Inositol pyrophosphates and Akt/PKB: Is the pancreatic β-cell the exception to the rule?

Among many cellular functions, inositol pyrophosphates (PP-InsPs) are metabolic messengers involved in the regulation of glucose uptake, insulin sensitivity, and weight gain. However, their mechanisms of action are still poorly understood. So far, the influence of PP-InsPs on cellular metabolism has been studied by overexpression or knockout/inhibition of relevant metabolizing kinases (IP6Ks, PPIP5Ks). These approaches are, inter alia, limited by time-resolution and potential compensation mechanisms. Here, we describe the synthesis of cell-permeant caged PP-InsPs as tools to rapidly modulate intracellular levels of defined isomers of PP-InsPs in a genetically non-perturbed cellular environment. We show that caged prometabolites readily enter live cells where they are enzymatically converted into still inactive, metabolically stable, photocaged PP-InsPs. Upon light-triggered release of 5-PP-InsP5, the major cellular inositol pyrophosphate, oscillations of intracellular Ca2+ levels in MIN6 cells were transiently reduced to spontaneously recover again. In contrast, uncaging of 1-PP-InsP5, a minor cellular isomer, was without effect. These results provide evidence that PP-InsPs play an active role in regulating [Ca2+]i oscillations, a key element in triggering exocytosis and secretion in β-cells.

Crosstalk between Ras and inositol phosphate signaling revealed by lithium action on inositol monophosphatase in Schizophyllum commune.

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi-loaded giant vacuoles prepared from WT or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ yeast strains or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.

Use of Protein Kinase–Focused Compound Libraries for the Discovery of New Inositol Phosphate Kinase Inhibitors

Inorganic phosphate (Pi) is essential for signal transduction and cell metabolism, and is also an essential structural component of the extracellular matrix of the skeleton. Pi is sensed in bacteria and yeast at the plasma membrane, which activates intracellular signal transduction to control the expression of Pi transporters and other genes that control intracellular Pi levels. In multicellular organisms, Pi homeostasis must be maintained in the organism and at the cellular level, requiring an endocrine and metabolic Pi-sensing mechanism, about which little is currently known. This Review will discuss the metabolic effects of Pi, which are mediated by Pi transporters, inositol pyrophosphates and SYG1-Pho81-XPR1 (SPX)-domain proteins to maintain cellular phosphate homeostasis in the musculoskeletal system. In addition, we will discuss how Pi is sensed by the human body to regulate the production of fibroblast growth factor 23 (FGF23), parathyroid hormone and calcitriol to maintain serum levels of Pi in a narrow range. New findings on the crosstalk between iron and Pi homeostasis in the regulation of FGF23 expression will also be outlined. Mutations in components of these metabolic and endocrine phosphate sensors result in genetic disorders of phosphate homeostasis, cardiomyopathy and familial basal ganglial calcifications, highlighting the importance of this newly emerging area of research.

Pseudohyphal growth is a nutrient-regulated program in which budding yeast form multicellular filaments of elongated and connected cells. Filamentous growth is required for virulence in pathogenic fungi and provides an informative model of stress-responsive signaling. The genetics and regulatory networks modulating pseudohyphal growth have been studied extensively, but little is known regarding the changes in metabolites that enable pseudohyphal filament formation. Inositol signaling molecules are an important class of metabolite messengers encompassing highly phosphorylated and diffusible inositol polyphosphates (InsPs). We report here that the InsP biosynthesis pathway is required for wild-type pseudohyphal growth. Under nitrogen-limiting conditions that can induce filamentation, InsPs exhibit characteristic profiles, distinguishing the InsP7 pyrophosphate isoforms 1PP-InsP5 and 5PP-InsP5. Deletion and overexpression analyses of InsP kinases identify elevated levels of 5PP-InsP5 relative to 1PP-InsP5 in mutants exhibiting hyper-filamentous growth. Overexpression of KCS1, which promotes formation of inositol pyrophosphates, is sufficient to drive pseudohyphal filamentation on medium with normal nitrogen levels. We find that the kinases Snf1p (AMPK), Kss1p, and Fus3p (MAPKs), required for wild-type pseudohyphal growth, are also required for wild-type InsP levels. Deletion analyses of the corresponding kinase genes indicate elevated InsP3 levels and an absence of exaggerated 5PP-InsP5 peaks in trace profiles from snf1Δ/Δ and kss1Δ/Δ mutants exhibiting decreased pseudohyphal filamentation. Elevated 5PP-InsP5:1PP-InsP5 ratios are present in the hyperfilamentous fus3 deletion mutant. Collectively, the data identify the presence of elevated 5PP-InsP5 levels relative to other inositol pyrophosphates as an in vivo marker of hyper-filamentous growth, while providing initial evidence for the regulation of InsP signaling by pseudohyphal growth kinases.

