Inositol Pyrophosphates as Versatile Metabolic Messengers.

Discovered in 1993, inositol pyrophosphates are evolutionarily conserved signaling metabolites whose versatile modes of action are being increasingly appreciated. These include their emerging roles as energy regulators, phosphodonors, steric/allosteric regulators, and G protein-coupled receptor messengers. Through studying enzymes that metabolize inositol pyrophosphates, progress has also been made in elucidating the various cellular and physiological functions of these pyrophosphate-containing, energetic molecules. The two main forms of inositol pyrophosphates, 5-IP7 and IP8, synthesized respectively by inositol-hexakisphosphate kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks), regulate phosphate homeostasis, ATP synthesis, and several other metabolic processes ranging from insulin secretion to cellular energy utilization. Here, we review the current understanding of the catalytic and regulatory mechanisms of IP6Ks and PPIP5Ks, as well as their counteracting phosphatases. We also highlight the genetic and cellular evidence implicating inositol pyrophosphates as essential mediators of mammalian metabolic homeostasis.

Effect of dietary calcium source, exogenous phytase, and formic acid on inositol phosphate degradation, mineral and amino acid digestibility, and microbiota in growing pigs.

The choice of the calcium (Ca) source in pig diets and the addition of formic acid may affect the gastrointestinal inositol phosphate (InsP) degradation and thereby, phosphorus (P) digestibility in pigs. This study assessed the effects of different Ca sources (Ca carbonate, Ca formate), exogenous phytase, and chemical acidification on InsP degradation, nutrient digestion and retention, blood metabolites, and microbiota composition in growing pigs. In a randomized design, 8 ileal-cannulated barrows (24 kg initial BW) were fed 5 diets containing Ca formate or Ca carbonate as the only mineral Ca addition, with or without 1,500 FTU/kg of an exogenous hybrid 6-phytase. A fifth diet was composed of Ca carbonate with phytase but with 8 g formic acid/kg diet. No mineral P was added to the diets. Prececal InsP6 disappearance and P digestibility were lower (P ≤ 0.032) in pigs fed diets containing Ca formate. In the presence of exogenous phytase, InsP5 and InsP4 concentrations in the ileal digesta were lower (P ≤ 0.019) with Ca carbonate than Ca formate. The addition of formic acid to Ca carbonate with phytase diet resulted in greater (P = 0.027) prececal InsP6 disappearance (87% vs. 80%), lower (P = 0.001) InsP5 concentration, and greater (P ≤ 0.031) InsP2 and myo-inositol concentrations in the ileal digesta. Prececal P digestibility was greater (P = 0.004) with the addition of formic acid compared to Ca carbonate with phytase alone. Prececal amino acid (AA) digestibility of some AA was greater with Ca formate compared to Ca carbonate but only in diets with phytase (P ≤ 0.048). The addition of formic acid to the diet with Ca carbonate and phytase increased (P ≤ 0.006) the prececal AA digestibility of most indispensable AA. Exogenous phytase affected more microbial genera in the feces when Ca formate was used compared to Ca carbonate. In the ileal digesta, the Ca carbonate diet supplemented with formic acid and phytase led to a similar microbial community as the Ca formate diets. In conclusion, Ca formate reduced prececal InsP6 degradation and P digestibility, but might be of advantage in regard to prececal AA digestibility in pigs compared to Ca carbonate when exogenous phytase is added. The addition of formic acid to Ca carbonate with phytase, however, resulted in greater InsP6 disappearance, P and AA digestibility values, and changed ileal microbiota composition compared to Ca carbonate with phytase alone.

The study aimed to investigate the effects of dietary calcium sources, exogenous phytase, and formic acid on inositol phosphate (InsP) degradation and nutrient digestibility in ileal-cannulated growing pigs. It also evaluated the concentrations of phosphorus, calcium, and myo-inositol in the blood, the composition of the microbiota in the ileal digesta and feces, and the concentrations of volatile fatty acids in the feces. Replacing calcium carbonate with calcium formate in the feed reduced prececal InsP6 disappearance and phosphorus digestibility. However, adding formic acid to a diet containing calcium carbonate and phytase enhanced prececal InsP6 disappearance and phosphorus digestibility, and increased InsP2 and myo-inositol concentrations in the ileal digesta. The dietary treatments resulted in more pronounced alterations of the microbiota in the feces than the ileal digesta. In ileal digesta, the shifts in relative abundance were primarily evident among low-abundant genera, while in feces, changes were observed in a larger number among genera with higher levels of abundance. The findings of this study suggest that calcium formate is not a suitable alternative to calcium carbonate for phosphorus digestibility in growing pigs. The release of phosphorus from InsP by exogenous phytase can be increased by adding formic acid.

