Search

Precise regulation of intracellular phosphate (Pi) is critical for cellular function, with xenotropic and polytropic retrovirus receptor 1 (XPR1) serving as the sole Pi exporter in humans. The mechanism of Pi efflux, activated by inositol pyrophosphates (PP-IPs), has remained unclear. This study presents cryo-electron microscopy structures of XPR1 in multiple conformations, revealing a transmembrane pathway for Pi export and a dual-binding activation pattern for PP-IPs. A canonical binding site is located at the dimeric interface of Syg1/Pho81/XPR1 (SPX) domains, and a second site, biased toward PP-IPs, is found between the transmembrane and SPX domains. By integrating structural studies with electrophysiological analyses, we characterized XPR1 as an inositol phosphates (IPs)/PP-IPs-activated phosphate channel. The interplay among its transmembrane domains, SPX domains, and IPs/PP-IPs orchestrates the conformational transition between its closed and open states.

Inositol pyrophosphates (PP-InsPs) are eukaryote-specific second messengers that regulate diverse cellular processes, including immunity, nutrient sensing, and hormone signaling pathways in plants. These energy-rich messengers exhibit high sensitivity to the cellular phosphate status, suggesting that the synthesis and degradation of PP-InsPs are tightly controlled within the cells. Notably, the molecular basis of PP-InsP hydrolysis in plants remains largely unexplored. In this study, we report the functional characterization of MpDDP1, a diadenosine and diphosphoinositol polyphosphate phosphohydrolase encoded by the genome of the liverwort, Marchantia polymorpha . We show that MpDDP1 functions as a PP-InsP phosphohydrolase in different heterologous organisms. Consistent with this finding, M. polymorpha plants defective in MpDDP1 exhibit elevated levels of 1/3-InsP 7 and 1/3,5-InsP 8 , highlighting the contribution of MpDDP1 in regulating PP-InsP homeostasis in planta . Furthermore, our study reveals that MpDDP1 controls thallus development and vegetative reproduction in M. polymorpha . Collectively, this study provides insights into the regulation of specific PP-InsP messengers by DDP1-type phosphohydrolases in land plants.

Combinatorial substitution of phosphate groups on the inositol ring gives rise to a plethora of inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs). These small molecules constitute an elaborate metabolic and signalling network that influences nearly every cellular function. This review delves into the knowledge accumulated over the past decades regarding the biochemical principles and significance of InsP metabolism. We focus on the biological actions of InsPs in mammals, with an emphasis on recent findings regarding specific target proteins. We further discuss the roles of InsP metabolism in contributing to physiological homeostasis and pathological conditions. A deeper understanding of InsPs and their metabolic pathways holds the potential to address unresolved questions and propel advances towards therapeutic applications., Competing Interests: Declaration of interests The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Inositol pyrophosphates (PP-InsPs) are a sub-family of water soluble inositol phosphates that possess one or more diphosphate groups. PP-InsPs can transfer their β-phosphate group to a phosphorylated Ser residue to generate pyrophosphorylated Ser. This unique post-translational modification occurs on Ser residues that lie in acidic stretches within an intrinsically disordered protein sequence. Serine pyrophosphorylation is dependent on the presence of Mg2+ ions, but does not require an enzyme for catalysis. The mechanisms by which cells regulate PP-InsP-mediated pyrophosphorylation are still unknown. We performed mass spectrometry to identify interactors of IP6K1, an enzyme responsible for the synthesis of the PP-InsP 5-InsP7. Interestingly, IP6K1 interacted with several proteins that are known to undergo 5-InsP7-mediated pyrophosphorylation, including the nucleolar proteins NOLC1, TCOF and UBF1, and AP3B1, the β subunit of the AP3 adaptor protein complex. The IP6K1 interactome also included CK2, a protein kinase that phosphorylates Ser residues prior to pyrophosphorylation. We observe the formation of a protein complex between IP6K1, AP3B1, and the catalytic α-subunit of CK2, and show that disrupting IP6K1 binding to AP3B1 lowers its in vivo pyrophosphorylation. We propose that assembly of a substrate-CK2-IP6K complex would allow for coordinated pre-phosphorylation and pyrophosphorylation of the target serine residue, and provide a mechanism to regulate this enzyme-independent modification., (© 2024 The Author(s).)

Background and Purpose: Pulmonary hypertension (PH) results from pulmonary vasculopathy, initially leading to a compensatory right ventricular (RV) hypertrophy, and eventually to RV failure. Hypoxia can trigger both pulmonary vasculopathy and RV failure. Therefore, we tested if myo-inositol trispyrophosphate (ITPP), which facilitates oxygen dissociation from haemoglobin, can relieve pulmonary vasculopathy and RV hypoxia, and eventually prevent RV failure and mortality in the rat model of monocrotaline-induced PH., Experimental Approach: Rats were injected with monocrotaline (PH) or saline (control) and received ITPP or placebo for 5 weeks. Serial echocardiograms were obtained to monitor the disease, pressure-volume loops were recorded and evaluated, myocardial pO 2 was measured using a fluorescent probe, and histological and molecular analyses were conducted at the conclusion of the experiment., Key Results and Conclusions: ITPP reduced PH-related mortality. It had no effect on progressive increase in pulmonary vascular resistance, yet significantly relieved intramyocardial RV hypoxia, which was associated with improvement of RV function and reduction of RV wall stress. ITPP also tended to prevent increased hypoxia inducible factor-1α expression in RV cardiac myocytes but did not affect RV capillary density., Implications: Our study suggests that strategies aimed at increasing oxygen delivery to hypoxic RV in PH could potentially be used as adjuncts to other therapies that target pulmonary vessels, thus increasing the ability of the RV to withstand increased afterload and reducing mortality. ITPP may be one such potential therapy., (© 2024 British Pharmacological Society.)

Clostridioides difficile is a bacterium that causes life-threatening intestinal infections. Infection symptoms are mediated by a toxin secreted by the bacterium. Toxin pathogenesis is modulated by the intracellular molecule, inositol-hexakisphosphate (IP6). IP6 binds to a cysteine protease domain (CPD) on the toxin, inducing autoproteolysis, which liberates a virulence factor in the cell cytosol. We developed second-generation IP6 analogs designed to induce autoproteolysis in the gut lumen, prior to toxin uptake, circumventing pathogenesis. We synthesized a panel of thiophosphate-/sulfate-containing IP6 analogs and characterized their toxin binding affinity, autoproteolysis induction, and cation interactions. Our top candidate was soluble in extracellular cation concentrations, unlike IP6. The IP6 analogs were more negatively charged than IP6, which improved affinity and stabilization of the CPD, enhancing toxin autoproteolysis. Our data illustrate the optimization of IP6 with thiophosphate biomimetic which are more capable of inducing toxin autoproteolysis than the native ligand, warranting further studies in vivo.

G protein-coupled receptors (GPCRs) play pivotal roles in cellular signaling and can regulate several cellular functions such as proliferation, secretion, protein expression, and cellular metabolism. Coupling of GPCRs to members of the Gq/11 protein family results in activation of inositol trisphosphate (IP3) and accumulation of calcium intracellularly. This protocol chapter outlines a step-by-step guide for utilizing the inositol phosphate-1 (IP 1 ) accumulation assay, a time-resolved fluorescence resonance energy transfer (TR-FRET) method, to investigate Gq-IP3 signaling. The assay serves as a valuable tool for those conducting pharmacological investigations and compound screening targeting this critical cellular pathway. This protocol chapter covers experimental setup, sample preparation, and data analysis, providing researchers with an in-depth guide to explore the pharmacology of Gq-coupled receptors., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD + -dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus Thermochaetoides thermophila . By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications., Competing Interests: Competing interests statement:The authors declare no competing interest.

