Abstract
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-InsP5) is the most abundant isoform, constituting more than 90% of intracellular inositol pyrophosphates. 5PP-InsP5 can be further phosphorylated to 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8). These two molecules, 5PP-InsP5 and InsP8, 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-InsP5/InsP8 signaling pathway by inhibiting the biosynthesis of 5PP-InsP5 demonstrates therapeutic benefits in preclinical studies, and thus holds promise as a therapeutic approach for certain diseases treatment, such as metabolic disorders.
Graphical Abstract
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Water soluble myo-inositol phosphates are a group of small molecules, with Ins(1,4,5)P3 being the most well-studied. Ins(1,4,5)P3 derives from the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Acting as a “second messenger”, Ins(1,4,5)P3 binds to its receptor, a Ca2+ channel in the endoplasmic reticulum. This binding opens the Ca2+ channel, leading to the release of Ca2+ from the intracellular store, which is necessary for the control of cellular and physiological processes including cell division, cell proliferation, apoptosis, fertilization, development, behavior, learning, and memory. Ins(1,4,5)P3 is further metabolized to generate a series of inositol phosphate molecules by inositol phosphate kinases (Fig. 1) or phosphatases to recycle these molecules back to inositol.
Metabolic pathway of Ins(1,4,5)P3 to Ins(1,2,3,4,5,6)P6. Ins(1,4,5)P3 is phosphorylated by IPMK to form Ins(1,3,4,5)P4, which can be further phosphorylated by IPMK to form Ins(1,3,4,5,6)P5. IPPK phosphorylates Ins(1,3,4,5,6)P5 to generate Ins(1,2,3,4,5,6)P6. IPMK, inositol polyphosphate multikinase. IPPK, inositol pentakisphosphate 2-kinase
Inositol polyphosphate multikinase (IPMK) phosphorylates Ins(1,4,5)P3 to synthesize inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) [1]. Both Ins(1,3,4,5)P4 and Ins(1,3,4,5,6)P5 are vital for regulating signaling pathways, for example, Ins(1,3,4,5)P4 antagonizes the PI3K pathway [2], and Ins(1,3,4,5,6)P5 regulates angiogenesis by modulating HIF1α/VEGF protein levels [3]. IPMK is essential for the synthesis of Ins(1,3,4,5,6)P5 but not Ins(1,3,4,5)P4, because deleting IPMK depletes only Ins(1,3,4,5,6)P5 [3, 4]. Knockout of IPMK in Drosophila causes developmental defects in the epidermis [5], and in mice leads to embryonic death [4], indicating that IPMK and Ins(1,3,4,5,6)P5 are essential for life development.
Ins(1,3,4,5,6)P5 is phosphorylated by inositol pentakisphosphate 2-kinase (IPPK) to form inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), which is the most abundant inositol phosphate in nature. InsP6 has a phosphate group attached to each of the inositol’s six hydroxyl groups and acts as an important phosphate store in plants. In mammalian cells, InsP6 mediates diverse cellular functions, including DNA reparation, endocytosis, mRNA export, and ion channel regulation [6]. Depleting InsP6 by knocking out IPPK is lethal in mice, highlighting its necessity for in vivo survival [7].
The “fully phosphorylated” InsP6 was once thought to be the highest inositol phosphate. The identification of higher inositol phosphates with more than six phosphate groups indicates that inositol polyphosphates containing diphosphate or pyrophosphate groups exist in cells [8]. The concentrations of inositol pyrophosphates are in the micromolar range in mammalian cells. In hepatocytes, the cellular pools of inositol pyrophosphates are normally turning over at least 10 times every 40 min [9]. Similarly, in pancreatoma cells, 50% of the InsP6 pool is converted to pyrophosphate derivatives within 60 min [10]. The distinctive features of these inositol pyrophosphates, such as highly energic pyrophosphate bonds and rapid turnover, suggest an important role in signaling transduction and metabolism [8]. Indeed, these molecules manifest diverse functions, such as regulation of energy homeostasis [11] and protein stability [12].
5-Diphosphoinositol 1,2,3,4,6-pentakisphosphate (5PP-InsP5 or 5-InsP7) is the first and most extensively studied inositol pyrophosphate. It is soluble in water and can be found in the nucleus, cytosol, and at the cell membrane. In mammalian cells, 5PP-InsP5 is synthesized by a family of three inositol hexakisphosphate kinases (IP6Ks) in its functioning areas. This molecule is involved in various cellular processes, including DNA repair, mRNA processing, vesicle trafficking, and cytoskeleton reorganization [13]. It can be further phosphorylated by diphosphoinositol pentakisphosphate kinases (PPIP5Ks) at the 1-position of the inositol ring to form 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8) (Fig. 2), which is the most phosphorylated and the “final metabolite” of inositol phosphate discovered so far. Although the concentration of InsP8 is only around 10% of that of the total 5PP-InsP5, InsP8 plays critical roles in cellular activities, such as the regulation of ATP synthesis [11].
Metabolic pathways of 5PP-InsP5 and InsP8. InsP6 is mainly phosphorylated by IP6Ks to form 5PP-InsP5, which is phosphorylated by PPIP5Ks to generate InsP8. InsP8 can be dephosphorylated back to 5PP-InsP5 by PPIP5Ks. A minor portion of InsP6 is phosphorylated by PPIP5Ks to form 1PP-InsP5, which can be dephosphorylated back to InsP6 by PPIP5Ks. 1PP-InsP5 can also be phosphorylated by IP6Ks to generate InsP8
InsP6 can also be pyrophosphorylated by PPIP5Ks at the 1-position of the inositol ring to form 1-diphosphoinositol 2,3,4,5,6-pentakisphosphate (1PP-InsP5 or 1-InsP7). Ins(1,3,4,5,6)P5 can be pyrophosphorylated by IP6Ks at the 5-position of the inositol ring to generate 5PP-Ins(1,3,4,6)P4. It is worth noting that both 1PP-InsP5 and 5PP-Ins(1,3,4,6)P4 are considered minor inositol pyrophosphates, as their cellular concentrations are far lower than those of 5PP-InsP5. 1PP-InsP5 is typically < 2% of total InsP7 in HCT116 cells [14]. Several functions of 1PP-InsP5 and 5PP-Ins(1,3,4,6)P4 have been demonstrated in budding yeast, where 1PP-InsP5 regulates gene transcription [15,16,17] and 5PP-Ins(1,3,4,6)P4 regulates cell death and telomere length [18, 19]. However, their roles in mammalian cells are yet to be determined.
Recently, with the development of a portfolio of detection methods that can assay mass levels of inositol pyrophosphates in cells from diverse species [20,21,22], new inositol pyrophosphate isomers such as the 3PP-InsP5 (or 3-InsP7), 4/6PP-InsP5 (or 4/6-InsP7) and 2PP-InsP5 (2-InsP7) have been identified in plant, mouse and in human peripheral blood mononuclear cells [23,24,25]. These findings suggest that inositol pyrophosphate signaling appears more complex than previously thought. The metabolic pathway and bioactive properties of these newly identified isomers, namely 3PP-InsP5, 4/6PP-InsP5, and 2PP-InsP5, are yet to be delineated and are not the focus of this review.
5PP-InsP5/InsP8 appears to be the major metabolic pathway of the inositol pyrophosphates in mammalian cells because the combined amount of 5PP-InsP5 and InsP8 surpasses 95% of total inositol pyrophosphates. InsP6 is mainly phosphorylated by IP6Ks to form 5PP-InsP5, which accounts for more than 90% of the total InsP7. Although InsP6 can be phosphorylated by PPIP5Ks to form 1PP-InsP5, PPIP5Ks prefer to phosphorylate 5PP-InsP5 in vivo to form InsP8, which can explain the low cellular levels of 1PP-InsP5. It should be noted that these molecules also have important roles in fungi and plants, but due to the focus of this review, they will not be discussed. For a comprehensive review of inositol pyrophosphates in plants and fungi, please refer to REF [26, 27].
5PP-InsP5 does not seem to freely move intracellularly. It is produced by IP6K1, IP6K2, and IP6K3 in compartmentalized subcellular areas where these kinases are located, and it functions at the sites where it is generated. Deletion of individual IP6K in the mouse does not cause noticeable defects. This characteristic makes the IP6K knockout mouse a valuable model for studying the in vivo functions of each IP6K and the 5PP-InsP5 it produces. However, whether 5PP-InsP5 is essential for embryonic development is currently unknown because there are no IP6K1/IP6K2/IP6K3 triple knockout animals available.
InsP8 is also produced in compartmentalized subcellular areas by PPIP5K1 and PPIP5K2 in mammalian cells. Since InsP8 is primarily derived from 5PP-InsP5, it likely operates in the same cellular microenvironments as 5PP-InsP5 and may participate in similar cellular processes. The role of InsP8 in embryonic development is currently not known, as there are no PPIP5K1/PPIP5K2 double KO animals available.
Several inhibitors of IP6K have been developed [28,29,30,31], and have demonstrated therapeutic benefits in animal models, such as lowering body weight [32], reducing blood glucose [30], and attenuating myocardial injury [29]. These findings suggest that targeting the 5PP-InsP5/InsP8 pathway by blocking 5PP-InsP5 biosynthesis may represent a viable therapeutic strategy for treating certain diseases. However, adverse effects are recorded in genetically IP6K knockout mice. For example, deletion of IP6K1 leads to neurodevelopmental and male reproductive disorders [33,34,35], deletion of IP6K2 affects neuronal development [36] and increases the risk of cancer development [37], and deletion of IP6K3 causes neurodevelopmental defects [38, 39]. Therefore, a comprehensive understanding of the functions and mechanisms of 5PP-InsP5, InsP8, and their synthetic enzymes is crucial before they can be considered druggable targets.
In this review, we focus on recent advancements in the biology of 5PP-InsP5, InsP8, and their synthetic enzymes in mammalian cells. These findings provide new insights into these molecules and may provide novel targets for future therapeutic targets.
Physiological Functions and Mechanisms of 5PP-InsP5 and InsP8
5PP-InsP5 and InsP8 were identified three decades ago. Myo-[3H]inositol and/or [32]Pi were applied to cells to label newly synthesized inositol phosphates, which were separated by HPLC. The inositol pyrophosphate compounds were eluted from various forms of anion exchange HPLC columns after InsP6. The detailed structures of these molecules were identified as diphosphoinositol pentakisphosphate (PP-InsP5 or InsP7) and bis-diphosphoinositol tetrakisphosphate (InsP8) [8]. The cytosol contains approximately 85% of 5PP-InsP5 and InsP8 [8]. In mammalian cells, the physiological concentration of 5PP-InsP5 is around 5 μM, while InsP8 is less than 0.5 μM [40]. Recently, the basal level of 5PP-InsP5 in rat blood has been determined to be 37.4 ng/mL [20].
Physiological Functions and Mechanisms of 5PP-InsP5
The functions and molecular mechanisms of 5PP-InsP5 have been elucidated through models involving genetic knockout of and pharmacological inhibition of IP6Ks (Table 1). Blocking the biosynthesis of 5PP-InsP5 reduces 90% of total InsP7 levels, suggesting that 5PP-InsP5 cannot be compensated by other PP-InsP5 isomers [28]. Furthermore, genetically knocking out of individual IP6K lowers total 5PP-InsP5 levels, indicating that 5PP-InsP5 does not freely diffuse but rather operates within its specific generating area. Additionally, the functions of individual IP6K cannot be compensated by other isoforms [39, 41, 42]. 5PP-InsP5 performs its functions by binding or pyrophosphorylating target proteins, which play crucial roles in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways [13, 43]. This posttranslational modification of proteins through pyrophosphorylation by 5PP-InsP5 is essential for regulating their functions. [44, 45]. Recent studies have highlighted the importance of 5PP-InsP5-mediated protein pyrophosphorylation in facilitating protein dimer formation, such as focal adhesion kinase (FAK) dimer formation and interferon regulatory factor 3 (IRF3) dimer formation. [46, 47]. While protein pyrophosphorylation was previously believed to be solely a non-enzymatic post-translational modification [48], a recent study has identified the metabolic enzyme UAP1 as a pyrophosphorylase responsible for catalyzing 5PP-InsP5-dependent pyrophosphorylation of S386 on IRF3 [47]. This finding challenges the previous notion and raises the possibility of other instances of enzymatic pyrophosphorylation. Further research is necessary to explore these potentials.
