Inositol Metabolites Research Articles

HIV-1 capsid stability enables inositol phosphate-independent infection of target cells and promotes integration into genes.

The mature HIV-1 capsid is stabilized by host and viral determinants. The capsid protein CA binds to the cellular metabolites inositol hexakisphosphate (IP6) and its precursor inositol (1, 3, 4, 5, 6) pentakisphosphate (IP5) to stabilize the mature capsid. In target cells, capsid destabilization by the antiviral compounds lenacapavir and PF74 reveals a HIV-1 infectivity defect due to IP5/IP6 (IP5/6) depletion. To test whether intrinsic HIV-1 capsid stability and/or host factor binding determines HIV-1 insensitivity to IP5/6 depletion, a panel of CA mutants was assayed for infection of IP5/6-depleted T cells and wildtype cells. Four CA mutants with unstable capsids exhibited dependence on host IP5/6 for infection and reverse transcription (RTN). Adaptation of one such mutant, Q219A, by spread in culture resulted in Vpu truncation and a capsid three-fold interface mutation, T200I. T200I increased intrinsic capsid stability as determined by in vitro uncoating of purified cores and partially reversed the IP5/6-dependence in target cells for each of the four CA mutants. T200I further rescued the changes to lenacapavir sensitivity associated with the parental mutation. The premature dissolution of the capsid caused by the IP5/6-dependent mutations imparted a unique defect in integration targeting that was rescued by T200I. Collectively, these results demonstrate that T200I restored other capsid functions after RTN for the panel of mutants. Thus, the hyperstable T200I mutation stabilized the instability defects imparted by the parental IP5/6-dependent CA mutation. The contribution of Vpu truncation to mutant adaptation was linked to BST-2 antagonization, suggesting that cell-to-cell transfer promoted replication of the mutants. We conclude that interactions at the three-fold interface are adaptable, key mediators of capsid stability in target cells and are able to antagonize even severe capsid instability to promote infection.

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.

Identification of the Antithrombotic Mechanism of Leonurine in Adrenalin Hydrochloride-Induced Thrombosis in Zebrafish via Regulating Oxidative Stress and Coagulation Cascade.

Thrombosis is a general pathological phenomenon during severe disturbances to homeostasis, which plays an essential role in cardiovascular and cerebrovascular diseases. Leonurine (LEO), isolated from Leonurus japonicus Houtt, showes a crucial role in anticoagulation and vasodilatation. However, the properties and therapeutic mechanisms of this effect have not yet been systematically elucidated. Therefore, the antithrombotic effect of LEO was investigated in this study. Hematoxylin-Eosin staining was used to detect the thrombosis of zebrafish tail. Fluorescence probe was used to detect the reactive oxygen species. The biochemical indexes related to oxidative stress (lactate dehydrogenase, malondialdehyde, superoxide dismutase and glutathione) and vasodilator factor (endothelin-1 and nitric oxide) were analyzed by specific commercial assay kits. Besides, we detected the expression of related genes (fga, fgb, fgg, pkcα, pkcβ, vwf, f2) and proteins (PI3K, phospho-PI3K, Akt, phospho-Akt, ERK, phospho-ERK FIB) related to the anticoagulation and fibrinolytic system by quantitative reverse transcription and western blot. Beyond that, metabolomic analyses were carried out to identify the expressions of metabolites associated with the anti-thrombosis mechanism of LEO. Our in vivo experimental results showed that LEO could improve the oxidative stress injury, abnormal platelet aggregation and coagulation dysfunction induced by adrenalin hydrochloride. Moreover, LEO restored the modulation of amino acids and inositol metabolites which are reported to alleviate the thrombus formation. Collectively, LEO attenuates adrenalin hydrochloride-induced thrombosis partly via modulating oxidative stress, coagulation cascade and platelet activation and amino acid and inositol metabolites.

