Inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs) are highly phosphorylated signaling molecules involved in diverse cellular processes. To resolve discrete signaling events mediated by these structurally related metabolites, a mass spectrometry-based approach was developed to derive apparent binding constants on a proteome-wide scale. The method employs chemically synthesized affinity reagents for inositol hexakisphosphate (InsP6) and the inositol pyrophosphates 1PP-InsP5, 5PP-InsP5, and 1,5(PP)2-InsP4 (InsP8). Concentration-dependent affinity enrichment combined with tandem mass tag (TMT) labeling enabled identification and quantification of ligand-protein interactions for hundreds of proteins from mammalian cell lysates. Biochemical and functional validation of selected targets demonstrated engagement with endogenous ligands. Comparison of enrichment conditions revealed a strong dependence of PP-InsP binding on Mg2+ ions. Additionally, gene ontology analysis linked PP-InsP interactors to nuclear and nucleolar RNA processing, and subsequent analyses could identify several pyrophosphorylation sites, previously uncharacterized. In summary, these datasets provide valuable resources for exploring PP-InsP-dependent signaling pathways across biological systems.

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis, phosphate (Pi) signaling, and phytohormone perception. Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis NUDIX-type hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange high-performance liquid chromatography (SAX-HPLC), polyacrylamide gel electrophoresis (PAGE), and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer selective and Mg2+ dependent. Specifically, Subclade I NUDTs preferentially hydrolyze 4-InsP7, while Subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher order mutants of Subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs), while RNA-seq analysis comparing wild-type (WT) and Subclade II nudt12/13/16 loss-of-function plants indicates additional PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Consistently, nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond Subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to Subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP-InsP signaling, plant nutrient and immune responses, and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.

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-InsP(7) ) 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-InsP(7) to stabilize adiponectin/ERp44 and DsbA-L/Ero1-L alpha interactions, driving adiponectin intracellular degradation. Depleting 5-InsP(7) 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-InsP(7) biosynthesis in wild-type but not adiponectin knockout mice attenuated myocardial ischaemia-reperfusion injury. Conclusion Our findings revealed that 5-InsP(7) is a physiological regulator of adiponectin biosynthesis that is amenable to pharmacological intervention for cardioprotection. [GRAPHICS] .

Background and aims Atherosclerotic cardiovascular diseases are the leading cause of death. Apolipoprotein A-I (apoA-I) mediates cholesterol efflux to lower the risks of atherosclerosis. Elevating circulating apoA-I is an effective strategy for atheroprotection. However, the regulatory mechanisms of apoA-I have been elusive. Methods Protein-protein interactions were examined by co-immunoprecipitations. Chemical biology tools were used to determine the binding of 5PP-InsP5 to its target proteins and its roles in mediating protein-protein interactions. The mouse atherosclerotic model was generated by injecting AAV-PCSK9 and feeding a Western diet. Atherosclerotic plaques were determined by Oil Red O and H&E staining. Results We show that blocking IP6K1 activity increases apoA-I production in hepatocytes. IP6K1 binds to apoA-I and via its product 5PP-InsP5 to induce apoA-I degradation, which requires ubiquitination factor E4A (UBE4A). Depleting 5PP-InsP5 by deleting IP6K1 or blocking IP6K1 activity disrupts the interaction between UBE4A and apoA-I, preventing apoA-I degradation, leading to increased production of apoA-I. Hepatocyte-specific deletion of IP6K1 elevates circulating apoA-I levels, which augments cholesterol efflux and lowers the burden of atherosclerosis. Mice with both apoA-I KO and hepatocyte-specific IP6K1 KO were generated to validate that IP6K1 deletion-induced atheroprotection requires apoA-I. Conclusions Our findings reveal a mechanism by which blocking IP6K1 boosts apoA-I production. Blocking IP6K1 represents a potential treatment strategy to elevate circulating apoA-I for atheroprotection.

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis and phosphate (Pi) signaling. However, the enzymes responsible for their turnover in plants remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis Nudix hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher-order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange (SAX)-HPLC, PAGE, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer-selective and Mg2+-dependent. Specifically, subclade I NUDTs preferentially hydrolyze 4-InsP7, while subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher-order mutants of subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Furthermore, ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs). However, RNA-seq analysis of WT and nudt12/13/16 plants suggests that these NUDTs have PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Expanding beyond subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we found that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to subclade I NUDTs. Collectively, our study provides new insights into the roles of NUDTs in regulating PP-InsP signaling pathways in plants and highlights cross-kingdom conservation of enzymes involved in PP-InsP metabolism. ### Competing Interest Statement The authors have declared no competing interest.

