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Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses
Chemical biology and the application of small molecules has proven to be a potent perturbation strategy, especially for the functional elucidation of proteins, their networks, and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9147192/ https://www.ncbi.nlm.nih.gov/pubmed/35628420 http://dx.doi.org/10.3390/ijms23105605 |
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author | Lenz, Thomas Stühler, Kai |
author_facet | Lenz, Thomas Stühler, Kai |
author_sort | Lenz, Thomas |
collection | PubMed |
description | Chemical biology and the application of small molecules has proven to be a potent perturbation strategy, especially for the functional elucidation of proteins, their networks, and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome profiling (TPP), have proven to be effective tools for identifying interactions of small molecules with their target proteins, as well as off-targets in living cells. Here, we asked the question whether isothermal dose–response (ITDR) CETSA can be exploited to characterize secondary effects downstream of the primary binding event, such as changes in post-translational modifications or protein–protein interactions (PPI). By applying ITDR-CETSA to MAPK14 kinase inhibitor treatment of living HL-60 cells, we found similar dose–responses for the direct inhibitor target and its known interaction partners MAPKAPK2 and MAPKAPK3. Extension of the dose–response similarity comparison to the proteome wide level using TPP with compound concentration range (TPP-CCR) revealed not only the known MAPK14 interaction partners MAPKAPK2 and MAPKAPK3, but also the potentially new intracellular interaction partner MYLK. We are confident that dose-dependent small molecule treatment in combination with ITDR-CETSA or TPP-CCR similarity assessment will not only allow discrimination between primary and secondary effects, but will also provide a novel method to study PPI in living cells without perturbation by protein modification, which we named “small molecule arranged thermal proximity coaggregation” (smarTPCA). |
format | Online Article Text |
id | pubmed-9147192 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-91471922022-05-29 Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses Lenz, Thomas Stühler, Kai Int J Mol Sci Article Chemical biology and the application of small molecules has proven to be a potent perturbation strategy, especially for the functional elucidation of proteins, their networks, and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome profiling (TPP), have proven to be effective tools for identifying interactions of small molecules with their target proteins, as well as off-targets in living cells. Here, we asked the question whether isothermal dose–response (ITDR) CETSA can be exploited to characterize secondary effects downstream of the primary binding event, such as changes in post-translational modifications or protein–protein interactions (PPI). By applying ITDR-CETSA to MAPK14 kinase inhibitor treatment of living HL-60 cells, we found similar dose–responses for the direct inhibitor target and its known interaction partners MAPKAPK2 and MAPKAPK3. Extension of the dose–response similarity comparison to the proteome wide level using TPP with compound concentration range (TPP-CCR) revealed not only the known MAPK14 interaction partners MAPKAPK2 and MAPKAPK3, but also the potentially new intracellular interaction partner MYLK. We are confident that dose-dependent small molecule treatment in combination with ITDR-CETSA or TPP-CCR similarity assessment will not only allow discrimination between primary and secondary effects, but will also provide a novel method to study PPI in living cells without perturbation by protein modification, which we named “small molecule arranged thermal proximity coaggregation” (smarTPCA). MDPI 2022-05-17 /pmc/articles/PMC9147192/ /pubmed/35628420 http://dx.doi.org/10.3390/ijms23105605 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Lenz, Thomas Stühler, Kai Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses |
title | Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses |
title_full | Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses |
title_fullStr | Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses |
title_full_unstemmed | Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses |
title_short | Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses |
title_sort | small molecule arranged thermal proximity coaggregation (smartpca)—a novel approach to characterize protein–protein interactions in living cells by similar isothermal dose–responses |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9147192/ https://www.ncbi.nlm.nih.gov/pubmed/35628420 http://dx.doi.org/10.3390/ijms23105605 |
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