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Solute carrier family 20 member 2 (SLC20A2) and xenotropic and polytropic retrovirus receptor 1 (XPR1) are transporters with phosphate uptake and efflux functions, respectively. Both are associated with primary familial brain calcification (PFBC), a genetic disease characterized by cerebral calcium-phosphate deposition and associated with neuropsychiatric symptoms. The association of the two transporters with the same disease suggests that they jointly regulate phosphate fluxes and cellular homeostasis, but direct evidence is missing. Here, we found that cross-talk between SLC20A2 and XPR1 regulates phosphate homeostasis, and we identified XPR1 as a key inositol polyphosphate (IP)-dependent regulator of this process. We found that overexpression of WT SLC20A2 increased phosphate uptake, as expected, but also unexpectedly increased phosphate efflux, whereas PFBC-associated SLC20A2 variants did not. Conversely, SLC20A2 depletion decreased phosphate uptake only slightly, most likely compensated for by the related SLC20A1 transporter, but strongly decreased XPR1-mediated phosphate efflux. The SLC20A2-XPR1 axis maintained constant intracellular phosphate and ATP levels, which both increased in XPR1 KO cells. Elevated ATP levels are a hallmark of altered inositol pyrophosphate (PP-IP) synthesis, and basal ATP levels were restored after phosphate efflux rescue with WT XPR1 but not with XPR1 harboring a mutated PP-IP–binding pocket. Accordingly, inositol hexakisphosphate kinase 1-2 (IP6K1-2) gene inactivation or IP6K inhibitor treatment abolished XPR1-mediated phosphate efflux regulation and homeostasis. Our findings unveil an SLC20A2-XPR1 interplay that depends on IPs such as PP-IPs and controls cellular phosphate homeostasis via the efflux route, and alteration of this interplay likely contributes to PFBC.

Phosphate's central role in most biochemical reactions in a living organism requires carefully maintained homeostasis. Although phosphate homeostasis in mammals has long been studied at the organismal level, the intracellular mechanisms controlling phosphate metabolism are not well-understood. Inositol pyrophosphates have emerged as important regulatory elements controlling yeast phosphate homeostasis. To verify whether inositol pyrophosphates also regulate mammalian cellular phosphate homeostasis, here we knocked out inositol hexakisphosphate kinase (IP6K) 1 and IP6K2 to generate human HCT116 cells devoid of any inositol pyrophosphates. Using PAGE and HPLC analysis, we observed that the IP6K1/2-knockout cells have nondetectable levels of the IP6-derived IP7 and IP8 and also exhibit reduced synthesis of the IP5-derived PP-IP4. Nucleotide analysis showed that the knockout cells contain increased amounts of ATP, whereas the Malachite green assay found elevated levels of free intracellular phosphate. Furthermore, [32Pi] pulse labeling experiments uncovered alterations in phosphate flux, with both import and export of phosphate being decreased in the knockout cells. Functional analysis of the phosphate exporter xenotropic and polytropic retrovirus receptor 1 (XPR1) revealed that it is regulated by inositol pyrophosphates, which can bind to its SPX domain. We conclude that IP6K1 and -2 together control inositol pyrophosphate metabolism and thereby physiologically regulate phosphate export and other aspects of mammalian cellular phosphate homeostasis.

HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.

Inositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the multiple inositol-polyphosphate phosphatase 1 gene (MINPP1). Patients are found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We find that patient-derived and genome edited MINPP1−/− induced stem cells exhibit an inefficient neuronal differentiation combined with an increased cell death. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakisphosphate (IP6), detected in HEK293 cells, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6 level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis., Tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, the authors describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in the MINPP1 gene, characterised by intracellular imbalance of inositol polyphosphate metabolism.

The analysis ofmyo-inositol phosphates (InsPs) andmyo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is highly desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables for the first time the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we uncover that there must be unknown inositol synthesis pathways in mammals, highlighting the unique potential of this method to dissect inositol phosphate metabolism and signalling.