A novel raffinose biological pathway is observed by symbionts of cotton≡Verticillium dahliae to improve salt tolerance genetically on cotton

AbstractVerticillium dahliae is a soil‐borne, phytopathogenic fungus that causes Verticillium wilt in cotton. Salt tolerance of cotton plants suffering from Verticillium wilt was significantly improved, but the mechanism is unknown. In this study, we built a symbiont named Cotton≡VD by immersing cotton plant roots into Verticillium dahliae liquid for four days. This resulted in a strong interaction between hyphae and the cotton roots. Under NaCl treatment for 24 h, the Cotton≡VD displayed less leaf wilting and improved root growth compared with the control. Biochemical tests of SOD, POD and CAT showed significant overexpression in Cotton≡VD compared with CK. RNA‐seq data analysis of Cotton≡VD indicated a novel ‘galactose metabolism pathway’ in the KEGG database, in which two key genes, GolS2 and RFS5, were significantly differentially expressed. Symbiotic metabolites inositol galactosyl could be transformed into raffinose to scavenge free radicals and participate in plant osmotic regulation to alleviate salt stress in Cotton≡VD. Functional analysis indicated that silencing of the target gene GhRFS (RFS5) led to a decrease of raffinose content and salt tolerance as compared with CK. Cotton≡VD can significantly improve cotton seedling salt tolerance through improving raffinose content, providing new insights into the salt tolerance mechanism and new salt‐tolerant material in the future.

Effect of hypoxia factors gene silencing on ROS production and metabolic status of A375 malignant melanoma cells

The innate response of melanocytes to exogenous or endogenous stress stimuli like extreme pH and temperature, metabolite and oxygen deficiency or a high UV dose initiates a cellular stress response. This process activates adaptive processes to minimize the negative impact of the stressor on the pigment cell. Under physiological conditions, a non-cancer cell is directed to apoptosis if the stressor persists. However, malignant melanoma cells will survive persistent stress thanks to distinct "cancerous" signaling pathways (e.g. MEK) and transcription factors that regulate the expression of so-called "survival genes" (e.g. HIF, MITF). In this survival response of cancer cells, MEK pathway directs melanoma cells to deregulate mitochondrial metabolism, to accumulate reduced species (NADH), and to centralize metabolism in the cytosol. The aim of this work was to study the effect of gene silencing in malignant melanoma A375 cells on metabolic processes in cytosol and mitochondria. Gene silencing of HIF-1α, and miR-210 in normoxia and pseudohypoxia, and analysis of its effect on MITF-M, and PDHA1 expression. Detection of cytosolic NADH by Peredox-mCherry Assay. Detection of OCR, and ECAR using Seahorse XF96. Measurement of produced O2•− with MitoTracker Red CMXRos. 1H NMR analysis of metabolites present in cell suspension, and medium. By gene silencing of HIF-1α and miR-210 the expression of PDHA1 was upregulated while that of MITF-M was downregulated, yielding acceleration of mitochondrial respiratory activity and thus elimination of ROS. Hence, we detected a significantly reduced A375 cell viability, an increase in alanine, inositol, nucleotides, and other metabolites that together define apoptosis. Based on the results of measurements of mitochondrial resipiratory activity, ROS production, and changes in the metabolites obtained in cells under the observed conditions, we concluded that silencing of HIF-1α and miR-210 yields apoptosis and, ultimately, apoptotic cell death in A375 melanoma cells.

Inositol Pyrophosphates: Signaling Molecules with Pleiotropic Actions in Mammals.

Inositol pyrophosphates (PP-IPs) such as 5-diphosphoinositol pentakisphosphate (5-IP7) are inositol metabolites containing high-energy phosphoanhydride bonds. Biosynthesis of PP-IPs is mediated by IP6 kinases (IP6Ks) and PPIP5 kinases (PPIP5Ks), which transfer phosphate to inositol hexakisphosphate (IP6). Pleiotropic actions of PP-IPs are involved in many key biological processes, including growth, vesicular remodeling, and energy homeostasis. PP-IPs function to regulate their target proteins through allosteric interactions or protein pyrophosphorylation. This review summarizes the current understanding of how PP-IPs control mammalian cellular signaling networks in physiology and disease.

Raffia palm (Raphia hookeri) wine inhibits glucose diffusion; improves antioxidative activities; and modulates dysregulated pathways and metabolites in oxidative pancreatic injury.

