Histidine-Rich Glycoprotein Modulates the Toxic Effects of High-Dose Polyphosphate in Mice.

BACKGROUND: Polyphosphate (polyP), a procoagulant released from platelets, activates coagulation via the contact system and modulates cardiomyocyte viability. High-dose intravenous polyP is lethal in mice, presumably because of thrombosis. Previously, we showed that HRG (histidine-rich glycoprotein) binds polyP and attenuates its procoagulant effects. In this study, we investigated the mechanisms responsible for the lethality of intravenous polyP in mice and the impact of HRG on this process. METHODS: The survival of wild-type or HRG-deficient mice given intravenous synthetic or platelet-derived polyP in doses up to 50 mg/kg or saline was compared. To determine the contribution of thrombosis, the effect of FXII (factor XII) knockdown or enoxaparin on polyP-induced fibrin deposition in the lungs was examined. To assess cardiotoxicity, the ECG was continuously monitored, the levels of troponin I and the myocardial band of creatine kinase were quantified, and the viability of a cultured murine cardiomyocyte cell line exposed to polyP in the absence or presence of HRG was determined. RESULTS: In HRG-deficient mice, polyP was lethal at 30 mg/kg, whereas it was lethal in wild-type mice at 50 mg/kg. Although FXII knockdown or enoxaparin administration attenuated polyP-induced fibrin deposition in the lungs, neither affected mortality. PolyP induced dose-dependent ECG abnormalities, including heart block and ST-segment changes, and increased the levels of troponin and myocardial band of creatine kinase, effects that were more pronounced in HRG-deficient mice than in wild-type mice and were attenuated when HRG-deficient mice were given supplemental HRG. Consistent with its cardiotoxicity, polyP reduced the viability of cultured cardiomyocytes in a dose-dependent manner, an effect attenuated with supplemental HRG. CONCLUSIONS: High-dose intravenous polyP is cardiotoxic in mice, and HRG modulates this effect.

Dynamic interactions between SPX proteins, the ubiquitination machinery, and signalling molecules for stress adaptation at a whole-plant level.

The plant macronutrient phosphorus is a scarce resource and plant-available phosphate is limiting in most soil types. Generally, a gene regulatory module called the phosphate starvation response (PSR) enables efficient phosphate acquisition by roots and translocation to other organs. Plants growing on moderate to nutrient-rich soils need to co-ordinate availability of different nutrients and repress the highly efficient PSR to adjust phosphate acquisition to the availability of other macro- and micronutrients, and in particular nitrogen. PSR repression is mediated by a small family of single SYG1/Pho81/XPR1 (SPX) domain proteins. The SPX domain binds higher order inositol pyrophosphates that signal cellular phosphorus status and modulate SPX protein interaction with PHOSPHATE STARVATION RESPONSE1 (PHR1), the central transcriptional regulator of PSR. Sequestration by SPX repressors restricts PHR1 access to PSR gene promoters. Here we focus on SPX4 that primarily acts in shoots and sequesters many transcription factors other than PHR1 in the cytosol to control processes beyond the classical PSR, such as nitrate, auxin, and jasmonic acid signalling. Unlike SPX1 and SPX2, SPX4 is subject to proteasomal degradation not only by singular E3 ligases, but also by SCF-CRL complexes. Emerging models for these different layers of control and their consequences for plant acclimation to the environment will be discussed.

Modulation of innate immunity related genes resulting in prophylactic antimicrobial and antiviral properties.