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-InsP7). We find that inositol hexakisphosphate kinase 1 (IP6K1) binds Arp2 and generates 5-InsP7 to recruit coronin, a negative regulator of the Arp2/3 complex. IP6K1 also produces focal adhesion-enriched 5-InsP7, which binds focal adhesion kinase (FAK) at the FERM domain to promote its dimerization and phosphorylation. Itraconazole treatment elicits displacement of IP6K1/5-InsP7, thus augments 5-InsP7-mediated inhibition of Arp2/3 complex and reduces 5-InsP7-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-InsP7 in regulating focal adhesion dynamics and actin cytoskeleton remodeling and reveal functional mechanisms by which itraconazole inhibits cell motility.

Inositol pyrophosphates (PP-InsPs) constitute a group of highly charged messengers, which regulate central biological processes in health and disease, such as cellular phosphate and general energy homeostasis. Deciphering the molecular mechanisms underlying PP-InsP-mediated signaling remains a challenge due to the unique properties of these molecules, the different modes of action they can access, and a somewhat limited chemical and analytical toolset. Herein, we summarize the most recent mechanistic insights into PP-InsP signaling, which illustrate our progress in connecting mechanism and function of PP-InsPs.

This protocol describes an affinity enrichment approach from mammalian cell extracts to identify protein binding partners of inositol hexakisphosphate (InsP6) and 5-diphosphoinositol pentakisphosphate (5PP-InsP5), two important eukaryotic metabolites. The interactomes are annotated using mass spectrometry-based proteomics, and comparison against a control resin can uncover hundreds of protein targets. Quantitative analysis of InsP6- versus 5PP-InsP5-binding proteins highlights specific protein-ligand interactions. The approach is applicable to different cells and organisms and will contribute to a mechanistic understanding of inositol poly- and pyrophosphate signaling. For complete details on the use and execution of this protocol, please refer to Furkert et al. (2020).

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are densely phosphorylated eukaryotic messengers, which are involved in numerous cellular processes. To elucidate their signaling functions at the molecular level, non-hydrolyzable bisphosphonate analogs of inositol pyrophosphates, PCP-InsPs, have been instrumental. Here, an efficient synthetic strategy to obtain these analogs in unprecedented quantities is described - relying on the use of combined phosphate ester-phosphoramidite reagents. The PCP-analogs, alongside their natural counterparts, were applied to investigate their regulatory effect on insulin-degrading enzyme (IDE), using a range of biochemical, biophysical and computational methods. A unique interplay between IDE, its substrates and the PP-InsPs was uncovered, in which the PP-InsPs differentially modulated the activity of the enzyme towards short peptide substrates. Aided by molecular docking and molecular dynamics simulations, a flexible binding mode for the InsPs/PP-InsPs was identified at the anion binding site of IDE. Targeting IDE for therapeutic purposes should thus take regulation by endogenous PP-InsP metabolites into account.

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated molecules that have emerged as central nutrient messengers in eukaryotic organisms. They can bind to structurally diverse target proteins to regulate biological functions, such as protein-protein interactions. PP-InsPs are strongly negatively charged and interact with highly basic surface patches in proteins, making their quantitative biochemical analysis challenging. Here, we present the synthesis of biotinylated myo-inositol hexakisphosphates and their application in surface plasmon resonance and grating-coupled interferometry assays, to enable the rapid identification, validation, and kinetic characterization of InsP- and PP-InsP-protein interactions.

Visual arrestin (Arr1) terminates rhodopsin signaling by blocking its interaction with transducin. To do this, Arr1 translocates from the inner to the outer segment of photoreceptors upon light stimulation. Mounting evidence indicates that inositol phosphates (InsPs) affect Arr1 activity, but the Arr1-InsP molecular interaction remains poorly defined. We report the structure of bovine Arr1 in a ligand-free state featuring a near-complete model of the previously unresolved C-tail, which plays a crucial role in regulating Arr1 activity. InsPs bind to the N-domain basic patch thus displacing the C-tail, suggesting that they prime Arr1 for interaction with rhodopsin and help direct Arr1 translocation. These structures exhibit intact polar cores, suggesting that C-tail removal by InsP binding is insufficient to activate Arr1. These results show how Arr1 activity can be controlled by endogenous InsPs in molecular detail.

