D., and Fields, G. B. (2003) Characterization of peptide-amphiphiles possessing cellular activation sequences. Biomacromolecules 4, 518–528.

    Article  PubMed  CAS  Google Scholar 

  74. Bhaskaran, R., Palmier, M. O., Lauer-Fields, J. L., Fields, G. B., and Van Doren, S. R. (2008) MMP-12 catalytic domain recognizes triple-helical peptide models of collagen V with exosites and high activity. J Biol Chem 283, 21779–21788.

    Article  PubMed  CAS  Google Scholar 

  75. Schullek, J. R., Butler, J. H., Zhi-Jie, N., Chen, D., and Yuan, Z. (1997) A high-density screening format for encoded combinatorial libraries: assay miniaturization and its application to enzymatic reactions. Anal Biochem 246, 20–29.

    Article  PubMed  CAS  Google Scholar 

  76. Szardenings, A. K., Antonenko, V., Campbell, D. A., DeFrancisco, N., Ida, S., Shi, L., Sharkov, N., Tien, D., Wang, Y., and Navre, M. (1999) Identification of highly selective inhibitors of collagenase-1 from combinatorial libraries of diketopiperazines. J Med Chem 42, 1348–1357.

    Article  PubMed  CAS  Google Scholar 

  77. Vassiliou, S., Mucha, A., Cuniasse, P., Georgiadis, D., Lucet-Levannier, K., Beau, F., Kannan, R., Murphy, G., Knauper, V., Rio, M. C., Basset, P., Yiotakis, A., and Dive, V. (1999) Phosphinic pseudo-tripeptides as potent inhibitors of matrix metalloproteinases: a structure–activity study. J Med Chem 42, 2610–2620.

    Article  PubMed  CAS  Google Scholar 

  78. Makaritis, A., Georgiadis, D., Dive, V., and Yiotakis, A. (2003) Diastereoselective solution and multipin-based combinatorial array synthesis of a novel class of potent phosphinic metalloprotease inhibitors. Chem Eur J 9, 2079–2094.

    Article  PubMed  CAS  Google Scholar 

  79. Uttamchandani, M., Wang, J., Li, J., Hu, M., Sun, H., Chen, K. Y.-T., Liu, K., and Yao, S. Q. (2007) Inhibitor fingerprinting of matrix metalloproteases using a combinatorial peptide hydroxamate library. J Am Chem Soc 129, 7848–7858.

    Article  PubMed  CAS  Google Scholar 

  80. Schiodt, C. B., Buchardt, J., Terp, G. E., Christensen, U., Brink, M., Larsen, Y. B., Meldal, M., and Foged, N. T. (2001) Phosphinic peptide inhibitors of macrophage metalloelastase (MMP-12): selectivity and mechanism of binding. Curr Med Chem 8, 967–976.

    Article  PubMed  CAS  Google Scholar 

  81. Baronas-Lowell, D., Lauer-Fields, J. L., Borgia, J. A., Sferrazza, G. F., Al-Ghoul, M., Minond, D., and Fields, G. B. (2004) Differential modulation of human melanoma cell metalloproteinase expression by α2β1 integrin and CD44 triple-helical ligands derived from type IV collagen. J Biol Chem 279, 43503–43513.

    Article  PubMed  CAS  Google Scholar 

  82. Lauer-Fields, J. L., Minond, D., Baronas-Lowell, D., Chalmers, M. J., Busby, S. A., Griffin, P. R., Nagase, H., and Fields, G. B. (2006) Target-based proteolytic profiling for characterizing cancer progression. In Blondelle, S. E. (ed.), Understanding biology using peptides. San Diego: American Peptide Society, pp. 315–319.

    Chapter  Google Scholar 

  83. Lauer-Fields, J. L., Minond, D., Chase, P. S., Baillargeon, P. E., Saldanha, S. A., Stawikowska, R., Hodder, P., and Fields, G. B. (2009) High throughput screening of potentially selective MMP-13 exosite inhibitors utilizing a triple-helical FRET substrate. Bioorg Med Chem 17, 990–1005.