BACKGROUND: The innate immunity acts during the early phases of infection and its failure in response to a multilayer network of co-infections is cause of immune system dysregulation. Epidemiological SARS-CoV-2 infections data, show that Influenza Virus (FLU-A-B-C) and Respiratory Syncytial Virus (RSV) are co-habiting those respiratory traits. These viruses, especially in children (mostly affected by 'multi-system inflammatory syndrome in children' [MIS-C] and the winter pandemic FLU), in the aged population, and in 'fragile' patients are causing alteration in immune response. Then, bacterial and fungal pathogens are also co-habiting the upper respiratory traits (e.g., Staphylococcus aureus and Candida albicans), thus contributing to morbidity in those COVID-19 affected patients. METHODS: Liquid chromatography coupled with high-resolution mass spectrometry using the quadrupole orbital ion trap analyser (i.e., UHPLC-Q-Orbitrap HRMS) was adopted to measure the polyphenols content of a new nutraceutical formula (Solution-3). Viral infections with SARS-CoV-2 (EG.5), FLU-A and RSV-A viruses (as performed in BLS3 authorised laboratory) and real time RT-PCR (qPCR) assay were used to test the antiviral action of the nutraceutical formula. Dilution susceptibility tests have been used to estimate the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) of Solution-3 on a variety of microorganisms belonging to Gram positive/ negative bacteria and fungi. Transcriptomic data analyses and functional genomics (i.e., RNAseq and data mining), coupled to qPCR and ELISA assays have been used to investigate the mechanisms of action of the nutraceutical formula on those processes involved in innate immune response. RESULTS: Here, we have tested the combination of natural products containing higher amounts of polyphenols (i.e., propolis, Verbascum thapsus L., and Thymus vulgaris L.), together with the inorganic long chain polyphosphates 'polyPs' with antiviral, antibacterial, and antifungal behaviours, against SARS-CoV-2, FLU-A, RSV-A, Gram positive/ negative bacteria and fungi (i.e., Candida albicans). These components synergistically exert an immunomodulatory action by enhancing those processes involved in innate immune response (e.g., cytokines: IFNγ, TNFα, IL-10, IL-6/12; chemokines: CXCL1; antimicrobial peptides: HBD-2, LL-37; complement system: C3). CONCLUSION: The prophylactic antimicrobial success of this nutraceutical formula against SARS-CoV-2, FLU-A and RSV-A viruses, together with the common bacteria and fungi co-infections as present in human oral cavity, is expected to be valuable.

Simple, fast, and simultaneous determination of orthophosphate, pyrophosphate, and tripolyphosphate by capillary electrophoresis with capacitively coupled contactless conductivity detection.

This work describes a novel analytical method using capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4D) for simultaneous, simple, and rapid determination of three inorganic phosphates (orthophosphate, pyrophosphate, and tripolyphosphate) widely used as food additives and in pharmaceutical formulations. A background electrolyte composed of 0.5 mol L-1 acetic acid provided fast separation (around 3.0 min) and good separation efficiency and peak resolution. Linearity in the concentration range of 10-500 mg L-1 was confirmed by the coefficients of determination (R2) higher than 0.99. The limits of detection varied from 0.41 to 0.58 mg L-1. The accuracy of the proposed method was assessed by recovery tests conducted at three concentration levels in tap water samples, food, and personal hygiene products. Recovery values varying from 81% to 118% were achieved, indicating an acceptable accuracy. The proposed CE-C4D successfully determined the three inorganic phosphates in the analyzed samples.

María Teresa Miras Portugal: a pioneer in the study of purinoceptors in chromaffin cells.

María Teresa Miras Portugal devoted most of her scientific life to the study of purinergic signalling. In an important part of her work, she used a model system: the chromaffin cells of the adrenal medulla. It was in these cells that she identified diadenosine polyphosphates, from which she proceeded to the study of adrenomedullary purinome: nucleotide synthesis and degradation, adenosine transport, nucleotide uptake into chromaffin granules, exocytotic release of nucleotides and autocrine regulation of chromaffin cell function via purinoceptors. This short review will focus on the current state of knowledge of the purinoceptors of adrenal chromaffin cells, a subject to which María Teresa made seminal contributions and which she continued to study until the end of her scientific life.

Synthesis, kinetic studies, and QSAR of dinucleoside polyphosphate derivatives as human AK1 inhibitors.