Inositol pyrophosphate 1,5-IP 8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1 , pho84 , and tgp1 , via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP 8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP 7 to 1,5-IP 8 and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP 7 , 1-IP 7 , and 1,5-IP 8 . Aps1 displays a ~twofold preference for hydrolysis of 1-IP 7 versus 5-IP 7 and aps1 ∆ cells have twofold higher levels of 1-IP 7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1 ∆ siw14 ∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP 8 toxicosis, whereby excessive 1,5-IP 8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1 ∆ siw14 ∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1 ∆ siw14 ∆ on YES by deleting tgp1 . However, the severe growth defect of an aps1 ∆ asp1-H397A strain could not be alleviated by deleting tgp1 , suggesting that 1,5-IP 8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity., Importance: Repression of the fission yeast PHO genes tgp1 , pho1 , and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP 8 , a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP 8 is formed by phosphorylation of 5-IP 7 and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo . Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP 7 , 1-IP 7 , and 1,5-IP 8 in vitro , with a ~twofold preference for 1-IP 7 over 5-IP 7 . aps1 ∆ cells have twofold higher levels of 1-IP 7 than wild-type cells. An aps1 ∆ siw14 ∆ double mutation is lethal because excessive 1,5-IP 8 triggers derepression of tgp1 , leading to toxic uptake of glycerophosphocholine., Competing Interests: The authors declare no conflict of interest.

Discovered in 1993, inositol pyrophosphates are evolutionarily conserved signaling metabolites whose versatile modes of action are being increasingly appreciated. These include their emerging roles as energy regulators, phosphodonors, steric/allosteric regulators, and G protein-coupled receptor messengers. Through studying enzymes that metabolize inositol pyrophosphates, progress has also been made in elucidating the various cellular and physiological functions of these pyrophosphate-containing, energetic molecules. The two main forms of inositol pyrophosphates, 5-IP 7 and IP 8 , synthesized respectively by inositol-hexakisphosphate kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks), regulate phosphate homeostasis, ATP synthesis, and several other metabolic processes ranging from insulin secretion to cellular energy utilization. Here, we review the current understanding of the catalytic and regulatory mechanisms of IP6Ks and PPIP5Ks, as well as their counteracting phosphatases. We also highlight the genetic and cellular evidence implicating inositol pyrophosphates as essential mediators of mammalian metabolic homeostasis.

Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1 , pho84 , and tgp1 ) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP 8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP 8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP 8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP 8 toxicity is alleviated by deletion of snf22 + , the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22- ( D996A-E997A ) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22 + hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO -repressive lncRNAs. Transcription profiling of snf22 ∆ and snf22- ( D996A-E997A ) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1 , pho1 , and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP 8 as an agonist of precocious lncRNA 3'-processing/termination. IP 8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP 8 levels on termination. In this study, a forward genetic screen revealed that IP 8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO -activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters., Competing Interests: The authors declare no conflict of interest.

We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV.IMPORTANCEInositol hexakisphosphate (IP6) is crucial for the assembly and replication of HIV-1. IP6 is packaged in HIV-1 particles and stabilizes the viral core enabling it to synthesize viral DNA early in viral infection. While its importance for HIV-1 is well established, its significance for other retroviruses is unknown. Here we report the role of IP6 in the gammaretrovirus, murine leukemia virus (MLV). We found that like HIV-1, MLV packages IP6, and as in HIV-1, IP6 stabilizes the MLV core thus promoting reverse transcription. Interestingly, we discovered a key difference in the role of IP6 in MLV versus HIV-1: while HIV-1 is not dependent upon IP6 levels in target cells, MLV replication is significantly reduced in IP6-deficient cell lines. We suggest that this difference in IP6 requirements reflects key differences between HIV-1 and MLV replication., Competing Interests: The authors declare no conflict of interest.

Aims: Adiponectin is an adipocyte-derived circulating protein that exerts cardiovascular and metabolic protection. Due to the futile degradation of endogenous adiponectin and the challenges of exogenous administration, regulatory mechanisms of adiponectin biosynthesis are of significant pharmacological interest., Methods and Results: Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) generated by inositol hexakisphosphate kinase 1 (IP6K1) governed circulating adiponectin levels via thiol-mediated protein quality control in the secretory pathway. IP6K1 bound to adiponectin and DsbA-L and generated 5-InsP7 to stabilize adiponectin/ERp44 and DsbA-L/Ero1-Lα interactions, driving adiponectin intracellular degradation. Depleting 5-InsP7 by either IP6K1 deletion or pharmacological inhibition blocked intracellular adiponectin degradation. Whole-body and adipocyte-specific deletion of IP6K1 boosted plasma adiponectin levels, especially its high molecular weight forms, and activated AMPK-mediated protection against myocardial ischaemia-reperfusion injury. Pharmacological inhibition of 5-InsP7 biosynthesis in wild-type but not adiponectin knockout mice attenuated myocardial ischaemia-reperfusion injury., Conclusion: Our findings revealed that 5-InsP7 is a physiological regulator of adiponectin biosynthesis that is amenable to pharmacological intervention for cardioprotection., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Phosphate (Pi) serves countless metabolic pathways and is involved in macromolecule synthesis, energy storage, cellular signaling, and bone maintenance. Herein, we describe the coordination of Pi uptake and efflux pathways to maintain mammalian cell Pi homeostasis. We discover that XPR1, the presumed Pi efflux transporter, separately supervises rates of Pi uptake. This direct, regulatory interplay arises from XPR1 being a binding partner for the Pi uptake transporter PiT1, involving a predicted transmembrane helix/extramembrane loop in XPR1, and its hitherto unknown localization in a subset of intracellular LAMP1-positive puncta (named "XLPVs"). A pharmacological mimic of Pi homeostatic challenge is sensed by the inositol pyrophosphate IP 8 , which functionalizes XPR1 to respond in a temporally hierarchal manner, initially adjusting the rate of Pi efflux, followed subsequently by independent modulation of PiT1 turnover to reset the rate of Pi uptake. These observations generate a unifying model of mammalian cellular Pi homeostasis, expanding opportunities for therapeutic intervention., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Inositol hexaphosphate (InsP 6 ) is the major storage form of phosphorus in seeds. Reducing seed InsP 6 content is a breeding objective in agriculture, as InsP 6 negatively impacts animal nutrition and the environment. Nevertheless, how InsP 6 accumulation is regulated remains largely unknown. Here, we identify a clade of receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP 6 accumulation. The InsP 6 concentration is dramatically reduced in seeds of ipck quadruple (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs recruit IPK1 involved in InsP 6 synthesis, and facilitate its binding and activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s and PI-PLCs required for InsP 4 /InsP 5 and InsP 3 biosynthesis respectively, to form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover a regulatory mechanism of InsP 6 accumulation governed by IPCKs, shedding light on the mechanisms of InsP biosynthesis in eukaryotes., (© 2024. The Author(s).)