Physiological Functions and Mechanisms of 5PP-InsP5 at the Plasma Membrane
A prominent function of 5PP-InsP5 at the cell membrane is to antagonize phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3). Structurally resembling the inositol head group of PtdIns(3,4,5)P3, 5PP-InsP5 competes with it for binding to the pleckstrin homology (PH) domain of its target proteins [49]. One well-characterized signaling pathway regulated by 5PP-InsP5 is the PDK1-dependent activation of Akt [41]. Binding to the PH domain of Akt, 5PP-InsP5 prevents PtdIns(3,4,5)P3-induced membrane translocation of Akt, and thus Akt remains unphosphorylated [49]. Depleting 5PP-InsP5 strengthens Akt activation [41]. Synthetic methylene-bisphosphonate analogues of 5PP-InsP5, which structurally mimic it but cannot donate its β-phosphoryl groups to protein substrates, can inhibit Akt activation to the same extent as 5PP-InsP5, indicating that 5PP-InsP5 does not inhibit Akt through pyrophosphorylation [50].
Transporting molecules in or out of cells is a fundamental cellular process. In neuronal cells, 5PP-InsP5 regulates synaptic membrane vesicle trafficking [51,52,53]. Specifically, 5PP-InsP5 inhibits synaptotagmin-dependent exocytosis by interfering with the fusogenic activity of Ca2+ and blocking adaptor protein-mediated synaptic vesicle recycling [52, 53]. In addition, 5PP-InsP5 is required for insulin release in pancreatic β-cells [54,55,56,57]. 5PP-InsP5 competes with PtdIns(4,5)P2 for binding to synaptotagmin-7, Ca2+ selectively binds 5PP-InsP5 with high affinity, freeing synaptotagmin-7 to enable fusion of insulin-containing vesicles with the cell membrane [57]. Besides, 5PP-InsP5 regulates Ca2+ oscillations, a key element in triggering exocytosis and secretion in β-cells [58]. 5PP-InsP5 also inhibits the release of HIV-1 virus-like particles from HeLa cells by modulating the interaction of adaptor protein complex AP-3 with Kif3A, a motor protein of the kinesin superfamily [59]. Specifically, 5PP-InsP5 pyrophosphorylates the β subunit of AP-3, blocking its interaction with Kif3A [59].
Protein activities and stabilities are crucial for cell functions. 5PP-InsP5 regulates certain plasma membrane proteins' activity and stability [12, 60]. Xenotropic and polytropic retrovirus receptor 1 (XPR1) is an 8-pass transmembrane molecule that mediates phosphate export from the cell. 5PP-InsP5 enhances the activity of XPR1 by binding to its SPX domain [61]. Depleting 5PP-InsP5 leads to an increase in intracellular phosphate by inhibiting XPR1-mediated phosphate export [60]. Pharmacological inhibition of 5PP-InsP5 synthesis in vivo blocks cellular phosphate export and subsequently lowers plasma phosphate levels [31]. Na+/K+-ATPase is universally expressed in the plasma membrane of animal cells, and it is essential for cell volume maintenance, signal transduction, and secondary transport of various nutrients. 5PP-InsP5 determines the protein levels of sodium/potassium-transporting ATPase (Na+/K+-ATPase) at the cell membrane [12]. Depleting 5PP-InsP5 elicits a two-fold enrichment of Na+/K+-ATPase in the plasma membrane. 5PP-InsP5 binds the RhoGAP domain of PI3K p85α to disinhibit its interaction with Na+/K+-ATPase, subsequently recruiting the adaptor protein 2 and triggering the clathrin-mediated endocytosis of Na+/K+-ATPase and its downstream degradation [12].
Focal adhesions are plasma membrane-associated protein complexes responsible for engaging with the extracellular matrix. The dynamics of these adhesions are regulated by 5PP-InsP5 [33, 39, 42, 62]. Depleting 5PP-InsP5 delays focal adhesion turnover by reducing the phosphorylation levels of focal adhesion kinase (FAK). 5PP-InsP5 binds to the FERM domain of FAK, promoting its dimer formation and leading to FAK phosphorylation [46]. Synthetic analogues of 5PP-InsP5 cannot promote FAK dimer formation, indicating that pyrophosphorylation is required for this process [46]. The antifungal drug itraconazole inhibits angiogenesis by disrupting the 5PP-InsP5-mediated focal adhesion dynamics and cytoskeletal remodeling [46].
Physiological Functions and Mechanisms of 5PP-InsP5 in the Cytosol
5PP-InsP5 participates in regulating intracellular ATP concentration [60, 63]. It affects the activity of the major glycolytic transcription factor GCR1. Depleting 5PP-InsP5 augments glycolysis and elicits approximately a three-fold increase of ATP in mouse embryonic fibroblast cells [63]. Conversely, higher cellular ATP level is associated with increased concentration of 5PP-InsP5 [11, 56]. The mechanisms behind this reciprocal regulation of ATP and 5PP-InsP5 remain unknown, and their physiological and pathophysiological relevance is yet to be demonstrated.
Mitochondria are the primary source of intracellular ATP. 5PP-InsP5 plays a critical role in regulating mitochondria biogenesis and function. In mouse embryonic fibroblast cells, depleting 5PP-InsP5 by deleting IP6K1 reduces oxygen consumption rates, decreases mitochondrial mass, and lowers membrane potential [63]. In contrast, animal studies reveal that depleting 5PP-InsP5 in hepatocytes by knocking out IP6K1 elevates oxygen consumption rates, upregulates mitochondrial oxidative phosphorylation proteins, and increases mitochondrial oxidative capacity [64]. Similarly, in adipocytes, depleting 5PP-InsP5 by knocking out IP6K1 boosts mitochondrial oxygen consumption rates, enhances mitochondria function to augment thermogenic energy expenditure [65, 66], and elevates PGC1-α expression levels under low-temperature conditions [65]. Depleting 5PP-InsP5 also enhances mitochondrial biogenesis and function in cardiomyocytes by activating the AMPK pathway and decreasing the acetylation state of PGC-1α [29]. IP6K2 is also responsible for 5PP-InsP5 production. Deletion of IP6K2 in N2A neuronal cells impairs mitochondrial function and represses the cytochrome c1 subunit of the mitochondrial electron transport chain, complex III. Loss of IP6K2 causes mitochondrial oxidative stress in the cerebellum [67]. IP6K2 is also involved in the attenuation of PINK1-mediated mitochondrial autophagy in the brain in a catalytically independent manner. Deletion of IP6K2 in N2A and PC12 cells results in increased mitochondrial fission and mitophagy. The expression levels of dynamin-related protein 1, PGC1-α, and mitophagy markers (PINK1, Parkin, and LC3-II) are upregulated in the cerebellum of IP6K2 KO brains [68]. The above evidence indicates that 5PP-InsP5 generated by IP6K1 plays opposite roles than that generated by IP6K2 in regulating mitochondria. It is worth noting that depleting 5PP-InsP5 also depletes InsP8. In both HCT116 cells and HEK293 cells, depleting InsP8 increases mitochondrial mass, elevates oxygen consumption rates, and increases mitochondrial oxidative phosphorylation [11]. The mitochondria are more tubular and less fragmented in InsP8-depleted HCT116 cells [11]. The mechanisms by which 5PP-InsP5 and InsP8 regulate mitochondria have not been delineated.
Cytoskeletal proteins are structural proteins that are critical for regulating cell shape, migration, division, etc. 5PP-InsP5 participates in cytoskeletal reorganization [46]. The Arp2/3 complex generates a dendritic actin network at the leading edge of motile cells to form lamellipodia. 5PP-InsP5 binds to the Arp2/3 complex and recruits coronin, which promotes the disassembly of branched actin networks, and it is required for Arp2/3-mediated actin dynamics in vivo [46]. 5PP-InsP5 is required for cytoplasmic dynein-driven vesicle transport in mammalian cells [45]. 5PP-InsP5 promotes the assembly of the dynein complex by strengthening the binding between the dynein intermediate chain and p150glued [39, 45]. 5PP-InsP5 pyrophosphorylates dynein intermediate chain. Depleting 5PP-InsP5 causes defects in dynein-dependent trafficking pathways, including endosomal sorting, vesicle movement, and Golgi maintenance [45]. Depleting 5PP-InsP5 also affects dynein-mediated cell polarity and neuronal migration [39].
Messenger RNA (mRNA) is essential for protein synthesis within the cytosol. 5PP-InsP5 regulates mRNA stability and dynamics of processing-body, cytoplasmic ribonucleoprotein granules containing translationally repressed mRNAs [69]. 5PP-InsP5 competes with 5′-capped mRNA for hydrolysis by NUDT3 (also named diphosphoinositol polyphosphate phosphatase 1, DIPP1), therefore inhibiting NUDT3-mediated mRNA decapping. Elevating cellular 5PP-InsP5 levels increases amounts of NUDT3 mRNA substrates and raises processing-body abundance [69]. A recent study showed that the IP6K1 protein itself can upregulate the formation of processing-bodies [70]. In that model, IP6K1 acts as a scaffolding protein to promote the interactions of DDX6 and 4E-T with the cap binding protein eIF4E, without requiring IP6K1’s product 5PP-InsP5 [70]. Additionally, 5PP-InsP5 is involved in regulating protein activities within the cytosol, for example, 5PP-InsP5 binds to insulin-degrading enzyme (IDE) and promotes IDE-catalyzed cleavage of bradykinin [71].
Physiological Functions of 5PP-InsP5 in the Nucleus
A major function of nuclear 5PP-InsP5 in mammalian cells is to regulate p53-mediated apoptosis. Depleting nuclear 5PP-InsP5 by knocking out IP6K2 impairs p53-mediated apoptosis, instead of favoring cell-cycle arrest [72]. These 5PP-InsP5 depleted cells show some resistance to ionizing radiation and the antiproliferative effects of interferon-β [37]. In contrast, direct microinjection of 5PP-InsP5 induces cell death in SCC22A squamous carcinoma cells [37]. 5PP-InsP5 binds CK2 to enhance its phosphorylation of the Tel2/Tti1/Tti2 complex, thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine 15 to activate the cell death program in HCT116 cells [73]. 5PP-InsP5 also affects cell survival and growth by regulating MYC protein stability. 5PP-InsP5 pyrophosphorylates MYC protein at its PEST domain (enriched in Pro, Glu/Asp, Ser, and Thr residues), recruiting the E3 ubiquitin ligase FBW7. This process causes ubiquitination and degradation of MYC protein [44]. Cigarette smoking disrupts 5PP-InsP5-mediated aged neutrophil death and thus exacerbates chronic obstructive pulmonary disease by elevating the neutrophil-to-lymphocyte ratio [74]. Hypoxic injury increases 5PP-InsP5 production in bone marrow-derived mesenchymal stem cells. The elevated 5PP-InsP5 induces autophagy and mediates hypoxic injury-induced apoptosis [75].
In addition to promoting apoptosis and cell death, nuclear 5PP-InsP5 in mammalian cells also participates in DNA damage response [76, 77]. 5PP-InsP5 mediates DNA damage repair via homologous recombination [76]. The Cullin-RING ubiquitin ligase 4 (CRL4) plays a critical role in nucleotide excision repair, and its basal activity is inhibited by binding to the COP9 signalosome (CSN). Ultra-violet radiation induces the synthesis of 5PP-InsP5 to promote the disassembly of the CRL4-CSN and thus activates CRL4 [77].