Raffia palm wine is a natural drink from the stem of Raffia palm (Raphia hookeri) tree with nutritional and medicinal properties. The effect of fermentation was investigated on its antidiabetic and antioxidative effects in yeast cells and pancreatic tissues, respectively. Both unfermented and fermented palm wine significantly increased glucose uptake, reduced glutathione level (GSH), superoxide dismutase, and catalase activities. They also inhibited glucose diffusion, myeloperoxidase, and ATPase activities as well as decreased malondialdehyde and nitric oxide levels. They also led to the inactivation of oxidative metabolic pathways in oxidative pancreas with the generation of adenosine, sugar and inositol metabolites, selenium (enzyme co-factor) and vitamin metabolites owing to concomitant activation of vitamins, lipid, steroids, inositol, and sulfate/sulfite metabolic pathways. The results suggest the antidiabetic and antioxidative potentials of unfermented and fermented palm wine and may be attributed to the LC-MS-identified compounds which were mainly polyphenols and its glycosides, vitamins, and amino acids. PRACTICAL APPLICATIONS: Raffia palm wine is among the natural beverages employed for social, nutritional, and medicinal purposes. However, there are limited studies on its medicinal properties. This study reports for the first time, the ability of Raffia palm wine to stimulate glucose uptake, inhibit glucose diffusion, and ameliorate pancreatic oxidative injury, as well as the possible associated metabolic pathways that may be involved. These findings will further contribute in understanding the antidiabetic effect of Raffia palm wine, and the possible metabolic pathways involved.

IP3 accumulation and/or inositol depletion: two downstream lithium's effects that may mediate its behavioral and cellular changes.

Lithium is the prototype mood stabilizer but its mechanism is still unresolved. Two hypotheses dominate—the consequences of lithium's inhibition of inositol monophosphatase at therapeutically relevant concentrations (the ‘inositol depletion' hypothesis), and of glycogen-synthase kinase-3. To further elaborate the inositol depletion hypothesis that did not decisively determine whether inositol depletion per se, or phosphoinositols accumulation induces the beneficial effects, we utilized knockout mice of either of two inositol metabolism-related genes—IMPA1 or SMIT1, both mimic several lithium's behavioral and biochemical effects. We assessed in vivo, under non-agonist-stimulated conditions, 3H-inositol incorporation into brain phosphoinositols and phosphoinositides in wild-type, lithium-treated, IMPA1 and SMIT1 knockout mice. Lithium treatment increased frontal cortex and hippocampal phosphoinositols labeling by several fold, but decreased phosphoinositides labeling in the frontal cortex of the wild-type mice of the IMPA1 colony strain by ~50%. Inositol metabolites were differently affected by IMPA1 and SMIT1 knockout. Inositoltrisphosphate administered intracerebroventricularly affected bipolar-related behaviors and autophagy markers in a lithium-like manner. Namely, IP3 but not IP1 reduced the immobility time of wild-type mice in the forced swim test model of antidepressant action by 30%, an effect that was reversed by an antagonist of all three IP3 receptors; amphetamine-induced hyperlocomotion of wild-type mice (distance traveled) was 35% reduced by IP3 administration; IP3 administration increased hippocampal messenger RNA levels of Beclin-1 (required for autophagy execution) and hippocampal and frontal cortex protein levels ratio of Beclin-1/p62 by about threefold (p62 is degraded by autophagy). To conclude, lithium affects the phosphatidylinositol signaling system in two ways: depleting inositol, consequently decreasing phosphoinositides; elevating inositol monophosphate levels followed by phosphoinositols accumulation. Each or both may mediate lithium-induced behavior.

Endoplasmic reticulum stress-induced apoptosis accompanies enhanced expression of multiple inositol polyphosphate phosphatase 1 (Minpp1): a possible role for Minpp1 in cellular stress response.

Inositol polyphosphates represent a group of differentially phosphorylated inositol metabolites, many of which are implicated to regulate diverse cellular processes such as calcium mobilization, vesicular trafficking, differentiation, apoptosis, etc. The metabolic network of these compounds is complex and tightly regulated by various kinases and phosphatases present predominantly in the cytosol. Multiple inositol polyphosphate phosphatase 1 (Minpp1) is the only known endoplasmic reticulum (ER) luminal enzyme that hydrolyzes various inositol polyphosphates in vitro as well as in vivo conditions. However, access of the Minpp1 to cytosolic substrates has not yet been demonstrated clearly and hence its physiological function. In this study, we examined a potential role for Minpp1 in ER stress-induced apoptosis. We generated a custom antibody and characterized its specificity to study the expression of Minpp1 protein in multiple mammalian cells under experimentally induced cellular stress conditions. Our results demonstrate a significant increase in the expression of Minpp1 in response to a variety of cellular stress conditions. The protein expression was corroborated with the expression of its mRNA and enzymatic activity. Further, in an attempt to link the role of Minpp1 to apoptotic stress, we studied the effect of Minpp1 expression on apoptosis following silencing of the Minpp1 gene by its specific siRNA. Our results suggest an attenuation of apoptotic parameters following knockdown of Minpp1. Thus, in addition to its known role in inositol polyphosphate metabolism, we have identified a novel role for Minpp1 as a stress-responsive protein. In summary, our results provide, for the first time, a probable link between ER stress-induced apoptosis and Minpp1 expression.

