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In Vitro Assay for Protease Activity of Proprotein Convertase Subtilisin Kexins (PCSKs): An Overall Review of Existing and New Methodologies

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Book cover Proprotein Convertases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 768))

Abstract

The mammalian proprotein convertase subtilisin kexins (PCSKs) previously called proprotein or prohormone convertases (PCs) are a family of Ca+2-dependent endoproteases in the subtilisin family. These proteolytic enzymes exert their many crucial physiological and biological functions in vivo via their ability to cleave larger inactive precursor proteins into their biologically active mature forms. This event takes place in a highly efficient and selective manner. Such actions of PCSKs either alone or in combination to cleave specific protein bonds are the hallmark events that not only define the normal functions and metabolism of the body but also may lead to a variety of diseases or disorders with associated conditions. These include among others, diabetes, obesity, cancer, cardiovascular diseases, reproduction abnormalities as well as viral bacterial infections. These conditions were the direct consequences of an enhanced level of enzymatic activity of one or more PCSKs except only PCSK9, whose protease activity in relation to its physiological substrate has yet to be characterized. Owing to this finding, a large number of research studies have been exclusively devoted to develop rapid, efficient and reliable in vitro methods for examining the protease activity of these enzymes. Several assays have been developed to monitor PCSK activity and these are widely used in chemical, biochemical, cellular and animal studies. This review will cover various methodologies and protocols that are currently available in the literature for PCSK activity assays. These include liquid phase methods using fluorogenic, chromogenic and intramolecularly quenched fluorescent substrates as well as a newly developed novel solid phase fluorescence method. This review will also highlight the usefulness of these methodologies and finally a comparative analysis has been made to examine their merits and demerits with some key examples.

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References

  1. Seidah, N. G., and Prat, A. (2002) Precursor convertases in the secretory pathway, cytosol and extracellular milieu Essays Biochem 38, 79–94.

    PubMed  CAS  Google Scholar 

  2. Seidah, N. G., and Chrétien, M. (1999) Proprotein and prohormone convertases: A family of subtilases generating diverse bioactive polypeptides Brain Res 848, 45–62.

    PubMed  CAS  Google Scholar 

  3. Steiner, D. F. (1998) The proprotein convertases Curr Opin Chem Biol 2, 31–9.

    PubMed  CAS  Google Scholar 

  4. Steiner, D. F. (1967) Evidence for a precursor in the biosynthesis of insulin Trans NY Acad Sci 30, 60–8.

    CAS  Google Scholar 

  5. Chrétien, M., and Li, C. H. (1967) Isolation, purification, and characterization of gamma-lipotropic hormone from sheep pituitary glands Can J Biochem 45, 1163–74.

    PubMed  Google Scholar 

  6. Kovac, S., Shulkes, A., and Baldwin, G. S. (2009) Peptide processing and biology in human disease Curr Opin Endocrinol Diabetes Obes 16, 79–85.

    PubMed  CAS  Google Scholar 

  7. Seidah, N. G., Mayer, G., Zaid, A., Rousselet, E., Nassoury, N. et al. (2008) The activation and physiological functions of the proprotein convertases Int J Biochem Cell Biol 40, 1111–25.

    PubMed  CAS  Google Scholar 

  8. Seidah, N. G., Gaspar, L., Mion, P., Marcinkiewicz, M., Mbikay, M., and Chrétien, M. (1990) cDNA sequence of two distinct pituitary proteins homologous to Kex2 and furin gene products: Tissue-specific mRNAs encoding candidates for pro-hormone processing proteinases DNA Cell Biol 9, 415–24.

    PubMed  CAS  Google Scholar 

  9. Smeekens, S. P., and Steiner, D. F. (1990) Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2 J Biol Chem 265, 2997–3000.

    PubMed  CAS  Google Scholar 

  10. Kiefer, M. C., Tucker, J. E., Joh, R., Landsberg, K. E., Saltman, D., and Barr, P. J. (1991) Identification of a second human subtilisin-like protease gene in the fes/fps region of chromosome DNA Cell Biol 10, 757–69.

    PubMed  CAS  Google Scholar 

  11. van de Ven, W. J., Voorberg, J., Fontijn, R., Pannekoek, H., van den Ouweland, A. M., van Duijnhoven, H. L., Roebroek, A. J., and Siezen, R. J. (1990) Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes Mol Biol Rep 14, 265–75.