Adenylate kinase (AK) plays a crucial role in the metabolic monitoring of cellular adenine nucleotide homeostasis by catalyzing the reversible transfer of a phosphate group between ATP and AMP, yielding two ADP molecules. By regulating the nucleotide levels and energy metabolism, the enzyme is considered a disease modifier and potential therapeutic target for various human diseases, including malignancies and inflammatory and neurodegenerative disorders. However, lacking approved drugs targeting AK hinders broad studies on this enzyme's pathological importance and therapeutic potential. In this work, we determined the effect of a series of dinucleoside polyphosphate derivatives, commercially available (11 compounds) and newly synthesized (8 compounds), on the catalytic activity of human adenylate kinase isoenzyme 1 (hAK1). The tested compounds belonged to the following groups: (1) diadenosine polyphosphates with different phosphate chain lengths, (2) base-modified derivatives, and (3) phosphate-modified derivatives. We found that all the investigated compounds inhibited the catalytic activity of hAK1, yet with different efficiencies. Three dinucleoside polyphosphates showed IC50 values below 1 µM, and the most significant inhibitory effect was observed for P1-(5'-adenosyl) P5-(5'-adenosyl) pentaphosphate (Ap5A). To understand the observed differences in the inhibition efficiency of the tested dinucleoside polyphosphates, the molecular docking of these compounds to hAK1 was performed. Finally, we conducted a quantitative structure-activity relationship (QSAR) analysis to establish a computational prediction model for hAK1 modulators. Two PLS-regression-based models were built using kinetic data obtained from the AK1 activity analysis performed in both directions of the enzymatic reaction. Model 1 (AMP and ATP synthesis) had a good prediction power (R2 = 0.931, Q2 = 0.854, and MAE = 0.286), while Model 2 (ADP synthesis) exhibited a moderate quality (R2 = 0.913, Q2 = 0.848, and MAE = 0.370). These studies can help better understand the interactions between dinucleoside polyphosphates and adenylate kinase to attain more effective and selective inhibitors in the future.

Diversification in the inositol tris/tetrakisphosphate kinase (ITPK) family: crystal structure and enzymology of the outlier AtITPK4.

Myo-inositol tris/tetrakisphosphate kinases (ITPKs) catalyze diverse phosphotransfer reactions with myo-inositol phosphate and myo-inositol pyrophosphate substrates. However, the lack of structures of nucleotide-coordinated plant ITPKs thwarts a rational understanding of phosphotransfer reactions of the family. Arabidopsis possesses a family of four ITPKs of which two isoforms, ITPK1 and ITPK4, control inositol hexakisphosphate and inositol pyrophosphate levels directly or by provision of precursors. Here, we describe the specificity of Arabidopsis ITPK4 to pairs of enantiomers of diverse inositol polyphosphates and show how substrate specificity differs from Arabidopsis ITPK1. Moreover, we provide a description of the crystal structure of ATP-coordinated AtITPK4 at 2.11 Šresolution that, along with a description of the enantiospecificity of the enzyme, affords a molecular explanation for the diverse phosphotransferase activity of this enzyme. That Arabidopsis ITPK4 has a KM for ATP in the tens of micromolar range, potentially explains how, despite the large-scale abolition of InsP6, InsP7 and InsP8 synthesis in Atitpk4 mutants, Atitpk4 lacks the phosphate starvation responses of Atitpk1 mutants. We further demonstrate that Arabidopsis ITPK4 and its homologues in other plants possess an N-terminal haloacid dehalogenase-like fold not previously described. The structural and enzymological information revealed will guide elucidation of ITPK4 function in diverse physiological contexts, including InsP8-dependent aspects of plant biology.

Importance of Non-Covalent Interactions in Yeast Cell Wall Molecular Organization.

This review covers a group of non-covalently associated molecules, particularly proteins (NCAp), incorporated in the yeast cell wall (CW) with neither disulfide bridges with proteins covalently attached to polysaccharides nor other covalent bonds. Most NCAp, particularly Bgl2, are polysaccharide-remodeling enzymes. Either directly contacting their substrate or appearing as CW lipid-associated molecules, such as in vesicles, they represent the most movable enzymes and may play a central role in CW biogenesis. The absence of the covalent anchoring of NCAp allows them to be there where and when it is necessary. Another group of non-covalently attached to CW molecules are polyphosphates (polyP), the universal regulators of the activity of many enzymes. These anionic polymers are able to form complexes with metal ions and increase the diversity of non-covalent interactions through charged functional groups with both proteins and polysaccharides. The mechanism of regulation of polysaccharide-remodeling enzyme activity in the CW is unknown. We hypothesize that polyP content in the CW is regulated by another NCAp of the CW-acid phosphatase-which, along with post-translational modifications, may thus affect the activity, conformation and compartmentalization of Bgl2 and, possibly, some other polysaccharide-remodeling enzymes.

Inositol polyphosphates-regulated polyubiquitination of PHR1 by NLA E3 ligase during phosphate starvation response in Arabidopsis.

