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Mechanism-based traps enable protease and hydrolase substrate discovery

Hydrolase enzymes, including proteases, are encoded by 2–3% of the genes in the human genome and 14% of these enzymes are active drug targets(1). However, the activities and substrate specificities of many proteases—especially those embedded in membranes—and other hydrolases remain unknown. Here we...

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Autores principales: Tang, Shan, Beattie, Adam T., Kafkova, Lucie, Petris, Gianluca, Huguenin-Dezot, Nicolas, Fiedler, Marc, Freeman, Matthew, Chin, Jason W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8866121/
https://www.ncbi.nlm.nih.gov/pubmed/35173328
http://dx.doi.org/10.1038/s41586-022-04414-9
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author Tang, Shan
Beattie, Adam T.
Kafkova, Lucie
Petris, Gianluca
Huguenin-Dezot, Nicolas
Fiedler, Marc
Freeman, Matthew
Chin, Jason W.
author_facet Tang, Shan
Beattie, Adam T.
Kafkova, Lucie
Petris, Gianluca
Huguenin-Dezot, Nicolas
Fiedler, Marc
Freeman, Matthew
Chin, Jason W.
author_sort Tang, Shan
collection PubMed
description Hydrolase enzymes, including proteases, are encoded by 2–3% of the genes in the human genome and 14% of these enzymes are active drug targets(1). However, the activities and substrate specificities of many proteases—especially those embedded in membranes—and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond(2); this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. (3,4)). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. (5)) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes.
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spelling pubmed-88661212022-03-15 Mechanism-based traps enable protease and hydrolase substrate discovery Tang, Shan Beattie, Adam T. Kafkova, Lucie Petris, Gianluca Huguenin-Dezot, Nicolas Fiedler, Marc Freeman, Matthew Chin, Jason W. Nature Article Hydrolase enzymes, including proteases, are encoded by 2–3% of the genes in the human genome and 14% of these enzymes are active drug targets(1). However, the activities and substrate specificities of many proteases—especially those embedded in membranes—and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond(2); this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. (3,4)). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. (5)) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes. Nature Publishing Group UK 2022-02-16 2022 /pmc/articles/PMC8866121/ /pubmed/35173328 http://dx.doi.org/10.1038/s41586-022-04414-9 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/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/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Tang, Shan
Beattie, Adam T.
Kafkova, Lucie
Petris, Gianluca
Huguenin-Dezot, Nicolas
Fiedler, Marc
Freeman, Matthew
Chin, Jason W.
Mechanism-based traps enable protease and hydrolase substrate discovery
title Mechanism-based traps enable protease and hydrolase substrate discovery
title_full Mechanism-based traps enable protease and hydrolase substrate discovery
title_fullStr Mechanism-based traps enable protease and hydrolase substrate discovery
title_full_unstemmed Mechanism-based traps enable protease and hydrolase substrate discovery
title_short Mechanism-based traps enable protease and hydrolase substrate discovery
title_sort mechanism-based traps enable protease and hydrolase substrate discovery
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8866121/
https://www.ncbi.nlm.nih.gov/pubmed/35173328
http://dx.doi.org/10.1038/s41586-022-04414-9
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