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Nienaturalne aminokwasy jako strategia do otrzymywania substratów, inhibitorów i niskocząsteczkowych sond aktywności dla enzymów proteolitycznych

Poręba Marcin, Kasperkiewicz-Wasilewska Paulina, Rut Wioletta, Drąg Marcin

Wiadomości Chemiczne

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2022

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[Z] 76, 5-6

433--454

EN

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.

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Narzędzia chemiczne do badania enzymów proteolitycznych przy użyciu cytometrii masowej

Groborz Katarzyna

Wiadomości Chemiczne

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2022

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[Z] 76, 11-12

864-880

EN

Mass cytometry is one of the newest and most high-throughput technologies that allows for the investigation of complex biological systems at single cell level. It relies on the use of stable metal isotopes as labels of specific cell markers and therefore, allows for simultaneous analysis of more than 40 parameters at single cell level. In order to fully explore the potential of mass cytometry, researchers are trying to develop new experimental setups based on the application of pure metal isotopes in biological studies. The incorporation of antibodies into mass cytometry setups, while extremely selective and well-validated, limits the analysis as it shows the whole protein pool present in the cell. In our group, we developed new technology that allows for the identification of active forms of proteins-the ones that actively participate in cell signaling pathways. Activity-based probes are the most valuable tools for enzyme activity profiling and for years now they have been in the center of the method called Activity-Based Protein Profiling. Classic activity-based probes consist of three parts: a warhead (electrophilic binding group that covalently modifies enzyme active site), linker (specific peptide sequence or non-specific carbon chain) and the fluorescent tag that allows for enzyme detection and localization inside the cell. Spectral properties of commercially available fluorophores allow for the detection of up to dozen different cell parameters, with the use of various techniques such as confocal microscopy or flow cytometry. To increase the number of analyzed parameters, we designed activity-based probes that possess DOTA chelating moiety that is able to trap one metal atom per one probe. The combination of mass cytometry with highly selective activity-based probes allowed for the development of new technology that grants the possibility of multiparametric analysis of complex biological samples such as blood or cancer tissue. The new type of activity-based probes (so-called TOF-probes) incorporate various inhibitor scaffolds designed with HyCoSuL technology (Hybrid Combinatorial Substrate Libraries). These compounds possess a variety of unnatural amino acids in their structures, which significantly increases their selectivity toward proteases of interest.

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Monitorowanie aktywności proteolitycznej jako element diagnostyki chorób cywilizacyjnych

Gruba Natalia, Romanowska Anita, Rejmak Wiktoria, Sikora Honorata, Wysocka Magdalena, Lesner Adam

Wiadomości Chemiczne

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2022

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[Z] 76, 5-6

335--347

EN

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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Wizualizacja enzymów proteolitycznych za pomocą sond chemicznych oraz cytometrii masowej

Groborz Katarzyna, Poręba Marcin

Wiadomości Chemiczne

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2021

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[Z] 75, 11-12

1171--1191

EN

Proteolytic enzymes, also known as peptidases or proteases, are protein catalysts that are primarily responsible for the hydrolysis of a peptide (amide) bond in peptide and protein substrates. By selective hydrolysis of selected substrates, these enzymes control many physiologically important processes including programmed cell death, blood coagulation cascade, protein maturation, fibrinolysis and many others. On the other hand, however, the imbalance in proteases activity leads to the development of diseases, including cancer, neurodegenerative diseases and coronary diseases etc.. In recent decades there has been great progress in studying the biological functions of many proteolytic enzymes. These observations were made possible through the use of various research techniques including genomics, epigenomics and proteomics. However, a major limitation of these techniques is the lack of information about the exact catalytic activity of the enzymes. For this reason, chemical probes are the most convenient toll for functional investigation of proteolytic enzymes. According to the generally accepted convention, chemical probes are compounds that can detect the catalytic activity of proteolytic enzymes. In general, chemical-based probes (activity-based probes, ABPs) consist of three main components: (1) a reactive binding group that binds permanently to the enzyme active site, (2) a recognition sequence (usually a peptide), which is responsible for the selective binding of a given probe to an individual enzyme or group of enzymes, and (3) a tag, mainly a fluorophore, enabling for detection of the probe-enzyme complex. However, the current limitation of ABPs is that only up to four enzymes can be detected and visualized in parallel, which significantly impedes their application for multi-parametric analysis. To date, the detection of proteases with the use of ABPs was limited to individual enzymes being investigated one by one, thus the obtained picture was far from being complete. In this review we describe the development of a new type of enzyme ABPs, so called TOF-probes that are compatible with mass cytometry format. The application of metal isotopes instead of fluorophores, makes possible to significantly increase the number of enzymes, which can be simultaneously visualized using chemical probes. Mass cytometry is a revolutionary technology that adopts atomic mass spectrometry into flow cytometry applications. The excellence of this method is that each metal isotope (mostly from lanthanides) has its own peak on mass spectrum, which eliminates the problem of signal overlap, thus allows for monitoring of more than 40 parameters at single cell level.