The maintenance of phosphate homeostasis serves as a foundation for energy metabolism and signal transduction processes in all living organisms. Inositol pyrophosphates (PP-InsPs), composed of an inositol ring decorated with monophosphate and diphosphate moieties, and inorganic polyphosphate (polyP), chains of orthophosphate residues linked by phosphoanhydride bonds, are energy-rich biomolecules that play critical roles in phosphate homeostasis. There is a complex interplay between these two phosphate-rich molecules, and they share an interdependent relationship with cellular adenosine triphosphate (ATP) and inorganic phosphate (Pi). In eukaryotes, the enzymes involved in PP-InsP synthesis show some degree of conservation across species, whereas distinct enzymology exists for polyP synthesis among different organisms. In fact, the mechanism of polyP synthesis in metazoans, including mammals, is still unclear. Early studies on PP-InsP and polyP synthesis were conducted in the slime mould Dictyostelium discoideum, but it is in the budding yeast Saccharomyces cerevisiae that a clear understanding of the interplay between polyP, PP-InsPs, and Pi homeostasis has now been established. Recent research has shed more light on the influence of PP-InsPs on polyP in mammals, and the regulation of both these molecules by cellular ATP and Pi levels. In this review we will discuss the cross-talk between PP-InsPs, polyP, ATP, and Pi in the context of budding yeast, slime mould, and mammals. We will also highlight the similarities and differences in the relationship between these phosphate-rich biomolecules among this group of organisms., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

MshA is a GT-B glycosyltransferase catalyzing the first step in the biosynthesis of mycothiol. While many GT-B enzymes undergo an open-to-closed transition, MshA is unique because its 97° rotation is beyond the usual range of 10-25°. Molecular dynamics (MD) simulations were carried out for MshA in both ligand bound and unbound states to investigate the effect of ligand binding on localized protein dynamics and its conformational free energy landscape. Simulations showed that both the unliganded "opened" and liganded "closed" forms of the enzyme sample a wide degree of dihedral angles and interdomain distances with relatively low overlapping populations. Calculation of the free energy surface using replica exchange MD for the apo "opened" and an artificial generated apo "closed" structure revealed overlaps in the geometries sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed transition in the absence of ligands. MD simulations of fully liganded MshA revealed a smaller sampling of the dihedral angles. The localized protein fluctuation changes suggest that UDP-GlcNAc binding activates the motions of loops in the 1-l- myo -inositol-1-phosphate (I1P)-binding site despite little change in the interactions with UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy, and mutagenesis studies were used to confirm the ligand-induced structural changes in MshA. The results support a proposed mechanism where UDP-GlcNAc binds with rigid interactions to the C-terminal domain of MshA and activates flexible loops in the N-terminal domain for binding and positioning of I1P. This model can be used for future structure-based drug development of inhibitors of the mycothiol biosynthetic pathway.

Inositol phosphates and their metabolites play a significant role in several biochemical pathways, gene expression regulation, and phosphate homeostasis. Among the different inositol phosphates, inositol hexakisphosphate (IP6) is a substrate of inositol hexakisphosphate kinases (IP6Ks), which phosphorylate one or more of the IP6 phosphate groups. Pyrophosphorylation of IP6 leads to the formation of inositol pyrophosphates, high-energy signaling molecules that mediate physiological processes through their ability to modify target protein activities, either by directly binding to their target protein or by pyrophosphorylating protein serine residues. 5-diphosphoinositol pentakisphosphate, the most abundant inositol pyrophosphate in mammals, has been extensively studied and found to be significantly involved in a wide range of physiological processes. Three IP6K (IP6K1, IP6K2, and IP6K3) isoforms regulate IP7 synthesis in mammals. Here, we summarize our current understanding of IP6K1's roles in cytoskeletal remodeling, trafficking, cellular migration, metabolism, gene expression, DNA repair, and immunity. We also briefly discuss current gaps in knowledge, highlighting the need for further investigation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

D-myo-inositol 1,4,5-trisphosphate (InsP 3 ) is a fundamental second messenger in cellular Ca 2+ mobilization. InsP 3 3-kinase, a highly specific enzyme binding InsP 3 in just one mode, phosphorylates InsP 3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP 3 , we have surveyed the limits of InsP 3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP 3 3-kinase plasticity and substrate tolerance that may be more widely exploitable., (© 2024. The Author(s).)

The inositol pyrophosphate signaling molecule 1,5-IP 8 is an agonist of RNA 3 ' -processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1 , pho84 , and tgp1 . IP 8 is synthesized from 5-IP 7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1 + cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP 6 kinase Kcs1 that generates 5-IP 7 , the immediate precursor to IP 8 . Transcription profiling of the kcs1 mutants in an asp1 + background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP 3 , an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP 8 governs repression of fission yeast phosphate homeostasis genes pho1 , pho84 , and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP 8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP 8 agonism of transcription termination. It was assumed that IP 8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP 8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP 3 and the essential Kcs1 kinase that converts IP 6 to 5-IP 7 , the immediate precursor of IP 8 ., Competing Interests: The authors declare no conflict of interest.

Kinases that synthesize inositol phosphates (IPs) and pyrophosphates (PP-IPs) control numerous biological processes in eukaryotic cells. Herein, we extend this cellular signaling repertoire to viruses. We have biochemically and structurally characterized a minimalist inositol phosphate kinase (i.e., TvIPK) encoded by Terrestrivirus, a nucleocytoplasmic large ("giant") DNA virus (NCLDV). We show that TvIPK can synthesize inositol pyrophosphates from a range of scyllo- and myo-IPs, both in vitro and when expressed in yeast cells. We present multiple crystal structures of enzyme/substrate/nucleotide complexes with individual resolutions from 1.95 to 2.6 Å. We find a heart-shaped ligand binding pocket comprising an array of positively charged and flexible side chains, underlying the observed substrate diversity. A crucial arginine residue in a conserved "G-loop" orients the γ-phosphate of ATP to allow substrate pyrophosphorylation. We highlight additional conserved catalytic and architectural features in TvIPK, and support their importance through site-directed mutagenesis. We propose that NCLDV inositol phosphate kinases may have assisted evolution of inositol pyrophosphate signaling, and we discuss the potential biogeochemical significance of TvIPK in soil niches., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Water-soluble myo-inositol phosphates have long been characterized as second messengers. The signaling properties of these compounds are determined by the number and arrangement of phosphate groups on the myo-inositol backbone. Recently, higher inositol phosphates with pyrophosphate groups were recognized as signaling molecules. 5-Diphosphoinositol 1,2,3,4,6-pentakisphosphate (5PP-InsP 5 ) is the most abundant isoform, constituting more than 90% of intracellular inositol pyrophosphates. 5PP-InsP 5 can be further phosphorylated to 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP 8 ). These two molecules, 5PP-InsP 5 and InsP 8 , are present in various subcellular compartments, where they participate in regulating diverse cellular processes such as cell death, energy homeostasis, and cytoskeletal dynamics. The synthesis and metabolism of inositol pyrophosphates are subjected to tight regulation, allowing for their highly specific functions. Blocking the 5PP-InsP 5 /InsP 8 signaling pathway by inhibiting the biosynthesis of 5PP-InsP 5 demonstrates therapeutic benefits in preclinical studies, and thus holds promise as a therapeutic approach for certain diseases treatment, such as metabolic disorders., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The inositol pyrophosphate pathway, a complex cell signaling network, plays a pivotal role in orchestrating vital cellular processes in the budding yeast, where it regulates cell cycle progression, growth, endocytosis, exocytosis, apoptosis, telomere elongation, ribosome biogenesis, and stress responses. This pathway has gained significant attention in pharmacology and medicine due to its role in generating inositol pyrophosphates, which serve as crucial signaling molecules not only in yeast, but also in higher eukaryotes. As targets for therapeutic development, genetic modifications within this pathway hold promise for disease treatment strategies, offering practical applications in biotechnology. The model organism Saccharomyces cerevisiae , renowned for its genetic tractability, has been instrumental in various studies related to the inositol pyrophosphate pathway. This review is focused on the Kcs1 and Vip1, the two enzymes involved in the biosynthesis of inositol pyrophosphate in S. cerevisiae , highlighting their roles in various cell processes, and providing an up-to-date overview of their relationship with phosphate homeostasis. Moreover, the review underscores the potential applications of these findings in the realms of medicine and biotechnology, highlighting the profound implications of comprehending this intricate signaling network.