Phosphate (Pi) availability is a major factor limiting plant growth and development. The key transcription factor controlling Pi-starvation response (PSR) is PHOSPHATE STARVATION RESPONSE 1 (PHR1) whose transcript levels do not change with changes in Pi levels. However, how PHR1 stability is regulated at the post-translational level is relatively unexplored in Arabidopsis thaliana. Inositol polyphosphates (InsPn) are important signal molecules that promote the association of stand-alone SPX domain proteins with PHR1 to regulate PSR. Here, we show that NITROGEN LIMITATION ADAPTATION (NLA) E3 ligase can associate with PHR1 through its conserved SPX domain and polyubiquitinate PHR1 in vitro. The association with PHR1 and its ubiquitination is enhanced by InsP6 but not by InsP5. Analysis of InsPn-related mutants and an overexpression plant shows PHR1 levels are more stable in itpk4-1 and vih2-4/VIH1amiRNA but less stable in ITPK4 overexpression plants. Under Pi-deficient conditions, nla seedlings contain high PHR1 levels, display long root hair and accumulate anthocyanin in shoots phenocopying PHR1 overexpression plants. By contrast, NLA overexpression plants phenocopy phr1 whose phenotypes are opposite to those of nla. Our results suggest NLA functions as a negative regulator of Pi response by modulating PHR1 stability and the NLA/PHR1 association depends on InsPn levels.

Short-chain polyphosphates: Extraction effects on migration and size estimation of Saccharomyces cerevisiae extracts with polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis is commonly used to characterize the chain length of polyphosphates (polyP), more generally called condensed phosphates. After separation, nonradioactive, optical polyP staining is limited to chain lengths greater than 15 PO 3 - ${\rm{PO}}_3^ - $ monomers with toluidine blue or 4',6-diamidino-2-phenylindole. PolyP chain lengths longer than 62 PO 3 - $\;{\rm{PO}}_3^ - $ monomers were correlated to the shortest DNA ladders. In this study, synthetic linear polyPs (Sigma-Aldrich "Type 45", estimated mean length of 45 PO 3 - ${\rm{PO}}_3^ - $ monomers), trimetaphosphate (trimetaP: 3 PO 3 - ${\rm{PO}}_3^ - $ ring), tripolyphosphate (tripolyP), pyrophosphate (PPi ), and inorganic orthophosphate (o-Pi ) were visualized after separation by an in situ hydrolytic degradation process to o-Pi that was subsequently stained with methyl green. Statistically insignificant migration reduction of synthetic short-chain polyP after perchloric acid or phenol-chloroform extraction was confirmed with the Friedman test. 31 P diffusion-ordered NMR spectroscopy confirmed that extraction also reduced PPi diffusivity by <10%. Linear regression between the Rf peak migration value and the logarithm of synthetic polyP molecular weights enabled estimation of extracted polyP chain lengths from 2 to 45 PO 3 - ${\rm{PO}}_3^ - $ monomers. Linear polyP extracts from Saccharomyces cerevisiae grown in aerobic conditions were generally shorter than extracts cultured in anaerobic conditions. Extractions from both aerobic and anaerobic S. cerevisiae included tripolyP and o-Pi , but no PPi .

A three-step process of manganese acquisition and storage in the microalga Chlorella sorokiniana.

Metabolism of metals in microalgae and adaptation to metal excess are of significant environmental importance. We report a three-step mechanism that the green microalga Chlorella sorokiniana activates during the acquisition of and adaptation to manganese (Mn), which is both an essential trace metal and a pollutant of waters. In the early stage, Mn2+ was mainly bound to membrane phospholipids and phosphates in released mucilage. The outer cell wall was reorganized and lipids were accumulated, with a relative increase in lipid saturation. Intracellular redox settings were rapidly altered in the presence of Mn excess, with increased production of reactive oxygen species that resulted in lipid peroxidation and a decrease in the concentration of thiols. In the later stage, Mn2+ was chelated by polyphosphates and accumulated in the cells. The structure of the inner cell wall was modified and the redox milieu established a new balance. Polyphosphates serve as a transient Mn2+ storage ligand, as proposed previously. In the final stage, Mn was stored in multivalent Mn clusters that resemble the structure of the tetramanganese-calcium core of the oxygen-evolving complex. The present findings elucidate the bioinorganic chemistry and metabolism of Mn in microalgae, and may shed new light on water-splitting Mn clusters.