5PP-InsP5 may also regulate gene transcription directly or through epigenetic modifications [78,79,80]. Depletion of 5PP-InsP5 alters the mRNA levels of genes associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as certain genes modulating neuronal differentiation and functioning [80]. It also regulates the Jumonji domain-containing 2C (JMJD2C), a histone lysine demethylase, by inducing the disassociation of JMJD2C with chromatin [79]. Depleting 5PP-InsP5 reduces levels of trimethyl-histone H3 lysine 9 (H3K9me3) and increases levels of acetyl-H3K9, leading to changes in JMJD2C-target gene transcription [79].
The nucleolus is a spherical structure that lies in the cell’s nucleus and is the center of ribosomal RNA synthesis and processing [81]. Its architecture is modulated by 5PP-InsP5 as it binds to multiple nucleolar proteins [82]. Elevated levels of 5PP-InsP5 enlarge the outer nucleolar granular region while leaving the inner fibrillar volume of the nucleolus unaffected [82].
Physiological Functions and Mechanisms of InsP8
Experimental evidence demonstrates that InsP8 participates in 5PP-InsP5-related signaling pathways in mammalian cells (Table 2) [11, 60, 83, 84]. In certain signaling pathways, InsP8 displays weaker activities than 5PP-InsP5, and thus converting 5PP-InsP5 to InsP8 may negate the effects of 5PP-InsP5 [85]. For example, InsP8 binds the PH domain of Akt, but the affinity of Akt for InsP8 (IC50 > 50 μM) is several folds lower than that for 5PP-InsP5 [85]. Converting 5PP-InsP5 to InsP8 relieves the inhibitory effects of 5PP-InsP5 upon the PtdIns(3,4,5)P3 signaling pathway, and thus favors the association of PH domain proteins with PtdIns(3,4,5)P3 [85].
At the plasma membrane, InsP8 functions similarly to 5PP-InsP5, regulating the cellular phosphate levels by affecting both the import and export of phosphate [83, 86]. InsP8 facilitates the efflux of inorganic phosphate from mammalian cells by binding to the N-terminus of xenotropic and polytropic retrovirus receptor 1 (XPR1), which is required for the activity of XPR1-mediated cellular phosphate efflux [83]. Phosphate efflux is also regulated in a dose-dependent manner by liposomal delivery of a metabolically resistant methylene bisphosphonate (PCP) analog of InsP8, indicating the independence of protein pyrophosphorylation [83]. InsP8 also induces cellular phosphate uptake. Delivering InsP8 into HCT116 cells shows it dose-dependently stimulates the rate of phosphate uptake [86]. Intriguingly, both InsP8 and 5PP-InsP5 affect phosphate homeostasis by targeting XPR1 [60, 83]. Whether InsP8 competes with 5PP-InsP5 in regulating XPR1 remains to be determined. In pancreatic β cells, granuphilin is a crucial component of the docking machinery of insulin-containing vesicles to the plasma membrane. Insulin release is triggered by an increase in calcium concentration. InsP8 elicits a direct reduction in calcium oscillations and causes immediate translocation of the C2AB domain of granuphilin from the plasma membrane into the cytosol [84]. 5PP-InsP5 also reduces cytosolic calcium oscillations [58], but 5PP-InsP5 does not cause the translocation of the C2AB domain of granuphilin [84]. The direct targets of 5PP-InsP5 and InsP8 remain to be characterized. The in vivo effects of InsP8 upon insulin release warrant future investigation.
In the cytosol, InsP8 serves as a regulator of ATP production but has weaker activity compared to 5PP-InsP5. Depleting InsP8 by deletion of the PPIP5Ks results in a 35% increase in ATP in HCT116 colon cancer cells by enhancing mitochondrial oxidative phosphorylation and glycolysis [11]. In contrast, depleting 5PP-InsP5 elevates ATP by approximately three-fold in mouse embryonic fibroblast cells [63]. InsP8 also functions similarly to 5PP-InsP5 in regulating insulin-degrading enzyme-dependent cleavage of bradykinin [71]. InsP8 and 5PP-InsP5 are catabolized by NUDT3 (also name DIPP1) in animal cells [87]. Future studies are needed to determine whether InsP8 performs similar functions as 5PP-InsP5 in inhibiting NUDT3-mediated mRNA decapping by competing with 5′-capped mRNA for hydrolysis by NUDT3.
In the nucleus, InsP8 antagonizes the 5PP-InsP5-mediated P53 signaling pathway in HCT116 colon cancer cells. Depleting InsP8 strengthens the P53-dependent growth-inhibited phenotype [11].
More complicated genetic and pharmacological studies are required to specify the functions of 5PP-InsP5 and InsP8.
Regulation of 5PP-InsP5 and InsP8
The intracellular levels of 5PP-InsP5 and InsP8 are primarily regulated by the cellular energy status, as their synthesis is critically dependent on ATP [88]. Increasing the cellular ATP/ADP ratio by either glucose stimulation or direct delivery of ATP leads to a rapid rise in 5PP-InsP5 concentration [11, 56]. InsP8 is more sensitive than 5PP-InsP5 to changes in environmental glucose levels. In low-glucose conditions, InsP8 concentrations decrease even before ATP levels are affected. Restoration of glucose levels quickly rescues InsP8 synthesis [89]. Thus, 5PP-InsP5 and InsP8 may act as indicators of cellular energy status in mammalian cells.
Animal experiments demonstrate that the levels of 5PP-InsP5 are affected by multiple factors such as aging and metabolic disorders. In a mouse model, hepatocytes from 10-month-old mice express higher levels of 5PP-InsP5 than those from 2-month-old [41]. Similarly, bone marrow mesenchymal stem cells isolated from 18-month-old mice display a two-fold increase in 5PP-InsP5 production than those isolated from 2-month-old mice [90]. Furthermore, a 1.5-fold increase of 5PP-InsP5 levels is recorded in the pancreatic β-cells of the obese diabetic ob/ob mice compared to those of controls [54].
InsP8 biosynthesis is tightly regulated by cellular levels of inorganic phosphate. InsP8 levels decrease upon phosphate starvation and subsequently recover during phosphate replenishment. InsP8 is more sensitive than 5PP-InsP5 in reflecting the intracellular inorganic phosphate levels and has a wider dynamic range of fluctuation [91]. Besides, InsP8 levels are modestly elevated when cells are activated by growth factors. For example, 45–70% increases in InsP8 were observed in EGF-treated DDT1MF-2 cells and PDGF-treated NIH3T3 cells [85, 92]. PDGF and insulin treatment also increase InsP8 levels in L6 myoblast cells [93]. However, the physiological relevance and the mechanism of the growth factor-induced elevation of InsP8 remains to be determined.
InsP8 is the major product of PPIP5Ks and is generated from 5PP-InsP5. The cellular concentration of InsP8 is typically less than 10% of 5PP-InsP5. This indicates that (1) the protein levels of PPIP5Ks are less than 10% of IP6Ks, or (2) the activities of PPIP5Ks are less than 10% of IP6Ks, or (3) 90% of IP6Ks are not surrounded by PPIP5Ks. However, further studies are required to substantiate these conjectures. It is noteworthy that PPIP5Ks are also able to hydrolyze InsP8 back down to 5PP-InsP5, which consequently reduces the pool of InsP8. This process is dependent on the regulatory mechanisms of the phosphatase domain of PPIP5Ks.
Functions of IP6Ks and their Roles in Diseases
Mammalian cells express a family of three IP6Ks that share identical kinase activity domains but differ in their N-terminal and C-terminal sequences [39]. The functions of IP6Ks are non-redundant, with different isoforms performing specific roles within their subcellular region without compensating for each other (Table 3). In both murine and human cells, IP6K1 and IP6K3 are primarily located in the cytosol and at the plasma membrane; however, IP6K1 is also present in the nucleolus. In contrast, IP6K2 is primarily a nuclear protein [39]. The specific N-terminal and C-terminal sequences of individual IP6Ks mediate protein–protein interactions and post-translational modifications, such as phosphorylation [57, 94], which may contribute to the specificity of subcellular distribution and distinct in vivo functions of IP6Ks. While IP6K1 and IP6K2 are ubiquitously expressed, IP6K3 is highly expressed in specific tissues such as the brain and muscle [38, 39, 95,96,97]. Thus, in most tissues, the cytosol 5PP-InsP5 is mainly produced by IP6K1, and the nuclear 5PP-InsP5 is primarily generated by IP6K2. The in vivo functions of IP6Ks are revealed by utilizing individual IP6K KO mice.
Functions and Mechanisms of IP6K1
IP6K1 generates up to 70% of cellular 5PP-InsP5, making it the dominant isoform [41]. The protein expression levels of IP6K1 increase with age [98]. Various factors can regulate the IP6K1 protein expression, such as consumption of lean meat increases IP6K1 in muscle [99], whereas high-intensity exercise decreases IP6K1 in muscle [100]. Additionally, the cold temperature reduces IP6K1 expression in white adipose tissue [65, 66]. IP6K1 activity can also be regulated by phosphorylation [57, 94]. PKD and PKC can phosphorylate IP6K1 at serine 118 and serine 121 respectively, and double-phosphorylation has been shown to enhance its catalytic activity [57].
Global deletion of IP6K1 is viable and does not affect life expectancy in mice, with no significance between IP6K1 KO mice and their WT littermates. Notably, the IP6K1 KO mice confer greater resistance to certain diseases such as metabolic disorders [41] but may also result in developmental defects [33,34,35].
Deletion of IP6K1 causes Neuronal Developmental Defects
IP6K1 plays important roles in neuronal cell migration, it binds to α-actinin, which brings it into proximity with FAK that, together with α-actinin, is part of the focal adhesion complex [33]. IP6K1 generates 5PP-InsP5 in focal adhesions to enhance FAK phosphorylation [33, 46], which in turn augments neuronal migration. Deletion of IP6K1 leads to impaired neuronal migration, brain malformation, and a neonatal death rate of around 40% [33]. P6K1 also influences presynaptic vesicle release and short-term facilitation of glutamatergic synapses in mice hippocampal neurons [51]. It binds to rab3A-interacting-like protein 1, a guanine nucleotide exchange factor for Rab-3A, which localizes to synaptic vesicles to limit the amount of neurotransmitter release [101]. Knocking out IP6K1 augments action potential-driven synaptic vesicle exocytosis at synapses [52]. IP6K1 plays an important role in synaptic vesicle recycling, with its deletion in neurons leading to increased synaptic facilitation and both the exocytosis and endocytosis of synaptic vesicle [53]. Mechanistically, IP6K1 via its product 5PP-InsP5 inhibits synaptic vesicle exocytosis by binding synaptotagmin and inhibiting synaptotagmin-dependent synaptic membrane fusion [52]. The IP6K1 KO animals exhibit decreased prepulse inhibition, impaired short-term memory formation [102], and abnormal locomotor and social behavior [103].
Deletion of IP6K1 Causes Azoospermia
Deletion of IP6K1 severely disrupts sperm development but does not affect mating behavior. Male IP6K1 KO mice are completely infertile, while females can become pregnant and produce progenies [34, 35, 104]. IP6K1 is essential for the formation of chromatoid body, a cytoplasmic granule found in round spermatids that is composed of RNA and RNA-binding proteins [35]. Besides, IP6K1 is required for multiple aspects of male germ cell development [34]. Deletion of IP6K1 elicited several aberrations, such as sloughing off of round germ cells; disorientation and malformation of elongating/elongated spermatids; degeneration of acrosomes; defects in germ-Sertoli cell interactions and failure of spermiation. Eventually, sperm cells are not released and are instead phagocytosed by Sertoli cells, leading to an absence of sperm in the epididymis [34, 35]. Evolutionary analyses of single-nucleus transcriptomics of testes from 11 species, including humans and mice, revealed that IP6K1 expression increases towards the end of spermiogenesis in all amniotes except primates. In primates, high IP6K1 expression occurs in early spermiogenesis, before decreasing later, suggesting a shift in the spermatogenic function of IP6K1 during primate evolution [105].
