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Inositol phosphates (IPs) comprise a network of phosphorylated molecules that play multiple signaling roles in eukaryotes. IPs synthesis is believed to originate with IP3 generated from PIP2 by phospholipase C (PLC). Here, we report that in mammalian cells PLC-generated IPs are rapidly recycled to inositol, and uncover the enzymology behind an alternative “soluble” route to synthesis of IPs. Inositol tetrakisphosphate 1-kinase 1 (ITPK1)—found in Asgard archaea, social amoeba, plants, and animals—phosphorylates I(3)P1 originating from glucose-6-phosphate, and I(1)P1 generated from sphingolipids, to enable synthesis of IP6. We also found using PAGE mass assay that metabolic blockage by phosphate starvation surprisingly increased IP6 levels in a ITPK1-dependentmanner, establishing a route to IP6 controlled by cellular metabolic status, that is not detectable by traditional [³H]-inositol labeling. The presence of ITPK1 in archaeal clades thought to define eukaryogenesis indicates that IPs had functional roles before the appearance of the eukaryote. [ABSTRACT FROM AUTHOR]

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 IP 3 to higher phosphorylated forms, such as the fully phosphorylated IP 6 and the inositol pyrophosphates IP 7 and IP 8 . Unicellular organisms have a reduced array of the kinases for synthesis of higher phosphorylated inositol polyphosphates, while human cells possess two metabolic routes to IP 6 . 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. [ABSTRACT FROM AUTHOR]

Phosphorus, as phosphate, plays a paramount role in biology. Since phosphate transfer reactions are an integral part of contemporary life, phosphate may have been incorporated into the initial molecules at the very beginning. To facilitate the studies into early phosphate utilization, we should look retrospectively to phosphate-rich molecules present in today's cells. Overlooked by origin of life studies until now, inositol and the inositol phosphates, of which some species possess more phosphate groups that carbon atoms, represent ideal molecules to consider in this context. The current sophisticated association of inositol with phosphate and the roles that some inositol phosphates play in regulating cellular phosphate homeostasis, intriguingly suggest that inositol might have played some role in the prebiotic process of phosphate exploitation. Inositol can be synthesized abiotically and unlike glucose or ribose, is chemically stable. This stability makes inositol the ideal candidate for the earliest organophosphate molecules, as primitive inositol phosphates. I also present arguments suggesting roles for some inositol phosphates in early chemical evolution events. Finally, the possible prebiotic synthesis of inositol pyrophosphates could have generated high-energy molecules to be utilized in primitive trans-phosphorylating processes. [ABSTRACT FROM AUTHOR]

Phosphate, as a cellular energy currency, essentially drives most biochemical reactions defining living organisms, and thus its homeostasis must be tightly regulated. Investigation into the role of inositol pyrophosphates (PP-IPs) has provided a novel perspective on the regulation of phosphate homeostasis. Recent data suggest that metabolic and signaling interplay between PP-IPs, ATP, and inorganic polyphosphate (polyP) influences and is influenced by cellular phosphate homeostasis. Different studies have demonstrated that the SPX protein domain is a key component of proteins involved in phosphate metabolism. How PP-IPs control some aspects of phosphate homeostasis has become clearer with the recently acquired crystal structures of SPX domains. We review here recent studies on eukaryote phosphate homeostasis and provide insights into future research. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Diphospho- myo-inositol phosphates (PP-InsP y) are an important class of cellular messengers. Thus far, no method for the transport of PP-InsP y into living cells is available. Owing to their high negative charge density, PP-InsP y will not cross the cell membrane. A strategy to circumvent this issue involves the generation of precursors in which the negative charges are masked with biolabile groups. A PP-InsP y prometabolite would require twelve to thirteen biolabile groups, which need to be cleaved by cellular enzymes to release the parent molecules. Such densely modified prometabolites of phosphate esters and anhydrides have never been reported to date. This study discloses the synthesis of such agents and an analysis of their metabolism in tissue homogenates by gel electrophoresis. The acetoxybenzyl-protected system is capable of releasing 5-PP-InsP5 in mammalian cell/tissue homogenates within a few minutes and can be used to release 5-PP-InsP5 inside cells. These molecules will serve as a platform for the development of fundamental tools required to study PP-InsP y physiology. [ABSTRACT FROM AUTHOR]