Disorder-to-order active site capping regulates the rate-limiting step of the inositol pathway.

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD+-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus Thermochaetoides thermophila. By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications.

RP-HPLC-RID analysis of InsP6 to InsP3 in Indian cereals, legumes, and their products: A comparative evaluation of PRP-1 Vs C18 column.

Phytic acid or inositol hexakisphosphate (InsP6) and its dephosphorylated forms (InsP5, InsP4 & InsP3) are integral to cellular functions and confer several health benefits. The present study was aimed to develop a cost effective and high sample throughput RP-HPLC-RID method for routine quantification of lower inositol phosphates in both raw and processed cereals and pulses. For this asuitable mobile phase composition was formulated and two columns (MacroporusHamilton PRP-1 Vs Waters Symmetry C18) were compared in terms ofsystem specificity,linearity, accuracy and precision. Separation ofInsP3, InsP4, InsP5 and InsP6 were recorded at 2.39, 2.93, 3.83 and 5.37 min usingPRP-1column while the RT were 4.67, 5.64, 6.99 and 9.14 min with C18column.Linearity of standards (R2 > 0.99), with an accuracy and precision ranging from 1 to 5 % was achieved. The LOD and LOQ of all InsPs were 5 and 15 µg/ml, respectively. In quality control sample InsP6 was found in highest concentration (446 ± 14.71 mg/100 g) followed by InsP5 (162 ± 8.00 mg/100 g) and InsP4 with the least concentration of 11.63 ± 1.06 mg/100 g whereas InsP3 was below detectable limit (BDL). The optimised method was used for profiling of InsPs in the raw and processed cereals and pulses consumed as staple foods in India. Processed foods contained lesser InsP6 and more of lower InsP compared to raw foods. The optimised method using unique mobile phase composition was found to yield accurate results and can used for large scale analysis of cereals and pulses and estimation of mineral nutrition potential and allied health benefits.

Activities and genetic interactions of fission yeast Aps1, a Nudix-type inositol pyrophosphatase and inorganic polyphosphatase.

Inositol pyrophosphate 1,5-IP8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1, pho84, and tgp1, via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP7 to 1,5-IP8 and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8. Aps1 displays a ~twofold preference for hydrolysis of 1-IP7 versus 5-IP7 and aps1∆ cells have twofold higher levels of 1-IP7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1∆ siw14∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP8 toxicosis, whereby excessive 1,5-IP8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1∆ siw14∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1∆ siw14∆ on YES by deleting tgp1. However, the severe growth defect of an aps1∆ asp1-H397A strain could not be alleviated by deleting tgp1, suggesting that 1,5-IP8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity. IMPORTANCE: Repression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP8, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP8 is formed by phosphorylation of 5-IP7 and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo. Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8 in vitro, with a ~twofold preference for 1-IP7 over 5-IP7. aps1∆ cells have twofold higher levels of 1-IP7 than wild-type cells. An aps1∆ siw14∆ double mutation is lethal because excessive 1,5-IP8 triggers derepression of tgp1, leading to toxic uptake of glycerophosphocholine.

Essential functions of inositol hexakisphosphate (IP6) in murine leukemia virus replication.

We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV.IMPORTANCEInositol hexakisphosphate (IP6) is crucial for the assembly and replication of HIV-1. IP6 is packaged in HIV-1 particles and stabilizes the viral core enabling it to synthesize viral DNA early in viral infection. While its importance for HIV-1 is well established, its significance for other retroviruses is unknown. Here we report the role of IP6 in the gammaretrovirus, murine leukemia virus (MLV). We found that like HIV-1, MLV packages IP6, and as in HIV-1, IP6 stabilizes the MLV core thus promoting reverse transcription. Interestingly, we discovered a key difference in the role of IP6 in MLV versus HIV-1: while HIV-1 is not dependent upon IP6 levels in target cells, MLV replication is significantly reduced in IP6-deficient cell lines. We suggest that this difference in IP6 requirements reflects key differences between HIV-1 and MLV replication.