    Article  PubMed  CAS  Google Scholar 

  84. Dennis, M. S., Eigenbrot, C., Skelton, N. J., Ultsch, M. H., Santell, L., Dwyer, M. A., O’Connell, M. P., and Lazarus, R. A. (2000) Peptide exosite inhibitors of factor VIIa as anticoagulants. Nature 404, 465–470.

    Article  PubMed  CAS  Google Scholar 

  85. Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) A novel exosite on coagulation factor VIIa and its molecular interactions with a new class of peptide inhibitors. Biochemistry 40, 9522–9531.

    Article  PubMed  CAS  Google Scholar 

  86. Saghatelian, A., Jessani, N., Joseph, A., Humphrey, M., and Cravatt, B. F. (2004) Activity-based probes for the proteomic profiling of metalloproteases. Proc Natl Acad Sci USA 101, 10000–10005.

    Article  PubMed  CAS  Google Scholar 

  87. Rao, B. G. (2005) Recent developments in the design of specific matrix metalloproteinase inhibitors aided by structural and computational studies. Curr Pharm Design 11, 295–322.

    Article  CAS  Google Scholar 

  88. Kokame, K., Nobe, Y., Kokubo, Y., Okayama, A., and Miyata, T. (2005) FRETS-VWF73, a first fluorogenic substrate for ADAMTS13 assay. Br J Haematol 129, 93–100.

    Article  PubMed  CAS  Google Scholar 

  89. Hills, R., Mazzarella, R., Fok, K., Liu, M., Nemirovskiy, O., Leone, J., Zack, M. D., Arner, E. C., Viswanathan, M., Abujoub, A., Muruganandam, A., Sexton, D. J., Bassill, G. J., Sato, A. K., Malfait, A.-M., and Tortorella, M. D. (2007) Identification of an ADAMTS-4 cleavage motif using phase display leads to the development of fluorogenic peptide substrates and reveals matrillin-3 as a novel substrate. J Biol Chem 282, 11101–11109.

    Article  PubMed  CAS  Google Scholar 

  90. Wayne, G. J., Deng, S.-J., Amour, A., Borman, S., Matico, R., Carter, H. L., and Murphy, G. (2007) TIMP-3 inhibition of ADAMTS-4 (aggrecanase-1) is modulated by interactions between aggrecan and the C-terminal domain of ADAMTS-4. J Biol Chem 282, 20991–20998.

    Article  PubMed  CAS  Google Scholar 

  91. Lauer-Fields, J. L., Spicer, T. P., Chase, P. S., Cudic, M., Burstein, G. D., Nagase, H., Hodder, P., and Fields, G. B. (2008) Screening of potential ADAMTS-4 inhibitors utilizing a collagen-model FRET substrate. Anal Biochem 373, 43–51.

    Article  PubMed  CAS  Google Scholar 

  92. Wittwer, A. J., Hills, R. L., Keith, R. H., Munie, G. E., Arner, E. C., Anglin, C. P., Malfait, A.-M., and Tortorella, M. D. (2007) Substrate-dependent inhibition kinetics of an active site-directed inhibitor of ADAMTS-4 (aggrecanase 1). Biochemistry 46, 6393–6401.

    Article  PubMed  CAS  Google Scholar 

  93. Shieh, H.-S., Mathis, K. J., Williams, J. M., Hills, R. L., Wiese, J. F., Benson, T. E., Kiefer, J. R., Marino, M. H., Carroll, J. N., Leone, J. W., Malfait, A.-M., Arner, E. C., Tortorella, M. D., and Tomasselli, A. (2008) High resolution crystal structure of the catalytic domain of ADAMTS-5 (aggrecanase-2). J Biol Chem 283, 1501–1507.

    Article  PubMed  CAS  Google Scholar 

  94. Anastasi, A., Knight, C. G., and Barrett, A. J. (1993) Characterization of the bacterial metalloendopeptidase pitrilysin by use of a continuous fluorescence assay. Biochem J 290, 601–607.

    PubMed  CAS  Google Scholar 

  95. Fields, G. B., Lauer-Fields, J. L., Liu, R.-Q., and Barany, G. (2001) Principles and practice of solid-phase peptide synthesis. In Grant, G. A., (ed.), Synthetic peptides: a user’s guide (2nd ed.). New York: W.H. Freeman & Co., pp. 93–219.

    Google Scholar 

  96. Knight, C. G. (1998) Stereospecific synthesis of L-2-amino-3(7-methoxy-4-coumaryl)propionic acid, an alternative to tryptophan in quenched fluorescent substrates for peptidases. Lett Pept Sci 5, 1–4.

    CAS  Google Scholar 

  97. Kele, P., Sui, G., Huo, Q., and Leblanc, R. M. (2000) Highly enantioselective synthesis of a fluorescent amino acid. Tetrahedron Asymmetry 11, 4959–4963.

    Article  CAS  Google Scholar 

  98. Maggiora, L. L., Smith, C. W., and Zhang, Z. Y. (1992) A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase synthesis. J Med Chem 35, 3727–3730.

    Article  PubMed  CAS  Google Scholar 

  99. Drijfhout, J. W., Nagel, J., Beekman, B., Te Koppele, J. M., and Bloemhoff, W. (1996) Solid phase synthesis of peptides containing the fluorescence energy transfer Dabcyl–Edans couple. In Kaumaya, P. T. P. and Hodges, R. S., (eds.), Peptides: chemistry, structure and biology. Kingswinford: Mayflower Scientific Ltd., pp. 129–131.

    Google Scholar 

  100. Fields, C. G., and Fields, G. B. (1993) Minimization of tryptophan alkylation following 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. Tetrahedron Lett 34, 6661–6664.

    Article  CAS  Google Scholar 

  101. Lyttle, M. H., and Hudson, D. (1992) Allyl based side-chain protection for SPPS. In Smith, J. A. and Rivier, J. E., (eds.), Peptides: chemistry and biology. Leiden: Escom, pp. 583–584.

    Google Scholar 

  102. Albericio, F., Barany, G., Fields, G. B., Hudson, D., Kates, S. A., Lyttle, M. H., and Solé, N. A. (1993) Allyl-based orthogonal solid-phase peptide synthesis. In Schneider, C. H. and Eberle, A. N., (eds.), Peptides 1992. Leiden: Escom, pp. 191–193.

    Google Scholar 

  103. Bycroft, B. W., Chan, W. C., Chhabra, S. R., and Hone, N. D. (1993) A novel lysine-protecting procedure for continuous flow solid phase synthesis of branched peptides. J Chem Soc Chem Commun, 778–779.

    Google Scholar 

  104. Chhabra, S. R., Hothi, B., Evans, D. J., White, P. D., Bycroft, B. W., and Chan, W. C. (1998) An appraisal of new variants of Dde amine protecting group for solid phase peptide synthesis. Tetrahedron Lett 39, 1603–1606.

    Article  CAS  Google Scholar 

  105. Aletras, A., Barlos, K., Gatos, D., Koutsogianni, S., and Mamos, P. (1995) Preparation of the very acid-sensitive Fmoc-Lys(Mtt)-OH. Int J Pept Protein Res 45, 488–500.

    Article  PubMed  CAS  Google Scholar 

  106. King, D. S., Fields, C. G., and Fields, G. B. (1990) A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int J Pept Protein Res 36, 255–266.

    Article  PubMed  CAS  Google Scholar 

  107. Liu, Y., Kati, W., Chen, C.-M., Tripathi, R., Molla, A., and Kohlbrenner, W. (1999) Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction. Anal Biochem 267, 331–335.

    Article  PubMed  CAS  Google Scholar 

  108. Itoh, M., Osaka, M., Chiba, T., Masuda, K., Akizawa, T., Yoshioka, M., and Seiki, M. (1997) Flow injection analysis for measurement of activity of matrix metalloproteinase-7 (MMP-7). J Pharmaceut Biomed Anal 15, 1417–1426.

    Article  CAS  Google Scholar 

  109. Beekman, B., van El, B., Drijfhout, J. W., Ronday, H. K., and TeKoppele, J. M. (1997) Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay. FEBS Lett 418, 305–309.

    Article  PubMed  CAS  Google Scholar 

  110. Müller, J. C. D., Ottl, J., and Moroder, L. (2000) Heterotrimeric collagen peptides as fluorogenic collagenase substrates: synthesis, conformational properties and enzymatic digestion. Biochemistry 39, 5111–5116.