Lipid profile of human synovial fluid following intra-articular ankle fracture.

This study characterizes the metabolic profile of synovial fluid after intra-articular ankle fracture with an emphasis on changes in the lipid profile. Bilateral ankle synovial fluid from 19 patients with unilateral intra-articular ankle fracture was submitted for metabolic profiling. Contralateral ankle synovial fluid from each patient served as a matched control. Seven patients participated in a second bilateral synovial fluid collection after 6 months. Random forest classification, matched pairs t-tests (α < 0.01), repeated measures ANOVA with post-test contrasts (α < 0.01), correlation to cytokines and matrix metalloproteinases, and fracture and injury classification analyses yielded key lipid biomarkers in synovial fluid following intra-articular fracture. Free fatty acids, sphingomyelins, and lysolipids demonstrated significant elevation in fractured ankles at baseline. Fatty acids and sphingomyelins showed a significant decrease 6 months post-surgery. Random forest analysis showed predominantly fatty acids differentiating between groups. Significant correlations included fatty acids, sphingomyelins, and lysolipids with inflammatory cytokines and matrix metalloproteinases. Fracture classification showed increased fatty acids, lysolipids, and inositol metabolites as fracture severity increased. Fatty acid and sn-1 lysolipid elevation could be detrimental to the joint, as these strongly correlated with matrix metalloproteinases and TNF-α. This elevation also suggests involvement of phospholipase A2 , a potential target for therapeutic intervention. Together with elevated 2-hydroxyl fatty acids, these findings suggest elevated sn-1 lysolipids, sphingomyelins, and subsequent lipid metabolites in synovial fluid as biomarkers of ankle injury. Reversal of this signature after 6 months suggests temporary involvement of these metabolites in disease progression, although they may activate signaling pathways which drive progression to osteoarthritis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:657-666, 2017.

Alterations in Lipid and Inositol Metabolisms in Two Dopaminergic Disorders.

BackgroundSerum metabolite profiling can be used to identify pathways involved in the pathogenesis of and potential biomarkers for a given disease. Both restless legs syndrome (RLS) and Parkinson`s disease (PD) represent movement disorders for which currently no blood-based biomarkers are available and whose pathogenesis has not been uncovered conclusively. We performed unbiased serum metabolite profiling in search of signature metabolic changes for both diseases.Methods456 metabolites were quantified in serum samples of 1272 general population controls belonging to the KORA cohort, 82 PD cases and 95 RLS cases by liquid-phase chromatography and gas chromatography separation coupled with tandem mass spectrometry. Genetically determined metabotypes were calculated using genome-wide genotyping data for the 1272 general population controls.ResultsAfter stringent quality control, we identified decreased levels of long-chain (polyunsaturated) fatty acids of individuals with PD compared to both RLS (PD vs. RLS: p = 0.0001 to 5.80x10-9) and general population controls (PD vs. KORA: p = 6.09x10-5 to 3.45x10-32). In RLS, inositol metabolites were increased specifically (RLS vs. KORA: p = 1.35x10-6 to 3.96x10-7). The impact of dopaminergic drugs was reflected in changes in the phenylalanine/tyrosine/dopamine metabolism observed in both individuals with RLS and PD.ConclusionsA first discovery approach using serum metabolite profiling in two dopamine-related movement disorders compared to a large general population sample identified significant alterations in the polyunsaturated fatty acid metabolism in PD and implicated the inositol metabolism in RLS. These results provide a starting point for further studies investigating new perspectives on factors involved in the pathogenesis of the two diseases as well as possible points of therapeutic intervention.

Metabolomics analysis identifies intestinal microbiota-derived biomarkers of colonization resistance in clindamycin-treated mice.