Homeostatic coordination of cellular phosphate uptake and efflux requires an organelle-based receptor for the inositol pyrophosphate IP8.

Phosphate (Pi) serves countless metabolic pathways and is involved in macromolecule synthesis, energy storage, cellular signaling, and bone maintenance. Herein, we describe the coordination of Pi uptake and efflux pathways to maintain mammalian cell Pi homeostasis. We discover that XPR1, the presumed Pi efflux transporter, separately supervises rates of Pi uptake. This direct, regulatory interplay arises from XPR1 being a binding partner for the Pi uptake transporter PiT1, involving a predicted transmembrane helix/extramembrane loop in XPR1, and its hitherto unknown localization in a subset of intracellular LAMP1-positive puncta (named "XLPVs"). A pharmacological mimic of Pi homeostatic challenge is sensed by the inositol pyrophosphate IP8, which functionalizes XPR1 to respond in a temporally hierarchal manner, initially adjusting the rate of Pi efflux, followed subsequently by independent modulation of PiT1 turnover to reset the rate of Pi uptake. These observations generate a unifying model of mammalian cellular Pi homeostasis, expanding opportunities for therapeutic intervention.

A clade of receptor-like cytoplasmic kinases and 14-3-3 proteins coordinate inositol hexaphosphate accumulation.

Inositol hexaphosphate (InsP6) is the major storage form of phosphorus in seeds. Reducing seed InsP6 content is a breeding objective in agriculture, as InsP6 negatively impacts animal nutrition and the environment. Nevertheless, how InsP6 accumulation is regulated remains largely unknown. Here, we identify a clade of receptor-like cytoplasmic kinases (RLCKs), named Inositol Polyphosphate-related Cytoplasmic Kinases 1-6 (IPCK1-IPCK6), deeply involved in InsP6 accumulation. The InsP6 concentration is dramatically reduced in seeds of ipck quadruple (T-4m/C-4m) and quintuple (C-5m) mutants, accompanied with the obviously increase of phosphate (Pi) concentration. The plasma membrane-localized IPCKs recruit IPK1 involved in InsP6 synthesis, and facilitate its binding and activity via phosphorylation of GRF 14-3-3 proteins. IPCKs also recruit IPK2s and PI-PLCs required for InsP4/InsP5 and InsP3 biosynthesis respectively, to form a potential IPCK-GRF-PLC-IPK2-IPK1 complex. Our findings therefore uncover a regulatory mechanism of InsP6 accumulation governed by IPCKs, shedding light on the mechanisms of InsP biosynthesis in eukaryotes.

Suppression of inositol pyrophosphate toxicosis and hyper-repression of the fission yeast PHO regulon by loss-of-function mutations in chromatin remodelers Snf22 and Sol1.

Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1, pho84, and tgp1) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP8 toxicity is alleviated by deletion of snf22+, the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22-(D996A-E997A) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22+ hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO-repressive lncRNAs. Transcription profiling of snf22∆ and snf22-(D996A-E997A) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP8 as an agonist of precocious lncRNA 3'-processing/termination. IP8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP8 levels on termination. In this study, a forward genetic screen revealed that IP8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO-activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters.

The ring rules the chain - inositol pyrophosphates and the regulation of inorganic polyphosphate.

The maintenance of phosphate homeostasis serves as a foundation for energy metabolism and signal transduction processes in all living organisms. Inositol pyrophosphates (PP-InsPs), composed of an inositol ring decorated with monophosphate and diphosphate moieties, and inorganic polyphosphate (polyP), chains of orthophosphate residues linked by phosphoanhydride bonds, are energy-rich biomolecules that play critical roles in phosphate homeostasis. There is a complex interplay between these two phosphate-rich molecules, and they share an interdependent relationship with cellular adenosine triphosphate (ATP) and inorganic phosphate (Pi). In eukaryotes, the enzymes involved in PP-InsP synthesis show some degree of conservation across species, whereas distinct enzymology exists for polyP synthesis among different organisms. In fact, the mechanism of polyP synthesis in metazoans, including mammals, is still unclear. Early studies on PP-InsP and polyP synthesis were conducted in the slime mould Dictyostelium discoideum, but it is in the budding yeast Saccharomyces cerevisiae that a clear understanding of the interplay between polyP, PP-InsPs, and Pi homeostasis has now been established. Recent research has shed more light on the influence of PP-InsPs on polyP in mammals, and the regulation of both these molecules by cellular ATP and Pi levels. In this review we will discuss the cross-talk between PP-InsPs, polyP, ATP, and Pi in the context of budding yeast, slime mould, and mammals. We will also highlight the similarities and differences in the relationship between these phosphate-rich biomolecules among this group of organisms.