The natural cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), is biosynthesized from prostaglandin E (PGE) and activated inositol phosphate (n-Ins-P), which is synthesized by a particulate rat-liver-enzyme from GTP and a precursor named inositol phosphate (pr-Ins-P), whose 5-ring phosphodiester structure is essential for n-Ins-P synthesis. Aortic myocytes, preincubated with [ 3 H] myo-inositol, synthesize after angiotensin II stimulation (30 s) [ 3 H] pr-Ins-P (65% yield), which is converted to [ 3 H] n-Ins-P and [ 3 H] cyclic PIP. Acid-treated (1 min) [ 3 H] pr-Ins-P co-elutes with inositol (1,4)-bisphosphate in high performance ion chromatography, indicating that pr-Ins-P is inositol (1:2-cyclic,4)-bisphosphate. Incubation of [ 3 H]-GTP with unlabeled pr-Ins-P gave [ 3 H]-guanosine-labeled n-Ins-P. Cyclic PIP synthase binds the inositol (1:2-cyclic)-phosphate part of n-Ins-P to PGE and releases the [ 3 H]-labeled guanosine as [ 3 H]-GDP. Thus, n-Ins-P is most likely guanosine diphospho-4-inositol (1:2-cyclic)-phosphate. Inositol feeding helps patients with metabolic conditions related to insulin resistance, but explanations for this finding are missing. Cyclic PIP appears to be the key for explaining the curative effect of inositol supplementation: (1) inositol is a molecular constituent of cyclic PIP; (2) cyclic PIP triggers many of insulin's actions intracellularly; and (3) the synthesis of cyclic PIP is decreased in diabetes as shown in rodents.

The choice of the calcium (Ca) source in pig diets and the addition of formic acid may affect the gastrointestinal inositol phosphate (InsP) degradation and thereby, phosphorus (P) digestibility in pigs. This study assessed the effects of different Ca sources (Ca carbonate, Ca formate), exogenous phytase, and chemical acidification on InsP degradation, nutrient digestion and retention, blood metabolites, and microbiota composition in growing pigs. In a randomized design, 8 ileal-cannulated barrows (24 kg initial BW) were fed 5 diets containing Ca formate or Ca carbonate as the only mineral Ca addition, with or without 1,500 FTU/kg of an exogenous hybrid 6-phytase. A fifth diet was composed of Ca carbonate with phytase but with 8 g formic acid/kg diet. No mineral P was added to the diets. Prececal InsP6 disappearance and P digestibility were lower (P ≤ 0.032) in pigs fed diets containing Ca formate. In the presence of exogenous phytase, InsP5 and InsP4 concentrations in the ileal digesta were lower (P ≤ 0.019) with Ca carbonate than Ca formate. The addition of formic acid to Ca carbonate with phytase diet resulted in greater (P = 0.027) prececal InsP6 disappearance (87% vs. 80%), lower (P = 0.001) InsP5 concentration, and greater (P ≤ 0.031) InsP2 and myo-inositol concentrations in the ileal digesta. Prececal P digestibility was greater (P = 0.004) with the addition of formic acid compared to Ca carbonate with phytase alone. Prececal amino acid (AA) digestibility of some AA was greater with Ca formate compared to Ca carbonate but only in diets with phytase (P ≤ 0.048). The addition of formic acid to the diet with Ca carbonate and phytase increased (P ≤ 0.006) the prececal AA digestibility of most indispensable AA. Exogenous phytase affected more microbial genera in the feces when Ca formate was used compared to Ca carbonate. In the ileal digesta, the Ca carbonate diet supplemented with formic acid and phytase led to a similar microbial community as the Ca formate diets. In conclusion, Ca formate reduced prececal InsP6 degradation and P digestibility, but might be of advantage in regard to prececal AA digestibility in pigs compared to Ca carbonate when exogenous phytase is added. The addition of formic acid to Ca carbonate with phytase, however, resulted in greater InsP6 disappearance, P and AA digestibility values, and changed ileal microbiota composition compared to Ca carbonate with phytase alone., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Inositol hexakisphosphate (IP6), a naturally occurring metabolite of inositol with specific functions in different organelles or tissues, participates in numerous physiological processes and plays a key role in mammalian metabolic regulation. However, current IP6 detection methods, i.e., high-performance liquid chromatography and gel electrophoresis, require sample destruction and lack spatiotemporal resolution. Here, we construct and characterize a genetically encoded fluorescence biosensor named HIPSer that enables ratiometric quantitative IP6 detection in HEK293T cells and subcellular compartments. We demonstrate that HIPSer has a high sensitivity and relative selectivity for IP6 in vitro. We also provide proof-of-concept evidence that HIPSer can monitor IP6 levels in real time in HEK293T cells and can be targeted for IP6 detection in the nucleus of HEK293T cells. Moreover, HIPSer could also detect changes in IP6 content induced by chemical inhibition of IP6-metabolizing enzymes in HEK293T cells. Thus, HIPSer achieves spatiotemporally precise detection of fluctuations in endogenous IP6 in live cells and provides a versatile tool for mechanistic investigations of inositol phosphate functions in metabolism and signaling.

The fast envelope stress responses play a key role in the transmission and pathogenesis of Yersinia enterocolitica, one of the most common foodborne pathogens. Our previous study showed that deletion of the waaF gene, essential for the biosynthesis of lipopolysaccharide (LPS) core polysaccharides, led to the formation of a truncated LPS structure and induced cell envelope stress. This envelope stress may disturb the intracellular signal transduction, thereby affecting the physiological functions of Y. enterocolitica. In this study, truncated LPS caused by waaF deletion was used as a model of envelope stress in Y. enterocolitica. We investigated the mechanisms of envelope stress responses and the cellular functions affected by truncated LPS. Transcriptome analysis and phenotypic validation showed that LPS truncation reduced flagellar assembly, bacterial chemotaxis, and inositol phosphate metabolism, presenting lower pathogenicity and viability both in vivo and in vitro environments. Further 4D label-free phosphorylation analysis confirmed that truncated LPS perturbed multiple intracellular signal transduction pathways. Specifically, a comprehensive discussion was conducted on the mechanisms by which chemotactic signal transduction and Rcs system contribute to the inhibition of chemotaxis. Finally, the pathogenicity of Y. enterocolitica with truncated LPS was evaluated in vitro using IPEC-J2 cells as models, and it was found that truncated LPS exhibited reduced adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. Our research provides an understanding of LPS in the regulation of Y. enterocolitica viability and pathogenicity and, thus, opening new avenues to develop novel food safety strategies or drugs to prevent and control Y. enterocolitica infections. KEY POINTS: • Truncated LPS reduces flagellar assembly, chemotaxis, and inositol phosphate metabolism in Y. enterocolitica. • Truncated LPS reduces adhesion, invasion, and toxicity of Y. enterocolitica to IPEC-J2 cells. • Truncated LPS regulates intracellular signal transduction of Y. enterocolitica., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Importance: The inositol pyrophosphate signaling molecule 1,5-IP 8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular 1,5-IP 8 levels are determined by a balance between the activities of the inositol polyphosphate kinase Asp1 and several inositol pyrophosphatase enzymes. Here, we characterize Schizosaccharomyces pombe Siw14 (SpSiw14) as a cysteinyl-phosphatase-family pyrophosphatase enzyme capable of hydrolyzing the phosphoanhydride substrates inorganic pyrophosphate, inorganic polyphosphate, and inositol pyrophosphates 5-IP 7 , 1-IP 7 , and 1,5-IP 8 . Genetic analyses implicate SpSiw14 in 1,5-IP 8 catabolism in vivo , insofar as: loss of SpSiw14 activity is lethal in the absence of the Nudix-type inositol pyrophosphatase enzyme Aps1; and siw14 ∆ aps1 ∆ lethality depends on synthesis of 1,5-IP 8 by the Asp1 kinase. Suppression of siw14 ∆ aps1 ∆ lethality by loss-of-function mutations of 3'-processing/termination factors points to precocious transcription termination as the cause of 1,5-IP 8 toxicosis., Competing Interests: The authors declare no conflicts of interest.

