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Affinity‐based proteomics reveals novel targets of inositol pyrophosphate (5‐IP 7 )‐dependent phosphorylation and binding in Trypanosoma cruzi replicative stages
Author(s) -
Mantilla Brian S.,
Kalesh Karunakaran,
Brown Nathaniel W.,
Fiedler Dorothea,
Docampo Roberto
Publication year - 2021
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.14672
Subject(s) - biology , phosphorylation , phosphoprotein , trypanosoma cruzi , biochemistry , proteomics , pyrophosphate , serine , inositol , trypanosoma brucei , microbiology and biotechnology , receptor , enzyme , parasite hosting , world wide web , computer science , gene
Diphosphoinositol‐5‐pentakisphosphate (5‐PP‐IP 5 ), also known as inositol heptakisphosphate (5‐IP 7 ), 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‐IP 7 affinity reagent, we performed pull‐down experiments coupled with mass spectrometry identification, and bioinformatic analysis, to reveal 5‐IP 7 ‐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‐IP 7 ‐driven phosphorylation is conserved in T. cruzi . Among the most interesting targets, we identified a choline/ o ‐acetyltransferase domain‐containing phosphoprotein that undergoes 5‐IP 7 ‐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‐IP 7 in this divergent eukaryote, and provided potential new targets for chemotherapy.