Proteases are known to participate in many cellular processes due to their ability to process the peptide bond. They are key players in several cascades taking place in the cell with apoptosis, fibrinolysis, blood clotting or general cell cycle being only a few examples. However, proteases can be also bad guys and participate in the cellular events, which lead to severe diseases such as cancer, diabetes or pathogens infections [1].
Each protease recognizes only substrates, which can fit into the recognition pockets. There are several methods of determination of substrate specificity [2]. Recently we have introduced Hybrid Combinatorial Substrate Library (HyCoSuL) for investigation of substrate specificity of endopeptidases in non-prime positions [3,4,5]. To get even better insight into substrate specificity of endopeptidases, we have applied approach with natural and unnatural amino acids for rational design of library of internally quenched substrates to investigate both prime and non-prime positions. To increase solubility and sensitivity of the assay we have also successfully introduced a new type of fluorophore and quencher pair. This allowed us to obtain for several different endopeptidases much better substrates. A strategy for profiling of substrate specificity for serine, cysteine and metalloproteases will be presented.
This work was supported by the National Science Centre grant 2014/13/B/ST5/00240 in Poland
[1] M. Drag, G. S. Salvesen, Nature Reviews Drug Discovery. 2010; 9 (9): 690-701.
[2] M. Poreba, M. Drag. Current Medicinal Chemistry 2010; 17(33): 3968-95.
[3] W. Rut, P. Kasperkiewicz, A. Byzia, M. Poreba, K. Groborz, M. Drag, Biological Chemistry 2015 Apr; 396(4):329-37
[4] P. Kasperkiewicz , M. Poreba, S.J. Snipas, H. Parker, C.C. Winterbourn, G.S. Salvesen, M. Drag, Proc Natl Acad Sci U S A. 2014 111(7): 2518-23
[5] M. Poreba, P. Kasperkiewicz, S.J. Snipas, D. Fasci, G.S. Salvesen, M. Drag M., Cell Death & Differentiation, 2014, (9): 1482-92