Inositol hexakisphosphate kinases (IP6Ks) are emerging as relevant pharmacological targets because a multitude of disease-related phenotypes has been associated with their function. While the development of potent IP6K inhibitors is gaining momentum, a pharmacological tool to distinguish the mammalian isozymes is still lacking. Here, we implemented an analog-sensitive approach for IP6Ks and performed a high-throughput screen to identify suitable lead compounds. The most promising hit, FMP-201300, exhibited high potency and selectivity toward the unique valine gatekeeper mutants of IP6K1 and IP6K2, compared to the respective wild-type (WT) kinases. Biochemical validation experiments revealed an allosteric mechanism of action that was corroborated by hydrogen deuterium exchange mass spectrometry measurements. The latter analysis suggested that displacement of the α C helix, caused by the gatekeeper mutation, facilitates the binding of FMP-201300 to an allosteric pocket adjacent to the ATP-binding site. FMP-201300 therefore serves as a valuable springboard for the further development of compounds that can selectively target the three mammalian IP6Ks; either as analog-sensitive kinase inhibitors or as an allosteric lead compound for the WT kinases., Competing Interests: TA, GD, SH, MN, CS, VH, AS, Jv, DF No competing interests declared, (© 2023, Aguirre et al.)

Intercellular calcium waves (ICW) are complex signalling phenomena that control many essential biological activities, including smooth muscle contraction, vesicle secretion, gene expression and changes in neuronal excitability. Accordingly, the remote stimulation of ICW could result in versatile biomodulation and therapeutic strategies. Here we demonstrate that light-activated molecular machines (MM)-molecules that perform mechanical work on the molecular scale-can remotely stimulate ICW. MM consist of a polycyclic rotor and stator that rotate around a central alkene when activated with visible light. Live-cell calcium-tracking and pharmacological experiments reveal that MM-induced ICW are driven by the activation of inositol-triphosphate-mediated signalling pathways by unidirectional, fast-rotating MM. Our data suggest that MM-induced ICW can control muscle contraction in vitro in cardiomyocytes and animal behaviour in vivo in Hydra vulgaris. This work demonstrates a strategy for directly controlling cell signalling and downstream biological function using molecular-scale devices., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Inert digestibility index markers such as titanium dioxide are universally accepted to provide simple measurement of digestive tract retention and relative digestibility in poultry feeding trials. Their use underpins industry practice: specifically dosing regimens for adjunct enzymes added to animal feed. Among these, phytases, enzymes that degrade dietary phytate, inositol hexakisphosphate, represent a billion-dollar sector in an industry that raises ca. 70 billion chickens/annum. Unbeknown to the feed enzyme sector, is the growth in cell biology of use of titanium dioxide for enrichment of inositol phosphates from extracts of cells and tissues. The adoption of titanium dioxide in cell biology arises from its affinity under acid conditions for phosphates, suggesting that in feeding trial contexts that target phytate degradation this marker may not be as inert as assumed. We show that feed grade titanium dioxide enriches a mixed population of higher and lower inositol phosphates from acid solutions. Additionally, we compared the extractable inositol phosphates in gizzard and ileal digesta of 21day old male Ross 308 broilers fed three phytase doses (0, 500 and 6000 FTU/kg feed) and one inositol dose (2g/kg feed). This experiment was performed with or without titanium dioxide added as a digestibility index marker at a level of 0.5%, with all diets fed for 21 days. Analysis yielded no significant difference in effect of phytase inclusion in the presence or absence of titanium dioxide. Thus, despite the utility of titanium dioxide for recovery of inositol phosphates from biological samples, it seems that its use as an inert marker in digestibility trials is justified-as its inclusion in mash diets does not interfere with the recovery of inositol phosphates from digesta samples., Competing Interests: The feeding trials described in this study were commissioned at Nottingham Trent University by AB Vista. AB Vista had no role in conduct of the trial, data collection and analysis, decision to publish or preparation of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Sprigg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The mature HIV-1 capsid is stabilized by host and viral determinants. The capsid protein CA binds to the cellular metabolites inositol hexakisphosphate (IP6) and its precursor inositol (1, 3, 4, 5, 6) pentakisphosphate (IP5) to stabilize the mature capsid. In target cells, capsid destabilization by the antiviral compounds lenacapavir and PF74 reveals a HIV-1 infectivity defect due to IP5/IP6 (IP5/6) depletion. To test whether intrinsic HIV-1 capsid stability and/or host factor binding determines HIV-1 insensitivity to IP5/6 depletion, a panel of CA mutants was assayed for infection of IP5/6-depleted T cells and wildtype cells. Four CA mutants with unstable capsids exhibited dependence on host IP5/6 for infection and reverse transcription (RTN). Adaptation of one such mutant, Q219A, by spread in culture resulted in Vpu truncation and a capsid three-fold interface mutation, T200I. T200I increased intrinsic capsid stability as determined by in vitro uncoating of purified cores and partially reversed the IP5/6-dependence in target cells for each of the four CA mutants. T200I further rescued the changes to lenacapavir sensitivity associated with the parental mutation. The premature dissolution of the capsid caused by the IP5/6-dependent mutations imparted a unique defect in integration targeting that was rescued by T200I. Collectively, these results demonstrate that T200I restored other capsid functions after RTN for the panel of mutants. Thus, the hyperstable T200I mutation stabilized the instability defects imparted by the parental IP5/6-dependent CA mutation. The contribution of Vpu truncation to mutant adaptation was linked to BST-2 antagonization, suggesting that cell-to-cell transfer promoted replication of the mutants. We conclude that interactions at the three-fold interface are adaptable, key mediators of capsid stability in target cells and are able to antagonize even severe capsid instability to promote infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Sowd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

STIM1 and Orai1 are the central core of the Store Operated Calcium Entry (SOCE). This calcium influx mechanism is triggered after the activation of Gq protein-coupled receptors at the plasma membrane (PM) that activate phospholipase C. The phospholipase C produces Inositol triphosphate (IP3) which rapidly diffuses throughout the cytosol, resulting in the binding and activation of IP3 receptors (IP3R) and the rapid efflux of calcium from the endoplasmic reticulum (ER) to the cytosol. The calcium depletion in the ER is sensed by the stromal interaction molecule 1 (STIM1) a single-pass transmembrane protein at the ER that binds intraluminal calcium through an EF-hand domain in its amino terminal region (Fig. 1A). The cytosolic portion of STIM1 contains multiple domains. The region that interacts and activates Orai channels is known as SOAR (the STIM1 Orai activating region) [1]. For SOAR be accessible to Orai1, STIM1 must get an extended conformation that unlocks SOAR from its coiled-coil 1 (CC1) region [2]. The extended conformation is triggered by calcium depletion at the ER that oligomerizes STIM1. The oligomers of STIM1 then translocate to a close distance between two opposing membranes, forming what is known as ER-PM junctions. STIM1 accumulates at ER-PM junctions conforming the denominated STIM1 puncta., Competing Interests: Declaration of Competing Interest All authors declare no financial interests in the present work., (Copyright © 2023. Published by Elsevier Ltd.)

