Proteolytic enzymes are molecular scissors that are responsible for the amide bond breakdown in peptide and protein substrates. Over the years, the view on proteases has been considerably changed from non-specific digestive enzymes to sophisticated biocatalysts, which by performing limited proteolysis control virtually all biological processes. In order to better understand how proteases work and what are their biologically relevant target substrates, it is indispensable to determine their catalytic preferences. This knowledge can be further utilized to develop selective substrates, inhibitors and activity-based probes (ABPs) enabling the monitoring of proteases activity in various settings, from in vitro analysis on recombinant enzymes or cell lysates to ex vivo and in vivo imaging at the single cell level. Among many chemical-based approaches that have been developed and applied over the years, the Hybrid Combinatorial Substrate Library (HyCoSuL) technology has emerged as one of the most powerful one. HyCoSuL is a combinatorial peptide-based library of fluorogenic substrates, that comprise natural and unnatural amino acids, that can deeply explore the chemical space in proteases active site, providing a structural framework for the development of highly-selective chemical tools. In this review we present the most prominent examples of proteolytic enzymes that have been profiled with HyCoSuL approach yielding selective substrates, potent inhibitors, and very sensitive activity-based probes.
Proteolytic enzymes are essential for the proper functioning of every living cell. Due to their great importance in controlling metabolic changes in living organisms, they could be used in the diagnosis of civilization diseases. Hence, the search for new methods of determining and controlling their activity is extremely important. Our team, has been studying substrates of proteases and their potential use in detection of biomarkers activity for many years.
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Określono spektrum aktywności zewnątrzkomórkowej depolimerazy produkowanej przez wyizolowany z gleby szczep Gliocladium solani oraz zbadano przebieg mechanizmów degradacji alifatycznego poliestru "Bionolle®" typ # 3001 z udziałem tego enzymu. Optymalna wartość pH odpowiadająca jego maksymalnej aktywności wynosi 5. Stwierdzono, że enzym charakteryzuje się szerokim spektrum aktywności hydrolitycznej zarówno wobec wiązań estrowych naturalnych oraz syntetycznych polimerów, jak i estrowych pochodnych krótko- i długołańcuchowych kwasów tłuszczowych. Wydajniej atakuje fazę amorficzną niż krystaliczną tworzywa oraz preferencyjnie hydrolizuje przede wszystkim mery adypinianowe poliestru pozostawiając w nim mery bursztynianowe.
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The range of activity of extracellular depolymerase secreted by Gliocladium solani strain (Fig. 2), isolated from the soil, was determined. The course of degradation of aliphatic "Bionolle®" polyester, type # 3001, under the influence of this enzyme (Table 1) was studied. Optimum pH value related to its maximal activity equals 5 (Fig. 1). It was found that the enzyme discussed shows wide range of hydrolytic activity, being active towards ester bonds of natural and synthetic polymers as well as towards ester derivatives of short- or long-chain fatty acids (Table 1). Depolymerase attacks better the amorphous phase of polymer than the crystalline one (Fig. 3). It preferentially caused hydrolysis of mainly adipate mers of polyester leaving succinate ones.
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