BackgroundThe intestinal microbiota protect the host against enteric pathogens through a defense mechanism termed colonization resistance. Antibiotics excreted into the intestinal tract may disrupt colonization resistance and alter normal metabolic functions of the microbiota. We used a mouse model to test the hypothesis that alterations in levels of bacterial metabolites in fecal specimens could provide useful biomarkers indicating disrupted or intact colonization resistance after antibiotic treatment.MethodsTo assess in vivo colonization resistance, mice were challenged with oral vancomycin-resistant Enterococcus or Clostridium difficile spores at varying time points after treatment with the lincosamide antibiotic clindamycin. For concurrent groups of antibiotic-treated mice, stool samples were analyzed using quantitative real-time polymerase chain reaction to assess changes in the microbiota and using non-targeted metabolic profiling. To assess whether the findings were applicable to another antibiotic class that suppresses intestinal anaerobes, similar experiments were conducted with piperacillin/tazobactam.ResultsColonization resistance began to recover within 5 days and was intact by 12 days after clindamycin treatment, coinciding with the recovery bacteria from the families Lachnospiraceae and Ruminococcaceae, both part of the phylum Firmicutes. Clindamycin treatment caused marked changes in metabolites present in fecal specimens. Of 484 compounds analyzed, 146 (30%) exhibited a significant increase or decrease in concentration during clindamycin treatment followed by recovery to baseline that coincided with restoration of in vivo colonization resistance. Identified as potential biomarkers of colonization resistance, these compounds included intermediates in carbohydrate or protein metabolism that increased (pentitols, gamma-glutamyl amino acids and inositol metabolites) or decreased (pentoses, dipeptides) with clindamycin treatment. Piperacillin/tazobactam treatment caused similar alterations in the intestinal microbiota and fecal metabolites.ConclusionsRecovery of colonization resistance after antibiotic treatment coincided with restoration of several fecal bacterial metabolites. These metabolites could provide useful biomarkers indicating intact or disrupted colonization resistance during and after antibiotic treatment.

Inositol Pyrophosphates Inhibit Synaptotagmin-Dependent Exocytosis

Inositol pyrophosphates such as IP7 (5-diphosphoinositol pentakisphosphate) are highly energetic inositol metabolites containing pyrophosphate bonds. While IP7 are known to regulate various biological events like metabolism, the molecular site of IP7 action in vesicle trafficking remains elusive. Here, we showed that IP7 potently inhibits synaptic vesicle exocytosis in both invitro reconstitution and cell culture settings. IP7 was identified as a high-affinity ligand for synaptotagmin 1 (Syt1), a calcium sensor essential for membrane fusion. Interactions of IP7 with Syt1 reduced the binding between Syt1 and Ca2+ as well as endowed Syt1 with its own negative effect, thereby suppressing Syt1 activation. These findings reveal a role of IP7 as an inhibitor of the exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

Abstract P137: Metabolomic Analysis of the Early and Late Hypertrophic Heart

The metabolic adaptations to acute myocardial pressure overload are characterized by alterations in metabolism that drive the hypertrophic response and that balance workload with energy demand. Under conditions of chronic pressure overload, it is known that substrate utilization becomes less flexible and that the heart shifts energy preference from fatty acids to glucose. Nevertheless, the metabolic changes that underlie the progression of compensated hypertrophy to heart failure are incompletely understood and attempts to correct the known metabolic defects to delay decompensation have been largely unsuccessful. To identify key changes in metabolic phenotype that could underlie progression to heart failure, we measured metabolites in a transverse aortic constriction (TAC) mouse model using an unbiased metabolomic approach. Hearts were harvested 1 d, 1 wk and 8 wks after sham or TAC operation, and metabolites were extracted from the hearts and analyzed via GC/MS and LC/MS/MS. The signal intensities of 288 named metabolites were re-scaled to median values. Welch’s t-test and two-way ANOVA were used to identify metabolites that changed significantly with pressure overload and progression to heart failure. Echocardiographic measurements showed a significant decrease in ejection fraction after 1 d (65±2% vs. 49±5%) and 8 wks (61±2% vs. 34±8%) of TAC; 1 wk of TAC showed a compensated phenotype characterized by a largely preserved ejection fraction. One day after TAC, only 1.7% of the metabolites changed significantly; however, nearly all amino acids measured were increased by 1 wk. By 8 wks, amino acids returned to near sham levels and a significant and robust decrease in phospholipid, carnitine, inositol, sterol, and fatty acid metabolites occurred. These findings demonstrate that the temporal changes in metabolic phenotype are more complex than previously thought. The preservation of pathways involved in lipid and amino acid metabolism may be important for maintaining myocardial energetics and preventing pump failure under conditions of chronic pressure overload.