Multiple inositol polyphosphate phosphatase (MINPP1) is an enigmatic enzyme that is responsible for the metabolism of inositol hexakisphosphate (Ins P 6 ) and inositol 1,3,4,5,6 pentakisphosphate (Ins(1,3,4,5,6) P 5 in mammalian cells, despite being restricted to the confines of the ER. The reason for this compartmentalization is unclear. In our previous studies in the insulin-secreting HIT cell line, we expressed MINPP1 in the cytosol to artificially reduce the concentration of these higher inositol phosphates. Undocumented at the time, we noted cytosolic MINPP1 expression reduced cell growth. We were struck by the similarities in substrate preference between a number of different enzymes that are able to metabolize both inositol phosphates and lipids, notably IPMK and PTEN. MINPP1 was first characterized as a phosphatase that could remove the 3-phosphate from inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5) P 4 ) . This molecule shares strong structural homology with the major product of the growth-promoting Phosphatidyl 3-kinase (PI3K), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5) P 3 ) and PTEN can degrade both this lipid and Ins(1,3,4,5) P 4 . Because of this similar substrate preference, we postulated that the cytosolic version of MINPP1 (cyt-MINPP1) may not only attack inositol polyphosphates but also PtdIns(3,4,5) P 3 , a key signal in mitogenesis. Our experiments show that expression of cyt-MINPP1 in HIT cells lowers the concentration of PtdIns(3,4,5) P 3 . We conclude this reflects a direct effect of MINPP1 upon the lipid because cyt-MINPP1 actively dephosphorylates synthetic, di(C4:0)PtdIns(3,4,5) P 3 in vitro. These data illustrate the importance of MINPP1's confinement to the ER whereby important aspects of inositol phosphate metabolism and inositol lipid signaling can be separately regulated and give one important clarification for MINPP1's ER seclusion.

Many proteins involved in eukaryotic phosphate homeostasis are regulated by SPX domains. In yeast, the vacuolar transporter chaperone (VTC) complex contains two such domains, but mechanistic details of its regulation are not well understood. Here, we show at the atomic level how inositol pyrophosphates interact with SPX domains of subunits Vtc2 and Vtc3 to control the activity of the VTC complex. Vtc2 inhibits the catalytically active VTC subunit Vtc4 by homotypic SPX-SPX interactions via the conserved helix α1 and the previously undescribed helix α7. Binding of inositol pyrophosphates to Vtc2 abrogates this interaction, thus activating the VTC complex. Accordingly, VTC activation is also achieved by site-specific point mutations that disrupt the SPX-SPX interface. Structural data suggest that ligand binding induces reorientation of helix α1 and exposes the modifiable helix α7, which might facilitate its post-translational modification in vivo. The variable composition of these regions within the SPX domain family might contribute to the diversified SPX functions in eukaryotic phosphate homeostasis., (© 2023. The Author(s).)

The antifungal drug itraconazole has been repurposed to anti-angiogenic agent, but the mechanisms of action have been elusive. Here we report that itraconazole disrupts focal adhesion dynamics and cytoskeletal remodeling, which requires 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP 7 ). We find that inositol hexakisphosphate kinase 1 (IP6K1) binds Arp2 and generates 5-InsP 7 to recruit coronin, a negative regulator of the Arp2/3 complex. IP6K1 also produces focal adhesion-enriched 5-InsP 7 , which binds focal adhesion kinase (FAK) at the FERM domain to promote its dimerization and phosphorylation. Itraconazole treatment elicits displacement of IP6K1/5-InsP 7 , thus augments 5-InsP 7 -mediated inhibition of Arp2/3 complex and reduces 5-InsP 7 -mediated FAK dimerization. Itraconazole-treated cells display reduced focal adhesion dynamics and actin cytoskeleton remodeling. Accordingly, itraconazole severely disrupts cell motility, an essential component of angiogenesis. These results demonstrate critical roles of IP6K1-generated 5-InsP 7 in regulating focal adhesion dynamics and actin cytoskeleton remodeling and reveal functional mechanisms by which itraconazole inhibits cell motility., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are central eukaryotic messengers. These very highly phosphorylated molecules can exist in two distinct conformations, a canonical one with five phosphoryl groups in equatorial positions, and a "flipped" conformation with five axial substituents. Using 13 C-labeled InsPs/PP-InsPs, the behavior of these molecules was investigated by 2D-NMR under solution conditions reminiscent of a cytosolic environment. Remarkably, the most highly phosphorylated messenger 1,5(PP) 2 -InsP 4 (also termed InsP 8 ) readily adopts both conformations at physiological conditions. Environmental factors-such as pH, metal cation composition, and temperature-strongly influence the conformational equilibrium. Thermodynamic data revealed that the transition of InsP 8 from the equatorial to the axial conformation is, in fact, an exothermic process. The speciation of InsPs and PP-InsPs also affects their interaction with protein binding partners; addition of Mg 2+ decreased the binding constant K d of InsP 8 to an SPX protein domain. The results illustrate that PP-InsP speciation reacts very sensitively to solution conditions, suggesting it might act as an environment-responsive molecular switch.

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP 7 ) and its precursor inositol hexakisphosphate (IP 6 ). Although their absolute levels in the GIT are diet dependent, elevated IP 7 metabolism still exists under dietary regimens devoid of exogenous IP 7 . Of the major GIT cells, enteric neurons selectively express the IP 7 -synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP 7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP 7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP 7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP 7 -a highly active IP6K2-IP 7 pathway is conducive to the enteric nervous system., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