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Charakterystyka biochemiczna proteazy syntetyzowanej przez Streptomyces rimosus

Jankiewicz U., Koblak S., Wierzchowski P., Russel S.

Woda-Środowisko-Obszary Wiejskie

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2018

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T. 18, z. 2

5--14

PL

Badania prezentowane w niniejszej pracy miały na celu charakterystykę biochemiczną zewnątrzkomórkowej proteazy syntetyzowanej przez wyizolowany z gleby szczep Streptomyces rimosus i ocenę możliwości praktycznego wykorzystania tego enzymu w przemyśle. Badany enzym wyizolowano z 7-dniowych hodowli bakterii Streptomyces rimosus. Oczyszczony dwukrotnie enzym wykorzystano do charakterystyki biochemicznej w zakresie optymalnych warunków jego działania oraz wpływu aktywatorów i inhibitorów. Proteaza syntetyzowana przez S. rimosus wykazywała najwyższą aktywność w temperaturze 50°C i pH 7,5 oraz wysoką termostabilność w temperaturze 50°C. Dwuwartościowe jony Zn, Mo, Ni, Cd, Co hamowały aktywność enzymu, natomiast Ca i Mg – aktywowały. Silna inhibicja aktywności enzymu w obecności diizopropylofluorofosforanu (DFP) świadczy o tym, że jest to proteaza serynowa. Aktywność badanego enzymu była stabilna w obecności takich detergentów, jak Triton X-100, Tween 20, Tween 80, bromek heksadecylotrimetyloamoniowy (CTAB) oraz dodecylosiarczanu sodu (SDS).

EN

The research was aimed at isolation and biochemical characterization of the extracellular protease synthesized by the soil Streptomyces rimosus and assessment of the possibilities of practical use of this enzyme in the industry. For this purpose, the test enzyme was isolated from 7 day old culture of S. rimosus. The enzyme two-fold purified was used for biochemical characterization for optimal temperature and pH of activity and activators and inhibitors of activity. The S. rimosus protease showed the highest activity at 50°C and at pH 7.5 and high thermostability at 50°C. Divalent ions such as Zn, Mo, Ni, Cd, Co caused inhibition while Ca and Mg stimulated activity. Strong inhibition of activity in the presence of diisopropylfluorophosphate (DFP) indicates that it is a serine protease. The activity of the test enzyme was stable in the presence of such detergents as Triton X-100, Tween 20, Tween 80, hexadecyl trimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS).

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Karczoch zwyczajny - innowacyjny surowiec o szerokim zastosowaniu

Michalak-Majewska M., Teterycz D.

Przemysł Spożywczy

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2018

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T. 72, nr 7

30--33

PL

Karczoch zwyczajny jest uprawiany głównie w krajach śródziemnomorskich i tam jest także najczęściej spożywany. Dzięki wyjątkowym właściwościom można go stosować nie tylko w gastronomii. Z karczocha uzyskuje się inulinę o wysokim stopniu polimeryzacji oraz neutralnym smaku, co pozwala na jej wykorzystanie w wielu produktach spożywczych w celu wzbogacenia ich w błonnik lub jako zamiennik tłuszczu. Ze względu na wysoką zawartość inuliny karczoch po zaszczepieniu bakteriami probiotycznymi może być także synbiotykiem. Spożywanie takiego karczocha poprawia pracę układu pokarmowego. Z karczocha produkowane są ekstrakty enzymatyczne, zawierające enzymy proteolityczne (kardozyna A i B). Są one wykorzystywane w produkcji serów jako zamiennik podpuszczki. Ekstrakty takie, ze względu na wysoką zawartość antyoksydantów (kwas chlorogenowy, cynaryna), mogą być stosowane jako przeciwutleniacz w żywności, mogą także przyczyniać się do ograniczenia powstawania heterocyklicznych amin aromatycznych (HCA) w mięsie przetworzonym termicznie.

