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 β-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 (Ser578-580). We also identified a novel SPX domain-containing phosphoribosyltransferase [EC 18.104.22.168] 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.
Bibliographical noteFunding Information:
This work was supported by U.S. National Institutes of Health grants AI077538 and AI108222 (to R.D.).
This work was supported by U.S. National Institutes of Health grants AI077538 and AI108222 (to R.D.). We thank Dennis Philips, head of the Proteomic and Mass Spectrometry (PAMS) Core Facility at UGA, for technical support in LC-MS and Chau-Wen Chou for proteomic sample processing, Robert Puschmann for support in purification of 5?[32P]5-IP7, Belen Cassera for assistance in LC-MS analysis, Guozhong Huang for providing the pMoTag-sm-cMyc construct used for tagging, Rodrigo P. Baptista for assistance in use Blast2Go software, Melissa Storey for technical assistance with amastigote isolation, Henning J. Jessen for 5-IP7, and Muthugapatti Kandasamy and the Biomedical Microscopy Core of the University of Georgia for the use of microscopes.
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