HIV immature lattice stability affects capsid maturation by altering IP6 packaging., HIV virion assembly begins with the construction of an immature lattice consisting of Gag hexamers. Upon virion release, protease-mediated Gag cleavage leads to a maturation event in which the immature lattice disassembles and the mature capsid assembles. The cellular metabolite inositiol hexakisphosphate (IP6) and maturation inhibitors (MIs) both bind and stabilize immature Gag hexamers, but whereas IP6 promotes virus maturation, MIs inhibit it. Here we show that HIV is evolutionarily constrained to maintain an immature lattice stability that ensures IP6 packaging without preventing maturation. Replication-deficient mutant viruses with reduced IP6 recruitment display increased infectivity upon treatment with the MI PF46396 (PF96) or the acquisition of second-site compensatory mutations. Both PF96 and second-site mutations stabilise the immature lattice and restore IP6 incorporation, suggesting that immature lattice stability and IP6 binding are interdependent. This IP6 dependence suggests that modifying MIs to compete with IP6 for Gag hexamer binding could substantially improve MI antiviral potency.

Inositol polyphosphates are vital metabolic and secondary messengers, involved in diverse cellular functions. Therefore, tight regulation of inositol polyphosphate metabolism is essential for proper cell physiology. Here, we describe an early-onset neurodegenerative syndrome caused by loss-of-function mutations in themultiple inositol polyphosphate phosphatase 1gene (MINPP1). Patients were found to have a distinct type of Pontocerebellar Hypoplasia with typical basal ganglia involvement on neuroimaging. We found that patient-derived and genome editedMINPP1-/-induced pluripotent stem cells (iPSCs) are not able to differentiate efficiently into neurons. MINPP1 deficiency results in an intracellular imbalance of the inositol polyphosphate metabolism. This metabolic defect is characterized by an accumulation of highly phosphorylated inositols, mostly inositol hexakiphosphate (IP6), detected in HEK293, fibroblasts, iPSCs and differentiating neurons lacking MINPP1. In mutant cells, higher IP6level is expected to be associated with an increased chelation of intracellular cations, such as iron or calcium, resulting in decreased levels of available ions. These data suggest the involvement of IP6-mediated chelation on Pontocerebellar Hypoplasia disease pathology and thereby highlight the critical role of MINPP1 in the regulation of human brain development and homeostasis.

Inositol polyphosphates are a diverse and multifaceted class of intracellular messengers omnipresent in eukaryotic cells. These water-soluble molecules regulate many aspects of fundamental cell physiology. Removing this metabolic pathway is deleterious: inositol phosphate kinase null mutations can result in lethality or substantial growth phenotypes. Inositol polyphosphate synthesis occurs through the actions of a set of kinases that phosphorylate phospholipase-generated IP3 to higher phosphorylated forms, such as the fully phosphorylated IP6 and the inositol pyrophosphates IP7 and IP8. Unicellular organisms have a reduced array of the kinases for synthesis of higher phosphorylated inositol polyphosphates, while human cells possess two metabolic routes to IP6. The enzymes responsible for inositol polyphosphate synthesis have been identified in all eukaryote genomes, although their amino acid sequence homology is often barely detectable by common search algorithms. Homology between human and microbial inositol phosphate kinases is restricted to a few catalytically important residues. Recent studies of the inositol phosphate metabolic pathways in pathogenic fungi (Cryptococcus neoformans) and protozoa (Trypanosome brucei) have revealed the importance of the highly phosphorylated inositol polyphosphates to the fitness and thus virulence of these pathogens. Given this, identification of inositol kinase inhibitors specifically targeting the kinases of pathogenic microorganisms is desirable and achievable.

Eukaryotic cells have ubiquitously utilized the myo-inositol backbone to generate a diverse array of signalling molecules. This is achieved by arranging phosphate groups around the six-carbon inositol ring. There is virtually no biological process that does not take advantage of the uniquely variable architecture of phosphorylated inositol. In inositol biology, phosphates are able to form three distinct covalent bonds: phosphoester, phosphodiester and phosphoanhydride bonds, with each providing different properties. The phosphoester bond links phosphate groups to the inositol ring, the variable arrangement of which forms the basis of the signalling capacity of the inositol phosphates. Phosphate groups can also form the structural bridge between myo-inositol and diacylglycerol through the phosphodiester bond. The resulting lipid-bound inositol phosphates, or phosphoinositides, further expand the signalling potential of this family of molecules. Finally, inositol is also notable for its ability to host more phosphates than it has carbons. These unusual organic molecules are commonly referred to as the inositol pyrophosphates (PP-IPs), due to the presence of high-energy phosphoanhydride bonds (pyro- or diphospho-). PP-IPs themselves constitute a varied family of molecules with one or more pyrophosphate moiety/ies located around the inositol. Considering the relationship between phosphate and inositol, it is no surprise that members of the inositol phosphate family also regulate cellular phosphate homoeostasis. Notably, the PP-IPs play a fundamental role in controlling the metabolism of the ancient polymeric form of phosphate, inorganic polyphosphate (polyP). Here we explore the intimate links between phosphate, inositol phosphates and polyP, speculating on the evolution of these relationships.