In Vitro and In Silico Explorations of the Protein Conformational Changes of Corynebacterium glutamicum MshA, a Model Retaining GT-B Glycosyltransferase.

MshA is a GT-B glycosyltransferase catalyzing the first step in the biosynthesis of mycothiol. While many GT-B enzymes undergo an open-to-closed transition, MshA is unique because its 97° rotation is beyond the usual range of 10-25°. Molecular dynamics (MD) simulations were carried out for MshA in both ligand bound and unbound states to investigate the effect of ligand binding on localized protein dynamics and its conformational free energy landscape. Simulations showed that both the unliganded "opened" and liganded "closed" forms of the enzyme sample a wide degree of dihedral angles and interdomain distances with relatively low overlapping populations. Calculation of the free energy surface using replica exchange MD for the apo "opened" and an artificial generated apo "closed" structure revealed overlaps in the geometries sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed transition in the absence of ligands. MD simulations of fully liganded MshA revealed a smaller sampling of the dihedral angles. The localized protein fluctuation changes suggest that UDP-GlcNAc binding activates the motions of loops in the 1-l-myo-inositol-1-phosphate (I1P)-binding site despite little change in the interactions with UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy, and mutagenesis studies were used to confirm the ligand-induced structural changes in MshA. The results support a proposed mechanism where UDP-GlcNAc binds with rigid interactions to the C-terminal domain of MshA and activates flexible loops in the N-terminal domain for binding and positioning of I1P. This model can be used for future structure-based drug development of inhibitors of the mycothiol biosynthetic pathway.

Activated Inositol Phosphate, Substrate for Synthesis of Prostaglandylinositol Cyclic Phosphate (Cyclic PIP)-The Key for the Effectiveness of Inositol-Feeding.

The natural cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), is biosynthesized from prostaglandin E (PGE) and activated inositol phosphate (n-Ins-P), which is synthesized by a particulate rat-liver-enzyme from GTP and a precursor named inositol phosphate (pr-Ins-P), whose 5-ring phosphodiester structure is essential for n-Ins-P synthesis. Aortic myocytes, preincubated with [3H] myo-inositol, synthesize after angiotensin II stimulation (30 s) [3H] pr-Ins-P (65% yield), which is converted to [3H] n-Ins-P and [3H] cyclic PIP. Acid-treated (1 min) [3H] pr-Ins-P co-elutes with inositol (1,4)-bisphosphate in high performance ion chromatography, indicating that pr-Ins-P is inositol (1:2-cyclic,4)-bisphosphate. Incubation of [3H]-GTP with unlabeled pr-Ins-P gave [3H]-guanosine-labeled n-Ins-P. Cyclic PIP synthase binds the inositol (1:2-cyclic)-phosphate part of n-Ins-P to PGE and releases the [3H]-labeled guanosine as [3H]-GDP. Thus, n-Ins-P is most likely guanosine diphospho-4-inositol (1:2-cyclic)-phosphate. Inositol feeding helps patients with metabolic conditions related to insulin resistance, but explanations for this finding are missing. Cyclic PIP appears to be the key for explaining the curative effect of inositol supplementation: (1) inositol is a molecular constituent of cyclic PIP; (2) cyclic PIP triggers many of insulin's actions intracellularly; and (3) the synthesis of cyclic PIP is decreased in diabetes as shown in rodents.

Insights into the roles of inositol hexakisphosphate kinase 1 (IP6K1) in mammalian cellular processes.

