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Kinases that synthesize inositol phosphates (IPs) and pyrophosphates (PP-IPs) control numerous biological processes in eukaryotic cells. Herein, we extend this cellular signaling repertoire to viruses. We have biochemically and structurally characterized a minimalist inositol phosphate kinase (i.e., TvIPK) encoded by Terrestrivirus, a nucleocytoplasmic large ("giant") DNA virus (NCLDV). We show that TvIPK can synthesize inositol pyrophosphates from a range of scyllo- and myo-IPs, both in vitro and when expressed in yeast cells. We present multiple crystal structures of enzyme/substrate/nucleotide complexes with individual resolutions from 1.95 to 2.6 Å. We find a heart-shaped ligand binding pocket comprising an array of positively charged and flexible side chains, underlying the observed substrate diversity. A crucial arginine residue in a conserved "G-loop" orients the γ-phosphate of ATP to allow substrate pyrophosphorylation. We highlight additional conserved catalytic and architectural features in TvIPK, and support their importance through site-directed mutagenesis. We propose that NCLDV inositol phosphate kinases may have assisted evolution of inositol pyrophosphate signaling, and we discuss the potential biogeochemical significance of TvIPK in soil niches. Synopsis: Inositol phosphate kinases regulate many biological processes in eukaryotes, but recent discoveries of archaeal homologs suggest an ancient evolutionary origin. Here, characterization of a primordial ATP-dependent inositol pyrophosphate kinase encoded by the giant virus genome of Terrestrivirus provides insights into the evolution of inositol phosphate kinases. Terrestrivirus TvIPK specifically pyrophosphorylates a range of myo- and scyllo-based inositol polyphosphates both in vitro and when ectopically expressed in yeast. X-ray crystallographic analysis of TvIPK in complex with its substrates or products reveals conserved catalytic and architectural features. Positively charged and flexible side chains allow recognition of the diverse range of substrates. Pyrophosphorylation depends on ATP γ-phosphate orientation via an Arg residues in a conserved "G-loop." TvIPK, an ATP-dependent inositol pyrophosphate kinase from Terrestrivirus, a putative member of the Mimiviridae family of giant viruses, offers insights into the evolution of inositol phosphate signaling. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background and Purpose: Cannabidiol (CBD) has been shown to differentially regulate the mechanistic target of rapamycin complex 1 (mTORC1) in preclinical models of disease, where it reduces activity in models of epilepsies and cancer and increases it in models of multiple sclerosis (MS) and psychosis. Here, we investigate the effects of phytocannabinoids on mTORC1 and define a molecular mechanism. Experimental Approach A novel mechanism for phytocannabinoids was identified using the tractable model system, Dictyostelium discoideum. Using mouse embryonic fibroblasts, we further validate this new mechanism of action. We demonstrate clinical relevance using cells derived from healthy individuals and from people with MS (pwMS). Key Results: Both CBD and the more abundant cannabigerol (CBG) enhance mTORC1 activity in D. discoideum. We identify a mechanism for this effect involving inositol polyphosphate multikinase (IPMK), where elevated IPMK expression reverses the response to phytocannabinoids, decreasing mTORC1 activity upon treatment, providing new insight on phytocannabinoids' actions. We further validated this mechanism using mouse embryonic fibroblasts. Clinical relevance of this effect was shown in primary human peripheral blood mononuclear cells, where CBD and CBG treatment increased mTORC1 activity in cells derived from healthy individuals and decreased mTORC1 activity in cells derived from pwMS. Conclusion and Implications: Our findings suggest that both CBD and the abundant CBG differentially regulate mTORC1 signalling through a mechanism dependent on the activity of the upstream IPMK signalling pathway, with potential relevance to the treatment of mTOR‐related disorders, including MS. [ABSTRACT FROM AUTHOR]

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. [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]