    PubMed  Google Scholar 

  12. Seidah, N. G., Day, R., Hamelin, J., Gaspar, A., Collard, M. W., and Chrétien, M. (1992) Testicular expression of PC4 in the rat: Molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase Mol Endocrinol 6, 1559–70.

    PubMed  CAS  Google Scholar 

  13. Nakayama, K., Kim, W. S., Torii, S., Hosaka, M., Nakagawa, T., Ikemizu, J., Baba, T., and Murakami, K. (1992) Identification of the fourth member of the mammalian endoprotease family homologous to the yeast Kex2 protease. Its testis-specific expression J Biol Chem 267, 5897–900.

    PubMed  CAS  Google Scholar 

  14. Lusson, J., Vieau, D., Hamelin, J., Day, R., Chrétien, M., and Seidah, N. G. (1993) cDNA structure of the mouse and rat subtilisin/kexin-like PC5: A candidate proprotein convertase expressed in endocrine and nonendocrine cells Proc Natl Acad Sci USA 90, 6691–5.

    PubMed  CAS  Google Scholar 

  15. Miranda, L., Wolf, J., Pichuantes, S., Duke, R., and Franzusoff, A. (1996) Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes Proc Natl Acad Sci USA 93, 7695–700.

    PubMed  CAS  Google Scholar 

  16. Rehemtulla, A., Barr, P. J., Rhodes, C. J., and Kaufman, R. J. (1993) PACE4 is a member of the mammalian propeptidase family that has overlapping but not identical substrate specificity to PACE Biochemistry 32, 11586–90.

    PubMed  CAS  Google Scholar 

  17. Bruzzaniti, A., Goodge, K., Jay, P., Taviaux, S. A., Lam, M. H., Berta, P., Martin, T. J., Moseley, J. M., and Gillespie, M. T. (1996) PC8 [corrected], a new member of the convertase family Biochem J 314, 727–31.

    PubMed  CAS  Google Scholar 

  18. Meerabux, J., Yaspo, M. L., Roebroek, A. J., Van de Ven, W. J., Lister, T. A., and Young, B. D. (1996) A new member of the proprotein convertase gene family (LPC) is located at a chromosome translocation breakpoint in lymphomas Cancer Res 56, 448–51.

    PubMed  CAS  Google Scholar 

  19. Seidah, N. G., Hamelin, J., Mamarbachi, M., Dong, W., Tardos, H., Mbikay, M., Chrétien, M., and Day, R. (1996) cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases Proc Natl Acad Sci USA 93, 3388–93.

    PubMed  CAS  Google Scholar 

  20. Seidah, N. G., Hamelin, J., Mamarbachi, A. M., Benjannet, S., Toure, B. B., Basak, A., Munzer, J. S., Marcinkiewicz, M., Zhong, M., Barale, J.-C., Lazure, C., Murphy, R. A., Chrétien, M., and Marcinkiewicz, M. (1999) Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization Proc Natl Acad Sci USA 96, 1321–6.

    PubMed  CAS  Google Scholar 

  21. Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999) Secreted site-1 protease cleaves peptides corresponding to luminal loop of sterol regulatory element-binding proteins J Biol Chem 274, 22805–12.

    PubMed  CAS  Google Scholar 

  22. Seidah, N. G., Benjannet, S., Wickham, L., Marcinkiewicz, J., Bélanger Jasmin, S., Stifani, S., Basak, A., Prat, A., and Chrétien, M. (2003) The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation Proc Natl Acad Sci USA 100, 928–33.

    PubMed  CAS  Google Scholar 

  23. Khatib, A. M., Siegfried, G., Chrétien, M., Metrakos, P., and Seidah, N. G. (2002) Proprotein convertases in tumor progression and malignancy: Novel targets in cancer therapy Am J Pathol 160, 1921–35.

    PubMed  CAS  Google Scholar 

  24. Bassi, D. E., Fu, J., Lopez de Cicco, R., and Klein-Szanto, A. J. (2005) Proprotein convertases: “master switches” in the regulation of tumor growth and progression Mol Carcinog 44, 151–61.

    PubMed  CAS  Google Scholar 

  25. Chrétien, M., Seidah, N. G., Basak, A., and Mbikay, M. (2008) Proprotein convertases as therapeutic targets Expert Opin Ther Targets 12, 1289–300.

