Affinity‐based proteomics reveals novel targets of inositol pyrophosphate (5‐IP7)‐dependent phosphorylation and binding in Trypanosoma cruzi replicative stages

Mantilla, Brian S.; Karunakaran, Kalesh; Brown, Nathaniel W.; Fiedler, Dorothea; Docampo, Roberto

Abstract

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 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.

Citation

Mantilla, B. S., Karunakaran, K., Brown, N. W., Fiedler, D., & Docampo, R. (2021). Affinity‐based proteomics reveals novel targets of inositol pyrophosphate (5‐IP7)‐dependent phosphorylation and binding in Trypanosoma cruzi replicative stages. Molecular Microbiology, 115(5), 986-1004. https://doi.org/10.1111/mmi.14672

• Journal Article Type: Article
• Acceptance Date: Dec 15, 2020
• Online Publication Date: Jan 21, 2021
• Publication Date: 2021-05
• Deposit Date: Jan 2, 2021
• Journal: Molecular Microbiology
• Print ISSN: 0950-382X
• Electronic ISSN: 1365-2958
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 115
• Issue: 5
• Pages: 986-1004
• DOI: https://doi.org/10.1111/mmi.14672
• Public URL: https://durham-repository.worktribe.com/output/1248418
• Related Public URLs: https://onlinelibrary.wiley.com/doi/abs/10.1111/mmi.14672