The generation of two daughter cells with the same genetic information requires error-free chromosome segregation during mitosis. Chromosome transmission fidelity is dependent on spindle structure/function, which requires Asp1 in the fission yeast Schizosaccharomyces pombe Asp1 belongs to the diphosphoinositol pentakisphosphate kinase (PPIP5K)/Vip1 family which generates high-energy inositol pyrophosphate (IPP) molecules. Here, we show that Asp1 is a bifunctional enzyme in vivo: Asp1 kinase generates specific IPPs which are the substrates of the Asp1 pyrophosphatase. Intracellular levels of these IPPs directly correlate with microtubule stability: pyrophosphatase loss-of-function mutants raised Asp1-made IPP levels 2-fold, thus increasing microtubule stability, while overexpression of the pyrophosphatase decreased microtubule stability. Absence of Asp1-generated IPPs resulted in an aberrant, increased spindle association of the S. pombe kinesin-5 family member Cut7, which led to spindle collapse. Thus, chromosome transmission is controlled via intracellular IPP levels. Intriguingly, identification of the mitochondrion-associated Met10 protein as the first pyrophosphatase inhibitor revealed that IPPs also regulate mitochondrial distribution.

Inositol pyrophosphates (PP-InsPs) are "energetic" intracellular signals that are ubiquitous in animals, plants, and fungi; structural and biochemical characterization of PP-InsP metabolic enzymes provides insight into their evolution, reaction mechanisms, and regulation. Here, we describe the 2.35-Å-resolution structure of the catalytic core of Siw14, a 5-PP-InsP phosphatase from Saccharomyces cerevisiae and a member of the protein tyrosine-phosphatase (PTP) superfamily. Conclusions that we derive from structural data are supported by extensive site-directed mutagenesis and kinetic analyses, thereby attributing new functional significance to several key residues. We demonstrate the high activity and exquisite specificity of Siw14 for the 5-diphosphate group of PP-InsPs. The three structural elements that demarcate a 9.2-Å-deep substrate-binding pocket each have spatial equivalents in PTPs, but we identify how these are specialized for Siw14 to bind and hydrolyze the intensely negatively charged PP-InsPs. (a) The catalytic P-loop with the CX5R(S/T) PTP motif contains additional, positively charged residues. (b) A loop between the α5 and α6 helices, corresponding to the Q-loop in PTPs, contains a lysine and an arginine that extend into the catalytic pocket due to displacement of the α5 helix orientation through intramolecular crowding caused by three bulky, hydrophobic residues. (c) The general-acid loop in PTPs is replaced in Siw14 with a flexible loop that does not use an aspartate or glutamate as a general acid. We propose that an acidic residue is not required for phosphoanhydride hydrolysis.