PolyP (inorganic polyphosphate) is a linear polymer of tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. This polymer is present in all living organisms from bacteria to mammals. Until recently, most of the studies on polyP have focused on its function in prokaryotes. In prokaryotes, polyP has been implicated in many unrelated processes ranging from basic metabolism to structural functions. However, polyP analysis and function in higher eukaryotes has been gainingmomentum recently. In the present review, we mainly aim to discuss the proposed intracellular functions of polyP in higher eukaryotes and its detection methods. [ABSTRACT FROM AUTHOR]

The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP6 or Phytic acid) and its derivative inositol pyrophosphates, IP7 and IP8. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP9 in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP5) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP8 was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP5K, doesn’t’ play a major role in the IP8 developmental increase. [ABSTRACT FROM AUTHOR]

Epigenetic modifications of chromatin represent a fundamental mechanism by which eukaryotic cells adapt their transcriptional response to developmental and environmental cues. Although an increasing number of molecules have been linked to epigenetic changes, the intracellular pathways that lead to their activation! repression have just begun to be characterized. Here, we demonstrate that inositol hexakisphosphate kinase 1 (IP6K1), the enzyme responsible for the synthesis of the high-energy inositol pyrophos- phates (1P7), is associated with chromatin and interacts with Jumonji domain containing 2C (JMJD2C), a recently identified histone lysine demethylase. Reducing IP6KI levels by RNAi or using mouse embryonic fibroblasts derived from ip6k1-/-knockout mice results in a decreased lP7 concentration that epigenetically translates to reduced levels of trimethyl-histone H3 lysine 9 (H3K9me3) and increased levels of acetyl-H3K9. Conversely, expression of IP6K1 induces JMJD2C dissociation from chromatin and increases H3K9me3 levels, which depend on IP6K1 catalytic activity. Importantly, these effects lead to changes in JMJD2C-target gene transcription. Our findings demonstrate that inositol pyrophosphate signaling influen- ces nuclear functions by regulating histone modifications. [ABSTRACT FROM AUTHOR]

The present review will explore the insights gained into inositol pyrophosphates in the 20 years since their discovery in 1993. These molecules are defined by the presence of the characteristic 'high energy' pyrophosphate moiety and can be found ubiquitously in eukaryotic cells. The enzymes that synthesize them are similarly well distributed and can be found encoded in any eukaryote genome. Rapid progress has been made in characterizing inositol pyrophosphate metabolism and they have been linked to a surprisingly diverse range of cellular functions. Two decades of work is now beginning to present a view of inositol pyrophosphates as fundamental, conserved and highly important agents in the regulation of cellular homoeostasis. In particular it is emerging that energy metabolism, and thus ATP production, is closely regulated by these molecules. Much of the early work on these molecules was performed in the yeast Saccharomyces cerevisiae and the social amoeba Dictyostelium discoideum, but the development of mouse knockouts for IP6K1 and IP6K2 [IP6K is IP6 (inositol hexakisphosphate) kinase] in the last 5 years has provided very welcome tools to better understand the physiological roles of inositol pyrophosphates. Another recent innovation has been the use of gel electrophoresis to detect and purify inositol pyrophosphates. Despite the advances that have been made, many aspects of inositol pyrophosphate biology remain far from clear. By evaluating the literature, the present review hopes to promote further research in this absorbing area of biology. [ABSTRACT FROM AUTHOR]

