Identification of phosphosites that alter protein thermal stability

Smith, Ian R.; Hess, Kyle; Bakhtina, Anna; Valente, Anthony S.; Rodriguez‐Mias, Ricard A.; Villén, Judit

doi:10.1038/s41592-021-01178-4

Cited by 39 publications

(21 citation statements)

References 23 publications

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“…Interestingly, in the context of our CETSA experiments, the phosphorylated and dephosphorylated MAPKAPK2 (MAPKAPK2p and MAPKAPK2) exhibited the same melting characteristics under the same treatment conditions, respectively ( Figure 1 and respective p values >> 0.05 in Supplementary Table S6 ). This finding is consistent with the reported lack of effect on melting behavior for the vast majority of phosphosites: Although the impact of protein phosphorylation on protein thermal stability is still under investigation and subject of a current scientific debate, it seems that influences of phosphorylation on thermal protein stability are rather low, and that only 1.5–3% of the phosphosites lead to a significant thermal shift [ 51 , 52 , 53 , 54 ].…”

Section: Discussionsupporting

confidence: 87%

Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses

Lenz

2022

IJMS

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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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Section: Discussionsupporting

confidence: 87%

Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses

Lenz

2022

IJMS

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“…However, for a few proteins, bikinin had an impact on both the phosphorylation and melting behaviors, indicating that the phosphorylation status might affect the thermal stability of some proteins. Although some studies in human cells reported that phosphorylation affected the protein thermal stability ( 52 ), others demonstrated that for most of the proteins, the melting behavior of phosphorylated and nonphosphorylated forms was concordant ( 53 , 54 ).…”

Section: Discussionmentioning

confidence: 99%

Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling

Lü

Zhang

Hellner

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Significance Chemical genetics, which investigates biological processes using small molecules, is gaining interest in plant research. However, a major challenge is to uncover the mode of action of the small molecules. Here, we applied the cellular thermal shift assay coupled with mass spectrometry (CETSA MS) to intact Arabidopsis cells and showed that bikinin, the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, changed the thermal stability of some of its direct targets and putative GSK3-interacting proteins. In combination with phosphoproteomics, we also revealed that GSK3s phosphorylated the auxin carrier PIN-FORMED1 and regulated its polarity that is required for the vascular patterning in the leaf.

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“…However, it has been realized that these methods can also detect other sources of protein biophysical variation which are difficult to quantify with other proteomics methods, including protein interactions with other biomolecules 53 . Since the adaptation of the method to infer functional phosphorylation sites 22,23 , it has also become clear that TPP bears the potential for detecting post-translationally modified proteoforms. Here we have performed TPP with unprecedented peptide coverage and generalized this concept to enable unbiased detection of co-existing functional proteoforms.…”

Section: Discussionmentioning

confidence: 99%

“…It is implemented by applying the cellular thermal shift assay (CETSA) 13,14 on a proteome-wide scale using multiplexed quantitative mass spectrometry 15 . Recent work has shown that TPP can not only inform on drug-target engagement 16,17 but also on protein-nucleic acid 18 , protein-protein 19 and protein-metabolite interactions 20 as well as metabolic pathway activity 21 and the functional relevance of post-translational modifications 22,23 . Moreover, it has been found that cell type-specific physiology is reflected in characteristic proteome thermal stability profiles and can be predictive of drug responses 24 .…”

Section: Introductionmentioning

confidence: 99%

Deep thermal proteome profiling for detection of proteoforms and drug sensitivity biomarkers

Kurzawa

Stahl

Leo

et al. 2022

Preprint

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The complexity of the functional proteome extends significantly beyond the protein coding genome resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases, but challenging to perform systematically. Here, we apply thermal proteome profiling with deep peptide coverage to detect functional proteoforms in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detect 15,846 proteoforms, capturing differently spliced, post-translationally modified, and cleaved proteins expressed from 9,290 genes. We identify differential coaggregation of proteoform pairs and establish links to disease biology. Moreover, we systematically make use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach thus provides a powerful and unique tool for systematic detection and functional annotation of proteoforms.

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Identification of phosphosites that alter protein thermal stability

Cited by 39 publications

References 23 publications

Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses

Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses

Proteome-wide cellular thermal shift assay reveals unexpected cross-talk between brassinosteroid and auxin signaling

Deep thermal proteome profiling for detection of proteoforms and drug sensitivity biomarkers

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