Inositol phosphates and their metabolites play a significant role in several biochemical pathways, gene expression regulation, and phosphate homeostasis. Among the different inositol phosphates, inositol hexakisphosphate (IP6) is a substrate of inositol hexakisphosphate kinases (IP6Ks), which phosphorylate one or more of the IP6 phosphate groups. Pyrophosphorylation of IP6 leads to the formation of inositol pyrophosphates, high-energy signaling molecules that mediate physiological processes through their ability to modify target protein activities, either by directly binding to their target protein or by pyrophosphorylating protein serine residues. 5-diphosphoinositol pentakisphosphate, the most abundant inositol pyrophosphate in mammals, has been extensively studied and found to be significantly involved in a wide range of physiological processes. Three IP6K (IP6K1, IP6K2, and IP6K3) isoforms regulate IP7 synthesis in mammals. Here, we summarize our current understanding of IP6K1's roles in cytoskeletal remodeling, trafficking, cellular migration, metabolism, gene expression, DNA repair, and immunity. We also briefly discuss current gaps in knowledge, highlighting the need for further investigation.

Substrate promiscuity of inositol 1,4,5-trisphosphate kinase driven by structurally-modified ligands and active site plasticity.

D-myo-inositol 1,4,5-trisphosphate (InsP3) is a fundamental second messenger in cellular Ca2+ mobilization. InsP3 3-kinase, a highly specific enzyme binding InsP3 in just one mode, phosphorylates InsP3 specifically at its secondary 3-hydroxyl group to generate a tetrakisphosphate. Using a chemical biology approach with both synthetised and established ligands, combining synthesis, crystallography, computational docking, HPLC and fluorescence polarization binding assays using fluorescently-tagged InsP3, we have surveyed the limits of InsP3 3-kinase ligand specificity and uncovered surprisingly unforeseen biosynthetic capacity. Structurally-modified ligands exploit active site plasticity generating a helix-tilt. These facilitated uncovering of unexpected substrates phosphorylated at a surrogate extended primary hydroxyl at the inositol pseudo 3-position, applicable even to carbohydrate-based substrates. Crystallization experiments designed to allow reactions to proceed in situ facilitated unequivocal characterization of the atypical tetrakisphosphate products. In summary, we define features of InsP3 3-kinase plasticity and substrate tolerance that may be more widely exploitable.

Kcs1 and Vip1: The Key Enzymes behind Inositol Pyrophosphate Signaling in Saccharomyces cerevisiae.

The inositol pyrophosphate pathway, a complex cell signaling network, plays a pivotal role in orchestrating vital cellular processes in the budding yeast, where it regulates cell cycle progression, growth, endocytosis, exocytosis, apoptosis, telomere elongation, ribosome biogenesis, and stress responses. This pathway has gained significant attention in pharmacology and medicine due to its role in generating inositol pyrophosphates, which serve as crucial signaling molecules not only in yeast, but also in higher eukaryotes. As targets for therapeutic development, genetic modifications within this pathway hold promise for disease treatment strategies, offering practical applications in biotechnology. The model organism Saccharomyces cerevisiae, renowned for its genetic tractability, has been instrumental in various studies related to the inositol pyrophosphate pathway. This review is focused on the Kcs1 and Vip1, the two enzymes involved in the biosynthesis of inositol pyrophosphate in S. cerevisiae, highlighting their roles in various cell processes, and providing an up-to-date overview of their relationship with phosphate homeostasis. Moreover, the review underscores the potential applications of these findings in the realms of medicine and biotechnology, highlighting the profound implications of comprehending this intricate signaling network.

Integration of metabolomics and network pharmacology technology to explain the effect mechanisms of Danggui Buxue decoction in vascular dementia.

Danggui Buxue decoction (DBD) is a traditional Chinese medicine herbal decoction that has a good therapeutic effect on vascular dementia (VaD). However, its pharmacodynamic substances and underlying mechanisms are ambiguous. The work aimed to decipher the pharmacodynamic substances and molecular mechanisms of DBD against VaD rats based on gas chromatography-mass spectrometry metabonomics, network pharmacology, molecular docking, and experimental verification. The results indicated that DBD significantly improved the learning abilities and cognitive impairment in the VaD rat model. Integration analysis of the metabolomics and network pharmacology approach revealed that DBD might primarily affect arachidonic acid (AA) and inositol phosphate metabolic pathways by regulating the platelet activation signaling pathways. Six core targets (TNF [tumor necrosis factor], IL-6 [interleukin 6], PTGS2 [prostaglandin-endoperoxide synthase 2], MAPK1, MAPK3, and TP53) in the platelet activation signaling pathways also had a good affinity to seven main active components (saponins, organic acids, flavonoids, and phthalides) of DBD through the verification of molecular docking. Enzyme-linked immunosorbent assay results (ELISA) showed that the levels of TNF, IL-6, PTGS2, thromboxane B2, and caspase-3 in the platelet activation signaling pathway can be regulated by DBD. Our results indicated that DBD treated VaD mainly by modulating the platelet activation signaling pathway, and AA and inositol phosphate metabolism.

