Abstract
Changes in proteolytic activity are associated with several diseases, including cancer. Proteases are potential drug targets and targeting of proteases is used for treatment of various conditions/diseases, like high blood pressure and HIV. We present here detailed protocols for basic evaluation of the effects of peptides on the activity of proteases, using kallikrein-related peptidases KLK2 and KLK3 (also known as hK2 and PSA), and trypsin as examples. KLK2 and KLK3 are major prostatic proteases, and they are potential targets for prostate cancer treatment. KLK2 has trypsin-like activity and KLK3 chymotrypsin-like activity. By phage display technology, we have developed peptides that specifically stimulate KLK3-activity and other peptides that inhibit KLK2 or trypsin. The effect of the peptides on the proteolytic activity of proteases can be studied using substrates, the cleavage of which generates detectable signal, allowing rapid evaluation of protease activity. The cleavage of protein substrates can be detected by SDS-PAGE, followed by staining of the proteins. We also describe graphical analysis of the IC50-value, the effect of a peptide on Michaelis-Menten constant (K m) and the maximal reaction rate (V max).
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Acknowledgments
The authors wish to thank Dr Leena Valmu for the silver staining protocol and Ms. Helena Taskinen for technical assistance. Our original studies referred to have been supported by National Technology Agency of Finland, University of Helsinki, Helsinki University Central Hospital, the Finnish Cancer Foundation, the Academy of Finland, Juselius Foundation and Finska Läkaresällskapet.
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Koistinen, H., Hekim, C., Wu, P., Närvänen, A., Stenman, UH. (2014). Evaluation of Peptides as Protease Inhibitors and Stimulators. In: Nixon, A. (eds) Therapeutic Peptides. Methods in Molecular Biology, vol 1088. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-673-3_10
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DOI: https://doi.org/10.1007/978-1-62703-673-3_10
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-672-6
Online ISBN: 978-1-62703-673-3
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