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Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity
Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover inhibitors have been hindered by the lack of tools to rapidly screen for...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9574550/ https://www.ncbi.nlm.nih.gov/pubmed/36130221 http://dx.doi.org/10.1093/protein/gzac006 |
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author | Galera-Prat, Albert Alaviuhkola, Juho Alanen, Heli I Lehtiö, Lari |
author_facet | Galera-Prat, Albert Alaviuhkola, Juho Alanen, Heli I Lehtiö, Lari |
author_sort | Galera-Prat, Albert |
collection | PubMed |
description | Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover inhibitors have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different enzymes. We engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their activity using a homogeneous NAD(+) conversion assay, facilitating its automation to lower the assay volume and costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity. |
format | Online Article Text |
id | pubmed-9574550 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-95745502022-10-19 Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity Galera-Prat, Albert Alaviuhkola, Juho Alanen, Heli I Lehtiö, Lari Protein Eng Des Sel Original Article Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover inhibitors have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different enzymes. We engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their activity using a homogeneous NAD(+) conversion assay, facilitating its automation to lower the assay volume and costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity. Oxford University Press 2022-09-20 /pmc/articles/PMC9574550/ /pubmed/36130221 http://dx.doi.org/10.1093/protein/gzac006 Text en © The Author(s) 2022. Published by Oxford University Press. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Article Galera-Prat, Albert Alaviuhkola, Juho Alanen, Heli I Lehtiö, Lari Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity |
title | Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity |
title_full | Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity |
title_fullStr | Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity |
title_full_unstemmed | Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity |
title_short | Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity |
title_sort | protein engineering approach to enhance activity assays of mono-adp-ribosyltransferases through proximity |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9574550/ https://www.ncbi.nlm.nih.gov/pubmed/36130221 http://dx.doi.org/10.1093/protein/gzac006 |
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