The role of inositol signaling in the control of apoptosis

Inositol signaling reactions are very broad in scope affecting many cellular functions. In this report we describe experiments showing that two distinct parts of this system play pivotal roles in an important cellular event, namely apoptosis. Apoptosis is important for organ development and also for controlling cell survival after various stresses which include DNA damage and other proapoptotic stimuli such as tumor necrosis factor α. We show that the inositol phosphate InsP6 or one of its pyrophosphate metabolites determines the extent of apoptosis following tumor necrosis factor α treatment whereby increased cellular levels of InsP6 protect from apoptosis and decreased levels promote it. Cellular levels of InsP6 are determined by the activity of 5/6-kinase since this is the rate limiting enzyme in production of the highly phosphorylated inositol phosphates including InsP6. A lipid inositol metabolite PtdIns5P is also critical in regulating the activity of p53-dependent apoptosis. This phospholipid is formed in cells by the action of type I 4-phosphatase on PtdIns(4,5)P2. PtdIns5P stabilizes p53 by promoting its acetylation in complex with the nuclear factor ING2. Upon genotoxic stress type I 4-phosphatase migrates to the nucleus where it catalyzes the formation of PtdIns5P to stabilize p53 and increase apoptosis.

Functional Insights Revealed by the Crystal Structures of Escherichia coli Glucose-1-phosphatase

The Escherichia coli periplasmic glucose-1-phosphatase is a member of the histidine acid phosphatase family and acts primarily as a glucose scavenger. Previous substrate profiling studies have demonstrated some of the intriguing properties of the enzyme, including its unique and highly selective inositol phosphatase activity. The enzyme is also potentially involved in pathogenic inositol phosphate signal transduction pathways via type III secretion into the host cell. We have determined the crystal structure of E. coli glucose-1-phosphatase in an effort to unveil the structural mechanism underlying such unique substrate specificity. The structure was determined by the method of multiwavelength anomalous dispersion using a tungstate derivative together with the H18A inactive mutant complex structure with glucose 1-phosphate at 2.4-A resolution. In the active site of glucose-1-phosphatase, there are two unique gating residues, Glu-196 and Leu-24, in addition to the conserved features of histidine acid phosphatases. Together they create steric and electrostatic constraints responsible for the unique selectivity of the enzyme toward phytate and glucose-1-phosphate as well as its unusually high pH optimum for the latter. Based on the structural characterization, we were able to derive simple structural principles that not only precisely explains the substrate specificity of glucose-1-phosphatase and the hydrolysis products of various inositol phosphate substrates but also rationalizes similar general characteristics across the histidine acid phosphatase family.

Inositol derivatives modulate spontaneous transmitter release at the frog neuromuscular junction

One of the consequences of G-protein-coupled receptor activation is stimulation of phosphoinositol metabolism, leading to the generation of IP 3 and its metabolites 1,3,4,5-tetrakisphosphate (IP 4) and inositol 1,2,3,4,5,6-hexakisphosphate (IP 6). Previous reports indicate that high inositol polyphosphates (IP 4 and IP 6) are involved in clathrin-coated vesicular recycling. In this study, we examined the effects of IP 4 and IP 6 on spontaneous transmitter release in the form of miniature endplate potentials (MEPP) and on enhanced vesicular recycling by high K + at frog motor nerve endings. In resting conditions, IP 4 and IP 6 delivered intracellularly via liposomes, caused concentration-dependent increases in MEPP frequency and amplitude. Pretreatment with the protein kinase A (PKA) inhibitor H-89 or KT 5720 reduced the IP 4-mediated MEPP frequency increase by 60% and abolished the IP 6-mediated MEPP frequency increases as well as the enhancement in MEPP amplitude. Pretreatment with antibodies against phosphatidylinositol 3-kinase (PI 3-K), enzyme also associated with clathrin-coated vesicular recycling, did not alter the IP 4 and IP 6-mediated MEPP frequency increases, but reduced the MEPP amplitude increase by 50%. In our previous reports, IP 3, but not other second messengers releasing Ca 2+ from internal Ca 2+ stores, is able to enhance the MEPP amplitude. In order to dissociate the effect of Ca 2+ release vs. metabolism to IP 4 and IP 6, we evaluated the effects of 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate (3F-IP 3), which is not converted to IP 4 or IP 6. 3F-IP 3 produced an increase then decrease in MEPP frequency and a decrease in MEPP amplitude. In elevated vesicle recycling induced by high K +-Ringer solution, IP 4 and IP 6 have similar effects, except decreasing MEPP frequency at a higher concentration (10 −4 M). We conclude that (1) high inositol polyphosphates may represent a link between IP 3 and cAMP pathways; (2) the IP 3-induced increase of MEPP amplitude is likely to be due to its high inositol metabolites; (3) PI 3-K is not involved in the IP 4 and IP 6-mediated MEPP frequency increases, but may be involved in MEPP size.

