Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented

Four Phosphates at One Blow: Access to Pentaphosphorylated Magic Spot Nucleotides and Their Analysis by Capillary Electrophoresis

The complex phosphorylation pattern of natural and modified pentaphosphorylated magic spot nucleotides is generated in a highly efficient way. A cyclic pyrophosphoryl phosphoramidite (cPyPA) reagent is used to introduce four phosphates on nucleosides regioselectively in a one-flask key transformation. The obtained magic spot nucleotides are used to develop a capillary electrophoresis UV detection method, enabling nucleotide assignment in complex bacterial extracts

The phytase RipBL1 enables the assignment of a specific inositol phosphate isomer as a structural component of human kidney stones

Inositol phosphates (InsPs) are ubiquitous in all eukaryotes. However, since there are 63 possible different phosphate ester isomers, the analysis of InsPs is challenging. In particular, InsP1, InsP2, and InsP3 already amass 41 different isomers, of which some occur as enantiomers. Profiling of these “lower” inositol phosphates in mammalian tissues requires powerful analytical methods and reference compounds. Here, we report an analysis of InsP2 and InsP3 with capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS). Using this method, the bacterial effector RipBL1 was analyzed and found to degrade InsP6 to Ins(1,2,3)P3, an understudied InsP3 isomer. This new reference molecule then aided us in the assignment of the isomeric identity of an InsP3 while profiling human samples: in urine and kidney stones, we describe for the first time the presence of defined and abundant InsP3 isomers, namely Ins(1,2,3)P3, Ins(1,2,6)P3 and/or Ins(2,3,4)P

Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

A recently discovered protein post-translational modification, lysine polyphosphorylation (K-PPn), consists of the covalent attachment of inorganic polyphosphate (polyP) to lysine residues. The non-enzymatic 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 non-physiologically 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

The inositol pyrophosphate metabolism of Dictyostelium discoideum does not regulate inorganic polyphosphate (polyP) synthesis

Initial studies on the inositol phosphates metabolism were enabled by the social amoeba Dictyostelium discoideum. The abundant amount of inositol hexakisphosphate (IP6 also known as Phytic acid) present in the amoeba allowed the discovery of the more polar inositol pyrophosphates, IP7 and IP8, 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-IP7 in contrast to the 5-IP7 isomer synthesized by the mammalian homologue. The amoeba Ppip5k synthesizes the same 1/3-IP7 as the mammalian enzyme. In D. discoideum, the ip6k strain possesses residual amounts of IP7. The residual IP7 is also present in the ip6k-ppip5K strain, while the ppip5k single mutant shows a decrease in both IP7 and IP8 levels. This phenotype is in contrast to the increase in IP7 observable in the yeast vip1Δ strain. The presence of IP8 in ppip5k and the presence of IP7 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

Arabidopsis ITPK1 and ITPK2 Have an Evolutionarily Conserved Phytic Acid Kinase Activity

Diphospho-myo-inositol polyphosphates, also termed inositol pyrophosphates, are molecular messengers containing at least one high-energy phosphoanhydride bond and regulate a wide range of cellular processes in eukaryotes. While inositol pyrophosphates InsP7 and InsP8 are present in different plant species, both the identity of enzymes responsible for InsP7 synthesis and the isomer identity of plant InsP7 remain unknown. This study demonstrates that Arabidopsis ITPK1 and ITPK2 catalyze the phosphorylation of phytic acid (InsP6) to the symmetric InsP7 isomer 5-InsP7 and that the InsP6 kinase activity of ITPK enzymes is evolutionarily conserved from humans to plants. We also show by 31P nuclear magnetic resonance that plant InsP7 is structurally identical to the in vitro InsP6 kinase products of ITPK1 and ITPK2. Our findings lay the biochemical and genetic basis for uncovering physiological processes regulated by 5-InsP7 in plants

Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of O-18-Phosphoramidites**

A family of 18O2-phosphoramidites facilitates synthetic access on gram-scale to various isotopically pure 18O-labelled phosphate products, like nucleotides, inositol phosphates, polyphosphates, and DNA. The utility of these 18O-natural products is underlined in the assignment of various metabolites from biological matrices using capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry. Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for 18O-labelled phosphates is presented, based on a family of modified 18O2-phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. 18O-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the 18O-labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry

Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry

The analysis of myo-inositol phosphates (InsPs) and myo-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 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 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 provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling

Polyphosphate degradation by Nudt3-Zn²⁺ mediates oxidative stress response

Polyphosphate (polyP) is a polymer of hundreds of phosphate residues present in all organisms. In mammals, polyP is involved in crucial physiological processes, including coagulation, inflammation, and stress response. However, after decades of research, the metabolic enzymes are still unknown. Here, we purify and identify Nudt3, a NUDIX family member, as the enzyme responsible for polyP phosphatase activity in mammalian cells. We show that Nudt3 shifts its substrate specificity depending on the cation; specifically, Nudt3 is active on polyP when Zn2+ is present. Nudt3 has in vivo polyP phosphatase activity in human cells, and importantly, we show that cells with altered polyP levels by modifying Nudt3 protein amount present reduced viability upon oxidative stress and increased DNA damage, suggesting that polyP and Nudt3 play a role in oxidative stress protection. Finally, we show that Nudt3 is involved in the early stages of embryo development in zebrafish

ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

In plants, phosphate (Pi) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. Here, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by Pi and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to Pi than lower inositol phosphates, a response conserved across kingdoms. With CE-ESI-MS we could separate different InsP7 isomers in Arabidopsis and rice, and identify 4/6-InsP7 and a PP-InsP4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP7, 5-InsP7 and InsP8 increase severalfold in shoots after Pi resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP6 compartmentalization. Notably, ITPK1 is critical for Pi-dependent 5-InsP7 and InsP8 synthesis in planta and its activity regulates Pi starvation responses in a PHR-dependent manner. Furthermore, we demonstrate that ITPK1-mediated conversion of InsP6 to 5-InsP7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP7 under low ATP. Collectively, our study provides deeper insights into Pi-dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates