ß-lapachone regulates mammalian inositol pyrophosphate levels in an NQO1- and oxygen-dependent manner.

Inositol pyrophosphates (PP-InsPs) are energetic signaling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP-InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing number of studies involve PP-InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP-InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP-InsPs during cellular ROS signaling is unknown. Here, we report how mammalian PP-InsP levels are changing during exposure to exogenous (H2O2) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that PP-InsP levels decrease upon exposure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly ß-lapachone (ß-lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that ß-lap treatment caused PP-InsP changes that are oxygen-dependent. Experiments in MDA-MB-231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase-1 (NQO1) demonstrated that ß-lap requires NQO1 bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP-InsP levels as shown in NQO1-deficient MDA-MB-231 cells treated with ß-lap. The data presented here unveil unique aspects of ß-lap pharmacology and its impact on PP-InsP levels. The identification of different quinone drugs as modulators of PP-InsP synthesis will allow the overall impact on cellular function of such drugs to be better appreciated.

ITPK1 mediates the lipid-independent synthesis of inositol phosphates controlled by metabolism.

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-dependent manner, 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.

Phosphate, inositol and polyphosphates.

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.

Prometabolites of 5-Diphospho-myo-inositol Pentakisphosphate.

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.

Inositol pyrophosphates: between signalling and metabolism.

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.

A stable immature lattice packages IP6 for HIV capsid maturation.

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.

MINPP1 prevents intracellular accumulation of the chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia.

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.

FOXO transcription factors: from cell fate decisions to regulation of human female reproduction.

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.

Cellular IP6 Levels Limit HIV Production while Viruses that Cannot Efficiently Package IP6 Are Attenuated for Infection and Replication.

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.

Microbial inositol polyphosphate metabolic pathway as drug development target.

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.

Importance of Radioactive Labelling to Elucidate Inositol Polyphosphate Signalling.

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.

Inositol Pyrophosphate Profiling of Two HCT116 Cell Lines Uncovers Variation in InsP8 Levels.

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 <2% of total InsP7 in HCT116NIH and HCT116UCL lines. The elevated levels of InsP8 in the HCT116UCL lines were not due to an increase in expression of the PP-InsP kinases (IP6Ks and PPIP5Ks), nor to a decrease in the capacity to dephosphorylate InsP8. We discuss how the divergent PP-InsP profiles of the newly-designated HCT116NIH and HCT116UCL lines should be considered an important research opportunity: future studies using these two lines may uncover new features that regulate InsP8 turnover, and may also yield new directions for studying InsP8 function.

A novel method for the purification of inositol phosphates from biological samples reveals that no phytate is present in human plasma or urine.

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.

The transcription factor encyclopedia.

Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.

FOXO transcription factors and their role in disorders of the female reproductive tract.

All fundamental reproductive events in the human ovary and uterus, including ovulation, implantation and menstruation, are dependent upon profound tissue remodelling, characterized by cyclical waves of cell proliferation, differentiation, recruitment of inflammatory cells, apoptosis, tissue breakdown and regeneration. Although the rise and fall in ovarian hormones, estradiol and progesterone, orchestrate these reproductive events, FOXO transcription factors, an evolutionary conserved subfamily of forkhead transcription factors, have emerged major downstream effector molecules, capable of integrating hormonal cues with a variety of stress, growth factor and cytokine signal transduction pathways. 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 the reproductive tract. Aberrant expression or perturbed activity of FOXO transcription factors are increasingly linked to prevalent reproductive disorders, such as endometriosis, endometrial cancer, primary ovarian insufficiency and pregnancy failure, which in turn highlights their potential as therapeutic targets.

FOXO and FOXM1 in cancer: the FOXO-FOXM1 axis shapes the outcome of cancer chemotherapy.

FOXO transcription factors, functioning downstream of the PI3K-PTEN-AKT (PKB) signalling cascade, are essential for cell proliferation, differentiation, DNA damage repair, and apoptosis. Recent research indicates that the related transcription factor FOXM1 is a direct target of repression by FOXO proteins. Inactivation of FOXO or overexpression of FOXM1 is associated with tumorigenesis and cancer progression. In addition, the cytostatic and cytotoxic effects of a diverse spectrum of anti-cancer drugs, such as paclitaxel, doxorubicin, lapatinib, gefitinib, imatinib, and cisplatin, are mediated through the activation of FOXO3a and/or the inhibition of its target FOXM1. Paradoxically, FOXO proteins also contribute to drug resistance by driving the expression of genes important for drug efflux as well as DNA repair and cell survival pathways in drug resistant cancers. Given its pivotal roles of in drug sensitivity as well as resistance, targeting the FOXO-FOXM1 axis could be a viable strategy for treatment of cancer and for overcoming drug resistance. Studying the expression profiles of the components of the FOXO-FOXM1 axis, and their cofactors, in cancer patients might also help to predict and monitor their clinical response to chemotherapy. A better understanding of the mechanism by which FOXO and FOXM1 are regulated, as well as their roles in drug sensitivity and resistance, may render these proteins crucial prognostic markers and therapeutic targets for breast cancer and other malignancies.