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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...

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Autores principales: Lenz, Thomas, Stühler, Kai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
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).
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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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