Recent advances have revealed an inositol phosphate (InsP) metabolic network more diverse than previously thought. InsPs and inositol pyrophosphates (PP-InsPs) are responsive to receptor activation cascades and metabolic processes. InsPs and PP-InsPs act on proteins through multiple mechanisms, ranging from transient binding to serving as a structural cofactor, while PP-InsPs additionally transfer their high-energy β-phosphate to proteins. The development of mouse knockout models has emphasised the importance of InsP/PP-InsP kinases in the control of fundamental physiological processes and pathophysiological states. Combinatorial substitution of phosphate groups on the inositol ring gives rise to a plethora of inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs). These small molecules constitute an elaborate metabolic and signalling network that influences nearly every cellular function. This review delves into the knowledge accumulated over the past decades regarding the biochemical principles and significance of InsP metabolism. We focus on the biological actions of InsPs in mammals, with an emphasis on recent findings regarding specific target proteins. We further discuss the roles of InsP metabolism in contributing to physiological homeostasis and pathological conditions. A deeper understanding of InsPs and their metabolic pathways holds the potential to address unresolved questions and propel advances towards therapeutic applications. [ABSTRACT FROM AUTHOR]

Phosphorylation is the most common module of cellular signalling pathways. The dynamic nature of phosphorylation, which is conferred by the balancing acts of kinases and phosphatases, allows this modification to finely control crucial cellular events such as growth, differentiation, and cell cycle progression. Although most research to date has focussed on protein phosphorylation, non-protein phosphorylation substrates also play vital roles in signal transduction. The most well-established substrate of non-protein phosphorylation is inositol, whose phosphorylation generates many important signalling molecules such as the second messenger IP 3 , a key factor in calcium signalling. A fundamental question to our understanding of inositol phosphorylation is how the levels of cellular inositol are controlled. While the availability of protein phosphorylation substrates is known to be readily controlled at the levels of transcription, translation, and/or protein degradation, the regulatory mechanisms that control the uptake, synthesis, and removal of inositol are underexplored. Potentially, such mechanisms serve as an important layer of regulation of cellular signal transduction pathways. There are two ways in which mammalian cells acquire inositol. The historic use of radioactive 3H- myo -inositol revealed that inositol is promptly imported from the extracellular environment by three specific symporters SMIT1/2, and HMIT, coupling sodium or proton entry, respectively. Inositol can also be synthesized de novo from glucose-6P, thanks to the enzymatic activity of ISYNA1. Intriguingly, emerging evidence suggests that in mammalian cells, de novo myo -inositol synthesis occurs irrespective of inositol availability in the environment, prompting the question of whether the two sources of inositol go through independent metabolic pathways, thus serving distinct functions. Furthermore, the metabolic stability of myo -inositol, coupled with the uptake and endogenous synthesis, determines that there must be exit pathways to remove this extraordinary sugar from the cells to maintain its homeostasis. This essay aims to review our current knowledge of myo -inositol homeostatic metabolism, since they are critical to the signalling events played by its phosphorylated forms. [ABSTRACT FROM AUTHOR]

A recently-discovered protein post-translational modification, lysine polyphosphorylation (K-PPn), consists of the covalent attachment of inorganic polyphosphate (polyP) to lysine residues. The nonenzymatic nature of K-PPn means that the degree of this modification depends on both polyP abundance and the amino acids surrounding the modified lysine. K-PPn was originally discovered in budding yeast (Saccharomyces cerevisiae), in which polyP anabolism and catabolism are well-characterized. However, yeast vacuoles accumulate large amounts of polyP, and upon cell lysis, the release of the vacuolar polyP could nonphysiologically cause K-PPn of nuclear and cytosolic targets. Moreover, yeast vacuoles possess two very active endopolyphosphatases, Ppn1 and Ppn2, that could have opposing effects on the extent of K-PPn. Here, we characterized the contribution of vacuolar polyP metabolism to K-PPn of two yeast proteins, Top1 (DNA topoisomerase 1) and Nsr1 (nuclear signal recognition 1). We discovered that whereas Top1-targeting K-PPn is only marginally affected by vacuolar polyP metabolism, Nsr1-targeting K-PPn is highly sensitive to the release of polyP and of endopolyphosphatases from the vacuole. Therefore, to better study K-PPn of cytosolic and nuclear targets, we constructed a yeast strain devoid of vacuolar polyP by targeting the exopolyphosphatase Ppx1 to the vacuole and concomitantly depleting the two endopolyphosphatases (ppn1Δppn2Δ, vt-Ppx1). This strain enabled us to study K-PPn of cytosolic and nuclear targets without the interfering effects of cell lysis on vacuole polyP and of endopolyphosphatases. Furthermore, we also define the fundamental nature of the acidic amino acid residues to the K-PPn target domain. [ABSTRACT FROM AUTHOR]

