Dataset Information

Thermal proteome profiling of Toxoplasma gondii lysates treated with enhancer 1 (ENH1)

ABSTRACT: Previously, we characterized a chemical modulator, ENH1, that exhibited potent antiparasitic properties against apicomplexans and induced calcium oscillations, but the target eluded identification. To determine the target of ENH1, we employed thermal proteome profiling in parasites. We treated extracellular parasites with vehicle (DMSO) or ENH1 for 15 minutes at 37Â°C, under conditions previously determined to induce calcium fluxes in T. gondii.8 Parasites were then heated at 10 different temperatures to induce thermal denaturation. Following lysis and high-speed centrifugation, soluble protein was isolated and analyzed by mass spectrometry to generate thermal stability profiles from each condition across two biological replicates.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Toxoplasma Gondii Gt1

SUBMITTER: Alice Herneisen

LAB HEAD: Sebastian Lourido

PROVIDER: PXD018810 | Pride | 2020-07-13

REPOSITORIES: Pride

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Publications

Identifying the Target of an Antiparasitic Compound in Toxoplasma Using Thermal Proteome Profiling.

Herneisen Alice L AL Sidik Saima M SM Markus Benedikt M BM Drewry David H DH Zuercher William J WJ Lourido Sebastian S

ACS chemical biology 20200706 7

Apicomplexan parasites include the causative agents of malaria and toxoplasmosis. Cell-based screens in Toxoplasma previously identified a chemical modulator of calcium signaling (ENH1) that blocked parasite egress from host cells and exhibited potent antiparasitic activity. To identify the targets of ENH1, we adapted thermal proteome profiling to Toxoplasma, which revealed calcium-dependent protein kinase 1 (CDPK1) as a target. We confirmed the inhibition of CDPK1 by ENH1 in vitro ...[more]

PMID: 32597628

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Project description:Plants have evolved a two-layered immune system that mainly includes pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) against pathogen attack. PTI and ETI signaling are functionally linked, but also distinct due to specific perceived ligands and activation modes. Unraveling how PTI and ETI coordinate the immune responses against pathogens is crucial for understanding the regulatory mechanisms in plant immunity. To better understand the protein profiling and phosphorylation events during PTI and ETI, we employed integrated whole proteome and phosphoproteome analyses in the tomato-Pst pathosystem with different Pst DC3000 mutants that allow dissection of different layers of immune responses. A total of 225 proteins and 79 phosphopeptides were differentially regulated in tomato leaves during immune responses. Our proteomics results indicate that some overlapping immune responses are triggered by both PTI and ETI-inducing treatment, and ETI response is more robust than PTI response for most proteins. The change patterns of protein abundance and phosphorylation revealed some key regulators involved in Ca2+ signaling, mitogen-activated protein kinase cascades, and reversible protein phosphorylation, ROS and redox homeostasis, direct defense, transcription machinery and protein turnover, cell wall remodeling, hormone biosynthesis, as well as immune molecule accumulation, are modulated during PTI and/or ETI, suggesting their common or specific roles in plant immune responses. However, NAC domain protein and lipid particle serine esterase, two PTI-specific genes from previous transcriptomic work, have not been detected as differentially regulated in our proteomic analysis, and they were proved to be not PTI-specific inducible and therefore cannot be used as PTI-reporters through “overlapping circle” pattern assay. These results provide insights into the fine-tuned regulatory mechanisms between PTI and ETI in-Pst pathosystem, which will springboard further investigations into the sophisticated mechanisms in plant immunity.

Dataset Information

Thermal proteome profiling of Toxoplasma gondii lysates treated with enhancer 1 (ENH1)

Publications

Identifying the Target of an Antiparasitic Compound in <i>Toxoplasma</i> Using Thermal Proteome Profiling.

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