    PubMed  Google Scholar 

  26. Scamuffa, N., Calvo, F., Chrétien, M., Seidah, N. G., and Khatib, A. M. (2006) Proprotein convertases: Lessons from knockouts FASEB J 20, 1954–63.

    PubMed  CAS  Google Scholar 

  27. Kourimate, S., Chétiveaux, M., Jarnoux, A. L., Lalanne, F., and Costet, P. (2009) Cellular and secreted pro-protein convertase subtilisin/kexin type 9 catalytic activity in hepatocytes Atherosclerosis 206, 134–40.

    PubMed  CAS  Google Scholar 

  28. Mousavi, S. A., Berge, K. E., and Leren, T. P. (2009) The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis J Intern Med 266, 507–19.

    PubMed  CAS  Google Scholar 

  29. Stawowy, P., Blaschke, F., Kilimnik, A., Goetze, S., Kallisch, H., Chrétien, M., Marcinkiewicz, M., Fleck, E., and Graf, K. (2002) Proprotein convertase PC5 regulation by PDGF-BB involves PI3-kinase/p70s6-kinase activation in vascular smooth muscle cells Hypertension 39, 399–404.

    PubMed  CAS  Google Scholar 

  30. Ugleholdt, R., Poulsen, M. L., Holst, P. J., Irminger, J. C., Orskov, C., Pedersen, J., Rosenkilde, M. M., Zhu, X., Steiner, D. F., and Holst, J. J. (2006) Prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor J Biol Chem 281, 11050–7.

    PubMed  CAS  Google Scholar 

  31. Gyamera-Acheampong, C., and Mbikay, M. (2009) Proprotein convertase subtilisin/kexin type 4 in mammalian fertility: A review Hum Reprod Update 15, 237–47.

    PubMed  CAS  Google Scholar 

  32. Qiu, Q., Basak, A., Mbikay, M., Tsang, B. K., and Gruslin, A. (2005) Role of pro-IGF-II processing by proprotein convertase 4 in human placental development Proc Natl Acad Sci USA 102, 11047–52.

    PubMed  CAS  Google Scholar 

  33. Shiryaev, S. A., Remacle, A. G., Ratnikov, B. I., Nelson, N. A., Savinov, A. Y., Wei, G., Bottini, M., Rega, M. F., Parent, A., Desjardins, R., Fugere, M., Day, R., Sabet, M., Pellecchia, M., Liddington, R. C., Smith, J. W., Mustelin, T., Guiney, D. G., Lebl, M., and Strongin, A. Y. (2007) Targeting host cell furin proprotein convertases as a therapeutic strategy against bacterial toxins and viral pathogens J Biol Chem 282, 20847–53.

    PubMed  CAS  Google Scholar 

  34. Patra, D., Xing, X., Davies, S., Bryan, J., Franz, C., Hunziker, E. B., and Sandell, L. J. (2007) Site-1 protease is essential for endochondral bone formation in mice J Cell Biol 179, 687–700.

    PubMed  CAS  Google Scholar 

  35. Constam, D. B., Calfon, M., and Robertson, E. J. (1996) SPC4, SPC6, and the novel protease SPC7 are coexpressed with bone morphogenetic proteins at distinct sites during embryogenesis J Cell Biol 134, 181–91.

    PubMed  CAS  Google Scholar 

  36. Seidah, N. G., Khatib, A. M., and Prat, A. (2006) The proprotein convertases and their implication in sterol and/or lipid metabolism Biol Chem 387, 871–7.

    PubMed  CAS  Google Scholar 

  37. Seidah, N. G., and Prat, A. (2007) The proprotein convertases are potential targets in the treatment of dyslipidemia J Mol Med 85, 685–96.

    PubMed  CAS  Google Scholar 

  38. Gordon, V. M., and Leppla, S. H. (1994) Proteolytic activation of bacterial toxins: Role of bacterial and host cell proteases Infect Immun 62, 333–40.

    PubMed  CAS  Google Scholar 

  39. Jasinski, J. P., and Woudenberg, R. C. (1994) 7-Amino-4-methylcoumarin Acta Crystallogr C50, 1954–6.

    CAS  Google Scholar 

  40. Bissell, E. R., Mitchell, A. R., and Smith, R. E. (1980) Synthesis and chemistry of 7-amino-4-(trifluoromethyl) coumarin and its amino acid and peptide derivatives J Org Chem 45, 2283–7.