This study investigated the effects of phytase and monocalcium phosphate supplementation on the dephosphorylation of phytic acid [myo-inositol 1,2,3,4,5,6-hexakis (dihydrogen phosphate); InsP 6 ] in cecectomized laying hens using total excreta collection. Four corn-soybean meal-rapeseed meal-based diets were mixed with or without 6 g of monocalcium phosphate/kg, with or without supplementation of 1,500 FTU phytase/kg, and had the same calcium concentration at 39 g/kg of feed. Each diet was tested in 5 replicates using a row-column design with 10 cecectomized laying hens in 2 periods. The hens received 120 g/d of feed while being housed individually in metabolism units, and total excreta were collected for a period of 4 d. The monocalcium phosphate × phytase interaction was not significant for InsP 6 degradation (P = 0.054). Phytase increased InsP 6 disappearance from 13% to 83% (P < 0.001), whereas monocalcium phosphate had no effect. Concentrations of most of the lower inositol phosphate isomers in excreta were higher when monocalcium phosphate was added to the diets. The concentration of Ins(1,2,5,6)P 4 in excreta was the highest among the studied partially dephosphorylated inositol phosphates with phytase supplementation and was higher than in diets without phytase supplementation (P < 0.001). Supplementation with phytase increased myo-inositol concentration in excreta (P = 0.002), whereas monocalcium phosphate had no effect. Phosphorus utilization ranged from 4% to 18% and was not significantly affected by the treatments. These results suggest that phytase supplementation markedly increased InsP 6 degradation in laying hens. The cecectomized laying hen assay may be suitable for studying the effects of phytase supplementation on phytate dephosphorylation under dietary conditions when performance and phosphorus excretion are unlikely to be affected., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP 7 ) levels. 5-InsP 7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Main Conclusion: The IPK1 genes, which code for 2-kinases that can synthesize Ins(1,2,4,5,6)P 5 from Ins(1,4,5,6)P 4 , are expressed throughout cotton plants, resulting in the highest Ins(1,2,4,5,6)P 5 concentrations in young leaves and flower buds. Cotton leaves contain large amounts of Ins(1,2,4,5,6)P 5 and InsP 6 compared to plants not in the Malvaceae family. The inositol polyphosphate pathway has been linked to stress tolerance in numerous plant species. Accordingly, we sought to determine why cotton and other Malvaceae have such high levels of these inositol phosphates. We have quantified the levels of InsP 5 and InsP 6 in different tissues of cotton plants and determined the expression of IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene) in vegetative and reproductive tissues. Gossypium hirsutum was found to contain four IPK1 genes that were grouped into two pair (AB, CD) where each pair consists of very similar sequences that were measured together. More IPK1AB is expressed in leaves than in roots, whereas more IPK1CD is expressed in roots than in leaves. Leaves and flower buds have more InsP 5 and InsP 6 than stems and roots. Leaves and roots contain more InsP 5 than InsP 6 , whereas flower buds and stems contain more InsP 6 than InsP 5 . Dark-grown seedlings contain more InsP 5 and InsP 6 than those grown under lights, and the ratio of InsP 5 to InsP 6 is greater in the light-grown seedlings. During 35 days of the life cycle of the third true leaf, InsP 5 and InsP 6 gradually decreased by more than 50%. Silencing IPK1AB and IPK1CD with Cotton Leaf Crumple Virus-induced gene silencing (VIGS) resulted in plants with an intense viral phenotype, reduced IPK1AB expression and lowered amounts of InsP 5 . The results are consistent with Ins(1,2,4,5,6)P 5 synthesis from Ins(1,4,5,6)P 4 by IPK1. This study detailed the central role of IPK1 in cotton inositol polyphosphate metabolism, which has potential to be harnessed to improve the resistance of plants to different kinds of stress., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The toxicity of nanoplastics (NPs) at relatively low concentrations to soil fauna at different organismal levels is poorly understood. We investigated the responses of earthworm (Eisenia fetida) to polystyrene NPs (90-110 nm) contaminated soil at a relatively low concentration (0.02 % w:w) based on multi-omics, morphological, and intestinal microorganism analyses. Results showed that NPs accumulated in earthworms' intestinal tissues. The NPs damaged earthworms' digestive and immune systems based on injuries of the intestinal epithelium and chloragogenous tissues (tissue level) and increased the number of changed genes in the digestive and immune systems (transcriptome level). The NPs reduced gut microorganisms' diversity (Shannon index) and species richness (Chao 1 index). Proteomic, transcriptome, and histopathological analyses showed that earthworms suffered from oxidative and inflammatory stresses. Moreover, NPs influenced the osmoregulatory metabolism of earthworms as NPs damaged intestinal epithelium (tissue level), increased aldosterone-regulated sodium reabsorption (transcriptome level), inositol phosphate metabolism (proteomic level) and 2-hexyl-5-ethyl-furan-3-sulfonic acid, and decreased betaine and myo-inositol concentrations (metabolic level). Transcriptional-metabolic and transcriptional-proteomic analyses revealed that NPs disrupted earthworm carbohydrate and arachidonic acid metabolisms. Our multi-level investigation indicates that NPs at a relatively low concentration induced toxicity to earthworms and suggests that NPs pollution has significant environmental toxicity risks for soil fauna., Competing Interests: Conflict of interest There are no conflicts of interest to declare., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Hypothesis: Inositol phospholipids are well known to form clusters in the cytoplasmic leaflet of the plasma membrane that are responsible for the interaction and recruitment of proteins involved in key biological processes like endocytosis, ion channel activation and secondary messenger production. Although their phosphorylated inositol ring headgroup plays an important role in protein binding, its orientation with respect to the plane of the membrane and its lateral packing density has not been previously described experimentally., Experiments: Here, we study phosphatidylinositol 4,5-bisphosphate (PIP 2 ) planar model membranes in the form of Langmuir monolayers by surface pressure-area isotherms, Brewster angle microscopy and neutron reflectometry to elucidate the relation between lateral (in-plane) and perpendicular (out-of-plane) molecular organization of PIP 2 ., Findings: Different surface areas were explored through monolayer compression, allowing us to correlate the formation of transient PIP 2 clusters with the change in orientation of the inositol-biphosphate headgroup, which was experimentally determined by neutron reflectometry., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP 8 . IP 8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP 8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg 2+ or Mn 2+ ; IP 6 , 5-IP 7 , or 1,5-IP 8 ) and the other which was ligand-free. The ligand-free enzyme adopts an "open" conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP 6 or 5-IP 7 engagement above the nucleotide favors adoption of a "closed" conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP 7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine- N 6 . Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N 6 -benzyl-ATP as phosphate donor. IMPORTANCE The inositol pyrophosphate signaling molecule 1,5-IP 8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular IP 8 levels are determined by Asp1, a bifunctional enzyme composed of an N-terminal kinase and a C-terminal pyrophosphatase domain. Here, we present a series of crystal structures of the Asp1 kinase domain, in a ligand-free state and in complexes with nucleotides ADPNP, ADP, and ATP, divalent cations magnesium and manganese, and inositol polyphosphates IP 6 , 5-IP 7 , and 1,5-IP 8 . Substrate binding elicits a switch from open to closed conformations, entailing a disordered-to-ordered transition and a rearrangement or movement of two peptide segments that form a binding site for the phospho-acceptor. Our structures, along with structure-guided mutagenesis, fortify understanding of the mechanism and substrate specificity of Asp1 kinase, and they extend and complement structural and functional studies of the orthologous human kinase PPIP5K2.