Inositol hexakisphosphate kinases (IP6Ks) are responsible for the synthesis of the energy‐rich Inositol pyrophosphates (PP‐IPs) in mammals and regulate cellular functions including chemotaxis, telomere length, endocytic trafficking, exocytosis as well as apoptosis. Among such pyrophosphates, diphosphoinositol pentakisphosphate (IP7) and bis‐diphosphoinositol tetrakisphosphate (IP8) have been extensively characterized. IP7 is produced in mammals by a family of inositol hexakisphosphate kinases, namely IP6K1, IP6K2 and IP6K3, which have distinct biological functions. Of these, Inositol hexakisphosphate kinase‐2 (IP6K2), has been identified as a pro‐apoptotic factor with the ability to sensitize cells to apoptosis. To identify the protein interactome of IP6K2, we subjected wild‐type mouse brain tissue lysates to anti‐IP6K2 antibody‐mediated coimmuno‐precipitation followed by Liquid Chromatography Tandem Mass‐Spectrometry (LC‐MS/MS). This screen revealed robust binding of protein 4.1N with IP6K2. 4.1N is believed to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin‐actin‐based cytoskeleton, integral membrane channels and receptors as well as membrane‐associated guanylate kinases. To further assess the specificity of the observed interaction between IP6K2 and 4.1N, we separately immuno‐precipitated IP6K1 and IP6K3 from brain tissue lysates of mice and failed to detect any association with the 4.1N protein. Thus, the interaction of 4.1N is evidently specific to the IP6K2 form of inositol hexakisphosphate kinases. Moreover, nuclear translocation of 4.1N, which is required for its principal functions, was dependent on IP6K2. We also found that IP6K2 and 4.1N are expressed in the granule cells and that their interaction regulate Purkinje cell morphology and synapse in the cerebellum. This was determined through Golgi staining, double immunofluorescence staining and electron microscopy. Deletion of IP6K2 in mice led to substantial defects in synaptic influences of granule cells upon Purkinje cells. Rotarod, open field test and Gait analysis also revealed the defects of motor coordination in IP6K2 knockout mice indicating the functional consequences of these interaction. Overall, our study establishes the functional implication of the robust association between IP6K2‐4.1N in the brain.Support or Funding InformationThe present funding capabilities and strategies of our lab do not have provisions for covering the defined expenditure related to the current scientific meeting. Thus, to enrich my research skills and knowledge in neurophysiology as well as inositol phosphates, I would request possible financial support through the Postdoctoral Travel Award funds to help me attend this meeting.This would allow me to share my work and ideas with young scientists as well as researchers from US and other parts of the world thus helping me to get my research ideas as well as work, evaluated and advised on, by the best in the worldI would therefore entreat your good offices to consider my present submission for possible financial aid to help me attend the present meeting on Experimental Biology 2018This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound sensorineural hearing loss locus, DFNB100 on chromosome 5q13.2-q23.2. Exome enrichment followed by massive parallel sequencing revealed a c.2510G>A transition variant in PPIP5K2 that segregated with DFNB100-associated hearing loss in two large apparently unrelated Pakistani families. PPIP5Ks enzymes interconvert 5-IP7 and IP8, two key members of the inositol pyrophosphate (PP-IP) cell-signaling family. Their actions at the interface of cell signaling and bioenergetic homeostasis can impact many biological processes. The c.2510G>A transition variant is predicted to substitute a highly invariant arginine residue with histidine (p.Arg837His) in the phosphatase domain of PPIP5K2. Biochemical studies revealed that the p.Arg837His variant reduces the phosphatase activity of PPIP5K2 and elevates its kinase activity. We found that in mouse inner ear, 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. Our demonstration that PPIP5K2 has a role in hearing in humans indicates that PP-IP signaling is important to hair cell maintenance and function within inner ear.

The 5-diphosphoinositol pentakisphosphate (5-InsP7) and bisdiphosphoinositol tetrakisphosphate (InsP8) are “energetic” inositol pyrophosphate signaling molecules that regulate bioenergetic homeostasis. Inositol pyrophosphate levels are regulated by diphosphoinositol pentakisphosphate kinases (PPIP5Ks); these are large modular proteins that host a kinase domain (which phosphorylates 5-InsP7 to InsP8), a phosphatase domain that catalyzes the reverse reaction, and a polyphosphoinositide-binding domain (PBD). Here, we describe new interactions between these three domains in the context of full-length human PPIP5K1. We determine that InsP7 kinase activity is dominant when PPIP5K1 is expressed in intact cells; in contrast, we found that InsP8 phosphatase activity prevails when the enzyme is isolated from its cellular environment. We approach a reconciliation of this disparity by showing that cellular InsP8 phosphatase activity is inhibited by C8-PtdIns(4,5)P2 (IC50 ~40 μM). We recapitulate this phosphatase inhibition with natural PtdIns(4,5)P2 that was incorporated into large unilamellar vesicles. Additionally, PtdIns(4,5)P2 increases net InsP7 kinase activity 5-fold. We demonstrate that PtdIns(4,5)P2 is not itself a phosphatase substrate; its inhibition of InsP8 phosphatase activity results from an unusual, functional overlap between the phosphatase domain and the PBD. Finally, we discuss the significance of PtdIns(4,5)P2 as a novel regulator of PPIP5K1, in relation to compartmentalization of InsP7/InsP8 signaling in vivo.