EN

The artichoke is grown mainly in the Mediterranean countries and is also commonly consumed there. The rich artichoke properties allow for its use not only in gastronomy. Artichoke is a source of inulin with a high degree of polymerization and a neutral taste, which allows it to be used in many foods to enrich them with fiber or as a fat substitute. Due to the high content of inulin, artichoke, after being inoculated with probiotic bacteria, may be also a synbiotic. Consuming such artichoke improves the digestive system. Artichoke enzymatic extracts contain proteolytic enzymes (cardiosin A and B). They are used in cheese production as arennet replacement. Artichoke extracts, due to the high content of antioxidants (chlorogenic acid, cynarin), can be used as an antioxidant in food and may contribute to reduction of the level of HCA formation in thermally processed meat. Artichoke waste can be used to produce biogas with high methane content.

7

Proteazy: znaczenie, rola i oznaczanie

Tokarzewicz A, Gorodkiewicz E.

Chemik

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2015

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Vol. 69, nr 2

81--88

PL

Proteazy są związkami, które odgrywają bardzo istotną rolę w ludzkim organizmie. Regulują one wiele procesów przebiegających także w innych żywych organizmach, również w wirusach, bakteriach i pasożytach. Ich zwiększona lub obniżona ilość może wskazywać na nieprawidłowości w orgaznizmie, takie jak: rozwój zapalenia, nowotwory, nadciśnienie. W artykule opisano najistotniejsze z ludzkich proteaz, ich znaczenie, rolę w organizmie oraz pokrótce metody oznaczania.

EN

Proteases are compounds that play an important role in the human body. They regulate many processes inside the living organisms, also in viruses, bacteria and parasites. Their increased or decreased level may indicate irregularities in the body, such as: the development of inflammation, cancer and others like hypertension. This article describes the most important of human proteases, their significance, roles in the organism and shortly methods of determination.

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Comparison of Surface Modification of Wool Fibres Using Pronase, Trypsin, Papain and Pepsin

Goudarzi G., Sepehrizadeh Z., Yazdi M. T., Jamshidiha M.

Fibres & Textiles in Eastern Europe

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2008

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Vol. 16, no. 3 (68)

90--92

EN

Nowadays the uses of enzymes in textile industries are being developed because of their harmless effluents and good effectiveness. One of these uses is the shrink proofing of wool fabrics using proteolytic enzymes. In this research, some proteolytic enzymes, such as pronase, trypsin, papain and pepsin were used to treat wool fibers in optimum conditions for 30, 60 and 120 minutes. Afterwards, the effectiveness of these enzymes on the surface of wool was studied by scanning microscopy (SEM). Comparison of resulting micrographs showed that papain is more proteolytically efficient for wool fiber morphology.

PL

Zastosowanie enzymów w przemyśle tekstylnym jest coraz większe, gdyż mają one wysoką wydajność i są mało szkodliwe dla środowiska. Enzymy proteolityczne stosuje się np. w celu zwiększenia odporności na skurcz tkanin wełnianych. W pracy użyto niektórych enzymów proteolitycznych tj. proteinazy, trypsyny, papainy i pepsyny. Obrabiano nimi włókna wełny w warunkach optymalnych przez 30, 60 i 120 minut. Następnie wykorzystując mikroskopię skaningową (SEM) badano skuteczność działania tych enzymów na powierzchnię włókien. Porównując wyniki badań można stwierdzić, że papaina ma najwyższą skuteczność.

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Kinetyka katalitycznych reakcji zachodzących w obecności enzymów proteolitycznych

Balejko J. A.

LAB Laboratoria, Aparatura, Badania

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2004

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R. 9, nr 2

15-17

PL

Autoliza mięsa jest procesem enzymatycznym, a ten jak wiadomo jest szczególnym przypadkiem procesów katalitycznych. Reakcje, stanowiące istotę tych procesów, w układach homogennych przebiegają cyklicznie i składają się z kilku następczych lub następczo-równoległych etapów, w wyniku których katalizator (enzym), ulegający zużyciu na pierwszym etapie, jest regenerowany na etapie ostatnim.