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Phosphoproteome analysis of TgPP1 depleted Toxoplasma gondii tachyzoites

ABSTRACT: Virulence of apicomplexan parasites is based on their ability to divide rapidly to produce significant biomass. The regulation of their cell-cycle is therefore key to their pathogenesis. Phosphorylation is a crucial post-transcriptional modification that regulates many aspects of the cell cycle. The phosphatase PP1 is known to play a major role in the phosphorylation balance in eukaryotes. We explored the role of TgPP1 during the cell cycle of the tachyzoite form of the apicomplexan parasite Toxoplasma gondii. Using a conditional mutant strain, we show that TgPP1 regulates many aspects of the cell cycle including the production of the daughter cells inner membrane complex (IMC), the segregation of organelles and the nuclear division. Unexpectedly, depletion of TgPP1 also results in the accumulation of amylopectin, a storage polysaccharide that is normally found in the latent bradyzoite form of the parasite. Using transcriptomics and phosphoproteomics, we show that TgPP1 mainly acts through post-transcriptional mechanisms by dephosphorylating target proteins including IMC proteins. TgPP1 also dephosphorylate a protein bearing a starch binding domain. Mutagenesis analysis reveals that the targeted phosphor-sites are linked to the ability of the parasite to produce amylopectin in conditions inducing bradyzoite differentiation. Therefore, we show that TgPP1 have pleiotropic roles during the tachyzoite cell cycle regulation, but also regulates amylopectin accumulation.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human) Toxoplasma Gondii Gt1

SUBMITTER: Cerina Chhuon

LAB HEAD: Ida Chiara Guerrera

PROVIDER: PXD043539 | Pride | 2024-08-08

REPOSITORIES: Pride

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			Action	DRS
	201106_MaGI_Phospho_LFQ_1_NoAux_1_9973.raw	Raw
	201106_MaGI_Phospho_LFQ_1_NoAux_2_9974.raw	Raw
	201106_MaGI_Phospho_LFQ_1_NoAux_3_9975.raw	Raw
	201106_MaGI_Phospho_LFQ_2_Aux_1_9985.raw	Raw
	201106_MaGI_Phospho_LFQ_2_Aux_2_9986.raw	Raw

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Project description:Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a non-coding CTG repeat expansion in the DMPK gene. This mutation generates a toxic CUG RNA that interferes with the RNA processing of target genes in multiple tissues. Despite debilitating neurological impairment, the pathophysiological cascade of molecular and cellular events in the central nervous system has been less extensively characterized than the molecular pathogenesis of muscle/cardiac dysfunction. Particularly, the contribution of different cell types to DM1 brain disease is not clearly understood. We first used transcriptomics to compare the impact of expanded CUG RNA on the transcriptome of primary neurons, astrocytes and oligodendrocytes derived from DMSXL mice, a transgenic model of DM1. RNA sequencing revealed more frequent expression and splicing changes in glia than neuronal cells. In particular, primary DMSXL oligodendrocytes showed the highest number of transcripts differentially expressed, while DMSXL astrocytes displayed the most severe splicing dysregulation. Interestingly, the expression and splicing defects of DMSXL glia recreated molecular signatures suggestive of impaired cell differentiation: while DMSXL oligodendrocytes failed to upregulate a subset of genes that are naturally activated during the oligodendroglia differentiation, a significant proportion of missplicing events in DMSXL oligodendrocytes and astrocytes increased the expression of RNA isoforms typical of precursor cell stages. Together these data suggest that expanded CUG RNA in glial cells affects preferentially differentiation-regulated molecular events. This hypothesis was corroborated by gene ontology (GO) analyses, which revealed an enrichment for biological processes and cellular components with critical roles during cell differentiation. Finally, we combined exon ontology with phosphoproteomics and cell imaging to explore the functional impact of CUG-associated spliceopathy on downstream protein metabolism. Changes in phosphorylation, protein isoform expression and intracellular localization in DMSXL astrocytes demonstrate the far-reaching impact of the DM1 repeat expansion on cell metabolism. Our multi-omics approaches provide insight into the mechanisms of CUG RNA toxicity in the CNS with cell type resolution, and support the priority for future research on non-neuronal mechanisms and proteomic changes in DM1 brain disease.

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Phosphoproteome analysis of TgPP1 depleted Toxoplasma gondii tachyzoites

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