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  • Affinity-based proteomics reveals novel targets of inositol pyrophosphate (5-IP7)-dependent phosphorylation and binding in Trypanosoma cruzi replicative stages MOLECULAR MICROBIOLOGY Mantilla, B. S., Kalesh, K., Brown, N. W., Fiedler, D., Docampo, R. 2021; 115 (5): 986-1004

    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.

    View details for DOI 10.1111/mmi.14672

    View details for Web of Science ID 000609330400001

    View details for PubMedID 33354791

  • MLKL Requires the Inositol Phosphate Code to Execute Necroptosis. Molecular cell Dovey, C. M., Diep, J. n., Clarke, B. P., Hale, A. T., McNamara, D. E., Guo, H. n., Brown, N. W., Cao, J. Y., Grace, C. R., Gough, P. J., Bertin, J. n., Dixon, S. J., Fiedler, D. n., Mocarski, E. S., Kaiser, W. J., Moldoveanu, T. n., York, J. D., Carette, J. E. 2018; 70 (5): 936–48.e7

    Abstract

    Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

    View details for PubMedID 29883610