Spontaneous Ca 2+ local transients (SCaLTs) in isolated oligodendrocyte precursor cells are largely regulated by the following fluxes: store-operated Ca 2+ entry (SOCE), Na + /Ca 2+ exchange, Ca 2+ pumping through Ca 2+ -ATPases, and Ca 2+ -induced Ca 2+ -release through ryanodine receptors and inositol-trisphosphate receptors. However, the relative contributions of these fluxes in mediating fast spiking and the slow baseline oscillations seen in SCaLTs remain incompletely understood. Here, we developed a stochastic spatiotemporal computational model to simulate SCaLTs in a homogeneous medium with ionic flow between the extracellular, cytoplasmic, and endoplasmic-reticulum compartments. By simulating the model and plotting both the histograms of SCaLTs obtained experimentally and from the model as well as the standard deviation of inter-SCaLT intervals against inter-SCaLT interval averages of multiple model and experimental realizations, we revealed the following: (1) SCaLTs exhibit very similar characteristics between the two data sets, (2) they are mostly random, (3) they encode information in their frequency, and (4) their slow baseline oscillations could be due to the stochastic slow clustering of inositol-trisphosphate receptors (modeled as an Ornstein-Uhlenbeck noise process). Bifurcation analysis of a deterministic temporal version of the model showed that the contribution of fluxes to SCaLTs depends on the parameter regime and that the combination of excitability, stochasticity, and mixed-mode oscillations are responsible for irregular spiking and doublets in SCaLTs. Additionally, our results demonstrated that blocking each flux reduces SCaLTs' frequency and that the reverse (forward) mode of Na + /Ca 2+ exchange decreases (increases) SCaLTs. Taken together, these results provide a quantitative framework for SCaLT formation in oligodendrocyte precursor cells., Competing Interests: Declaration of interests Nothing to declare., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsPs), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also an important precursor of the higher phosphorylated inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recent work has shown that Arabidopsis (Arabidopsis thaliana) INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (ITPK1) and ITPK2 display in vitro InsP6 kinase activity and that, in planta, ITPK1 stimulates 5-InsP7 and InsP8 synthesis and regulates phosphate starvation responses. Here we report a critical role of ITPK1 in auxin-related processes that is independent of the ITPK1-controlled regulation of phosphate starvation responses. Those processes include primary root elongation, root hair development, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity to exogenously applied auxin. We found that the recombinant auxin receptor complex, consisting of the F-Box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1), ARABIDOPSIS SKP1 HOMOLOG 1 (ASK1), and the transcriptional repressor INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), binds to anionic inositol polyphosphates with high affinity. We further identified a physical interaction between ITPK1 and TIR1, suggesting a localized production of 5-InsP7, or another ITPK1-dependent InsP/PP-InsP isomer, to activate the auxin receptor complex. Finally, we demonstrate that ITPK1 and ITPK2 function redundantly to control auxin responses, as deduced from the auxin-insensitive phenotypes of itpk1 itpk2 double mutant plants. Our findings expand the mechanistic understanding of auxin perception and suggest that distinct inositol polyphosphates generated near auxin receptors help to fine-tune auxin sensitivity in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Inositol and inositol phosphates (IPx) are central metabolites. Their accurate quantitative analysis in complex biological samples is challenging due to lengthy sample preparation procedures, sample losses by strong adsorption to surfaces, and unpredictable matrix effects. Currently, U 13 C-inositol and U 13 C-IPx are not available from commercial sources. In this study, we developed a method that is capable of generating U 13 C-inositol and U 13 C-IPx. An inositol-independent cell line L929S was cultured in inositol-free medium supplemented with U 13 C-glucose. Inositol contamination in FBS was observed as the critical parameter for labeling efficiency (LE). A balance between cell growth and LE was achieved by adopting a two-step labeling strategy. In the first step, a LE of 90% could be obtained by normal cell growth in the long-term. Cells were then cultured in a second step in ultra-labeling medium for improved LE for a short duration before harvesting. The generated U 13 Canalogs were of high isotopic purity (>99%). Utilized as internal standards spiked before sample preparation in biological applications, U 13 Canalogs can effectively compensate sample loss during sample preparation as well as the matrix effect during electrospray ionization. An exemplary pharmacological study was conducted with phospholipase C inhibitor and activator to document the great utility of the prepared stable isotope-labeled internal standards in elucidating the PLC-dependent IP code. U 13 CIPx are used as internal standards to generate quantitative profiles of IPx in HeLa cell samples after treatment with PLC inhibitor and activator. This established method generating U 13 Canalogs is cost-effective, robust, and reproducible, which can facilitate quantitative studies of inositol and IPx in biological scenarios.

Phytases, enzymes that degrade phytate present in feedstuffs, are widely added to the diets of monogastric animals. Many studies have correlated phytase addition with improved animal productivity and a subset of these have sought to correlate animal performance with phytase-mediated generation of inositol phosphates in different parts of the gastro-intestinal tract or with release of inositol or of phosphate, the absorbable products of phytate degradation. Remarkably, the effect of dietary phytase on tissue inositol phosphates has not been studied. The objective of this study was to determine effect of phytase supplementation on liver and kidney myo-inositol and myo-inositol phosphates in broiler chickens. For this, methods were developed to measure inositol phosphates in chicken tissues. The study comprised wheat/soy-based diets containing one of three levels of phytase (0, 500 and 6,000 FTU/kg of modified E. coli 6-phytase). Diets were provided to broilers for 21 D and on day 21 digesta were collected from the gizzard and ileum. Liver and kidney tissue were harvested. Myo-inositol and inositol phosphates were measured in diet, digesta, liver and kidney. Gizzard and ileal content inositol was increased progressively, and total inositol phosphates reduced progressively, by phytase supplementation. The predominant higher inositol phosphates detected in tissues, D-and/or L-Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5, differed from those (D-and/or L-Ins(1,2,3,4)P4, D-and/or L-Ins(1,2,5,6)P4, Ins(1,2,3,4,6)P5, D-and/or L-Ins(1,2,3,4,5)P5 and D-and/or L-Ins(1,2,4,5,6)P5) generated from phytate (InsP6) degradation by E. coli 6-phytase or endogenous feed phytase, suggesting tissue inositol phosphates are not the result of direct absorption. Kidney inositol phosphates were reduced progressively by phytase supplementation. These data suggest that tissue inositol phosphate concentrations can be influenced by dietary phytase inclusion rate and that such effects are tissue specific, though the consequences for physiology of such changes have yet to be elucidated., Competing Interests: The feeding trials described in this study were commissioned at Nottingham Trent University by AB Vista. AB Vista had no role in conducting the research, generating the data, interpreting the results or writing the manuscript.

Phosphoinositides (PIs) are relatively rare lipid components of the cellular membranes. Their homeostasis is tightly controlled by specific PI kinases and PI phosphatases. PIs play essential roles in cellular signaling, cytoskeletal organization, and secretory processes in various diseases and normal physiology. Gene targeting experiments strongly suggest that in mice with deficiency of several PI phosphatases, such as Pten, Mtmrs, Inpp4b, and Inpp5b, spermatogenesis is affected, resulting in partial or complete infertility. Similarly, in men, loss of several of the PI phosphatases is observed in infertility characterized by the lack of mature sperm. Using available gene expression databases, we compare the expression of known PI phosphatases in various testicular cell types, infertility patients, and mouse age-dependent testicular gene expression, and discuss their potential roles in testis physiology and spermatogenesis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The cell wall integrity pathway (CWI) is a MAPK-mediated signaling route essential for yeast cell response to cell wall damage, regulating distinct aspects of fungal physiology. We have recently proven that the incorporation of a genetic circuit that operates as a signal amplifier into this pathway allows for the identification of novel elements involved in CWI signaling. Here, we show that the strong growth inhibition triggered by pathway hyperactivation in cells carrying the "Integrity Pathway Activation Circuit" (IPAC) also allows the easy identification of new stimuli. By using the IPAC, we have found various chemical agents that activate the CWI pathway, including the aminoglycoside neomycin. Cells lacking key components of this pathway are sensitive to this antibiotic, due to the disruption of signaling upon neomycin stimulation. Neomycin reduces both phosphatidylinositol-4,5-bisphosphate (PIP 2 ) availability at the plasma membrane and myriocin-induced TORC2-dependent Ypk1 phosphorylation, suggesting a strong interference with plasma membrane homeostasis, specifically with PIP 2 . The neomycin-induced transcriptional profile involves not only genes related to stress and cell wall biogenesis, but also to amino acid metabolism, reflecting the action of this antibiotic on the yeast ribosome.