In the human-pathogenic fungus Cryptococcus neoformans, the inositol polyphosphate signaling pathway is critical for virulence. We recently demonstrated the key role of the inositol pyrophosphate IP7 (isomer 5-PP-IP5) in driving fungal virulence; however, the mechanism of action remains elusive. Using genetic and biochemical approaches, and mouse infection models, we show that IP7 synthesized by Kcs1 regulates fungal virulence by binding to a conserved lysine surface cluster in the SPX domain of Pho81. Pho81 is the cyclin-dependent kinase (CDK) inhibitor of the phosphate signaling (PHO) pathway. We also provide novel mechanistic insight into the role of IP7 in PHO pathway regulation by demonstrating that IP7 functions as an intermolecular "glue" to stabilize Pho81 association with Pho85/Pho80 and, hence, promote PHO pathway activation and phosphate acquisition. Blocking IP7-Pho81 interaction using site-directed mutagenesis led to a dramatic loss of fungal virulence in a mouse infection model, and the effect was similar to that observed following PHO81 gene deletion, highlighting the key importance of Pho81 in fungal virulence. Furthermore, our findings provide additional evidence of evolutionary divergence in PHO pathway regulation in fungi by demonstrating that IP7 isomers have evolved different roles in PHO pathway control in C. neoformans and nonpathogenic yeast.IMPORTANCE Invasive fungal diseases pose a serious threat to human health globally with >1.5 million deaths occurring annually, 180,000 of which are attributable to the AIDS-related pathogen, Cryptococcus neoformans Here, we demonstrate that interaction of the inositol pyrophosphate, IP7, with the CDK inhibitor protein, Pho81, is instrumental in promoting fungal virulence. IP7-Pho81 interaction stabilizes Pho81 association with other CDK complex components to promote PHO pathway activation and phosphate acquisition. Our data demonstrating that blocking IP7-Pho81 interaction or preventing Pho81 production leads to a dramatic loss in fungal virulence, coupled with Pho81 having no homologue in humans, highlights Pho81 function as a potential target for the development of urgently needed antifungal drugs.

The inositol pyrophosphates (PP-InsPs) are a ubiquitous group of highly phosphorylated eukaryotic messengers. They have been linked to a panoply of central cellular processes, but a detailed understanding of the discrete signaling events is lacking in most cases. To create a more mechanistic picture of PP-InsP signaling, we sought to annotate the mammalian interactome of the most abundant inositol pyrophosphate 5PP-InsP5. To do so, triplexed affinity reagents were developed, in which a metabolically stable PP-InsP analog was immobilized in three different ways. Application of these triplexed reagents to mammalian lysates identified between 300 and 400 putative interacting proteins. These interactomes revealed connections between 5PP-InsP5 and central cellular regulators, such as lipid phosphatases, protein kinases, and GTPases, and identified protein domains commonly targeted by 5PP-InsP5. Both the triplexed affinity reagents, and the proteomic datasets, constitute powerful resources for the community, to launch future investigations into the multiple signaling modalities of inositol pyrophosphates.

Sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) is one of the most abundant cell membrane proteins and is essential for eukaryotes. Endogenous negative regulators have long been postulated to play an important role in regulating the activity and stability of Na+/K+-ATPase, but characterization of these regulators has been elusive. Mechanisms of regulating Na+/K+-ATPase homeostatic turnover are unknown. Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7), generated by inositol hexakisphosphate kinase 1 (IP6K1), promotes physiological endocytosis and downstream degradation of Na+/K+-ATPase-α1. Deletion of IP6K1 elicits a twofold enrichment of Na+/K+-ATPase-α1 in plasma membranes of multiple tissues and cell types. Using a suite of synthetic chemical biology tools, we found that 5-InsP7 binds the RhoGAP domain of phosphatidylinositol 3-kinase (PI3K) p85α to disinhibit its interaction with Na+/K+-ATPase-α1. This recruits adaptor protein 2 (AP2) and triggers the clathrin-mediated endocytosis of Na+/K+-ATPase-α1. Our study identifies 5-InsP7 as an endogenous negative regulator of Na+/K+-ATPase-α1.

Real-time monitoring of acid sphingomyelinase (ASM) activity is crucial for investigating its role in lipid-mediated signaling processes. In this study, we synthesized fluorescent phosphosphingolipids capable of FRET by phosphorodichloridate chemistry. These sphingomyelin analogues are substrates for recombinant human ASM and can be used to monitor ASM activity by fluorescence spectroscopy. Incubation with cell lysates from wild-type and knock-out mice further confirmed probe cleavage to be exclusive to ASM. We also systematically exploited the environmental sensitivity of the fluorophores to achieve significant increases in responsiveness. This concept may be transferred to other lipid probes in the future. The ASM activity in live cells was imaged by two-photon-excitation microscopy.

AbstractEine Echtzeit‐Beobachtung der Aktivität der Sauren Sphingomyelinase (ASM) ist notwendig zur Aufklärung ihrer Rolle in Lipid‐Signaltransduktionsprozessen. Mithilfe von Phosphorodichloridat‐Chemie gelang die Synthese von fluoreszierenden Phosphosphingolipiden mit der Fähigkeit zum FRET. Diese Sphingomyelin‐Analoga sind Substrate für die rekombinante humane ASM und ermöglichen die Verfolgung der ASM‐Aktivität mittels Fluoreszenzspektroskopie. Inkubation mit Zell‐Lysaten von Wildtyp‐ und Knockout‐Mäusen zeigte eine ausschließliche Spaltung durch ASM. Darüber hinaus nutzten wir systematisch die Umgebungsempfindlichkeit der Fluorophore, um signifikante Zuwächse in der Responsivität zu erzielen. Die dabei angewendeten Konzepte könnten auch für andere zukünftige Lipase‐Sonden zum Einsatz kommen. Eine mikroskopische Abbildung der ASM‐Aktivität in lebenden Zellen wurde mittels Zweiphotonenanregung realisiert.