    Article  PubMed  CAS  Google Scholar 

  111. Meldal, M. and Breddam, K. (1991) Anthranilamide and nitrotyrosine as a donor–acceptor pair in internally quenched fluorescent substrates for endopeptidases: multibolumn peptide synthesis of enzyme substrates for Subtilisin carlsberg and pepsin. Anal Biochem 195, 141–147.

    Article  PubMed  CAS  Google Scholar 

  112. Sun, H., Panicker, R. C., and Yao, S. Q. (2007) Activity based fingerprinting of proteases using FRET peptides. Biopolymers (Pept Sci) 88, 141–149.

    Article  CAS  Google Scholar 

  113. Palmier, M. O., and Van Doren, S. R. (2007) Rapid determination of enzyme kinetics from fluorescence: overcoming the inner filter effect. Anal Biochem 371, 43–51.

    Article  PubMed  CAS  Google Scholar 

  114. Sui, G., Kele, P., Orbulescu, J., Huo, Q., and Leblanc, R. M. (2002) Synthesis of a coumarin based fluorescent amino acid. Lett Pept Sci 8, 47–51.

    Google Scholar 

  115. Holtz, B., Cuniasse, P., Boulay, A., Kannan, R., Mucha, A., Beau, F., Basset, P., and Dive, V. (1999) Role of the S1’ subsite glutamine 215 in activity and specificity of stromelysin-3 by site-directed mutagenesis. Biochemistry 38, 12174–12179.

    Article  PubMed  CAS  Google Scholar 

  116. Mohan, M. J., Seaton, T., Mitchell, J. L., Howe, A., Blackburn, K., Burkhart, W., Moyer, M., Patel, I., Waitt, G. M., Becherer, J. D., Moss, M. L., and Milla, M. E. (2002) The tumor necrosis factor-α converting enzyme (TACE): a unique metalloproteinase with highly defined substrate selectivity. Biochemistry 41, 9462–9469.

    Article  PubMed  CAS  Google Scholar 

  117. Crabbe, T., Willenbrock, F., Eaton, D., Hynds, P., Carne, A. F., Murphy, G., and Docherty, A. J. P. (1992) Biochemical characterization of matrilysin: activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases. Biochemistry 31, 8500–8507.

    Article  PubMed  CAS  Google Scholar 

  118. Gronski, T. J., Jr., Martin, R. L., Kobayashi, D. K., Walsh, B. C., Holman, M. C., Huber, M., Van Wart, H. E., and Shapiro, S. D. (1997) Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase. J Biol Chem 272, 12189–12194.

    Article  PubMed  CAS  Google Scholar 

  119. Rasmussen, F. H., Yeung, N., Kiefer, L., Murphy, G., Lopez-Otin, C., Vitek, M. P., and Moss, M. L. (2004) Use of a multiple-enzyme/multiple reagent assay system to quantify activity levels in samples containing mixtures of matrix metalloproteinases. Biochemistry 43, 2987–2995.

    Article  PubMed  CAS  Google Scholar 

  120. Bennett, F. A., Barlow, D. J., Dodoo, A. N. O., Hider, R. C., Lansley, A. B., Lawrence, M. J., Marriott, C., and Bansai, S. S. (1997) L-(6,7-dimethoxy-4-coumaryl)alanine: an intrinsic probe for the labelling of peptides. Tetrahedron Lett 38, 7449–7452.

    Article  CAS  Google Scholar 

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Acknowledgments

I gratefully acknowledge the support of my laboratory’s MMP research by the National Institutes of Health grants CA 98799, EB 000289, and MH 078948.

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  1. Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL, USA

    Gregg B. Fields

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  1. School of Biological Sciences, University of East Anglia, Norwich, NR47TJ, United Kingdom

    Ian M. Clark

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Fields, G.B. (2010). Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases. In: Clark, I. (eds) Matrix Metalloproteinase Protocols. Methods in Molecular Biology, vol 622. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-299-5_24

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  • DOI: https://doi.org/10.1007/978-1-60327-299-5_24

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