Inositol pyrophosphates (PP-InsPs) are eukaryotic nutrient messengers. The N-terminal kinase domain of diphosphoinositol pentakisphosphate kinase (PPIP5K) generates the messenger 1,5-InsP8, the C-terminal phosphatase domain catalyzes PP-InsP breakdown. The balance between kinase and phosphatase activities regulates 1,5-InsP8 levels. Here, we present crystal structures of the apo and substrate-bound PPIP5K phosphatase domain from S. cerevisiae (ScVip1PD). ScVip1PD is a phytase-like inositol 1-pyrophosphate histidine phosphatase with two conserved catalytic motifs. The enzyme has a strong preference for 1,5-InsP8 and is inhibited by inorganic phosphate. It contains an α-helical insertion domain stabilized by a structural Zn2+ binding site, and a unique GAF domain that channels the substrate to the active site. Mutations that alter the active site, restrict the movement of the GAF domain, or change the substrate channel’s charge inhibit the enzyme activity in vitro, and Arabidopsis VIH2 in planta. Our work reveals the structure, enzymatic mechanism and regulation of eukaryotic PPIP5K phosphatases.

Phosphorus in crucial for all living organisms. In vertebrate, cellular phosphate homeostasis is partly controlled by XPR1, a poorly characterized inositol pyrophosphate-dependent phosphate exporter. Here, we report the cryo-EM structure of human XPR1, which forms a loose dimer with 10 transmembrane helices (TM) in each protomer. The structure consists of a scaffold domain (TM1, TM3-4) and a core domain (TM2, TM5-10) structurally related to ion-translocating rhodopsins. Bound phosphate is observed in a tunnel within the core domain at a narrow point that separates the tunnel into intracellular and extracellular vestibules. This site contains a cluster of basic residues that coordinate phosphate and a conserved W573 essential for export function. Loss of inositol pyrophosphate binding is accompanied by structural movements in TM9 and the W573 sidechain, closing the extracellular vestibule and blocking phosphate export. These findings provide insight into XPR1 mechanism and pave the way for further in-depth XPR1 studies.

The diverse chemical structures of inositol pyrophosphates attract growing interest toward this class of small molecule messengers. However, their highly charged nature, the lack of a chromophore or a fluorophore, their close structural relatedness, and the complexity of their metabolism pose serious challenges to inositol pyrophosphates studies. Here, we summarize how researchers have begun to overcome these challenges and how recent experimental advances are propelling inositol pyrophosphate research into the future.

The availability of well-defined andpure PP-InsP substrates in high quantities is pivotal to study their molecular mechanisms by means of biochemical or biophysical approaches. Here, we present a workflow employing enzymatic synthesis that allows for the quick and easy synthesis and isolation of mammalian PP-InsP isomers. With the respective enzymes, inositol pyrophosphates can be generated in a single reaction step. Isolation and purification of the desired products does not require special laboratory equipment and can be achieved by precipitation of PP-InsPs as magnesium salts. Having access to PP-InsPs will aid the field in discovering and characterizing their molecular mechanisms and binding modes.

Knockout mice have served as excellent model systems to investigate the functions of specific mammalian proteins at the organismal level. Several studies on tissue-specific or whole-body depletion of individual IP6 kinase (IP6K) paralogs have shed light on myriad roles for their product, the inositol pyrophosphate 5-InsP7, in different physiological processes and pathological states. The loss of Ip6k1, Ip6k2, or Ip6k3 leads predominantly to nonoverlapping phenotypes in mice, reflecting their differential tissue, cell type, and subcellular distribution. Using the example of Ip6k1 knockout mice, and the stark phenotype of male infertility resulting from the depletion of condensed spermatids in the testes of these mice, this chapter provides detailed methods for the use of knockout mice as a model to study the functions of IP6Ks. We begin with protocols for the maintenance and breeding of the mouse colony, with instructions to genotype offspring from the mating of heterozygous mice carrying one Ip6k1 knockout allele. We then provide methods for the histopathological comparison of tissues in wild-type versus knockout mice by hematoxylin and eosin staining, and the detection of IP6K1 expression in specific tissues and subcellular compartments by western blotting and immunofluorescence.

