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Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native enviro...

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Autores principales: Uliana, Federico, Vizovišek, Matej, Acquasaliente, Laura, Ciuffa, Rodolfo, Fossati, Andrea, Frommelt, Fabian, Goetze, Sandra, Wollscheid, Bernd, Gstaiger, Matthias, De Filippis, Vincenzo, auf dem Keller, Ulrich, Aebersold, Ruedi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966775/
https://www.ncbi.nlm.nih.gov/pubmed/33727531
http://dx.doi.org/10.1038/s41467-021-21754-8
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author Uliana, Federico
Vizovišek, Matej
Acquasaliente, Laura
Ciuffa, Rodolfo
Fossati, Andrea
Frommelt, Fabian
Goetze, Sandra
Wollscheid, Bernd
Gstaiger, Matthias
De Filippis, Vincenzo
auf dem Keller, Ulrich
Aebersold, Ruedi
author_facet Uliana, Federico
Vizovišek, Matej
Acquasaliente, Laura
Ciuffa, Rodolfo
Fossati, Andrea
Frommelt, Fabian
Goetze, Sandra
Wollscheid, Bernd
Gstaiger, Matthias
De Filippis, Vincenzo
auf dem Keller, Ulrich
Aebersold, Ruedi
author_sort Uliana, Federico
collection PubMed
description Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.
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spelling pubmed-79667752021-04-01 Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen Uliana, Federico Vizovišek, Matej Acquasaliente, Laura Ciuffa, Rodolfo Fossati, Andrea Frommelt, Fabian Goetze, Sandra Wollscheid, Bernd Gstaiger, Matthias De Filippis, Vincenzo auf dem Keller, Ulrich Aebersold, Ruedi Nat Commun Article Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays. Nature Publishing Group UK 2021-03-16 /pmc/articles/PMC7966775/ /pubmed/33727531 http://dx.doi.org/10.1038/s41467-021-21754-8 Text en © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Uliana, Federico
Vizovišek, Matej
Acquasaliente, Laura
Ciuffa, Rodolfo
Fossati, Andrea
Frommelt, Fabian
Goetze, Sandra
Wollscheid, Bernd
Gstaiger, Matthias
De Filippis, Vincenzo
auf dem Keller, Ulrich
Aebersold, Ruedi
Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen
title Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen
title_full Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen
title_fullStr Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen
title_full_unstemmed Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen
title_short Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen
title_sort mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7966775/
https://www.ncbi.nlm.nih.gov/pubmed/33727531
http://dx.doi.org/10.1038/s41467-021-21754-8
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