Journal
MOLECULAR MICROBIOLOGY
Volume 115, Issue 5, Pages 986-1004Publisher
WILEY
DOI: 10.1111/mmi.14672
Keywords
inositol phosphate; inositol pyrophosphate; pyrophosphorylation; SPX domain; Trypanosoma cruzi
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Funding
- U.S. National Institutes of Health [AI077538, AI108222]
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This study identified potential targets of 5-IP7 in Trypanosoma cruzi using pull-down experiments and mass spectrometry analysis, revealing new functional roles of 5-IP7 in this divergent eukaryote and providing potential new targets for chemotherapy.
Diphosphoinositol-5-pentakisphosphate (5-PP-IP5), also known as inositol heptakisphosphate (5-IP7), has been described as a high-energy phosphate metabolite that participates in the regulation of multiple cellular processes through protein binding or serine pyrophosphorylation, a posttranslational modification involving a beta-phosphoryl transfer. In this study, utilizing an immobilized 5-IP7 affinity reagent, we performed pull-down experiments coupled with mass spectrometry identification, and bioinformatic analysis, to reveal 5-IP7-regulated processes in the two proliferative stages of the unicellular parasite Trypanosoma cruzi. Our protein screen clearly defined two cohorts of putative targets either in the presence of magnesium ions or in metal-free conditions. We endogenously tagged four protein candidates and immunopurified them to assess whether 5-IP7-driven phosphorylation is conserved in T. cruzi. Among the most interesting targets, we identified a choline/o-acetyltransferase domain-containing phosphoprotein that undergoes 5-IP7-mediated phosphorylation events at a polyserine tract (Ser(578-580)). We also identified a novel SPX domain-containing phosphoribosyltransferase [EC 2.7.6.1] herein termed as TcPRPPS4. Our data revealed new possible functional roles of 5-IP7 in this divergent eukaryote, and provided potential new targets for chemotherapy.
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