Initial studies on the inositol phosphates metabolism were enabled by the social amoeba Dictyostelium discoideum. The abundant amount of inositol hexakisphosphate (IP 6 also known as Phytic acid) present in the amoeba allowed the discovery of the more polar inositol pyrophosphates, IP 7 and IP 8 , possessing one or two high energy phosphoanhydride bonds, respectively. Considering the contemporary growing interest in inositol pyrophosphates, it is surprising that in recent years D. discoideum , has contributed little to our understanding of their metabolism and function. This work fulfils this lacuna, by analysing the ip6k, ppip5k and ip6k-ppip5K amoeba null strains using PAGE, 13C-NMR and CE-MS analysis. Our study reveals an inositol pyrophosphate metabolism more complex than previously thought. The amoeba Ip6k synthesizes the 4/6-IP 7 in contrast to the 5-IP 7 isomer synthesized by the mammalian homologue. The amoeba Ppip5k synthesizes the same 1/3-IP 7 as the mammalian enzyme. In D. discoideum , the ip6k strain possesses residual amounts of IP 7. The residual IP 7 is also present in the ip6k-ppip5K strain, while the ppip5k single mutant shows a decrease in both IP 7 and IP 8 levels. This phenotype is in contrast to the increase in IP 7 observable in the yeast vip1 Δ strain. The presence of IP 8 in ppip5k and the presence of IP 7 in ip6k-ppip5K indicate the existence of an additional inositol pyrophosphate synthesizing enzyme. Additionally, we investigated the existence of a metabolic relationship between inositol pyrophosphate synthesis and inorganic polyphosphate (polyP) metabolism as observed in yeast. These studies reveal that contrary to the yeast, Ip6k and Ppip5k do not control polyP cellular level in amoeba. [ABSTRACT FROM AUTHOR]

Inositol phosphate encompasses a large multifaceted family of signalling molecules that originate from the combinatorial attachment of phosphate groups to the inositol ring. To date, four distinct inositol kinases have been identified, namely, IPK, ITPK, IPPK (IP5–2K), and PPIP5K. Although, ITPKs have recently been identified in archaea, eukaryotes have taken advantage of these enzymes to create a sophisticated signalling network based on inositol phosphates. However, it remains largely elusive what fundamental biochemical principles control the signalling cascade. Here, we present an evolutionary approach to understand the development of the 'inositol phosphate code' in eukaryotes. Distribution analyses of these four inositol kinase groups throughout the eukaryotic landscape reveal the loss of either ITPK, or of PPIP5K proteins in several species. Surprisingly, the loss of IPPK, an enzyme thought to catalyse the rate limiting step of IP 6 (phytic acid) synthesis, was also recorded. Furthermore, this study highlights a noteworthy difference between animal (metazoan) and plant (archaeplastida) lineages. While metazoan appears to have a substantial amplification of IPK enzymes, archaeplastida genomes show a considerable increase in ITPK members. Differential evolution of IPK and ITPK between plant and animal lineage is likely reflective of converging functional adaptation of these two types of inositol kinases. Since, the IPK family comprises three sub-types IPMK, IP6K, and IP3–3K each with dedicated enzymatic specificity in metazoan, we propose that the amplified ITPK group in plant could be classified in sub-types with distinct enzymology. [ABSTRACT FROM AUTHOR]