Deletion of IP6K1 Protects Mice from Metabolic Dysfunctions
Deletion of IP6K1 lowers blood glucose levels. The IP6K1 KO mice display higher activities of Akt and AMPK pathways, which have been proposed to mediate the glucose-lowering effects of IP6K1 deletion [41, 64, 65, 98]. It is worth mentioning that the deletion of IP6K1 also disrupts glucose-mediated first-phase insulin secretion in pancreatic β-cells. IP6K1 may act as a metabolic sensor in pancreatic β-cells. Upon glucose stimulation, which increases the ATP/ADP ratio, IP6K1 kinase activities in β-cells are activated [56, 57]. As a result, the blood insulin levels are lower in IP6K1 KO mice than in the WT littermates [54, 55].
Whole body deletion of IP6K1 enhances lipolysis [94] and protects animals from high-fat-diet-induced obesity. The IP6K1 KO mice display reduced fat accumulation and markedly lower blood levels of leptin, a hormone derived from adipocytes that regulates hunger and food intake [41]. The IP6K1 KO mice consume similar amounts of food as WTs, indicating that deletion of IP6K1 increases leptin sensitivity. Adipose tissue-specific deletion of IP6K1 leads to decreased fat mass and increased adipose tissue browning and enhanced energy expenditure [65]. Hepatocyte-specific IP6K1 KO mice display improved insulin sensitivity, although not resistant to body weight gain. Deletion of IP6K1 increases hepatocyte mitochondrial oxidative phosphorylation capacity [64]. IP6K1 is upregulated in human nonalcoholic steatohepatitis livers. Deletion of IP6K1 improves nonalcoholic fatty liver disease and steatohepatitis in animal models [64].
The protein levels of IP6K1 are upregulated in tissues of aged mice, and deletion of IP6K1 protects mice from age-induced weight gain, insulin resistance, and metabolic dysfunction [98]. Knocking out IP6K1 also prevents age-related adipogenesis in animals, as well as increasing mesenchymal stem cell yields from bone marrow while enhancing cell growth and survival. The IP6K1 KO mesenchymal stem cells exhibit enhanced osteogenesis and hematopoiesis-supporting activity. Furthermore, deletion of IP6K1 also prevents high-fat-diet-caused trabecular bone loss [106].
Deletion of IP6K1 Reduces Inflammatory Damages
Bacterial clearance is enhanced in IP6K1-deficient mice in a bacteria-induced pneumonia model. Knocking out IP6K1 reduces polyphosphate production in platelet, which in turn reduces pulmonary neutrophil accumulation in the pneumonia model and alleviates lung damage in LPS-induced lung inflammation [107]. Neutrophils lacking IP6K1 have greater phagocytic and bactericidal abilities, along with amplified NADPH oxidase-mediated superoxide production. Deletion of IP6K1 in neutrophils activates the Akt signaling pathway, which is responsible for the enhanced bactericidal ability [108]. Inhibiting IP6K1 kinase activity delays neutrophil spontaneous death, which may contribute to the pathogenesis of chronic obstructive pulmonary disease caused by cigarette smoking [74].
Deletion of IP6K1 lowers the Risks of Cardiovascular Disease
Risk factors for cardiovascular disease include obesity, metabolic disorders, diabetes, age, and inflammation. Knocking out of IP6K1 prevents high-fat diet-induced obesity and metabolic disorders [65], lowers blood glucose levels [41], protects mice from age-induced weight gain, insulin resistance, and metabolic dysfunction [98], and attenuates inflammatory reaction [107]. The above evidence indicates that deletion of IP6K1 may reduce the risks of developing cardiovascular disease. Additionally, knocking out IP6K1 could potentially lower the risk of thrombosis. IP6K1 is required for maintaining cellular polyphosphate levels. Deleting IP6K1 reduces polyphosphate levels in platelet, leading to slower platelet aggregation and lengthened plasma clotting time [109]. However, the exact roles of IP6K1 in cardiovascular diseases remain to be demonstrated.
Deletion of IP6K1 alters Tumor Behaviors
Deletion of IP6K1 in cancer cells, such as Hela cervical carcinoma cells and HCT116 human colorectal carcinoma cells, reduces migration, invasion, and anchorage-independent growth. When fed an oral carcinogen, mice lacking IP6K1 show reduced progression from epithelial dysplasia to invasive carcinoma [62]. IP6K1 also regulates the tumor microenvironment [110]. The MC38 mouse colon allograft tumors from IP6K1 KO host mice contained increased levels of CD11b+Gr1+ IL10+ cells, but reduced levels of the CD80+ IFN-γ M1 macrophages compared to those from WT host mice. The MC38 carcinomas grow faster in the IP6K1 KO host mice than in WT mice [110].
Deletion of IP6K1 activates the Akt pathway, a major contributor to cancer development. However, the deletion of IP6K1 does not cause spontaneous cancer development in animal models. One possible reason is that deletion of IP6K1 affects other mechanisms that counterbalance cancer development. The roles and mechanisms of IP6K1 in tumor growth and behavior remain to be delineated.
Functions and Mechanisms of IP6K2
IP6K2 is primarily found within the nucleus, where it is responsible for synthesizing 20–40% of intracellular 5PP-InsP5 in mouse fibroblast cells [37, 73]. Whole body deletion of IP6K2 in mice does not cause obvious abnormalities. Both male and female IP6K2 KO mice can produce progenies, and their lifespan in laboratory animal facilities is similar to that of WT mice.
IP6K2 protein levels can be regulated by phosphorylation and degradation. CK2 selectively phosphorylates IP6K2 at serine residues S347 and S356, which are located within the degradation-specific PEST motif. This enhances the protein’s ubiquitination and degradation [40, 111].
Roles and Mechanisms of IP6K2 in Neurological Diseases
Deletion of IP6K2 elicits alterations of the cerebellar Purkinje cell morphology and psychomotor behavior, without requiring IP6K2 kinase activity [36]. Additionally, IP6K2 deletion alters the transcription of genes associated with neuronal function and development, resulting in a neurodevelopmental imbalance in the mammalian gastrointestinal tract [80]. Furthermore, IP6K2 deletion increases glycolysis [68] but reduces mitochondria function, specifically impairing the expression of the cytochrome-c1 subunit of complex III of the electron transport chain in cerebellar neurons [67]. The IP6K2 KO cells produce less phosphocreatine and ATP, leading to elevated levels of reactive oxygen species and protein oxidative damage [67]. In addition, IP6K2 is involved in the attenuation of PINK1-mediated mitochondrial autophagy in the brain in a catalytically independent manner. Deletion of IP6K2 upregulates dynamin-related protein 1 and the expression of mitochondrial biogenesis markers (PGC1-α and NRF-1) in the cerebellum [68].
IP6K2 promotes cell death [112], which may be involved in the development of neurodegenerative diseases. The expression levels of IP6K2 were higher in the spinal cord of amyotrophic lateral sclerosis patients [113]. IP6K2 can induce cell death in the anterior horn cells of the spinal cord by binding with phosphorylated TDP-43 in the cytoplasm [114] and inhibiting Akt signaling [113]. It has been observed that IP6K2 can translocate from the nucleus to the cytoplasm to inhibit Akt signaling and trigger cell death in lymphoblast cells from individuals with Huntington’s disease [115].
Roles and Mechanisms of IP6K2 in Cancers
Deletion of IP6K2 predisposes animals to carcinogen-induced invasive squamous cell carcinoma formation in the oral cavity and esophagus, although no spontaneous tumors were observed in the IP6K2 KO mice [37]. When IP6K2 is disrupted in colorectal cancer cells, it selectively impairs p53-mediated apoptosis, favoring cell-cycle arrest instead. IP6K2 interacts with p53 directly, reducing expression of proarrest gene targets such as the cyclin-dependent kinase inhibitor p21 [72]. IP6K2 also generates 5PP-InsP5 to mediate the DNA-PK/ATM-p53 cell death pathway by enhancing casein kinase 2-mediated phosphorylation of Tti1/Tel2 [73]. Within the nucleus, IP6K2 can bind LKB1, which prevents LKB1 from translocating to the cytosol and blocking cancer cell metastasis by inhibiting the focal adhesion kinase [42]. IP6K2 may also regulate cancer cell behavior by affecting the hedgehog pathway, as its deletion diminishes the hedgehog signaling response [116].
The role of IP6K2 in cancer cell behavior may be tissue specific. IP6K2 predicts favorable clinical outcomes of primary breast cancer [117]. In contrast, in glioma, the elevated IP6K2 level facilitates the proliferation, migration, and invasion of cancer cells [118].
Functions and Mechanisms of IP6K3
The expression of IP6K3 is tissue-specific, with high expression in the brain and muscles [97]. IP6K3 generates less than 25% of intracellular 5PP-InsP5 in neuronal cells [39]. Global deletion of IP6K3 does not affect embryonic development and leads to normal fertility in mice. The lifespans of IP6K3 KO mice are comparable to that of WTs.
Roles and Mechanisms of IP6K3 in Neurological Diseases
Deletion of IP6K3 leads to deficits in motor learning and coordination [38]. In cerebellar Purkinje cells, IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin. When IP6K3 is deleted, the interactions between adducin and spectrin are disrupted, causing abnormalities in the structure of cerebellar Purkinje cells and decreased numbers of synapses [38]. IP6K3 also plays a critical role in synaptic vesicle recycling. Knocking out IP6K3 decreases synaptic vesicle release, but accelerates AP-3-dependent synaptic vesicle endocytosis. This leads to reduced synaptic facilitation in the IP6K3 KO mice [38, 53]. IP6K3 is also enriched at the migrating leading edge of cell membranes, where it physiologically interacts with protein dynein intermediate chain 2. These two proteins are mutually recruited to the leading edge of migrating cells. At the cell membrane, IP6K3 promotes cell motility via its effects on the turnover of focal adhesions. Deletion of IP6K3 results in defects in cell motility and neuronal dendritic growth, eventually leading to brain malformations [39].
In humans, analysis of the IP6K3 gene’s single nucleotide polymorphism (SNP) in patients with familial and sporadic late-onset Alzheimer's disease reveals that two SNPs in the 5'-flanking promoter region of the IP6K3 gene are associated with sporadic late-onset Alzheimer's disease. One of the SNPs increases IP6K3 promoter activity [96]. Intriguingly, the same SNP of IP6K3 that is associated with increased risk for late-onset Alzheimer's disease, increases the likelihood of longevity. The mechanisms behind this phenomenon are currently not known and warrant further studies [95].
Roles and Mechanisms of IP6K3 in Metabolic Dysfunctions
Systemic administration of dexamethasone elevates blood glucose and upregulates the expression of IP6K3 in the muscles. Consistently, diabetic mice show higher levels of IP6K3 protein in their muscles compared to control mice [97]. While deletion of IP6K3 does not affect muscle mass, it does lead to lower circulating insulin levels and a decrease in fat mass and as well as body weight [97]. Deletion of MyD88 reduces the transcriptional levels of IP6K3 and increases glucose uptake in muscle cells [119], implying that IP6K3 may play a role in regulating blood glucose. However, IP6K3 KO mice are not protected against diet-induced obesity [97]. The specific roles of IP6K3 in muscle and metabolic dysfunctions have not been elucidated.