Depleting inositol pyrophosphate 5-InsP7 protected the heart against ischaemia-reperfusion injury by elevating plasma adiponectin.

AIMS: Adiponectin is an adipocyte-derived circulating protein that exerts cardiovascular and metabolic protection. Due to the futile degradation of endogenous adiponectin and the challenges of exogenous administration, regulatory mechanisms of adiponectin biosynthesis are of significant pharmacological interest. METHODS AND RESULTS: Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) generated by inositol hexakisphosphate kinase 1 (IP6K1) governed circulating adiponectin levels via thiol-mediated protein quality control in the secretory pathway. IP6K1 bound to adiponectin and DsbA-L and generated 5-InsP7 to stabilize adiponectin/ERp44 and DsbA-L/Ero1-Lα interactions, driving adiponectin intracellular degradation. Depleting 5-InsP7 by either IP6K1 deletion or pharmacological inhibition blocked intracellular adiponectin degradation. Whole-body and adipocyte-specific deletion of IP6K1 boosted plasma adiponectin levels, especially its high molecular weight forms, and activated AMPK-mediated protection against myocardial ischaemia-reperfusion injury. Pharmacological inhibition of 5-InsP7 biosynthesis in wild-type but not adiponectin knockout mice attenuated myocardial ischaemia-reperfusion injury. CONCLUSION: Our findings revealed that 5-InsP7 is a physiological regulator of adiponectin biosynthesis that is amenable to pharmacological intervention for cardioprotection.

Precursor-directed biosynthesis and biological activity of tripropeptin Cpip, a new tripropeptin C analog containing pipecolic acid.

Tripropeptin C, a non-ribosomal cyclic lipopeptide containing three proline residues, exhibits excellent efficacy in the mouse-methicillin-resistant Staphylococcus aureus septicemia model. Since tripropeptins contain L-prolyl-D-proline and, as a result, are known to form a hairpin structure in proteins, it was of interest to determine whether this substructure contributes to their antibacterial activity. In this study, prolines in tripropeptin C were replaced with pipecolic acid(s) using precursor-directed biosynthesis. Only a new tripropeptin analog, tripropeptin Cpip, which has one L-pipecolic acid in place of L-proline, was isolated. The in vitro antimicrobial activity of the new analog was approximately two to four times weaker activity against Gram-positive bacteria, including drug-resistant species, compared with that of tripropeptin C. These results suggest that the L-prolyl-D-proline substructure plays an important role in the observed potency of tripropeptins.

Investigation of the potential of yeast strains for phytase biosynthesis in a two-step screening procedure.

Research into phytase production is useful for improving the efficiency of animal production, reducing environmental impact, and contributing to the development of sustainable and efficient animal production systems. This study aims to investigate the potential of yeast strains for phytase biosynthesis in nutrient media. Phytase is a phosphomonoesterase (E.C 3.1.3.8) catalyzing in a ladder-like manner the dephosphorylation of phytic acid and its salts, with various resulting myo-inositol phosphates and phosphoric acid. Yeasts of the genera Saccharomyces, Zygosaccharomyces, Candida, and Pichia were evaluated in a two-step screening procedure for phytase production. One hundred and eighteen strains were screened in the first stage, which was conducted on four types of solid culture media containing calcium phytate as the selected background. On PSM medium, many strains were found to form halos as early as the 24th hour of development. Several strains with significant potential for enzyme production were evaluated in the second step of the screening. It was conducted in a liquid culture medium. In conclusion, the strain C. melibiosica 2491 was selected for further studies when cultured in a YPglu culture medium. Further research will focus on finding suitable conditions that increase the biosynthesis of the enzyme, which is of significant technological and practical interest for animal nutrition.

Biochemical and structural characterization of an inositol pyrophosphate kinase from a giant virus.

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.