Summary HIV-1 hijacks host proteins to promote infection. Here we show that HIV is also dependent upon the host metabolite inositol hexakisphosphate (IP6) for viral production and primary cell replication. HIV-1 recruits IP6 into virions using two lysine rings in its immature hexamers. Mutation of either ring inhibits IP6 packaging and reduces viral production. Loss of IP6 also results in virions with highly unstable capsids, leading to a profound loss of reverse transcription and cell infection. Replacement of one ring with a hydrophobic isoleucine core restores viral production, but IP6 incorporation and infection remain impaired, consistent with an independent role for IP6 in stable capsid assembly. Genetic knockout of biosynthetic kinases IPMK and IPPK reveals that cellular IP6 availability limits the production of diverse lentiviruses, but in the absence of IP6, HIV-1 packages IP5 without loss of infectivity. Together, these data suggest that IP6 is a critical cofactor for HIV-1 replication., Graphical Abstract, Highlights • HIV needs cellular IP6 to efficiently produce and assemble virions • HIV packages IP6 using two rings of lysine residues in immature gag hexamers • Viruses that fail to package IP6 do not mature properly and have unstable capsids • Deficient IP6 packaging prevents HIV replicating in primary cells, Mallery et al. demonstrate that HIV is crucially dependent upon the host metabolite IP6 to produce infectious virions. Cells deficient in IP6 produce fewer virions, while virions that fail to package sufficient numbers of IP6 molecules are poorly infectious and fail to replicate in primary cells.

Inositol phosphates (IPs) comprise a family of ubiquitous eukaryotic signaling molecules. They have been linked to the regulation of a pleiotropy of important cellular activities, but low abundance and detection difficulties have hampered our understanding. Here we present a method to purify and enrich IPs or other phosphate-rich metabolites from mammalian cells or other sample types. Acid-extracted IPs from cells bind selectively via their phosphate groups to titanium dioxide beads. After washing, the IPs are easily eluted from the beads by increasing the pH. This technique, in combination with downstream analytical methods such as PAGE or SAX-HPLC, opens unprecedented investigative possibilities, allowing appropriate analysis of IPs from virtually any biological or non-biological source.

Inositol polyphosphates, in their water-soluble or lipid-bound forms, represent a large and multifaceted family of signalling molecules. Some inositol polyphosphates are well recognised as defining important signal transduction pathways, as in the case of the calcium release factor Ins(1,4,5)P3, generated by receptor activation-induced hydrolysis of the lipid PtdIns(4,5)P2 by phospholipase C. The birth of inositol polyphosphate research would not have occurred without the use of radioactive phosphate tracers that enabled the discovery of the “PI response”. Radioactive labels, mainly of phosphorus but also carbon and hydrogen (tritium), have been instrumental in the development of this research field and the establishment of the inositol polyphosphates as one of the most important networks of regulatory molecules present in eukaryotic cells. Advancements in microscopy and mass spectrometry and the development of colorimetric assays have facilitated inositol polyphosphate research, but have not eliminated the need for radioactive experimental approaches. In fact, such experiments have become easier with the cloning of the inositol polyphosphate kinases, enabling the systematic labelling of specific positions of the inositol ring with radioactive phosphate. This approach has been valuable for elucidating their metabolic pathways and identifying specific and novel functions for inositol polyphosphates. For example, the synthesis of radiolabelled inositol pyrophosphates has allowed the discovery of a new protein post-translational modification. Therefore, radioactive tracers have played and will continue to play an important role in dissecting the many complex aspects of inositol polyphosphate physiology. In this review we aim to highlight the historical importance of radioactivity in inositol polyphosphate research, as well as its modern usage.

