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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. [ABSTRACT FROM AUTHOR]

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., (© 2020 John Wiley & Sons Ltd.)

Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, specific roles of individual family members in each bacterial strain, as well as their protein-protein interactions, are yet to be understood. The apparent functional redundancy of the 4-18 PBPs that most eubacteria possess makes determination of their individual roles difficult. Existing techniques to study PBPs are not ideal because they do not directly visualize protein activity and can suffer from artifacts and perturbations of native PBP function. Therefore, development of new methods for studying the roles of individual PBPs in cell wall synthesis is required. We recently generated a library of fluorescent chemical probes containing a β-lactone scaffold that specifically targets the PBPs, enabling the visualization of their catalytic activity. Herein, we describe a general protocol to label and detect the activity of individual PBPs in Streptococcus pneumoniae using our fluorescent β-lactone probes., (© 2020 Elsevier Inc. All rights reserved.)

The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)