Role of the inositol pyrophosphate multikinase Kcs1 in Cryptococcus inositol metabolism

Inositol polyphosphates contribute to cellular circadian rhythms: Implications for understanding lithium's molecular mechanism

Inositol pyrophosphates (IPPs) are present in organisms ranging from plants, slime moulds and fungi to mammals. Distinct classes of kinases generate different forms of energetic diphosphate-containing IPPs from inositol phosphates (IPs). Conversely, polyphosphate phosphohydrolase enzymes dephosphorylate IPPs to regenerate the respective IPs. IPPs and/or their metabolizing enzymes regulate various cell biological processes by modulating many proteins via diverse mechanisms. In the last decade, extensive research has been conducted in mammalian systems, particularly in knockout mouse models of relevant enzymes. Results obtained from these studies suggest impacts of the IPP pathway on organ development, especially of brain and testis. Conversely, deletion of specific enzymes in the pathway protects mice from various diseases such as diet-induced obesity (DIO), type-2 diabetes (T2D), fatty liver, bacterial infection, thromboembolism, cancer metastasis and aging. Furthermore, pharmacological inhibition of the same class of enzymes in mice validates the therapeutic importance of this pathway in cardio-metabolic diseases. This review critically analyses these findings and summarizes the significance of the IPP pathway in mammalian health and diseases. It also evaluates benefits and risks of targeting this pathway in disease therapies. Finally, future directions of mammalian IPP research are discussed.

Inositol pyrophosphates have been implicated in a wide range of cellular processes. Inositol hexakisphosphate kinase 1 catalyzes the pyrophosphorylation of inositol hexakisphosphate into inositol 5-diphospho-1,2,3,4,6-pentakisphosphate which is important in numerous areas of cell physiology such as DNA repair and glucose homeostasis. Furthermore, inositol 5-diphospho-1,2,3,4,6-pentakisphosphate is implicated in the pathology of diabetes and other human diseases. As such there is a demonstrated need in the field for a robust chemical probe to better understand the role of inositol hexakisphosphate kinase 1 and inositol pyrophosphate in physiology and disease. To aid in this effort we developed a homogenous coupled bioluminescence assay for measuring inositol hexakisphosphate kinase 1 activity in a 384-well format (Z’ = 0.62±0.05). Using this assay we were able to confirm the activity of a known inositol hexakisphosphate kinase 1 inhibitor N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl)purine. We also screened the Sigma library of pharmacologically active compounds at 10μM concentration and found 24 hits. Two of the most potent compounds were found to have an IC50 less than 5μM. The use of this high-throughput assay will accelerate the field towards the discovery of a potent inositol hexakisphosphate kinase 1 inhibitor.

Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic β-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity

Crystal structure of inositol 1,3,4,5,6-pentakisphosphate 2-kinase from Cryptococcus neoformans.

Inositol pyrophosphates are novel signaling molecules possessing high-energy pyrophosphate bonds and involved in a number of biological functions. Here, we report the correct identification and characterization of the kinases involved in the inositol pyrophosphate biosynthetic pathway in Trypanosoma brucei: inositol polyphosphate multikinase (TbIPMK), inositol pentakisphosphate 2-kinase (TbIP5K) and inositol hexakisphosphate kinase (TbIP6K). TbIP5K and TbIP6K were not identifiable by sequence alone and their activities were validated by enzymatic assays with the recombinant proteins or by their complementation of yeast mutants. We also analyzed T. brucei extracts for the presence of inositol phosphates using polyacrylamide gel electrophoresis and high-performance liquid chromatography. Interestingly, we could detect inositol phosphate (IP), inositol 4,5-bisphosphate (IP2 ), inositol 1,4,5-trisphosphate (IP3 ), and inositol hexakisphosphate (IP6 ) in T. brucei different stages. Bloodstream forms unable to produce inositol pyrophosphates, due to downregulation of TbIPMK expression by conditional knockout, have reduced levels of polyphosphate and altered acidocalcisomes. Our study links the inositol pyrophosphate pathway to the synthesis of polyphosphate in acidocalcisomes, and may lead to better understanding of these organisms and provide new targets for drug discovery.

Retraction: A High-Throughput Screening-Compatible Strategy for the Identification of Inositol Pyrophosphate Kinase Inhibitors