Inositol pyrophosphates are diphosphate-containing inositol phosphate cellular messengers that orchestrate a large array of physiological processes in eukaryotes. In plants, these energy-rich second messengers are involved in hormone signaling and phosphate homeostasis. Notably, much of our understanding of the functional roles of inositol pyrophosphates in plants is based on research involving the model organism Arabidopsis thaliana. Emerging recent studies have highlighted the possible functional importance of these PP-InsP species across plant kingdom. Specifically, PP-InsPs are linked to nutrient sensing and thallus development in Marchantia polymorpha, a nonvascular bryophytethat first emerged on land around 500 million years ago. In this study, we employed strong anion-exchange (SAX)-HPLC techniques combined with[3H]-myo-inositol labeling to analyze the steady-state cellular levels of various InsP and PP-InsP species of M. polymorpha.

Inositol phosphates are signaling molecules crucial for cellular processes. To achieve specificity, their diverse phosphorylation patterns necessitate precise molecular identification. Phosphorylation of the myo-inositol scaffold can form enantiomeric pairs, but most analysis methods only resolve regioisomers. This chapter presents an approach for enantiomer assignment using 31P-NMR with L-arginine amide as a chiral solvating agent. Chemically synthesized standards enabled clear enantiomer differentiation. This method applies to both inositol phosphates as well as inositol pyrophosphates and their bisphosphonate analogs, aiding in the identification of biologically relevant isomers in natural samples.

In recent years, there has been substantial progress in the development of methods to analyze inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs). However, many of these techniques are labor- and cost-intensive and can usually only be carried out by laboratories specialized in InsPs/PP-InsPs analysis. In this chapter, we present a simple method that exploits the fact that phosphorylation and/or dephosphorylation of certain InsP/PP-InsP species induces the activation of promoters driving the expression of genes involved in phosphate starvation response (PSR). By linking PSR-inducible promoters to the RUBY reporter, which allows continuous and noninvasive visualization of promoter activation, we provide a new analytical tool to monitor enzymatic activities that alter PP-InsP levels, eliminating the need for complex biochemical analysis. We present a step-by-step protocol showing how simple coinfiltration of promoter-RUBY T-DNA constructs together with PP-InsP pyrophosphatase-encoding T-DNAs into Nicotiana benthamiana leaves enables evaluation of the enzymatic activity of expressed pyrophosphatases, and mention possible downstream analyses by methods described in other chapters of this special issue.

Inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), play crucial roles in various biological processes. Pharmacologic inhibition of IP6K has potential therapeutic benefits for treating type II diabetes, venous thrombosis, chronic kidney disease, and psychiatric disorders. This chapter describes the identification of IP6K inhibitors through kinase-activity-based high-throughput assays and dose-response assay. Additionally, it details cell-based assays on the determination of the effect of IP6K inhibitors on IP7 production in HCT116 cells and the binding of IP6K inhibitors to IP6K inside cells through cellular thermal shift assays (CETSAs). These robust assays provide a foundation for further exploration of IP6K inhibitors in various disease states.

Inositol pyrophosphates (PP-InsPs), which possess functionally significant diphosphate groups, regulate a wide range of biological processes across eukaryotic organisms. The presence and isomeric diversity of PP-InsPs suggest potential biological relevance and enable these molecules to serve as versatile regulators. Identification and characterization of the PP-InsP isomers require specialized approaches. We have successfully utilized X-ray crystallography to directly visualize and identify PP-InsP isomers, thereby informing biological studies and advancing our understanding of their functional roles. Here, we describe detailed methods for identifying PP-InsP isomers in Crystallo.