The inositol polyphosphate family of small, cytosolic molecules has a prominent place in the field of cell signalling, and inositol pyrophosphates are the most recent addition to this large family. First identified in 1993, they have since been found in all eukaryotic organisms studied. The defining feature of inositol pyrophosphates is the presence of the characteristic ‘high energy’ pyrophosphate group, which immediately attracted interest in them as possible signalling molecules. In addition to their unique ‘high energy’ pyrophosphate bond, their concentration in the cell is tightly regulated with an extremely rapid turnover. This, together with the history of other inositol polyphosphates, makes it likely that they have an important role in intracellular signalling involving some basic cellular processes. This hypothesis is supported by the surprisingly wide range of cellular functions where inositol pyrophosphates seem to be involved. A seminal finding was that inositol pyrophosphates are able to directly phosphorylate pre-phosphorylated proteins, thereby identifying an entirely new post-translational protein modification, namely serine-pyrophosphorylation. Rapid progress has been made in characterising the metabolism of these molecules in the 15 years since their first identification. However, their detailed signalling role in specific cellular processes and in the context of relevant physiological cues has developed more slowly, particularly in mammalian system. We will discuss inositol pyrophosphates from the cell signalling perspective, analysing how their intracellular concentration is modulated, what their possible molecular mechanisms of action are, together with the physiological consequences of this novel form of signalling. J. Cell. Physiol. 220: 8–15, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Keywords: capillary electrophoresis; mass spectrometry; nucleotides; phosphorylation; stable isotope labelling EN capillary electrophoresis mass spectrometry nucleotides phosphorylation stable isotope labelling 1 1 1 01/20/22 20220126 NES 220126 B SP 18 sp O-labelled condensed phosphates b can be synthesized with a family of SP 18 sp O-phosphoramidite reagents, as reported by Henning J. Jessen and co-workers in their Research Article (e202112457). Capillary electrophoresis, mass spectrometry, nucleotides, phosphorylation, stable isotope labelling Inside Cover: Stable Isotope Phosphate Labelling of Diverse Metabolites is Enabled by a Family of 18O-Phosphoramidites (Angew. [Extracted from the article]

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R2713 --> Phosphate (Pi) is an indispensable component of living organisms. In the body, Pi homeostasis is tightly regulated by four main tissues: intestine, kidney, bone and parathyroid glands. There are multiple complex mechanisms underlying the regulation of serum Pi including hormones and transporters. Although plenty of studies have analysed Pi regulation, the mechanism with which Pi levels are sensed by mammalian cells remains elusive. Molecular mechanisms of Pi‐sensing have been elucidated in detail in yeast and bacteria. However, most of the Pi‐sensing proteins identified in yeast and bacteria are encoded by genes that are not conserved in mammals. As a major exception, inositol polyphosphates—which respond to changes in ambient Pi in yeast and, in turn, regulate the expression of Pi transporters—are also found in mammals. Here, we addressed the role of inositol polyphosphates and their biogenic enzymes, the IP6Ks (especially IP6K1 and 2), in cellular Pi sensing in mammals. Human and mouse kidneys express the IP6K1 and IP6K2 isoforms. Likewise, Opossum Kidney (OK) cells, a model for renal proximal tubules express IP6K1 and 2. Pharmacological inhibition of IP6K1/2 or CRISPR/Cas9 mediated knockout in OK cells downregulates Pi transporter mRNA, particularly NaPi‐IIa/c. Ip6k1‐/‐2‐/‐ KO cells also exhibited decreased Pi uptake (by 70%) and an abrogated adaptation to ambient Pi. To study the role of IP6K1 and 2 in vivo, we generated renal tubule‐specific inducible ip6k1‐/‐2‐/‐ KO mice: the mice had strongly reduced expression of renal Pi transporters, at the protein and mRNA levels, and lower serum Pi (by 20%) together with the transcriptional downregulation of many other transporters. Serum intact fibroblast growth factor 23—a principal phosphaturic hormone—was also substantially reduced (10‐fold) in these mice. Our data indicate that the deletion of IP6K1 and 2 decreases transcription of renal Pi transporters both in vivo and in vitro, and that Ip6k1‐/‐2‐/‐ cells are unable to adapt to ambient Pi. Therefore, IP6K1 and 2 play a critical role in renal Pi metabolism and, potentially, sensing. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]