Proteome-wide quantification of inositol pyrophosphate-protein interactions

Article https://doi.org/10.1038/s41467-026-73804-8 Proteome-wide quantification of inositol pyrophosphate-protein interactions Received: 1 August 2024 Accepted: 21 May 2026 1234567890():,; 1234567890():,; Check for updates Annika Richter1,2,4, Jaime A. Isern 1,4, Max Ruwolt Abhirup Majumdar 1,2, Fan Liu 1,3, David Furkert Dorothea Fiedler 1,2 1 , Sarah Lampe 1,2 & 1,2 , 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 polyphosphates (InsPs) are soluble, myo-inositol–based signaling molecules that are found throughout eukaryotes. A widelystudied example is inositol-1,4,5-trisphosphate (InsP3), which is produced from membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP2) upon phospholipase C activation, and mediates calcium release from the endoplasmic reticulum1. Sequential phosphorylation of InsP3 gives rise to more densely phosphorylated InsPs, including inositol hexakisphosphate (InsP6), a molecule that can function as a structural cofactor in mammals, while serving as a major phosphate storage compound in plants2–6. Although InsPs are structurally more diverse than the related phosphoinositides (PIPs), they remain comparatively less well studied. In recent years, however, a subgroup of InsPs - the inositol pyrophosphates (PP-InsPs)—has drawn increasing attention. These metabolites are generated through the action of inositol hexakisphosphate kinases (IP6Ks), which phosphorylate InsP6 at the 5position, and diphosphoinositol pentakisphosphate kinases (PPIP5Ks), which target the 1-position. Together, these enzymes produce 5-diphosphoinositol pentakisphosphate (5PP-InsP5), 1-diphosphoinositol pentakisphosphate (1PP-InsP5), and bis-1,5diphosphoinositol tetrakisphosphate (1,5(PP)2-InsP4) (Fig. 1a)7–13. The dephosphorylation of these densely phosphorylated metabolites is driven primarily by diphosphoinositol polyphosphate phosphatases (DIPPs). A putative role in cellular signaling has been attributed to the PP-InsPs for a long time, as these molecules are rapidly turned 1 Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Straße 10, Berlin, Germany. 2Institut für Chemie, Humboldt-Universität zu Berlin, Brook-Taylor-Straße 2, Berlin, Germany. 3Charité - Universitätsmedizin Berlin, Charitéplatz 1, Berlin, Germany. 4These authors contributed equally: e-mail: ; fi[email protected] Annika Richter, Jaime A. Isern. Nature Communications | (2026)17:4967 1 Article https://doi.org/10.1038/s41467-026-73804-8 Fig. 1 | Inositol pyrophosphate biosynthesis and estimated levels in mammalian cells. a Pathway for the biosynthesis and turnover of inositol pyrophosphates: Inositol pyrophosphates are synthesized from InsP6 (inositol hexakisphosphate), which can be phosphorylated by IP6K1/2/3 to generate 5PP-InsP5 (5-diphosphoinositol pentakisphosphate) or by PPIP5K1/2 to produce 1PP-InsP5 (1diphosphoinositol pentakisphosphate). These molecules can be further phosphorylated to yield 1,5(PP)2-InsP4 (bis-1,5-diphosphoinositol tetrakisphosphate, also known as InsP8), with IP6Ks converting 1PP-InsP5 and PPIP5Ks converting 5PPInsP5. Dephosphorylation of these metabolites is predominantly mediated by diphosphoinositol polyphosphate phosphohydrolases, the DIPPs (DIPP1, DIPP2, DIPP3α, and DIPP3β). The main fluxes for the synthesis and turnover of PP-InsPs can differ between organisms and cell lines9,81. b Concentration ranges of (PP)-InsPs detected in a variety of human cell lines16–19. over in cells (up to ten times per hour)14,15. However, current analytical methods are not capable of resolving the detection and quantification of InsPs/PP-InsPs with subcellular resolution; therefore, many questions regarding local turnover and availability for signaling purposes have remained unanswered. Within cells, InsP6/PP-InsP levels have been reported to range from nanomolar to micromolar concentrations (Fig. 1b)16–19. Among the PP-InsPs, 5PP-InsP5 is the most abundant species in most cell lines, with concentrations typically ranging between 1–5 µM. Over the past years, the signaling functions of PP-InsPs have mainly been investigated using genetic methods, as well as pharmacological tools targeting IP6Ks10,20. These kinases, and in several cases by proxy the molecule 5PP-InsP5, have been associated with insulin secretion21,22, focal adhesion dynamics23,24, and apoptosis25. In these examples, 5PP-InsP5 accesses different modes of action for signal transduction, including competition for phospholipidbinding domains26. In addition, 5PP-InsP5 can transmit signals by transferring the β-phosphoryl group onto pre-phosphorylated proteins in a process termed protein pyrophosphorylation27–31. This unusual protein modification was demonstrated, for example, to regulate protein localization and protein degradation32–34. Compared to the functions of 5PP-InsP5, relatively little is known about the closely related messengers 1PP-InsP5 and 1,5(PP)2-InsP4. Nevertheless, recent analyses of cell lines lacking PPIP5Ks, and consequently 1,5(PP)2-InsP4, have sparked interest because these cell lines exhibit a growth-inhibited phenotype and a hypermetabolic state35,36. Deciphering the concrete signaling functions of individual PPInsPs remains challenging. Genetic or pharmacologic perturbation of IP6Ks not only reduces 5PP-InsP5 levels but also simultaneously diminishes 1,5(PP)2-InsP4 levels. Conversely, deletion of PPIP5Ks depletes 1,5(PP)2-InsP4 but concomitantly increases the cellular amounts of 5PP-InsP536. Therefore, phenotypic observations must be complemented by biochemical and/or biophysical data to assign function to a specific PP-InsP molecule. For example, a recent study demonstrated that the xenotropic and polytropic retrovirus receptor 1 (XP (...truncated)