Effect of Deletions of the Genes Encoding Pho3p and Bgl2p on Polyphosphate Level, Stress Adaptation, and Attachments of These Proteins to Saccharomyces cerevisiae Cell Wall.

Inorganic polyphosphates (polyP), according to literature data, are involved in the regulatory processes of molecular complex of the Saccharomyces cerevisiae cell wall (CW). The aim of the work was to reveal relationship between polyP, acid phosphatase Pho3p, and the major CW protein, glucanosyltransglycosylase Bgl2p, which is the main glucan-remodelling enzyme with amyloid properties. It has been shown that the yeast cells with deletion of the PHO3 gene contain more high molecular alkali-soluble polyP and are also more resistant to exposure to alkali and manganese ions compared to the wild type strain. This suggests that Pho3p is responsible for hydrolysis of the high molecular polyP on the surface of yeast cells, and these polyP belong to the stress resistance factors. The S. cerevisiae strain with deletion of the BGL2 gene is similar to the Δpho3 strain both in the level of high molecular alkali-soluble polyP and in the increased resistance to alkali and manganese. Comparative analysis of the CW proteins demonstrated correlation between the extractability of the acid phosphatase and Bgl2p, and also revealed a change in the mode of Bgl2p attachment to the CW of the strain lacking Pho3p. It has been suggested that Bgl2p and Pho3p are able to form a metabolon or its parts that connects biogenesis of the main structural polymer of the CW, glucan, and catabolism of an important regulatory polymer, polyphosphates.

Synthesis and biological evaluation of flavonoid-based IP6K2 inhibitors.

Inositol polyphosphates (IPs) are a group of inositol metabolites that act as secondary messengers for external signalling cues. They play various physiological roles such as insulin release, telomere length maintenance, cell metabolism, and aging. Inositol hexakisphosphate kinase 2 (IP6K2) is a key enzyme that produces 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-IP7), which influences the early stages of glucose-induced exocytosis. Therefore, regulation of IP6Ks may serve as a promising strategy for treating diseases such as diabetes and obesity. In this study, we designed, synthesised, and evaluated flavonoid-based compounds as new inhibitors of IP6K2. Structure-activity relationship studies identified compound 20s as the most potent IP6K2 inhibitor with an IC50 value of 0.55 µM, making it 5-fold more potent than quercetin, the reported flavonoid-based IP6K2 inhibitor. Compound 20s showed higher inhibitory potency against IP6K2 than IP6K1 and IP6K3. Compound 20s can be utilised as a hit compound for further structural modifications of IP6K2 inhibitors.

Maintenance of enamel properties after bleaching with high-concentrated hydrogen-peroxide gel containing calcium polyphosphate sub-microparticles.

OBJECTIVE: To assessed the physical and chemical properties of human-enamel after treatment with an experimental bleaching gel containing 35%-hydrogen peroxide (HP) and calcium polyphosphate sub-microparticles (CaPP). MATERIALS AND METHODS: Enamel/dentin specimens (4 × 4 × 3 mm) were obtained (n = 120) and allocated to different groups: control (saliva only); experimental (HP35%); commercial (whiteness-HP-Maxx); CaPP0.5% (HP35% + CaPP0.5wt%); CaPP1.5% (HP35% + CaPP1.5wt%). Three sessions were performed. The specimens' color was assessed using a spectrophotometer and the color (ΔE/ΔE00) and bleaching index (ΔWID) determined. The surface roughness and microhardness were assessed with a roughness tester and Knoop indenter. Raman spectroscopy was performed to obtain the ratios between the areas under the 431, 580, and 1070 cm-1 and the 960 cm-1 bands (430:960, 580:960, 1070:960). Kruskal-Wallis and Dunn compared the color, Ra, and SMH data. The Raman data was analyzed with Kruskal-Wallis and Dunn (α = 5%). RESULTS: The ΔE, ΔE00, and ΔWID were similar among the bleached groups (p > 0.05). The roughness was not different between the groups (p > 0.05). After the 3rd session, CaPP0.5% had higher microhardness than the experimental (p < 0.05). The 1070:960 was higher in the experimental than in the CaPP1.5% and control (p < 0.05). CONCLUSIONS: In human enamel, CaPP did not alter the bleaching effectiveness or roughness, and additionally, CaPP-containing gels increased the microhardness and preserved the mineral content when compared to the experimental without CaPP. CLINICAL RELEVANCE: Experimental bleaching gels containing calcium polyphosphate sub-microparticles as a mineral source reduce the mineral content alteration and superficial microhardness reduction, known potential side effects of the in-office bleaching treatments.