Functions and Mechanisms of PPIP5Ks
PPIP5Ks are proteins of large size, with PPIP5K1 and PPIP5K2 having molecular weights of around 160 and 140 kDa, respectively. PPIP5Ks are not only kinase but also host a phosphatase domain [120]. Experimental data show that PPIP5Ks preferentially phosphorylate 5PP-InsP5 to produce InsP8 in vivo. The concentration of cellular InsP8 is 5 to 10 times higher than that of 1PP-InsP5 [121, 122]. The phosphatase activity of PPIP5K displays positional specificity: only the 1-β-phosphate from either 1PP-InsP5 or InsP8 is hydrolyzed, indicating a futile cycle of 1-kinase/1-phosphatase is catalyzed by the PPIP5Ks [123]. Interestingly, PPIP5Ks prefer to dephosphorylate InsP8 over 1PP-InsP5 as phosphatases, resulting in InsP8 being dephosphorylated about tenfold faster than 1PP-InsP5 [123]. The activities of the kinase and phosphatase domain can be regulated by phosphate, with phosphate inhibiting the InsP8 phosphatase activity while stimulating the 5PP-InsP5 kinase activity [91].
All the aforementioned evidence indicates that the 5PP-InsP5/InsP8 cycle serves as the major metabolic pathway for PPIP5Ks. Based on this, it is plausible to assume that PPIP5Ks function within similar subcellular compartments as IP6Ks (Table 4), given that PPIP5Ks’ enzymatic activities rely on IP6K’s product. This “futile” cycle of 5PP-InsP5/InsP8 may be essential for the timely and precise modulation of the local concentration of 5PP-InsP5 and InsP8, possibly playing a critical role in signaling on and off.
Functions and Mechanisms of PPIP5K1
PPIP5K1 features a domain that binds to phosphatidylinositols such as PtdIns(4,5)P2 and, to a greater degree, PtdIns(3,4,5)P3. The activation of the PI3K pathway leads to the recruitment of PPIP5K1 to the plasma membrane [85, 124], where it metabolizes 5PP-InsP5 to InsP8. The conversion of 5PP-InsP5 to InsP8 relieves the inhibition of Akt by 5PP-InsP5 because InsP8 has a tenfold lower affinity for Akt. PPIP5K1 also possesses a phosphatase domain that can convert InsP8 back to 5PP-InsP5. However, its InsP8 phosphatase activity is inhibited by PtdIns(4,5)P2 and PtdIns(3,4,5)P3. These phosphatidylinositols are not phosphatase substrates of PPIP5K1, the inhibition of InsP8 phosphatase activity results from an unusual, functional overlap between the phosphatase domain and the polyphosphoinositide-binding domain [93]. Thus, PPIP5K1 may act as a switch by balancing the amount of 5PP-InsP5 and InsP8 to regulate the inhibitory effect of 5PP-InsP5 on PH-domain-containing proteins.
The plasma membrane localization of PPIP5K1 may also be regulated by 5PP-InsP5, which could potentially compete with PtdIns(3,4,5)P3 for binding to the polyphosphoinositide-binding domain, a cryptic PH domain. This may prevent the PtdIns(3,4,5)P3-mediated relocation of PPIP5K1 from the cytoplasm to the plasma membrane. However, additional experimental data is necessary to support this speculation.
PPIP5K1 contains an unusually long and evolutionarily conserved intrinsically disordered region. This region is responsible for the interaction with multiple proteins, involving cellular localization, vesicle-mediated transport, cell division, cellular component biogenesis, actin cytoskeleton organization, phosphatidylinositol metabolic processes, apoptotic DNA fragmentation, and cell migration [125]. It has been reported that deletion of PPIP5K1 leads to decreased motility of HeLa cells in a wound-healing assay [125].
PPIP5K1 overexpression decreases p53 phosphorylation on key residues, including Ser-15, -46, and -392, and thus decreases cell sensitivity to several cytotoxic agents, including etoposide, cisplatin, and sulindac [126]. This impact could be related to the kinase activity of PPIP5K1, since its phosphatase activity converts InsP8 to 5PP-InsP5, enhancing p53-mediated cell death induced by 5PP-InsP5.
Functions of PPIP5K2 and its Roles in Diseases
PPIP5K2 possesses a nuclear localization signal and thus may be responsible for the nuclear InsP8 synthesis. PPIP5K2 facilitates DNA homologous recombination repair, which promotes colorectal carcinoma pathogenesis [127]. PPIP5K2 deficiency has been found to reduce Akt activation in hematopoietic stem cells induced by a high-phosphate diet [128]. Due to the non-productive, substrate-stimulated ATPase activity, PPIP5K2 utilizes approximately 2 ATP molecules to synthesize each molecule of 1PP-InsP5 and 1.2 ATP molecules to synthesize InsP8 [122]. It is worth noting that PPIP5K2 is insensitive to physiological changes in either AMP or ATP/ADP ratios [122]. While the exact mechanisms are not fully understood, PPIP5K2 may play an important role in pancreatic β-cell and participate in type 1 and 2 diabetes in humans [129].
Two mutations in the phosphatase domain (amino acids 363–909) of the PPIP5K2 protein, namely S419A, and N843S, have been linked to keratoconus, a common corneal degenerative disorder that can cause high myopia, irregular astigmatism, and cornea scarring. These mutations may impact the dynamic balance of phosphatase and kinase activities, leading to increased production of InsP8 by the mutant PPIP5K2 in vivo [130].
A mutation in PPIP5K2's arginine 837 residue to histidine is associated with hearing loss in humans. This mutant reduces the phosphatase activity of PPIP5K2 and increases its kinase activity. PPIP5K2 is expressed in the cochlear and vestibular sensory hair cells, supporting cells, and spiral ganglion neurons. Mice homozygous for a targeted deletion of the PPIP5K2 phosphatase domain exhibit degeneration of cochlear outer hair cells and elevated hearing thresholds [131].
Therapeutic Applications of Targeting the 5PP-InsP5/InsP8 Pathway
Several IP6K inhibitors have been characterized and tested in animal models [28, 31, 132,133,134]. N2-(m-(trifluoromethy)lbenzyl) N6-(p-nitrobenzyl)purine (TNP) is the first characterized IP6K inhibitor [28]. Recently, a more potent orally available selective IP6K inhibitor SC-919 has been developed [31]. Pharmacological inhibition of IP6K kinase activity to block 5PP-InsP5 biosynthesis has shown beneficial effects in the treatment of several diseases (Table 5) [29,30,31, 135]. Depleting 5PP-InsP5 by administration of TNP did not show long-term damage on spermatogenesis in mice [135], although genetic deletion of IP6K1 causes azoospermia [34, 35].
Blocking 5PP-InsP5 Synthesis Prevents Metabolic Disorders
Pharmacological inhibition of IP6K by administration of TNP prevents high-fat-diet-induced metabolic disorders. TNP administration decelerates the initiation of high-fat-diet-induced obesity and insulin resistance. Inhibiting IP6K activity facilitates weight loss and restores metabolic parameters in obese animals [30]. Additionally, blocking the synthesis of 5PP-InsP5 can protect animals from obesity-induced bone loss [135].
Blocking 5PP-InsP5 Synthesis Protects Cardiomyocytes
In vivo administration of TNP activates the PI3K/Akt/BAD pathway in cardiac tissue, blocking cardiomyocyte apoptosis and reducing myocardial infarction in a mouse model of ischemic-reperfusion injury [29]. TNP also attenuates hypoxia-induced apoptosis of bone marrow-derived mesenchymal stem cells [75] and promotes mesenchymal stem cell engraftment and paracrine effect in infarcted hearts to preserve heart function and decrease fibrosis [136]. Blocking IP6K1 may also prevent coagulation, and thus prevent thrombosis [109].
Blocking 5PP-InsP5 Synthesis Reduces Inflammatory Damage
Inhibiting IP6K by TNP treatment enhances host bacterial killing and reduces pulmonary neutrophil accumulation, minimizing the lung damage caused by both Gram-positive and Gram-negative bacterial pneumonia [107].
Blocking 5PP-InsP5 Synthesis Alleviates Hyperphosphataemia
Oral administration of SC-919, which inhibits IP6K, has been shown to alleviate hyperphosphatemia, increase ATP levels in the kidneys, and improve kidney function in chronic kidney disease animal models. This in vivo treatment has been found to reduce plasma levels of creatinine, a biomarker for renal dysfunction, and corrects the imbalanced plasma levels of parathyroid hormone and 1,25 dihydroxyvitamin D [31].
Both TNP and SC-919 are pan IP6K inhibitors, targeting IP6K1, IP6K2 and IP6K3 simultaneously. It remains unclear whether the in vivo effects of these IP6K inhibitors are specific to one isoform or all three. Preclinical studies suggest that IP6K1 inhibition may prevent metabolic dysfunctions [41, 65], while pharmacological inhibition of IP6K2 could be useful in treating neurodegenerative diseases [113, 114]. The in vivo functions of IP6K3 remain mysterious, and thus there is insufficient evidence to support it as a useful therapeutic target for any disease. However, isoform-specific IP6K inhibitors will be necessary to clarify these questions.
Blocking 5PP-InsP5 biosynthesis by targeting IP6Ks in mammalian cells also reduces InsP8 [60], thus we cannot rule out the possibility that the depletion of InsP8 also contributes to the beneficial effects of IP6K inhibitors.
InsP8 might a be Potential Target for Tumor Therapy
PPIP5K2 is found to be associated with the survival risk of cervical cancer [137]. Deleting PPIP5Ks upregulates the expression of p53 and p21, and slows down cell proliferation and G1/S cell-cycle transition [11]. In xenograft models, the knockdown of PPIP5K2 inhibits ovarian tumor progression [138]. The PPIP5Ks KO HCT116 cancer cells show a significant reduction in colony formation in soft agar or on a solid surface, as well as a decrease in vivo tumorigenic capacity. Furthermore, the levels of precursors for de novo nucleotide synthesis are lower in the PPIP5Ks KO cells, indicating that deletion of PPIP5Ks inhibits de novo nucleotide biosynthesis [89]. Depleting InsP8 by deletion of PPIP5Ks elevates 5PP-InsP5 [11], which may also contribute to the inhibition of cancer development.
It is worth mentioning that the effects of PPIP5Ks deletion can be complicated, as these enzymes possess both kinase and phosphatase activities. It is not yet clear whether it is the kinase activity or the phosphatase activity of PPIP5Ks that is responsible for the observed phenotypes of genetic knockouts. Thus, it is too preliminary to assert that PPIPK5s are druggable targets for the treatment of diseases. Inhibitors of the kinase activity and the phosphatase activity are needed to resolve these issues.
Conclusion and Perspective
5PP-InsP5 and InsP8 are so far the “final metabolites” of inositol pyrophosphate biosynthesis. IP6Ks generate 5PP-InsP5 in their functional locations, while PPIP5Ks act by converting 5PP-InsP5 to InsP8 and InsP8 back to 5PP-InsP5, potentially operating in the same localized regions. Blocking the biosynthesis of InsP6, the precursor of 5PP-InsP5, and lower inositol phosphates causes severe adverse effects, such as embryonic lethality. In contrast, animals with genetic mutations that express lower levels of 5PP-InsP5 or InsP8 are viable and live with normal life span. Currently, the impact on the life of completely depleting 5PP-InsP5 or InsP8 is unknown, as there are no available IP6K1/IP6K2/IP6K3 triple knockout animals and PPIP5K1/PPIP5K2 double KO animals. Pharmacological inhibiting 5PP-InsP5 biosynthesis has proved therapeutic benefits, such as preventing metabolic disorder, attenuating infarction, reducing inflammatory injury, and alleviating hyperphosphatemia [29,30,31, 107, 135]. More specific and effective inhibitors of IP6K1 are currently being developed to treat obesity and related metabolic dysfunctions [139, 140]. Recently, a more selective IP6K2 inhibitor has been developed [141]. These compounds will facilitate the validation of IP6K inhibition in vivo and expedite future drug development.
