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Identification of proteins exhibiting TgDegP2-responsive thermal stability

ABSTRACT: 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

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Toxoplasma Gondii Gt1

SUBMITTER: Alice Herneisen

LAB HEAD: Sebastian Lourido

PROVIDER: PXD019917 | Pride | 2020-08-28

REPOSITORIES: pride

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Dataset's files

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			Action	DRS
	20200118_DegP2_CETSA_heavy.mzML	Mzml
	20200118_DegP2_CETSA_heavy.mzid	Mzid
	20200118_DegP2_CETSA_light.mzML	Mzml
	20200118_DegP2_CETSA_light.mzid	Mzid
	20200217_DegP2_CETSA_plusRapa_10.raw	Raw

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Publications

Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility.

Harding Clare R CR Sidik Saima M SM Petrova Boryana B Gnädig Nina F NF Okombo John J Herneisen Alice L AL Ward Kurt E KE Markus Benedikt M BM Boydston Elizabeth A EA Fidock David A DA Lourido Sebastian S

Nature communications 20200923 1

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibil ...[more]

PMID: 32968076

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

Project description:reviously, 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 37°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.

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De novo mapping of the apicomplexan Ca2+-responsive proteome: concentration range thermal profiling experiment 2

Project description:Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the transition between the replicative and lytic phases of the infectious cycle. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific molecular components in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.

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