Effect of incorporation of calcium polyphosphate sub-microparticles in low-concentration bleaching gels on physical properties of dental enamel.

AIM: To evaluate the bleaching efficacy and effects on enamel properties of experimental gels with carbamide peroxide (CP; 10%) or hydrogen peroxide (HP; 6%) containing calcium polyphosphate sub-microparticles (CaPPs). METHODS: A total of 216 bovine tooth specimens were divided for microhardness and color analyses (n = 108) and block randomized into nine groups (n = 12): (G1) commercial CP (Whiteness Perfect, FGM; Brazil); (G2) experimental CP; (G3) CP-0.5%CaPPs; (G4) CP-1.5%CaPPs; (G5) commercial HP (Potenza Bianco, PHS; Brazil); (G6) experimental HP; (G7) HP-0.5%CaPPs; (G8) HP-1.5%CaPPs; (G9) artificial saliva. The gels' pH values were determined with a bench pH meter. Color (ΔE, ΔE00, ΔWID) and microhardness variation were evaluated before and after the therapy. Part of the specimens used for microhardness was submitted to the scanning electron microscopy (SEM) (n = 3) and energy-dispersive X-ray spectroscopy EDX (n = 3) analyses. Statistical analyses were performed in the R statistical software (α = 0.05). Linear mixed models for repeated measures in time were used to analyze microhardness and L* values. Generalized linear models were used to analyze the a*, b*, ΔE, ΔE00, and ΔWID, considering a group effect. The EDX data were analyzed using a one-way ANOVA with Tukey's test. RESULTS: The gels' pH remained over 6,0. All gels effectively bleached the specimens and did not differ significantly. When compared to the control group, the hardness was significantly lower in the G1, G2, G6, and G7 groups. The G3, G4, G5, and G8 groups did not differ significantly (p > 0.05). CONCLUSION: The incorporation of CaPPs in low-concentration whitening gels reduces its negative effects on microhardness without interfering with their bleaching efficacy.

The Plasma Membrane Purinoreceptor P2K1/DORN1 Is Essential in Stomatal Closure Evoked by Extracellular Diadenosine Tetraphosphate (Ap4A) in Arabidopsis thaliana.

Dinucleoside polyphosphates (NpnNs) are considered novel signalling molecules involved in the induction of plant defence mechanisms. However, NpnN signal recognition and transduction are still enigmatic. Therefore, the aim of our research was the identification of the NpnN receptor and signal transduction pathways evoked by these nucleotides. Earlier, we proved that purine and pyrimidine NpnNs differentially affect the phenylpropanoid pathway in Vitis vinifera suspension-cultured cells. Here, we report, for the first time, that both diadenosine tetraphosphate (Ap4A) and dicytidine tetraphosphate (Cp4C)-induced stomatal closure in Arabidopsis thaliana. Moreover, we showed that plasma membrane purinoreceptor P2K1/DORN1 (does not respond to nucleotide 1) is essential for Ap4A-induced stomata movements but not for Cp4C. Wild-type Col-0 and the dorn1-3 A. thaliana knockout mutant were used. Examination of the leaf epidermis dorn1-3 mutant provided evidence that P2K1/DORN1 is a part of the signal transduction pathway in stomatal closure evoked by extracellular Ap4A but not by Cp4C. Reactive oxygen species (ROS) are involved in signal transduction caused by Ap4A and Cp4C, leading to stomatal closure. Ap4A induced and Cp4C suppressed the transcriptional response in wild-type plants. Moreover, in dorn1-3 leaves, the effect of Ap4A on gene expression was impaired. The interaction between P2K1/DORN1 and Ap4A leads to changes in the transcription of signalling hubs in signal transduction pathways.

Porphyromonas gingivalis initiates coagulation and secretes polyphosphates - A mechanism for sustaining chronic inflammation?