Maintenance of electric potential and synaptic transmission are energetically demanding tasks that neuronal metabolism must continually satisfy. Inability to fulfil these energy requirements leads to the development of neurodegenerative disorders, including Alzheimer's disease. A prominent feature of Alzheimer's disease is in fact neuronal glucose hypometabolism. Thus understanding the fine control of energetic metabolism might help to understand neurodegenerative disorders. Recent research has indicated that a novel class of signalling molecules, the inositol pyrophosphates, act as energy sensors. They are able to alter the balance between mitochondrial oxidative phosphorylation and glycolytic flux, ultimately affecting the cellular level of ATP. The neuronal inositol pyrophosphate synthesis relies on the activity of the neuron enriched inositol hexakisphosphate kinase 3 (IP6K3) enzyme. To verify an involvement of inositol pyrophosphate signalling in neurodegenerative disorders, we performed tagging single nucleotide polymorphism (SNP) analysis of the IP6K3 gene in patients with familial and sporadic late onset Alzheimer's disease (LOAD). Two SNPs in the 5'-flanking promoter region of the IP6K3 gene were found to be associated with sporadic LOAD. Characterizing the functionality of the two polymorphisms by luciferase assay revealed that one of them (rs28607030) affects IP6K3 promoter activity, with the G allele showing an increased activity. As the same allele has a beneficial effect on disease risk, this may be related to upregulation of IP6K3 expression, with a consequent increase in inositol pyrophosphate synthesis. In conclusion, we provide the first evidence for a contribution of genetic variability in the IP6K3 gene to LOAD pathogenesis.

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6(phytate), its pyrophosphate derivatives InsP7and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7and InsP8were visualized inDictyosteliumextracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2bead purification also enabled us to quantify InsP6in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6phosphatase in human plasma, and secondly, InsP6is undetectable in either fluid. These observations seriously question reports that InsP6is present in human biofluids and the advisability of using InsP6as a dietary supplement.

The HCT116 cell line, which has a pseudo-diploid karotype, is a popular model in the fields of cancer cell biology, intestinal immunity, and inflammation. In the current study, we describe two batches of diverged HCT116 cells, which we designate as HCT116NIH and HCT116UCL. Using both gel electrophoresis and HPLC, we show that HCT116UCL cells contain 6-fold higher levels of InsP8 than HCT116NIH cells. This observation is significant because InsP8 is one of a group of molecules collectively known as ‘inositol pyrophosphates’ (PP-InsPs)—highly ‘energetic’ and conserved regulators of cellular and organismal metabolism. Variability in the cellular levels of InsP8 within divergent HCT116 cell lines could have impacted the phenotypic data obtained in previous studies. This difference in InsP8 levels is more remarkable for being specific; levels of other inositol phosphates, and notably InsP6 and 5-InsP7, are very similar in both HCT116NIH and HCT116UCL lines. We also developed a new HPLC procedure to record 1-InsP7 levels directly (for the first time in any mammalian cell line); 1-InsP7 comprised

All key reproductive events in the human ovary and uterus, including follicle activation, ovulation, implantation, decidualization, luteolysis and menstruation, are dependent upon profound tissue remodelling, characterised by cyclical waves of cell proliferation, differentiation, apoptosis, tissue breakdown and regeneration. FOXO transcription factors, an evolutionarily conserved subfamily of the forkhead transcription factors, have emerged as master regulators of cell fate decision capable of integrating avariety of stress, growth factor and cytokine signaling pathways with the transcription machinery. The ability of FOXOs to regulate seemingly opposing cellular responses, ranging from cell cycle arrest and oxidative stress responses to differentiation and apoptosis, renders these transcription factors indispensable for cyclic tissue remodelling in female reproduction. Conversely, perturbations in the expression or activity of FOXO transcription factors are increasingly linked to common reproductive disorders, such as pregnancy loss, endometriosis, endometrial cancer and primary ovarian insufficiency.

Inositol phosphates are a large and diverse family of signalling molecules. While genetic studies have discovered important functions for them, the biochemistry behind these roles is often not fully characterized. A key obstacle in inositol phosphate research in mammalian cells has been the lack of straightforward techniques for their purification and analysis. Here we describe the ability of titanium dioxide (TiO2) beads to bind inositol phosphates. This discovery allowed the development of a new purification protocol that, coupled with gel analysis, permitted easy identification and quantification of InsP6 (phytate), its pyrophosphate derivatives InsP7 and InsP8, and the nucleotides ATP and GTP from cell or tissue extracts. Using this approach, InsP6, InsP7 and InsP8 were visualized in Dictyostelium extracts and a variety of mammalian cell lines and tissues, and the effects of metabolic perturbation on these were explored. TiO2 bead purification also enabled us to quantify InsP6 in human plasma and urine, which led to two distinct but related observations. Firstly, there is an active InsP6 phosphatase in human plasma, and secondly, InsP6 is undetectable in either fluid. These observations seriously question reports that InsP6 is present in human biofluids and the advisability of using InsP6 as a dietary supplement.