Although animal models have not shown any abnormal responses to pharmacological inhibitors of IP6K, genetic deletion of IP6K has been found to cause developmental defects and mental disorders in mice [33,34,35, 39, 103]. Additionally, mutations of PPIP5Ks are associated with diseases [130, 131]. Therefore, it is essential to further investigate the effects and molecular mechanisms of 5PP-InsP5 and InsP8 in different systems in vivo to evaluate their potential as druggable targets. This will be a productive direction for future research.
References
Lee B, Park SJ, Hong S, Kim K, Kim S. Inositol Polyphosphate Multikinase Signaling: Multifaceted Functions in Health and Disease. Mol Cells. 2021;44:187–94. https://doi.org/10.14348/molcells.2021.0045.
Eckmann L, et al. D-myo-Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-kinase signaling pathways. Proc Natl Acad Sci U S A. 1997;94:14456–60. https://doi.org/10.1073/pnas.94.26.14456.
Fu C, et al. Inositol Polyphosphate Multikinase Inhibits Angiogenesis via Inositol Pentakisphosphate-Induced HIF-1alpha Degradation. Circ Res. 2018;122:457–72. https://doi.org/10.1161/CIRCRESAHA.117.311983.
Frederick JP, et al. An essential role for an inositol polyphosphate multikinase, Ipk2, is in mouse embryogenesis and second messenger production. Proc Natl Acad Sci U S A. 2005;102:8454–9. https://doi.org/10.1073/pnas.0503706102.
Seeds AM, Tsui MM, Sunu C, Spana EP, York JD. Inositol phosphate kinase 2 is required for imaginal disc development in Drosophila. Proc Natl Acad Sci U S A. 2015;112:15660–5. https://doi.org/10.1073/pnas.1514684112.
Marolt G, Kolar M. Analytical Methods for Determination of Phytic Acid and Other Inositol Phosphates: A Review. Molecules. 2020; 26. https://doi.org/10.3390/molecules26010174
Verbsky J, Lavine K, Majerus PW. Disruption of the mouse inositol 1,3,4,5,6-pentakisphosphate 2-kinase gene, associated lethality, and tissue distribution of 2-kinase expression. Proc Natl Acad Sci U S A. 2005;102:8448–53. https://doi.org/10.1073/pnas.0503656102.
Stephens L, et al. The detection, purification, structural characterization, and metabolism of diphosphoinositol pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s). J Biol Chem. 1993;268:4009–15.
Glennon MC, Shears SB. Turnover of inositol pentakisphosphates, inositol hexakisphosphate and diphosphoinositol polyphosphates in primary cultured hepatocytes. Biochem J. 1993;293(Pt 2):583–90. https://doi.org/10.1042/bj2930583.
Menniti FS, Miller RN, Putney JW Jr, Shears SB. Turnover of inositol polyphosphate pyrophosphates in pancreatoma cells. J Biol Chem. 1993;268:3850–6.
Gu C, et al. KO of 5-InsP(7) kinase activity transforms the HCT116 colon cancer cell line into a hypermetabolic, growth-inhibited phenotype. Proc Natl Acad Sci U S A. 2017;114:11968–73. https://doi.org/10.1073/pnas.1702370114.
Chin AC et al. The inositol pyrophosphate 5-InsP(7) drives sodium-potassium pump degradation by relieving an autoinhibitory domain of PI3K p85alpha. Sci Adv. 2020; 6. https://doi.org/10.1126/sciadv.abb8542
Furkert D, Hostachy S, Nadler-Holly M, Fiedler D. Triplexed Affinity Reagents to Sample the Mammalian Inositol Pyrophosphate Interactome. Cell Chem Biol. 2020;27:1097-1108 e1094. https://doi.org/10.1016/j.chembiol.2020.07.017.
Gu C, Wilson MS, Jessen HJ, Saiardi A, Shears SB. Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels. PLoS One. 2016;11:e0165286. https://doi.org/10.1371/journal.pone.0165286.
Lee YS, Huang K, Quiocho FA, O’Shea EK. Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate. Nat Chem Biol. 2008;4:25–32. https://doi.org/10.1038/nchembio.2007.52.
Pulloor NK, et al. Human genome-wide RNAi screen identifies an essential role for inositol pyrophosphates in Type-I interferon response. PLoS Pathog. 2014;10:e1003981. https://doi.org/10.1371/journal.ppat.1003981.
Lee YS, Mulugu S, York JD, O’Shea EK. Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates. Science. 2007;316:109–12. https://doi.org/10.1126/science.1139080.
Saiardi A, Resnick AC, Snowman AM, Wendland B, Snyder SH. Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases. Proc Natl Acad Sci U S A. 2005;102:1911–4. https://doi.org/10.1073/pnas.0409322102.
York SJ, Armbruster BN, Greenwell P, Petes TD, York JD. Inositol diphosphate signaling regulates telomere length. J Biol Chem. 2005;280:4264–9. https://doi.org/10.1074/jbc.M412070200.
Kobayashi A, Abe SI, Watanabe M, Moritoh Y. Liquid chromatography-mass spectrometry measurements of blood diphosphoinositol pentakisphosphate levels. J Chromatogr A. 2022;1681:463450. https://doi.org/10.1016/j.chroma.2022.463450.
Harmel RK, et al. Harnessing (13)C-labeled myo-inositol to interrogate inositol phosphate messengers by NMR. Chem Sci. 2019;10:5267–74. https://doi.org/10.1039/c9sc00151d.
Qiu D, et al. Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry. Nat Commun. 2020;11:6035. https://doi.org/10.1038/s41467-020-19928-x.
Lin H, et al. Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases. J Biol Chem. 2009;284:1863–72. https://doi.org/10.1074/jbc.M805686200.
Riemer E, et al. ITPK1 is an InsP(6)/ADP phosphotransferase that controls phosphate signaling in Arabidopsis. Mol Plant. 2021;14:1864–80. https://doi.org/10.1016/j.molp.2021.07.011.
Qiu D, et al. Capillary electrophoresis mass spectrometry identifies new isomers of inositol pyrophosphates in mammalian tissues. Chem Sci. 2023;14:658–67. https://doi.org/10.1039/d2sc05147h.
Nguyen Trung M, Furkert D, Fiedler D. Versatile signaling mechanisms of inositol pyrophosphates. Curr Opin Chem Biol. 2022;70:102177. https://doi.org/10.1016/j.cbpa.2022.102177.
Cridland C, Gillaspy G. Inositol Pyrophosphate Pathways and Mechanisms: What Can We Learn from Plants? Molecules. 2020; 25. https://doi.org/10.3390/molecules25122789
Padmanabhan U, Dollins DE, Fridy PC, York JD, Downes CP. Characterization of a selective inhibitor of inositol hexakisphosphate kinases: use in defining biological roles and metabolic relationships of inositol pyrophosphates. J Biol Chem. 2009;284:10571–82. https://doi.org/10.1074/jbc.M900752200.
Sun D, et al. Oncostatin M (OSM) protects against cardiac ischaemia/reperfusion injury in diabetic mice by regulating apoptosis, mitochondrial biogenesis and insulin sensitivity. J Cell Mol Med. 2015;19:1296–307. https://doi.org/10.1111/jcmm.12501.
Ghoshal S, et al. TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] ameliorates diet induced obesity and insulin resistance via inhibition of the IP6K1 pathway. Mol Metab. 2016;5:903–17. https://doi.org/10.1016/j.molmet.2016.08.008.
Moritoh Y, et al. The enzymatic activity of inositol hexakisphosphate kinase controls circulating phosphate in mammals. Nat Commun. 2021;12:4847. https://doi.org/10.1038/s41467-021-24934-8.
Mukherjee S et al. The IP6K Inhibitor LI-2242 Ameliorates Diet-Induced Obesity, Hyperglycemia, and Hepatic Steatosis in Mice by Improving Cell Metabolism and Insulin Signaling. Biomolecules. 2023;13. https://doi.org/10.3390/biom13050868
Fu C, et al. Neuronal migration is mediated by inositol hexakisphosphate kinase 1 via alpha-actinin and focal adhesion kinase. Proc Natl Acad Sci U S A. 2017;114:2036–41. https://doi.org/10.1073/pnas.1700165114.
Fu C, et al. Multiple aspects of male germ cell development and interactions with Sertoli cells require inositol hexakisphosphate kinase-1. Sci Rep. 2018;8:7039. https://doi.org/10.1038/s41598-018-25468-8.
Malla AB, Bhandari R. IP6K1 is essential for chromatoid body formation and temporal regulation of Tnp2 and Prm2 expression in mouse spermatids. J Cell Sci. 2017;130:2854–66. https://doi.org/10.1242/jcs.204966.
Nagpal L, Fu C, Snyder SH. Inositol Hexakisphosphate Kinase-2 in Cerebellar Granule Cells Regulates Purkinje Cells and Motor Coordination via Protein 4.1N. J Neurosci. 2018;38:7409–19. https://doi.org/10.1523/JNEUROSCI.1165-18.2018.
Morrison BH, et al. Gene deletion of inositol hexakisphosphate kinase 2 predisposes to aerodigestive tract carcinoma. Oncogene. 2009;28:2383–92. https://doi.org/10.1038/onc.2009.113.
Fu C, et al. Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin. J Neurosci. 2015;35:11056–67. https://doi.org/10.1523/JNEUROSCI.1069-15.2015.
Rojas T, et al. Inositol hexakisphosphate kinase 3 promotes focal adhesion turnover via interactions with dynein intermediate chain 2. Proc Natl Acad Sci U S A. 2019;116:3278–87. https://doi.org/10.1073/pnas.1817001116.
Chakraborty A. The inositol pyrophosphate pathway in health and diseases. Biol Rev Camb Philos Soc. 2018;93:1203–27. https://doi.org/10.1111/brv.12392.
Chakraborty A, et al. Inositol pyrophosphates inhibit Akt signaling, thereby regulating insulin sensitivity and weight gain. Cell. 2010;143:897–910. https://doi.org/10.1016/j.cell.2010.11.032.
Rao F, et al. Inositol pyrophosphates promote tumor growth and metastasis by antagonizing liver kinase B1. Proc Natl Acad Sci U S A. 2015;112:1773–8. https://doi.org/10.1073/pnas.1424642112.
Wu M, Chong LS, Perlman DH, Resnick AC, Fiedler D. Inositol polyphosphates intersect with signaling and metabolic networks via two distinct mechanisms. Proc Natl Acad Sci U S A. 2016;113:E6757–65. https://doi.org/10.1073/pnas.1606853113.
Lolla P, Shah A, Unnikannan CP, Oddi V, Bhandari R. Inositol pyrophosphates promote MYC polyubiquitination by FBW7 to regulate cell survival. Biochem J. 2021;478:1647–61. https://doi.org/10.1042/BCJ20210081.
Chanduri M, et al. Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport. Biochem J. 2016;473:3031–47. https://doi.org/10.1042/BCJ20160610.
Qi J, et al. Itraconazole inhibits endothelial cell migration by disrupting inositol pyrophosphate-dependent focal adhesion dynamics and cytoskeletal remodeling. Biomed Pharmacother. 2023;161:114449. https://doi.org/10.1016/j.biopha.2023.114449.
Yang S, et al. Metabolic enzyme UAP1 mediates IRF3 pyrophosphorylation to facilitate innate immune response. Mol Cell. 2023;83:298-313 e298. https://doi.org/10.1016/j.molcel.2022.12.007.
Harmel R, Fiedler D. Features and regulation of non-enzymatic post-translational modifications. Nat Chem Biol. 2018;14:244–52. https://doi.org/10.1038/nchembio.2575.
Pavlovic I, et al. Cellular delivery and photochemical release of a caged inositol-pyrophosphate induces PH-domain translocation in cellulo. Nat Commun. 2016;7:10622. https://doi.org/10.1038/ncomms10622.
Wu M, Dul BE, Trevisan AJ, Fiedler D. Synthesis and characterization of non-hydrolysable diphosphoinositol polyphosphate second messengers. Chem Sci. 2013;4:405–10. https://doi.org/10.1039/C2SC21553E.
