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Homeostasis of cellular fluxes of inorganic phosphate (Pi) supervises its structural roles in bones and teeth, its pervasive regulation of cellular metabolism, and its functionalization of numerous organic compounds. Cellular Pi efflux is heavily reliant on Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), regulation of which is largely unknown. We demonstrate specificity of XPR1 regulation by a comparatively uncharacterized member of the inositol pyrophosphate (PP-InsP) signaling family: 1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8). XPR1-mediated Pi efflux was inhibited by reducing cellular InsP8 synthesis, either genetically (knockout [KO] of diphosphoinositol pentakisphosphate kinases [PPIP5Ks] that synthesize InsP8) or pharmacologically [cell treatment with 2.5 µM dietary flavonoid or 10 µM N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine], to inhibit inositol hexakisphosphate kinases upstream of PPIP5Ks. Attenuated Pi efflux from PPIP5K KO cells was quantitatively phenocopied by KO of XPR1 itself. Moreover, Pi efflux from PPIP5K KO cells was rescued by restoration of InsP8 levels through transfection of wild-type PPIP5K1; transfection of kinase-dead PPIP5K1 was ineffective. Pi efflux was also rescued in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP8; PCP analogs of other PP-InsP signaling molecules were ineffective. High-affinity binding of InsP8 to the XPR1 N-terminus (Kd = 180 nM) was demonstrated by isothermal titration calorimetry. To derive a cellular biology perspective, we studied biomineralization in the Soas-2 osteosarcoma cell line. KO of PPIP5Ks or XPR1 strongly reduced Pi efflux and accelerated differentiation to the mineralization end point. We propose that catalytically compromising PPIP5K mutations might extend an epistatic repertoire for XPR1 dysregulation, with pathological consequences for bone maintenance and ectopic calcification. [ABSTRACT FROM AUTHOR]

We present here the most active synthetic Ni superoxide dismutase (NiSOD) mimic reported to date. Reactive oxygen species are aggressive compounds, which concentrations are tightly regulated in vivo. Among them, the superoxide anion, O2⸱-, is controlled by superoxide dismutases. Capitalizing on the versatility of the Amino-Terminal CuII- and NiII-binding (ATCUN) peptide motif, we introduced positive charges around the NiII center to favor the interaction with the superoxide radical anion. At physiological pH, the pentapeptide H-Cys-His-Cys-Arg-Arg-NH2 coordinates NiII after the deprotonation of one thiol, two amides, and either the second thiol or the N-terminal ammonium, leading to an equilibrium between the two N3S1 and N2S2 coordination modes. Under catalytic conditions, a kcat value of 8.6(4) x 106 L.mol-1.s-1 was measured. Within the first second, the catalyst remained undegraded with quantitative consumption of O2⸱- (completed up to 37 catalytic cycles). An extra arginine (Arg) was introduced at the peptide C-terminus to increase the global charge of the NiII complex from +1 to + 2. This had no effect on the catalytic performance, highlighting the critical role of charge distribution in space as a determining factor influencing the reactivity., (© 2024 Wiley‐VCH GmbH.)

Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP 5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP 5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence-based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl-binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high-affinity uranyl-binding peptides. We finally validated our approach by re-evaluating the uranyl-binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)