Phosphorylation of a Synaptic Vesicle-associated Protein by an Inositol Hexakisphosphate-regulated Protein Kinase

Despite the fact that inositol hexakisphosphate (InsP(6)) is the most abundant inositol metabolite in cells, its cellular function has remained an enigma. In the present study, we present the first evidence of a protein kinase identified in rat cerebral cortex/hippocampus that is activated by InsP(6). The substrate for the InsP(6)-regulated protein kinase was found to be the synaptic vesicle-associated protein, pacsin/syndapin I. This brain-specific protein, which is highly enriched at nerve terminals, is proposed to act as a molecular link coupling components of the synaptic vesicle endocytic machinery to the cytoskeleton. We show here that the association between pacsin/syndapin I and dynamin I can be increased by InsP(6)-dependent phosphorylation of pacsin/syndapin I. These data provide a model by which InsP(6)-dependent phosphorylation regulates synaptic vesicle recycling by increasing the interaction between endocytic proteins at the synapse.

Overexpression of the inositol phosphatase SopB in human 293 cells stimulates cellular chloride influx and inhibits nuclear mRNA export.

SopB is an inositol phosphate phosphatase that is a virulence factor in Salmonella species. We have overexpressed SopB cDNA in a tetracycline-dependent system in human embryonic 293 cells, and used this model system to directly analyze the role of SopB in altering inositol metabolite levels in vivo. Addition of tetracycline to these cells resulted in the rapid induction of SopB expression, which was coincident with perturbations in the cellular levels of multiple soluble inositol phosphates. All of the changes induced by SopB expression were reversed within 24 h on removal of tetracycline from media. Specifically, cellular inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) levels were depleted within 4 to 6 h after inducing SopB expression. A transient rise in cellular inositol 1,4,5,6-tetrakisphosphate was also observed and was accompanied by increased chloride channel activity. This indicates that SopB alone is sufficient for changes in chloride channel function in cells infected with Salmonella organisms. Depletion of inositol phosphates, including InsP(5) and InsP(6) metabolites, was coincident with the accumulation of polyadenylated RNA in the nucleus. This suggested that a defect in nuclear export had occurred. Moreover, the penetrance of the export defect required localization of SopB to the nucleus. These results provide evidence that inositol phosphate productions may be required for efficient mRNA export in mammalian cells.

Overexpression of the inositol phosphatase SopB in human 293 cells stimulates cellular chloride influx and inhibits nuclear mRNA export

SopB is an inositol phosphate phosphatase that is a virulence factor in Salmonella species. We have overexpressed SopB cDNA in a tetracycline-dependent system in human embryonic 293 cells, and used this model system to directly analyze the role of SopB in altering inositol metabolite levels in vivo. Addition of tetracycline to these cells resulted in the rapid induction of SopB expression, which was coincident with perturbations in the cellular levels of multiple soluble inositol phosphates. All of the changes induced by SopB expression were reversed within 24 h on removal of tetracycline from media. Specifically, cellular inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) levels were depleted within 4 to 6 h after inducing SopB expression. A transient rise in cellular inositol 1,4,5,6-tetrakisphosphate was also observed and was accompanied by increased chloride channel activity. This indicates that SopB alone is sufficient for changes in chloride channel function in cells infected with Salmonella organisms. Depletion of inositol phosphates, including InsP(5) and InsP(6) metabolites, was coincident with the accumulation of polyadenylated RNA in the nucleus. This suggested that a defect in nuclear export had occurred. Moreover, the penetrance of the export defect required localization of SopB to the nucleus. These results provide evidence that inositol phosphate productions may be required for efficient mRNA export in mammalian cells.