    CAS  Google Scholar 

  41. Izquierdo, C., and Burguillo, J. (1989) Synthetic substrates for thrombin Int J Biochem 21, 579–92.

    PubMed  CAS  Google Scholar 

  42. Sedding, D. G., Homann, M., Seay, U., Tillmanns, H., Preissner, K. T., and Braun-Dullaeus, R. C. (2008) Calpain counteracts mechanosensitive apoptosis of vascular smooth muscle cells in vitro and in vivo FASEB J 22, 579–89.

    PubMed  CAS  Google Scholar 

  43. Onderwater, R. C., Venhorst, J., Commandeur, J. N., and Vermeulen, N. P. (1999) Design, Synthesis, and characterization of 7-methoxy-4-(aminomethyl) coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening Chem Res Toxicol 12, 555–9.

    PubMed  CAS  Google Scholar 

  44. Ledgerwood, E. C., Brennan, S. O., and George, P. M. (1997) Endoproteases other than furin have a role in hepatic proprotein processing IUBMB Life 42, 1131–42.

    CAS  Google Scholar 

  45. Lopez-Perez, E., Seidah, N. G., and Checler, F. (1999) Proprotein convertase activity contributes to the processing of the Alzheimer’s β-amyloid precursor protein in human cells: Evidence for a role of the prohormone convertase PC7 in the constitutive α-secretase pathway J Neurochem 73, 2056–62.

    PubMed  CAS  Google Scholar 

  46. Pasquato, A., Pullikotil, P., Asselin, M. C., Vacatello, M., Paolillo, L., Ghezzo, F., Basso, F., Di Bello, C., Dettin, M., and Seidah, N. G. (2006) The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors J Biol Chem 281, 23471–81.

    PubMed  CAS  Google Scholar 

  47. Bayer, E., and Mutter, M. (1972) Liquid phase synthesis of peptides Nature 237, 512–13.

    PubMed  CAS  Google Scholar 

  48. Basak, S., Stewart, N. A., Chrétien, M., and Basak, A. (2004) Aminoethyl benzenesulfonyl fluoride and its hexapeptide (Ac-VFRSLK) conjugate are both in vitro inhibitors of subtilisin kexin isozyme-1 FEBS Lett 573, 186–94.

    PubMed  CAS  Google Scholar 

  49. Scott, R. P. W. (1993) Silica Gel and Bonded Phases. Their Production, Properties and Use in LC. Wiley, Chichester, Separation Science Series.

    Google Scholar 

  50. Stewart, J. M., and Young, J. D. (1984) Solid Phase Peptide Synthesis (2nd ed.). Pierce Chemical Company, Rockford, IL.

    Google Scholar 

  51. Technical bulletin (1998, May) “Cleavage, Deprotection, and Isolation of Peptides after Fmoc Synthesis”. PE-Biosystems web site: http://www3.appliedbiosystems.com/cms/groups/psm_marketing/documents/generaldocuments/cms_040654.pdf.

  52. Thomas, G. (2002) Furin at the cutting edge: From protein traffic to embryogenesis and disease Nat Rev Mol Cell Biol 3, 753–66.

    PubMed  CAS  Google Scholar 

  53. Tanga, S. S., Zhangc, J. H., Liud, H. X., and Li, H. Z. (2009) PC2/CPE-mediated pro-protein processing in tumor cells and its differentiated cells or tissues Mol Cell Endocrinol 303, 43–9.

    Google Scholar 

  54. Hall, T., Kam, F., Min, F., Liu, M., Zobel, J. F., Marino, M. H., Malfait, A. M., Tortorella, M. D., and Tomasselli, A. G. (2007) A high performance liquid chromatography assay for monitoring proprotein convertase activity J Chromatogr A 1148, 46–54.

    PubMed  CAS  Google Scholar 

  55. Malfait, A. M., Arner, E. C., Song, R. H., Alston, J. T., Markosyan, S., Staten, M., Yang, Z., Griggs, D. W., and Tortorella, M. D. (2008) Proprotein convertase activation of aggrecanases in cartilage in situ Arch Biochem Biophys 478, 43–51.

    PubMed  CAS  Google Scholar 

  56. Park, K., Kuechle, M. K., Choe, Y., Craik, C. S., Lawrence, O. Y., and Presland, R. B. (2006) Expression and characterization of constitutively active human caspase-14 Biochem Biophys Res Commun 347, 941–8.

