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

Lü, Qing; Zhang, Yonghong; Hellner, Joakim; Giannini, Caterina; Xu, Xiangyu; Pauwels, Jarne; Ma, Qian; Dejonghe, Wim; Han, Huibin; B, van de Cotte; Impens, Francis; Gevaert, Kris; Smet, Ive De; Friml, Jiřı́; Molina, Daniel Martinez; Russinova, Eugenia

doi:10.1073/pnas.2118220119

Cited by 20 publications

(11 citation statements)

References 60 publications

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“…Intact cells with functional signaling pathways allow the investigation of not only the target protein alone, but also proteins that are modified downstream of the direct target. Meanwhile, cell lysate is more powerful when the direct target is to be spotted [ 80 , 81 ].…”

Section: Developing Chemical Tools With Novel Targets To Further Deli...mentioning

confidence: 99%

“…High-resolution mass spectrometry-based profiling of the proteome could reveal proteins whose stability has changed upon chemical treatment [ 77 ]. In Arabidopsis, CETSA was performed with intact PSB-D suspension cell cultures or cell lysates [ 80 , 81 ]. Intact cells with functional signaling pathways allow the investigation of not only the target protein alone, but also proteins that are modified downstream of the direct target.…”

Section: Developing Chemical Tools With Novel Targets To Further Deli...mentioning

confidence: 99%

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New Wine in an Old Bottle: Utilizing Chemical Genetics to Dissect Apical Hook Development

Aizezi

Xie

Guo

et al. 2022

Life

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The apical hook is formed by dicot seedlings to protect the tender shoot apical meristem during soil emergence. Regulated by many phytohormones, the apical hook has been taken as a model to study the crosstalk between individual signaling pathways. Over recent decades, the roles of different phytohormones and environmental signals in apical hook development have been illustrated. However, key regulators downstream of canonical hormone signaling have rarely been identified via classical genetics screening, possibly due to genetic redundancy and/or lethal mutation. Chemical genetics that utilize small molecules to perturb and elucidate biological processes could provide a complementary strategy to overcome the limitations in classical genetics. In this review, we summarize current progress in hormonal regulation of the apical hook, and previously reported chemical tools that could assist the understanding of this complex developmental process. We also provide insight into novel strategies for chemical screening and target identification, which could possibly lead to discoveries of new regulatory components in apical hook development, or unidentified signaling crosstalk that is overlooked by classical genetics screening.

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Section: Developing Chemical Tools With Novel Targets To Further Deli...mentioning

confidence: 99%

Section: Developing Chemical Tools With Novel Targets To Further Deli...mentioning

confidence: 99%

New Wine in an Old Bottle: Utilizing Chemical Genetics to Dissect Apical Hook Development

Aizezi

Xie

Guo

et al. 2022

Life

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“…TPP is known for its specificity, and analogous experiments in mammalian cells have successfully retrieved known 3′,5′‐cAMP binders (Lim et al., 2018). TPP is also slowly gaining traction in plants (Lu et al., 2022; Volkening et al., 2019). Here, using TPP, we report 51 proteins for which thermal stability was significantly changed upon 3′,5′‐cAMP treatment, indicating a change in their interaction status.…”

Section: Introductionmentioning

confidence: 99%

Protein interactome of 3′,5′‐cAMP reveals its role in regulating the actin cytoskeleton

Figueroa,

Franz,

Luzarowski

et al. 2023

The Plant Journal

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SUMMARYIdentification of protein interactors is ideally suited for the functional characterization of small molecules. 3′,5′‐cAMP is an evolutionary ancient signaling metabolite largely uncharacterized in plants. To tap into the physiological roles of 3′,5′‐cAMP, we used a chemo‐proteomics approach, thermal proteome profiling (TPP), for the unbiased identification of 3′,5′‐cAMP protein targets. TPP measures shifts in the protein thermal stability upon ligand binding. Comprehensive proteomics analysis yielded a list of 51 proteins significantly altered in their thermal stability upon incubation with 3′,5′‐cAMP. The list contained metabolic enzymes, ribosomal subunits, translation initiation factors, and proteins associated with the regulation of plant growth such as CELL DIVISION CYCLE 48. To functionally validate obtained results, we focused on the role of 3′,5′‐cAMP in regulating the actin cytoskeleton suggested by the presence of actin among the 51 identified proteins. 3′,5′‐cAMP supplementation affected actin organization by inducing actin‐bundling. Consistent with these results, the increase in 3′,5′‐cAMP levels, obtained either by feeding or by chemical modulation of 3′,5′‐cAMP metabolism, was sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. The observed rescue was specific to 3′,5′‐cAMP, as demonstrated using a positional isomer 2′,3′‐cAMP, and true for the nanomolar 3′,5′‐cAMP concentrations reported for plant cells. In vitro characterization of the 3′,5′‐cAMP–actin pairing argues against a direct interaction between actin and 3′,5′‐cAMP. Alternative mechanisms by which 3′,5′‐cAMP would affect actin dynamics, such as by interfering with calcium signaling, are discussed. In summary, our work provides a specific resource, 3′,5′‐cAMP interactome, as well as functional insight into 3′,5′‐cAMP‐mediated regulation in plants.

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“…The strategy to combine CETSA MS data in intact Arabidopsis cells with phosphoproteomics data was employed by Lu et al to generate novel insights into the downstream proteins of the plant-specific GSK3 inhibitor bikinin. 22 These pathway proteins were affected in both their phosphorylation levels as well as thermal stability. The change in thermal stability can be caused by protein phosphorylation but also protein recruitment by a partner protein, translocation, and the general change of the redox state of the cell.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

A Shift in Thinking: Cellular Thermal Shift Assay-Enabled Drug Discovery

Caballero

Lundgren

2023

ACS Med. Chem. Lett.

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A decade has passed since the cellular thermal shift assay (CETSA) was introduced to the drug discovery community. Over the years, the method has guided numerous projects by providing insights about, for example, target engagement, lead generation, target identification, lead optimization, and preclinical profiling. With this Microperspective, we intend to highlight recently published applications of CETSA and how the data generated can enable efficient decision-making and prioritization throughout the drug discovery and development value chain.

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

Cited by 20 publications

References 60 publications

New Wine in an Old Bottle: Utilizing Chemical Genetics to Dissect Apical Hook Development

New Wine in an Old Bottle: Utilizing Chemical Genetics to Dissect Apical Hook Development

Protein interactome of 3′,5′‐cAMP reveals its role in regulating the actin cytoskeleton

A Shift in Thinking: Cellular Thermal Shift Assay-Enabled Drug Discovery

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