Proteome-wide analysis of protein thermal stability in Arabidopsis
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis Thaliana
SUBMITTER: 
Chengchao Xu
PROVIDER: PXD033755 | iProX | Sat May 06 00:00:00 BST 2023
REPOSITORIES: iProX
Similar Datasets
Project description:In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to characterize compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified 1.498 million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell- and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.
2025-02-13 | PXD048009 | Pride
Project description:DZNep (3-deazaneplanocin A) is commonly used to reduce lysine methylation. DZNep inhibits S-adenosyl-L-homocysteine hydrolase (AHCY), preventing the conversion of S-adenosyl-L-homocysteine (SAH) into L-homocysteine and reducing the level of S-adenosylmethionine (SAM). As a result, the SAM to SAH ratio decreases, an indicator of the methylation potential within a cell. Many studies have characterized the impact of DZNep on histone lysine methylation or in specific cell or disease contexts. Recently, protein thermal stability has provided a new dimension for studying the mechanism of action of small molecule inhibitors. In addition to ligand binding, post-translational modifications and protein-protein interactions impact thermal stability. Here, we sought to characterize protein thermal stability changes induced by DZNep treatment in HEK293T cells using the Protein Integral Solubility Alteration (PISA) assay. DZNep treatment altered the thermal stability of 135 proteins, with over half previously reported to be methylated at lysine residues. In addition to thermal stability, we identify changes in transcript and protein abundance after DZNep treatment to distinguish between direct and indirect impacts on thermal stability. Nearly one-third of the proteins with altered thermal stability had no changes at the transcript or protein level. Of these thermally altered proteins, CDK6 had a stabilized methylated peptide, while its unmethylated counterpart was unaltered. Multiple methyltransferases were among the proteins with thermal stability alteration, including DNMT1, potentially due to changes in SAM/SAH levels. This study systematically evaluates DZNep’s impact on the transcriptome, the proteome, and the thermal stability of proteins.
2024-08-02 | GSE268629 | GEO
Project description:This project investigates the thermal stability of isolated mitochondrial proteins from Arabidopsis thaliana cell cultures following L-cysteine treatment, using Thermal Proteome Profiling (TPP). Shotgun proteomics was employed to identify and quantify proteins after thermal treatment. Quantitative data (iBAQ values) were subsequently analyzed using a custom R script (available on GitHub: “MeCuP”) to identify candidate proteins that exhibit changes in thermal stability due to interaction with L-cysteine.
2025-05-29 | PXD064096 | Pride
Project description:In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to characterize compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified 1.498 million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell- and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.
2025-02-13 | PXD053138 | Pride
Project description:We demonstrated that bikinin changed the thermal stability of some of its target proteins as well as several known or putative interacting proteins of Arabidopsis GSK3s. By combining the thermal- and the phospho-proteomes of bikinin we uncovered the auxin carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s. We showed that inhibition the kinase activity of GSK3s by brassinosteroids or the specific chemical inhibitor bikinin led to the loss of PIN1 polarity, revealing a novel crosstalk between brassinosteroid and auxin signaling. Hence, our study demonstrates the applicability of CETSA MS in plant intact cells for identification of small-molecule targets and for discovery of novel protein-protein interactions.
2021-12-08 | MSV000088545 | MassIVE
Project description:To broadly explore artemisinin susceptibility in apicomplexan parasites, we used genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. From these screens, we identified the mitochondrial protease DegP2, which appeared gDegP2-responsive thermal stability in a pathway leading to decreased DHA susceptibility. To identify proteins exhibiting DegP2-responsive thermal stability
2020-08-28 | PXD019917 | Pride
Project description:We demonstrated that bikinin changed the thermal stability of some of its target proteins as well as several known or putative interacting proteins of Arabidopsis GSK3s. By combining the thermal- and the phospho-proteomes of bikinin we uncovered the auxin carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s. We showed that inhibition the kinase activity of GSK3s by brassinosteroids or the specific chemical inhibitor bikinin led to the loss of PIN1 polarity, revealing a novel crosstalk between brassinosteroid and auxin signaling. Hence, our study demonstrates the applicability of CETSA MS in plant intact cells for identification of small-molecule targets and for discovery of novel protein-protein interactions.
2022-03-08 | PXD029936 | Pride
Project description:Despite the growing interest in using chemical genetics in plant research, small-molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution massspectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or post-translational modifications allows the identification of drug targets, or the study of protein-metabolite and protein-protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal- and the phospho-proteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf thus, revealing a novel crosstalk between brassinosteroid and auxin signaling.
2022-04-04 | PXD030309 | Pride
Project description:We developed a robust method termed Dali that identifies phosphorylation events that alter protein thermal stability as a proxy for function.
2021-06-18 | PXD016750 | Pride
Project description:Altered thermal solubility measurement techniques are emerging as powerful tools to assess ligand binding, post-translational modification, protein-protein interactions, and many other cellular processes that affect protein state under various cellular conditions. Thermal solubility or stability profiling techniques are enabled on a global proteomic scale by employing the latest generation of isobaric tagging reagents that facilitate multiplexing capacity required to measure the proteome across thermal gradients. Key among these is thermal proteomic profiling (TPP), which requires 8-10 isobaric tags per gradient and multiple sets of proteomic analyses to measure different replicates and conditions. Furthermore, using thermal profiling techniques to measure protein state across different conditions also requires measurements of difference in protein abundance. Here, we use PISA to assess protein abundance and thermal stability.
2024-02-14 | PXD031800 | Pride