    PubMed  CAS  Google Scholar 

  57. Cox, S. W., Cho, K., Eley, B. M., and Smith, R. E. (2006) A simple, combined fluorogenic and chromogenic method for the assay of proteases in gingival crevicular fluid J Periodontal Res 25, 164–71.

    Google Scholar 

  58. St. Leger, R. J., Joshi, L., Bidochka, M. J., Rizzo, N. W., and Roberts, D. W. (1996) Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles Appl Environ Microbiol 62, 1257–64.

    Google Scholar 

  59. Cox, S. W., and Eley, B. M. J. (1989) Detection of cathepsin B and L, elastase, tryptase, trypsin, and dipeptidyl peptidase IV-like activities in crevicular fluid from gingivitis and periodontitis patients with peptidyl derivatives of 7-amino-4-trifluoromethyl coumarin Periodontal Res 24, 353–1.

    CAS  Google Scholar 

  60. Lojda, Z. (1996) The use of substrates with 7-amino-3-trifluoromethylcoumarine (AFC) leaving group in the localization of protease activities in situ Acta Histochem 98, 215–28.

    PubMed  CAS  Google Scholar 

  61. Patent inventors: Chelsky, Daniel (Moylan, PA) Burbaum (Cranbury, NJ): http://www.freepatentsonline.com/5856083.html.

  62. Miller, J. N. (2008) Advances in Fluorescence Enzyme Detection Methods. In Standardization and Quality Assurance in Fluorescence Measurements II, Springer Series on Fluorescence. Springer, Berlin, Heidelberg.

    Google Scholar 

  63. Foley, J. D., Rosenbaum, H., and Griep, A. E. (2004) Temporal regulation of VEID-7-amino-4-trifluoromethylcoumarin cleavage activity and caspase-6 correlates with organelle loss during lens development J Biol Chem 279, 32142–50., and video journal: Cupp-Enyard, C. (2009), Use of the protease fluorescent detection kit to determine protease activity J Vis Exp 30; http://www.jove.com/index/Details.stp?ID=1514.

    PubMed  CAS  Google Scholar 

  64. Albani, J. R. (2007) Principles and Applications of Fluorescence Spectroscopy. Wiley-Blackwell, Oxford.

    Google Scholar 

  65. Scully, M. F., and Kakkar, V. V. (Eds) (1977) Chromogenic peptide substrates: chemistry and clinical usage/International Symposium on Chromogenic Substrates, King’s College Hospital Medical School.

    Google Scholar 

  66. Sharma, N., Liu, S., Tang, L., Irwin, J., Meng, G., and Rancourt, D. E. (2006) Implantation serine proteinases heterodimerize and are critical in hatching and implantation BMC Dev Biol 6, 61–5.

    PubMed  Google Scholar 

  67. Gore, M. G. (Ed) (2000) Spectrophotometry and Spectrofluorometry: A Practical Approach. Oxford University Press, Oxford.

    Google Scholar 

  68. Clegg, R. M. (1995) Fluorescence resonance energy transfer Curr Opin Biotechnol 6, 103–10.

    PubMed  CAS  Google Scholar 

  69. Cotrin, S. S., Puzer, L., de Souza, W. A., Juliano, J. L., Carmona, A. K., and Juliano, M. A. (2004) Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides to define substrate specificity of carboxydipeptidases: Assays with human cathepsin Anal Biochem 335, 244–52.

    PubMed  CAS  Google Scholar 

  70. Mitobe, J., Morita-Ishihara, T., Ishihama, A., and Watanabe, H. (2009) Involvement of RNA-binding protein Hfq in the osmotic-response regulation of invE gene expression in Shigella sonne BMC Microbiol 9, 110–16.

    Google Scholar 

  71. Basak, A., Zhong, M., Munzer, J. S., Chrétien, M., and Seidah, N. G. (2001) Implication of the proprotein convertases Furin, PC5 and PC7 in the cleavage of surface glycoproteins of Hong Kong, Ebola and Respiratory Syncytial viruses – a comparative analysis using fluorogenic peptides Biochem J 353, 537–45.

    PubMed  CAS  Google Scholar 

  72. Carmona, A. K., Schwager, S. L., Juliano, M. A., Juliano, L., and Sturrock, E. D. (2006) A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay Nat Protocol 1, 1971–6.

    CAS  Google Scholar 

  73. Basak, S., Mohottalage, D., and Basak, A. (2006) Multibranch and Pseudopeptide approach for design of novel inhibitors of Subtilisin Kexin Isozyme-1 Prot Pept Lett 13, 863–76.