BACKGROUND: Periodontitis is a chronic inflammation resulting in destruction of tooth-supporting bone. Chronic inflammation is characterized by extravascular fibrin deposition. Fibrin is central to destruction of bone; monocytes bind to fibrin and form osteoclasts, thus providing a link between coagulation and the tissue destructive processes in periodontitis. The oral microbiome is essential to oral health. However, local ecological changes, such as increased biofilm formation, result in a dysbiotic microbiome characterized by an increase of protease-producing species e.g. Porphyromonas gingivalis. Proteases initiate inflammation and may cleave coagulation factors. Polyphosphates (polyP) may also provide bacteria with procoagulant properties similar to platelet-released polyP. P. gingivalis has also been found in remote locations related to vascular pathology and Alzheimer's disease. OBJECTIVES: The aim of this study was to investigate procoagulant activity of ten different species of oral bacteria present in oral health and disease as well as presence of polyP and fibrin formation in planktonic and biofilm bacteria. METHODS: Oral bacteria were studied for protease production and procoagulant activity. The presence of polyP and formation of fibrin was observed using confocal microscopy. RESULTS: P. gingivalis showed strong protease activity and was the only species exerting procoagulant activity. Confocal microscopy showed polyP intracellularly in planktonic bacteria and extracellularly after biofilm formation. Fibrin formation emanated from planktonic bacteria and from both bacteria and polyP in biofilm cultures. CONCLUSIONS: The procoagulant activity of P. gingivalis could explain its role in chronic inflammation, locally in oral tissues as well as in remote locations.

Inositol polyphosphate-protein interactions: Implications for microbial pathogenicity.

Inositol polyphosphates (IPs) and inositol pyrophosphates (PP-IPs) regulate diverse cellular processes in eukaryotic cells. IPs and PP-IPs are highly negatively charged and exert their biological effects by interacting with specific protein targets. Studies performed predominantly in mammalian cells and model yeasts have shown that IPs and PP-IPs modulate target function through allosteric regulation, by promoting intra- and intermolecular stabilization and, in the case of PP-IPs, by donating a phosphate from their pyrophosphate (PP) group to the target protein. Technological advances in genetics have extended studies of IP function to microbial pathogens and demonstrated that disrupting PP-IP biosynthesis and PP-IP-protein interaction has a profound impact on pathogenicity. This review summarises the complexity of IP-mediated regulation in eukaryotes, including microbial pathogens. It also highlights examples of poor conservation of IP-protein interaction outcome despite the presence of conserved IP-binding domains in eukaryotic proteomes.

Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalk between SA-dependent immunity and phosphate-starvation responses.

KEY MESSAGE: Selective Arabidopsis thaliana inositol phosphate kinase functions modulate response amplitudes in innate immunity by balancing signalling adjustments with phosphate homeostasis networks. Pyrophosphorylation of InsP6 generates InsP7 and/or InsP8 containing high-energy phosphoanhydride bonds that are harnessed during energy requirements of a cell. As bona fide co-factors for several phytohormone networks, InsP7/InsP8 modulate key developmental processes. With requirements in transducing jasmonic acid (JA) and phosphate-starvation responses (PSR), InsP8 exemplifies a versatile metabolite for crosstalks between different cellular pathways during diverse stress exposures. Here we show that Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE 1 (IPK1), INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), and DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASE 2 (VIH2) implicated in InsP8 biosynthesis, suppress salicylic acid (SA)-dependent immunity. In ipk1, itpk1 or vih2 mutants, constitutive activation of defenses lead to enhanced resistance against the Pseudomonas syringae pv tomato DC3000 (PstDC3000) strain. Our data reveal that upregulated SA-signaling sectors potentiate increased expression of several phosphate-starvation inducible (PSI)-genes, previously known in these mutants. In reciprocation, upregulated PSI-genes moderate expression amplitudes of defense-associated markers. We demonstrate that SA is induced in phosphate-deprived plants, however its defense-promoting functions are likely diverted to PSR-supportive roles. Overall, our investigations reveal selective InsPs as crosstalk mediators in defense-phosphate homeostasis and in reprogramming stress-appropriate response intensities.

Depletion of mitochondrial inorganic polyphosphate (polyP) in mammalian cells causes metabolic shift from oxidative phosphorylation to glycolysis.

Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical with those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.