Polyacrylamide gel electrophoresis (PAGE) is a versatile technique widely used in molecular biology for the separation of biomolecules based on size and charge. While it is traditionally applied to proteins and nucleic acids, recently, PAGE has been adapted for the analysis of inositol polyphosphates and their pyrophosphate derivatives, which play multiple roles in cellular signaling. Studying these molecules presents analytical challenges due to their small size, high charge density, and low concentrations in biological samples. In this work, we describe an optimized PAGE protocol for the separation and detection of inositol polyphosphates and inositol pyrophosphate. The method uses high-concentration polyacrylamide gels and toluidine blue staining to achieve sensitive detection at nanomolar levels. The simplicity and cost-efficiency of the approach make it accessible to most laboratories. This method provides a reliable tool for investigating inositol-phosphate-based signaling pathways and their involvement in cellular processes.

Pulldown experiments isolate molecular interactions using a "bait" molecule on solid supports, often leveraging the biotin-streptavidin system. Here, a streamlined workflow is described, which employs biotinylated inositol phosphate (InsPs) and inositol pyrophosphate (PP-InsP) probes to enrich target proteins from complex proteomes. The reagents are first immobilized onto streptavidin-coated beads, then exposed to cell lysates, and subsequently washed to remove nonspecific interactions. The enriched proteins are then eluted and analyzed via western blot or quantitative mass spectrometry. This approach leverages biotin-tagged probes to enhance coupling efficiency, simplify workflows, and enable diverse applications.

The direct detection of InsP7 (diphosphoinositol pentakisphosphate) and InsP8 (bis-diphosphoinositol tetrakisphosphate), known as inositol pyrophosphates (PP-InsPs), in mammalian specimens faces technical difficulties owing to their characteristic chemical properties and minute quantities in mammalian tissues. We developed an analytical protocol to sensitively and directly detect PP-InsPs and their precursor, inositol hexakisphosphate (InsP6), using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). This analytical protocol, coupled with high-purity synthetic standards, allows for the absolute quantitation of these analytes in cellular samples as well as in various organs and tissues obtained by autopsies of animals and humans, offering an effective option to study PP-InsP functions in mammalian physiology and pathology. Herein, we describe this protocol in detail, from the sample preparation procedure throughout the column regeneration after analysis, along with several notable cautionary points.

High-performance anion-exchange chromatography with acid eluents allows for effective separation of myo-inositol tetrakisphosphates, myo-inositol pentakisphosphates, diphospho-myo-inositol phosphates (inositol pyrophosphates), inositol hexakisphosphates, inositol pyrophosphate analogs, and carbohydrate-based analogs of inositol phosphates. All are detectable by postcolumn addition of ferric ion. In the following, we provide examples drawn from characterization of ITPK, IPK1, and IP3 3K enzymes.

Inositol phosphate and pyrophosphate kinases (InsPKs) regulate cellular signaling by phosphorylating inositol phosphates, which play key roles in diverse physiological processes from insulin signaling to cell proliferation. Here, we review the structural biology of these enzymes and our understanding of their distinctive architectures. InsPKs can be classified into two structural families, which consist of InsPKs and other enzymes: the protein kinase-like and ATP-Grasp, each with distinct catalytic features. While crystal structures have been determined for representative catalytic domains through the InsPK synthetic pathways, structural information remains limited to these domains, with little information to guide our understanding of regulatory mechanisms or architectures of multidomain InsPKs. Here, we review the evolution of InsPK classification from early substrate-based naming to modern structural family groupings. We highlight how structural insights have revealed relationships between seemingly unrelated enzymes and what can be understood about InsPKs from features they share with other enzymes. We also provide an overview of the decision points in InsPK structural biology, from protein engineering and expression to crystallization, emphasizing how choices at each stage impact both the likelihood of success and the biological relevance of the results. We discuss the tools and recent advances, such as AlphaFold structure predictions, which have provided new insights into previously intractable regions of InsPKs and offer guidance for future exploration of these kinases.