    CAS  Google Scholar 

  74. Oliveira, M. C. F., Hirata, I. Y., Chagas, J. R., Boschcov, P., Gomes, R. A. S., Figueiredo, A. F. S., and Juliano, L. (1992) Intramolecularly quenched fluorogenic peptide substrates for human rennin Anal Biochem 203, 39–46.

    PubMed  CAS  Google Scholar 

  75. James, J., Schmidt, J., and Robert, G. (2003) Fluorigenic substrates for the protease activities of botulinum neurotoxins, serotypes A, B, and F Appl Environ Microbiol 69, 297–303.

    Google Scholar 

  76. Paschalidou, K., Neumann, U., Gerhartz, B., and Tzougraki, C. (2004) Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of L-2-amino-3-(7-methoxy-4-coumaryl) propionic acid with 2,4-dinitrophenyl groups at various positions Biochem J 382, 1031–8.

    PubMed  CAS  Google Scholar 

  77. Kennelly, P. J. (2001) Protein phosphatases – a phylogenetic perspective Chem Rev 101, 2291–312.

    PubMed  CAS  Google Scholar 

  78. Goudreau, N., Guis, C., Soleilhac, J. M., and Roques, B. P. (1994) Dns-Gly-(p-NO2)Phe-βAla, a specific fluorogenic substrate for neutral endopeptidase 24.11 Anal Biochem 219, 87–95.

    PubMed  CAS  Google Scholar 

  79. Rakhmanova, V., Po, P., and Meyer, R. Technical bulletin, A sensitive fluorimetric assay for detection of ß-secretase activity http://www.biocompare.com/Articles/ApplicationNote/1593/A-SensitiveFluorimetric-Assay-For-Detection-Of-Secretase-Activity.html.

  80. Diwu, Z., Xiang, Q., He, J., Zhang, J., Wang, H., Tang, Y., and Hong, A. (2005) Novel QXL-based protease substrates and their applications in drug discovery (http://www.anaspec.com/resources/publications.asp)

  81. Yu, X., Sainz, B., Jr., and Uprichard, S. L. (2009) Development of a cell-based hepatitis C virus infection fluorescent resonance energy transfer assay for high-throughput antiviral compound screening Antimicrob Agents Chemother 53, 4311–19.

    PubMed  CAS  Google Scholar 

  82. Yaron, A., Carmel, A., and Katchalski-Kazir, E. (1979) Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes Anal Biochem 95, 228–35.

    PubMed  CAS  Google Scholar 

  83. Tonna, E. A., Aronson, R. B., and Pavelec, M. (1974) Autoradiographic assessment of proteolytic enzyme action using skeletal connective tissue matrices Connect Tissue Res 2, 183–91.

    PubMed  CAS  Google Scholar 

  84. Kaberdin, V. R., and McDowall, K. J. (2003) Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix Genome Res 13, 1961–5.

    PubMed  CAS  Google Scholar 

  85. Basak, A., Shervani, N. J., Mbikay, M., and Kolajova, M. (2008) Recombinant proprotein convertase 4 (PC4) from Leishmania tarentolae expression system: Purification, biochemical study and inhibitor design Protein Expr Purif 60, 117–26.

    PubMed  CAS  Google Scholar 

  86. Varani, J., Johnson, K., and Kaplan, J. (1980) Development of a solid-phase assay for measurement of proteolytic enzyme activity Anal Biochem 107, 377–84.

    PubMed  CAS  Google Scholar 

  87. Guarise, C., Pasquato, L., De Filippis, V., and Scrimin, P. (2006) Gold nanoparticles-based protease assay Proc Natl Acad Sci USA 103, 3978–82.

    PubMed  CAS  Google Scholar 

  88. St Hilaire, P. M., Willert, M., Juliano, M. A., Juliano, L., and Meldal, M. (1999) Fluorescence-quenched solid phase combinatorial libraries in the characterization of cysteine protease substrate specificity J Comb Chem 1, 509–23.

    PubMed  CAS  Google Scholar 

  89. Eisenthal, R., and Danson, M. J. (Eds) (2002) Enzyme Assays: A Practical Approach (2nd ed.). Oxford University Press, Oxford.