Park SJ, et al. Inositol Pyrophosphate Metabolism Regulates Presynaptic Vesicle Cycling at Central Synapses. iScience. 2020;23:101000. https://doi.org/10.1016/j.isci.2020.101000.
Lee TS, et al. Inositol pyrophosphates inhibit synaptotagmin-dependent exocytosis. Proc Natl Acad Sci U S A. 2016;113:8314–9. https://doi.org/10.1073/pnas.1521600113.
Li H, Datunashvili M, Reyes RC, Voglmaier SM. Inositol hexakisphosphate kinases differentially regulate trafficking of vesicular glutamate transporters 1 and 2. Front Cell Neurosci. 2022;16:926794. https://doi.org/10.3389/fncel.2022.926794.
Illies C, et al. Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells. Science. 2007;318:1299–302. https://doi.org/10.1126/science.1146824.
Bhandari R, Juluri KR, Resnick AC, Snyder SH. Gene deletion of inositol hexakisphosphate kinase 1 reveals inositol pyrophosphate regulation of insulin secretion, growth, and spermiogenesis. Proc Natl Acad Sci U S A. 2008;105:2349–53. https://doi.org/10.1073/pnas.0712227105.
Rajasekaran SS, et al. Inositol hexakisphosphate kinase 1 is a metabolic sensor in pancreatic beta-cells. Cell Signal. 2018;46:120–8. https://doi.org/10.1016/j.cellsig.2018.03.001.
Zhang X, et al. 5-IP7 is a GPCR messenger mediating neural control of synaptotagmin-dependent insulin exocytosis and glucose homeostasis. Nat Metab. 2021;3:1400–14. https://doi.org/10.1038/s42255-021-00468-7.
Hauke S, et al. Photolysis of cell-permeant caged inositol pyrophosphates controls oscillations of cytosolic calcium in a beta-cell line. Chem Sci. 2019;10:2687–92. https://doi.org/10.1039/c8sc03479f.
Azevedo C, Burton A, Ruiz-Mateos E, Marsh M, Saiardi A. Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release. Proc Natl Acad Sci U S A. 2009;106:21161–6. https://doi.org/10.1073/pnas.0909176106.
Wilson MS, Jessen HJ, Saiardi A. The inositol hexakisphosphate kinases IP6K1 and -2 regulate human cellular phosphate homeostasis, including XPR1-mediated phosphate export. J Biol Chem. 2019;294:11597–608. https://doi.org/10.1074/jbc.RA119.007848.
Wild R, et al. Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains. Science. 2016;352:986–90. https://doi.org/10.1126/science.aad9858.
Jadav RS, et al. Deletion of inositol hexakisphosphate kinase 1 (IP6K1) reduces cell migration and invasion, conferring protection from aerodigestive tract carcinoma in mice. Cell Signal. 2016;28:1124–36. https://doi.org/10.1016/j.cellsig.2016.04.011.
Szijgyarto Z, Garedew A, Azevedo C, Saiardi A. Influence of inositol pyrophosphates on cellular energy dynamics. Science. 2011;334:802–5. https://doi.org/10.1126/science.1211908.
Mukherjee S, et al. Pleiotropic actions of IP6K1 mediate hepatic metabolic dysfunction to promote nonalcoholic fatty liver disease and steatohepatitis. Mol Metab. 2021;54:101364. https://doi.org/10.1016/j.molmet.2021.101364.
Zhu Q, et al. Adipocyte-specific deletion of Ip6k1 reduces diet-induced obesity by enhancing AMPK-mediated thermogenesis. J Clin Invest. 2016;126:4273–88. https://doi.org/10.1172/JCI85510.
Zhu Q, Ghoshal S, Tyagi R, Chakraborty A. Global IP6K1 deletion enhances temperature modulated energy expenditure which reduces carbohydrate and fat induced weight gain. Mol Metab. 2017;6:73–85. https://doi.org/10.1016/j.molmet.2016.11.010.
Nagpal L, Kornberg MD, Albacarys LK, Snyder SH. Inositol hexakisphosphate kinase-2 determines cellular energy dynamics by regulating creatine kinase-B. Proc Natl Acad Sci U S A. 2021; 118. https://doi.org/10.1073/pnas.2020695118.
Nagpal L, Kornberg MD, Snyder SH. Inositol hexakisphosphate kinase-2 non-catalytically regulates mitophagy by attenuating PINK1 signaling. Proc Natl Acad Sci U S A. 2022;119:e2121946119. https://doi.org/10.1073/pnas.2121946119.
Sahu S, et al. InsP(7) is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics. Proc Natl Acad Sci U S A. 2020;117:19245–53. https://doi.org/10.1073/pnas.1922284117.
Shah A, Bhandari R. IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap. J Cell Sci. 2020;134. https://doi.org/10.1242/jcs.259117
Hostachy S, et al. Dissecting the activation of insulin degrading enzyme by inositol pyrophosphates and their bisphosphonate analogs. Chem Sci. 2021;12:10696–702. https://doi.org/10.1039/d1sc02975d.
Koldobskiy MA, et al. p53-mediated apoptosis requires inositol hexakisphosphate kinase-2. Proc Natl Acad Sci U S A. 2010;107:20947–51. https://doi.org/10.1073/pnas.1015671107.
Rao F, et al. Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2. Mol Cell. 2014;54:119–32. https://doi.org/10.1016/j.molcel.2014.02.020.
Xu Y, et al. Cigarette smoke (CS) and nicotine delay neutrophil spontaneous death via suppressing production of diphosphoinositol pentakisphosphate. Proc Natl Acad Sci U S A. 2013;110:7726–31. https://doi.org/10.1073/pnas.1302906110.
Deng J, et al. Inositol pyrophosphates mediated the apoptosis induced by hypoxic injury in bone marrow-derived mesenchymal stem cells by autophagy. Stem Cell Res Ther. 2019;10:159. https://doi.org/10.1186/s13287-019-1256-3.
Jadav RS, Chanduri MV, Sengupta S, Bhandari R. Inositol pyrophosphate synthesis by inositol hexakisphosphate kinase 1 is required for homologous recombination repair. J Biol Chem. 2013;288:3312–21. https://doi.org/10.1074/jbc.M112.396556.
Rao F, et al. Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4-signalosome complex to regulate DNA repair and cell death. Proc Natl Acad Sci U S A. 2014;111:16005–10. https://doi.org/10.1073/pnas.1417900111.
Lazcano P, Schmidtke MW, Onu CJ, Greenberg ML. Phosphatidic acid inhibits inositol synthesis by inducing nuclear translocation of kinase IP6K1 and repression of myo-inositol-3-P synthase. J Biol Chem. 2022;298:102363. https://doi.org/10.1016/j.jbc.2022.102363.
Burton A, Azevedo C, Andreassi C, Riccio A, Saiardi A. Inositol pyrophosphates regulate JMJD2C-dependent histone demethylation. Proc Natl Acad Sci U S A. 2013;110:18970–5. https://doi.org/10.1073/pnas.1309699110.
Ito M, et al. Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP(7) metabolism in the enteric nervous system. J Biol Chem. 2023;299:102928. https://doi.org/10.1016/j.jbc.2023.102928.
Hori Y, Engel C, Kobayashi T. Regulation of ribosomal RNA gene copy number, transcription and nucleolus organization in eukaryotes. Nat Rev Mol Cell Biol. 2023. https://doi.org/10.1038/s41580-022-00573-9.
Sahu S et al. Nucleolar Architecture Is Modulated by a Small Molecule, the Inositol Pyrophosphate 5-InsP(7). Biomolecules. 2023;13. https://doi.org/10.3390/biom13010153
Li X, et al. Control of XPR1-dependent cellular phosphate efflux by InsP(8) is an exemplar for functionally-exclusive inositol pyrophosphate signaling. Proc Natl Acad Sci U S A. 2020;117:3568–74. https://doi.org/10.1073/pnas.1908830117.
Bittner T, et al. Photolysis of Caged Inositol Pyrophosphate InsP(8) Directly Modulates Intracellular Ca(2+) Oscillations and Controls C2AB Domain Localization. J Am Chem Soc. 2020;142:10606–11. https://doi.org/10.1021/jacs.0c01697.
Gokhale NA, Zaremba A, Janoshazi AK, Weaver JD, Shears SB. PPIP5K1 modulates ligand competition between diphosphoinositol polyphosphates and PtdIns(3,4,5)P3 for polyphosphoinositide-binding domains. Biochem J. 2013;453:413–26. https://doi.org/10.1042/BJ20121528.
Wang Z, et al. Rapid stimulation of cellular Pi uptake by the inositol pyrophosphate InsP(8) induced by its photothermal release from lipid nanocarriers using a near infra-red light-emitting diode. Chem Sci. 2020;11:10265–78. https://doi.org/10.1039/d0sc02144j.
Shears SB. Intimate connections: Inositol pyrophosphates at the interface of metabolic regulation and cell signaling. J Cell Physiol. 2018;233:1897–912. https://doi.org/10.1002/jcp.26017.
Wundenberg T, Mayr GW. Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis. Biol Chem. 2012;393:979–98. https://doi.org/10.1515/hsz-2012-0133.
Gu C. et al. Metabolic supervision by PPIP5K, an inositol pyrophosphate kinase/phosphatase, controls proliferation of the HCT116 tumor cell line. Proc Natl Acad Sci U S A. 2021;118. https://doi.org/10.1073/pnas.2020187118
Zhang Z, et al. Inositol pyrophosphates mediate the effects of aging on bone marrow mesenchymal stem cells by inhibiting Akt signaling. Stem Cell Res Ther. 2014;5:33. https://doi.org/10.1186/scrt431.
Gu C, et al. The Significance of the Bifunctional Kinase/Phosphatase Activities of Diphosphoinositol Pentakisphosphate Kinases (PPIP5Ks) for Coupling Inositol Pyrophosphate Cell Signaling to Cellular Phosphate Homeostasis. J Biol Chem. 2017;292:4544–55. https://doi.org/10.1074/jbc.M116.765743.
Choi JH, Williams J, Cho J, Falck JR, Shears SB. Purification, sequencing, and molecular identification of a mammalian PP-InsP5 kinase that is activated when cells are exposed to hyperosmotic stress. J Biol Chem. 2007;282:30763–75. https://doi.org/10.1074/jbc.M704655200.
Nair VS et al. Inositol Pyrophosphate Synthesis by Diphosphoinositol Pentakisphosphate Kinase-1 is Regulated by Phosphatidylinositol(4,5)bisphosphate. Biosci Rep. 2018;38 https://doi.org/10.1042/BSR20171549
Ghoshal S, Tyagi R, Zhu Q, Chakraborty A. Inositol hexakisphosphate kinase-1 interacts with perilipin1 to modulate lipolysis. Int J Biochem Cell Biol. 2016;78:149–55. https://doi.org/10.1016/j.biocel.2016.06.018.
Dato S, et al. IP6K3 and IPMK variations in LOAD and longevity: Evidence for a multifaceted signaling network at the crossroad between neurodegeneration and survival. Mech Ageing Dev. 2021;195:111439. https://doi.org/10.1016/j.mad.2021.111439.
Crocco P, et al. Contribution of polymorphic variation of inositol hexakisphosphate kinase 3 (IP6K3) gene promoter to the susceptibility to late onset Alzheimer’s disease. Biochim Biophys Acta. 1862;1766–1773:2016. https://doi.org/10.1016/j.bbadis.2016.06.014.
Moritoh Y, et al. Inositol Hexakisphosphate Kinase 3 Regulates Metabolism and Lifespan in Mice. Sci Rep. 2016;6:32072. https://doi.org/10.1038/srep32072.