Phosphate (Pi) homeostasis at the cellular level is crucial, requiring coordinated Pi uptake, storage, and export. However, the regulatory mechanisms, particularly those governing Pi export, remain elusive, despite their relevance to human diseases like primary familial brain calcification. While Xpr1, conserved across eukaryotes, is the only known Pi exporter, the existence of additional Pi exporting factors is evident; however, these factors have been poorly characterized. Using the fission yeast Schizosaccharomyces pombe as a model, we have aimed to better understand cellular Pi homeostasis mechanisms. Previously, we showed three Pi regulators with SPX domains to be critical: Pqr1 (Pi uptake restrictor), Xpr1/Spx2, and the VTC complex (polyphosphate synthase). SPX domains bind to inositol pyrophosphate, modulating Pi regulator functions. The double mutant Δpqr1Δxpr1 hyper-accumulates Pi and undergoes cell death under high Pi conditions, indicating the necessity of both Pi uptake restriction and export. Notably, Δpqr1Δxpr1 exhibits residual Pi export activity independent of Xpr1, suggesting the presence of unidentified Pi exporters. To uncover these cryptic Pi exporters and regulators of Pi homeostasis, we conducted suppressor screening for high Pi hypersensitivity in Δpqr1Δxpr1. Among the eight suppressors identified, Shp2, a plasma-membrane protein, showed Pi export-facilitating activity in an Xpr1-independent manner, supporting cell proliferation at high Pi. The present results provide the first evidence for Pi export facilitator other than the established Xpr1, unprecedented in eukaryotes. As Shp2 is orthologous to the budding yeast Tpo1, a spermidine/polyamine transporter, a potential link between Pi homeostasis and polyamine metabolism can be speculated.

Grating-coupled interferometry (GCI) is a biophysical method suitable to studying the binding kinetics of protein-ligand interactions. Here, we describe the application of biotinylated inositol pyrophosphate (PP-InsP) nutrient messengers to characterize PP-InsP-protein interactions using a Creoptix WAVEsystem.

Recent studies have established a key role of inositol pyrophosphates (PP-InsPs) in regulating phosphate (Pi) homeostasis across various organisms. In plants, PP-InsP levels are intricately linked to Pi status, with this reciprocal regulation being especially pronounced under fluctuating Pi conditions compared to stable nutrient sufficiency or deficiency. Here, we present a hydroponic cultivation protocol for Arabidopsis and rice, designed to simulate distinct and fluctuating Pi regimes. The composition of the nutrient solutions in these systems can be easily adjusted, enabling precise monitoring of changes in PP-InsP metabolism, under both steady-state and dynamic Pi conditions. Plants grown hydroponically under different Pi regimes can also be analyzed for phosphate starvation response (PSR) phenotypes and subjected to inositol phosphate (InsP)/PP-InsP extraction and quantification using methods detailed in other chapters of this special issue.

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 nutrient messengers whose cellular levels are precisely regulated. Diphosphoinositol pentakisphosphate kinases (PPIP5Ks) generate the active signaling molecule 1,5-InsP8. PPIP5Ks harbor phosphatase domains that hydrolyze PP-InsPs. Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSPs) and NUDIX phosphatases (NUDTs) are also involved in PP-InsP degradation. Here, we analyze the relative contributions of the three different phosphatase families to plant PP-InsP catabolism. We report the biochemical characterization of inositol pyrophosphate phosphatases from Arabidopsis and Marchantia polymorpha. Overexpression of different PFA-DSP and NUDT enzymes affects PP-InsP levels and leads to stunted growth phenotypes in Arabidopsis. nudt17/18/21 knock-out mutants have altered PP-InsP pools and gene expression patterns, but no apparent growth defects. In contrast, Marchantia polymorpha Mppfa-dsp1ge, Mpnudt1ge and Mpvip1ge mutants display severe growth and developmental phenotypes and associated changes in cellular PP-InsP levels. Analysis of Mppfa-dsp1geand Mpvip1ge mutants supports a role for PP-InsPs in Marchantia phosphate signaling, and additional functions in nitrate homeostasis and cell wall biogenesis. Simultaneous elimination of two phosphatase activities enhanced the observed growth phenotypes. Taken together, PPIP5K, PFA-DSP and NUDT inositol pyrophosphate phosphatases regulate growth and development by collectively shaping plant PP-InsP pools.