    Google Scholar 

  90. Reymond, J. L. (Ed) (2006) Enzyme Assays. Wiley-VCH, Weinheim.

    Google Scholar 

  91. Peláez, C., Mejía, A., and Planas, A. (2004) Development of a solid phase kinetic assay for determination of enzyme activities during composting Process Biochem 39, 971–5.

    Google Scholar 

  92. Weissleder, R., Zhao, M., Josephson, L., and Tang, T. (2003) Magnetic sensors for protease assays Angew Chem Int Ed 42, 1375–8.

    Google Scholar 

  93. Wondrak, E. W., Copeland, T. D., Louis, J. M., and Oroszlan, S. (1990) A solid phase assay for the protease of human immunodeficiency virus Anal Biochem 188, 82–5.

    PubMed  CAS  Google Scholar 

  94. Jean, F., Basak, A., Chrétien, M., and Lazure, C. (1991) Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay Anal Biochem 194, 399–406.

    PubMed  CAS  Google Scholar 

  95. Janelle, L., Fields, L., Hideaki, N., and Fields, G. B. (2004) Development of a solid-phase assay for analysis of matrix metalloproteinase activity J Biomol Tech 15, 305–16.

    Google Scholar 

  96. Meldal, M. (1998) Introduction to Combinatorial Solid-Phase Assays for Enzyme Activity and Inhibition Methods in Molecular Biology. Humana, Totowa, NJ, pp. 51–57.

    Google Scholar 

  97. Fearrari, S., Marin, O., Pagano, M. A., Meggio, F., Hess, D., and Shemerly, M. E. (2005) Aurora – A site specificity: Study with synthetic peptide substrates Biochem J 390, 293–302.

    Google Scholar 

  98. Kuramochi, K., Miyano, Y., Enomoto, Y., Takeuchi, R., Ishi, K., Takakusagi, Y., Saitoh, T., Fukudome, K., Manita, D., Takeda, Y., Kobayashi, S., Sakaguchi, K., and Sugawara, F. (2008) Identification of small molecule binding molecules by affinity purification using a specific ligand immobilized on PEGA resin Bioconjug Chem 19, 2417–26.

    PubMed  CAS  Google Scholar 

  99. Kostka, V., and Carpenter, F. H. (1964) Inhibition of chymotrypsin activity in crystalline trypsin preparations J Biol Chem 239, 2417–26.

    Google Scholar 

  100. Majumdar, S., Chen, A., Palmer-Smith, H., and Basak, A. (2011) Novel Circular, Cyclic and Acyclic Ψ(CH(2)O) Containing Peptide Inhibitors of SKI-1/S1P: Synthesis, Kinetic and Biochemical Evaluations. Curr Med Chem. 18, 2770–82.

    PubMed  CAS  Google Scholar 

  101. Ilekis, J. V. (2007) Review article: Preeclampsia: A pressing problem: An executive summary of a national institute of child health and human development Workshop Reprod Sci 14, 508–23.

    CAS  Google Scholar 

  102. Holeman, K. (2003) Fetal growth restriction and consequences for the offspring in animal models J Soc Gynecol Invest 10, 392–9.

    Google Scholar 

  103. Beynon, R. J., and Bond, J. S. (2001) Proteolytic Enzymes. A Practical Approach (2nd ed.). Oxford University Press, Oxford.

    Google Scholar 

  104. Reinharz, A., and Roth M. (1969) Studies on Renin with synthetic substrates Eurp J Biochem 7, 334–9.

    CAS  Google Scholar 

  105. Craik, C. S., Page, M. J., and Madison, E. L. (2011) Proteases as Therapeutics Biochem J 435, 1–16.

    PubMed  CAS  Google Scholar 

  106. Molloy, S. S., Bresnahan, P. A., Leppla, S. H., Klimpel, K. R., and Thomas, G. (1992) Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg and efficiently cleaves anthrax toxin protective antigen J Biol Chem 267, 16396–402.

    PubMed  CAS  Google Scholar 

  107. Komiyama, T., Coppola, J. M., Larsen, M. J., van Dort, M. E., Ross, B. D., Day, R., Rehemtulla, A., and Fuller, R. S. (2009) Inhibition of furin/proprotein convertase-catalyzed surface and intracellular processing by small molecules J Biol Chem 284, 15729–38.

    PubMed  CAS  Google Scholar 

  108. Munzer, J. S., Basak, A., Zhong, M., Mamarbachi, A., Hamelin, J., Savaria, D., Lazure, C., Hendy, G. N., Benjannet, S., Chrétien, M., and Seidah, N. G. (1997) In vitro characterization of the novel proprotein convertase PC7 J Biol Chem 272, 19672–81.