Ghoshal S et al. Whole Body Ip6k1 Deletion Protects Mice from Age-Induced Weight Gain, Insulin Resistance and Metabolic Dysfunction. Int J Mol Sci. 2022;23. https://doi.org/10.3390/ijms23042059
Barclay RD, et al. Ingestion of lean meat elevates muscle inositol hexakisphosphate kinase 1 protein content independent of a distinct post-prandial circulating proteome in young adults with obesity. Metabolism. 2020;102:153996. https://doi.org/10.1016/j.metabol.2019.153996.
Naufahu J, et al. High-Intensity Exercise Decreases IP6K1 Muscle Content and Improves Insulin Sensitivity (SI2*) in Glucose-Intolerant Individuals. J Clin Endocrinol Metab. 2018;103:1479–90. https://doi.org/10.1210/jc.2017-02019.
Luo HR, et al. GRAB: a physiologic guanine nucleotide exchange factor for Rab3A, which interacts with inositol hexakisphosphate kinase. Neuron. 2001;31:439–51. https://doi.org/10.1016/s0896-6273(01)00384-1.
Kim MG, et al. Inositol hexakisphosphate kinase-1 is a key mediator of prepulse inhibition and short-term fear memory. Mol Brain. 2020;13:72. https://doi.org/10.1186/s13041-020-00615-3.
Chakraborty A, Latapy C, Xu J, Snyder SH, Beaulieu JM. Inositol hexakisphosphate kinase-1 regulates behavioral responses via GSK3 signaling pathways. Mol Psychiatry. 2014;19:284–93. https://doi.org/10.1038/mp.2013.21.
Li K, et al. Panoramic transcriptome analysis and functional screening of long noncoding RNAs in mouse spermatogenesis. Genome Res. 2021;31:13–26. https://doi.org/10.1101/gr.264333.120.
Murat F, et al. The molecular evolution of spermatogenesis across mammals. Nature. 2023;613:308–16. https://doi.org/10.1038/s41586-022-05547-7.
Boregowda SV, et al. IP6K1 Reduces Mesenchymal Stem/Stromal Cell Fitness and Potentiates High Fat Diet-Induced Skeletal Involution. Stem Cells. 2017;35:1973–83. https://doi.org/10.1002/stem.2645.
Hou Q et al. Inhibition of IP6K1 suppresses neutrophil-mediated pulmonary damage in bacterial pneumonia. Sci Transl Med. 2018;10. https://doi.org/10.1126/scitranslmed.aal4045
Prasad A, et al. Inositol hexakisphosphate kinase 1 regulates neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-trisphosphate signaling. Nat Immunol. 2011;12:752–60. https://doi.org/10.1038/ni.2052.
Ghosh S, et al. Inositol hexakisphosphate kinase 1 maintains hemostasis in mice by regulating platelet polyphosphate levels. Blood. 2013;122:1478–86. https://doi.org/10.1182/blood-2013-01-481549.
Lee H, Park SJ, Hong S, Lim SW, Kim S. Deletion of IP6K1 in mice accelerates tumor growth by dysregulating the tumor-immune microenvironment. Anim Cells Syst (Seoul). 2022;26:19–27. https://doi.org/10.1080/19768354.2022.2029560.
Chakraborty A, et al. Casein kinase-2 mediates cell survival through phosphorylation and degradation of inositol hexakisphosphate kinase-2. Proc Natl Acad Sci U S A. 2011;108:2205–9. https://doi.org/10.1073/pnas.1019381108.
Nagata E, et al. Inositol hexakisphosphate kinase-2, a physiologic mediator of cell death. J Biol Chem. 2005;280:1634–40. https://doi.org/10.1074/jbc.M409416200.
Nagata E, et al. Inositol hexakisphosphate kinase 2 promotes cell death of anterior horn cells in the spinal cord of patients with amyotrophic lateral sclerosis. Mol Biol Rep. 2020;47:6479–85. https://doi.org/10.1007/s11033-020-05688-w.
Nagata E, et al. Inositol Hexakisphosphate Kinase 2 Promotes Cell Death in Cells with Cytoplasmic TDP-43 Aggregation. Mol Neurobiol. 2016;53:5377–83. https://doi.org/10.1007/s12035-015-9470-1.
Nagata E, et al. Inositol hexakisphosphate kinases induce cell death in Huntington disease. J Biol Chem. 2011;286:26680–6. https://doi.org/10.1074/jbc.M111.220749.
Sarmah B, Wente SR. Inositol hexakisphosphate kinase-2 acts as an effector of the vertebrate Hedgehog pathway. Proc Natl Acad Sci U S A. 2010;107:19921–6. https://doi.org/10.1073/pnas.1007256107.
Sandstrom J, et al. IP6K2 predicts favorable clinical outcome of primary breast cancer. Mol Clin Oncol. 2021;14:94. https://doi.org/10.3892/mco.2021.2256.
Zhang Y, et al. LINC00467 facilitates the proliferation, migration and invasion of glioma via promoting the expression of inositol hexakisphosphate kinase 2 by binding to miR-339-3p. Bioengineered. 2022;13:3370–82. https://doi.org/10.1080/21655979.2021.2018098.
Mahmassani ZS, et al. Absence of MyD88 from Skeletal Muscle Protects Female Mice from Inactivity-Induced Adiposity and Insulin Resistance. Obesity (Silver Spring). 2020;28:772–82. https://doi.org/10.1002/oby.22759.
Shears SB, Baughman BM, Gu C, Nair VS, Wang H. The significance of the 1-kinase/1-phosphatase activities of the PPIP5K family. Adv Biol Regul. 2017;63:98–106. https://doi.org/10.1016/j.jbior.2016.10.003.
Wang H, Falck JR, Hall TM, Shears SB. Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding. Nat Chem Biol. 2011;8:111–6. https://doi.org/10.1038/nchembio.733.
Weaver JD, Wang H, Shears SB. The kinetic properties of a human PPIP5K reveal that its kinase activities are protected against the consequences of a deteriorating cellular bioenergetic environment. Biosci Rep. 2013;33:e00022. https://doi.org/10.1042/BSR20120115.
Wang H, et al. Asp1 from Schizosaccharomyces pombe binds a [2Fe-2S](2+) cluster which inhibits inositol pyrophosphate 1-phosphatase activity. Biochemistry. 2015;54:6462–74. https://doi.org/10.1021/acs.biochem.5b00532.
Gokhale NA, Zaremba A, Shears SB. Receptor-dependent compartmentalization of PPIP5K1, a kinase with a cryptic polyphosphoinositide binding domain. Biochem J. 2011;434:415–26. https://doi.org/10.1042/BJ20101437.
Machkalyan G, Trieu P, Petrin D, Hebert TE, Miller GJ. PPIP5K1 interacts with the exocyst complex through a C-terminal intrinsically disordered domain and regulates cell motility. Cell Signal. 2016;28:401–11. https://doi.org/10.1016/j.cellsig.2016.02.002.
Machkalyan G, Hebert TE, Miller GJ. PPIP5K1 Suppresses Etoposide-triggered Apoptosis. J Mol Signal. 2016;11:4. https://doi.org/10.5334/1750-2187-11-4.
Cao CH, et al. PPIP5K2 promotes colorectal carcinoma pathogenesis through facilitating DNA homologous recombination repair. Oncogene. 2021;40:6680–91. https://doi.org/10.1038/s41388-021-02052-5.
Du C, et al. Renal Klotho and inorganic phosphate are extrinsic factors that antagonistically regulate hematopoietic stem cell maintenance. Cell Rep. 2022;38:110392. https://doi.org/10.1016/j.celrep.2022.110392.
Kaur S, Mirza AH, Overgaard AJ, Pociot F, Storling J. A Dual Systems Genetics Approach Identifies Common Genes, Networks, and Pathways for Type 1 and 2 Diabetes in Human Islets. Front Genet. 2021;12:630109. https://doi.org/10.3389/fgene.2021.630109.
Khaled ML, et al. PPIP5K2 and PCSK1 are Candidate Genetic Contributors to Familial Keratoconus. Sci Rep. 2019;9:19406. https://doi.org/10.1038/s41598-019-55866-5.
Yousaf, R. et al. Mutations in Diphosphoinositol-Pentakisphosphate Kinase PPIP5K2 are associated with hearing loss in human and mouse. PLoS Genet. 14; e1007297. https://doi.org/10.1371/journal.pgen.1007297 (2018).
Wormald MM, Ernst G, Wei H, Barrow JC. Synthesis and characterization of novel isoform-selective IP6K1 inhibitors. Bioorg Med Chem Lett. 2019;29:126628. https://doi.org/10.1016/j.bmcl.2019.126628.
Gu C, et al. Inhibition of Inositol Polyphosphate Kinases by Quercetin and Related Flavonoids: A Structure-Activity Analysis. J Med Chem. 2019;62:1443–54. https://doi.org/10.1021/acs.jmedchem.8b01593.
Liao G, et al. Identification of Small-Molecule Inhibitors of Human Inositol Hexakisphosphate Kinases by High-Throughput Screening. ACS Pharmacol Transl Sci. 2021;4:780–9. https://doi.org/10.1021/acsptsci.0c00218.
Boregowda SV et al. Pharmacological Inhibition of Inositol Hexakisphosphate Kinase 1 Protects Mice against Obesity-Induced Bone Loss. Biology (Basel). 2022;11. https://doi.org/10.3390/biology11091257
Zhang Z, et al. Selective inhibition of inositol hexakisphosphate kinases (IP6Ks) enhances mesenchymal stem cell engraftment and improves therapeutic efficacy for myocardial infarction. Basic Res Cardiol. 2014;109:417. https://doi.org/10.1007/s00395-014-0417-x.
Zhang J, et al. Leveraging Methylation Alterations to Discover Potential Causal Genes Associated With the Survival Risk of Cervical Cancer in TCGA Through a Two-Stage Inference Approach. Front Genet. 2021;12:667877. https://doi.org/10.3389/fgene.2021.667877.
Li Y, et al. Suppressing MDSC Infiltration in Tumor Microenvironment Serves as an Option for Treating Ovarian Cancer Metastasis. Int J Biol Sci. 2022;18:3697–713. https://doi.org/10.7150/ijbs.70013.
Zhou Y, et al. Development of Novel IP6K Inhibitors for the Treatment of Obesity and Obesity-Induced Metabolic Dysfunctions. J Med Chem. 2022;65:6869–87. https://doi.org/10.1021/acs.jmedchem.2c00220.
Puhl-Rubio AC, et al. Use of Protein Kinase-Focused Compound Libraries for the Discovery of New Inositol Phosphate Kinase Inhibitors. SLAS Discov. 2018;23:982–8. https://doi.org/10.1177/2472555218775323.
Ahn M, et al. Synthesis and biological evaluation of flavonoid-based IP6K2 inhibitors. J Enzyme Inhib Med Chem. 2023;38:2193866. https://doi.org/10.1080/14756366.2023.2193866.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (82220108021, 82070259) and the Natural Science Foundation of Shanghai (22ZR1440700). We thank the thoughtful input from Dr. Dorothea Fiedler. We also thank Alfred C. Chin for editing this manuscript.
Author information
Authors and Affiliations
Contributions
A.F.C. and C.F. conceptualized. C.F. wrote the manuscript. J.Q. drew the diagrams and completed the tables. J.Q., L.S., L.Z., Y.C., H.Z., and W.C. revised and edited the manuscript.
Corresponding authors
Ethics declarations
Competing interest
The authors declare that they have no conflicts of interest in this work.
Additional information
Associate Editor Yihua Bei oversaw the review of this article.
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Qi, J., Shi, L., Zhu, L. et al. Functions, Mechanisms, and therapeutic applications of the inositol pyrophosphates 5PP-InsP5 and InsP8 in mammalian cells. J. of Cardiovasc. Trans. Res. 17, 197–215 (2024). https://doi.org/10.1007/s12265-023-10427-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12265-023-10427-0