    PubMed  CAS  Google Scholar 

  109. Lee, S. N., Prodhomme, E., and Lindberg, I. (2004) Prohormone convertase 1 (PC1) processing and sorting: Effect of PC1 propeptide and proSAAS J Endocrinol 182, 353–64.

    PubMed  CAS  Google Scholar 

  110. Cornwall, G. A., Cameron, A., Lindberg, I., Hardy, D. M., Cormier, N., and Hsia, N. (2003) The cystatin-related epididymal spermatogenic protein inhibits the serine protease prohormone convertase Endocrinology 144(3), 901–8.

    PubMed  CAS  Google Scholar 

  111. Basak, S., Chrétien, M., Mbikay, M., and Basak, A. (2004) In vitro elucidation of substrate specificity and bioassay of proprotein convertase 4 using intramolecularly quenched fluorogenic peptides Biochem J 380, 505–14.

    PubMed  CAS  Google Scholar 

  112. Basak, A., Touré, B. B., Lazure, C., Mbikay, M., Chrétien, M., and Seidah, N. G. (1999) Enzymic characterization in vitro of recombinant proprotein convertase PC4 Biochem J 343, 29–37.

    PubMed  CAS  Google Scholar 

  113. Jean, F., Boudreault, A., Basak, A., Seidah, N. G., and Lazure, C. (1995) Fluorescent peptidyl substrates as an aid in studying the substrate specificity of human prohormone convertase PC1 and human furin and designing a potent irreversible inhibitor J Biol Chem 270, 19225–31.

    PubMed  CAS  Google Scholar 

  114. Komiyama, T., Swanson, J. A., and Fuller, R. S. (2005) Protection from anthrax toxin-mediated killing of macrophages by the combined effects of furin inhibitors and Chloroquine Antimicrob Agents Chemother 49, 3875–82.

    PubMed  CAS  Google Scholar 

  115. Basak, A., Chrétien, M., and Seidah, N. G. (2002) A rapid fluorometric assay for the proteolytic activity of SKI-1/S1P based on the surface glycoprotein of the hemorrhagic fever Lassa virus FEBS Let 514, 333–9.

    CAS  Google Scholar 

  116. Johanning, K., Juliano, M. A., Juliano, L., Lazure, C., Lamango, N. S., Steiner, D. F., and Lindberg, I. (1998) Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates J Biol Chem 273, 22672–80.

    PubMed  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank Alex Duchene, a co-op student, for carrying out some of the initial work involving the fluorescent solid phase assay method. The authors are thankful to Canadian Institutes of Health Research (CIHR) for CANADA-HOPE scholarship grant (AB and SM) and Team grant program (MOP-69093) as well as Center for Catalysis Research and Innovation, U Ottawa (AB) for financial assistance. The above funders for this study had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada, K1Y 4E9

    Ajoy Basak, Andrew Chen, Swapan Majumdar & Heather Palmer Smith

  2. Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada

    Ajoy Basak & Heather Palmer Smith

  3. Department of Chemistry, Tripura University, Suryamaninagar, India

    Swapan Majumdar

Authors
  1. Ajoy Basak
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  2. Andrew Chen
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  3. Swapan Majumdar
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  4. Heather Palmer Smith
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Correspondence to Ajoy Basak .

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Editors and Affiliations

  1. Chronic Disease Program, Ottawa Hospital Research Institute, Parkdale Avenue 725, Ottawa, K1Y 4E9, Ontario, Canada

    Majambu Mbikay

  2. (ICRM), Lab. Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Avenue des Pins Ouest 110, Montreal, H2W 1R7, Québec, Canada

    Nabil G. Seidah

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Basak, A., Chen, A., Majumdar, S., Smith, H.P. (2011). In Vitro Assay for Protease Activity of Proprotein Convertase Subtilisin Kexins (PCSKs): An Overall Review of Existing and New Methodologies. In: Mbikay, M., Seidah, N. (eds) Proprotein Convertases. Methods in Molecular Biology, vol 768. Humana Press. https://doi.org/10.1007/978-1-61779-204-5_6

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  • DOI: https://doi.org/10.1007/978-1-61779-204-5_6

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  • Print ISBN: 978-1-61779-203-8

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