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Phytate (myo-inositol hexakisphosphate or InsP6) is the main phosphorus reservoir that is present in almost all wholegrains, legumes, and oilseeds. It is a major component of the Mediterranean and Dietary Approaches to Stop Hypertension (DASH) diets. Phytate is recognized as a nutraceutical and is classified by the Food and Drug Administration (FDA) as Generally Recognized As Safe (GRAS). Phytate has been shown to be effective in treating or preventing certain diseases. Phytate has been shown to inhibit calcium salt crystallization and, therefore, to reduce vascular calcifications, calcium renal calculi and soft tissue calcifications. Moreover, the adsorption of phytate to the crystal faces can inhibit hydroxyapatite dissolution and bone resorption, thereby playing a role in the treatment/prevention of bone mass loss. Phytate has a potent antioxidation and anti-inflammatory action. It is capable of inhibiting lipid peroxidation through iron chelation, reducing iron-related free radical generation. As this has the effect of mitigating neuronal damage and loss, phytate shows promise in the treatment/prevention of neurodegenerative disease. It is reported that phytate improves lipid and carbohydrate metabolism, increases adiponectin, decreases leptin and reduces protein glycation, which is linked with macrovascular and microvascular diabetes complications. In this review, we summarize the benefits of phytate intake as seen in in vitro, animal model, epidemiological and clinical trials, and we also identify questions to answer in the future. [ABSTRACT FROM AUTHOR]

Removal of 59 cap on cellular mRNAs by the African swine fever virus (ASFV) decapping enzyme g5R protein (g5Rp) is beneficial to viral gene expression during the early stages of infection. As the only nucleoside diphosphate-linked moiety X (Nudix) decapping enzyme encoded in the ASFV genome, g5Rp works in both the degradation of cellular mRNA and the hydrolyzation of the diphosphoinositol polyphosphates. Here, we report the structures of dimeric g5Rp and its complex with inositol hexakisphosphate (InsP6). The two g5Rp protomers interact head to head to form a dimer, and the dimeric interface is formed by extensive polar and nonpolar interactions. Each protomer is composed of a unique N-terminal helical domain and a C-terminal classic Nudix domain. As g5Rp is an mRNA-decapping enzyme, we identified key residues, including K8, K94, K95, K98, K175, R221, and K243 located on the substrate RNA binding interfaces of g5Rp which are important to RNA binding and decapping enzyme activity. Furthermore, the g5Rp-mediated mRNA decapping was inhibited by InsP6. The g5Rp-InsP6 complex structure showed that the InsP6 molecules occupy the same regions that primarily mediate g5Rp-RNA interaction, elucidating the roles of InsP6 in the regulation of the viral decapping activity of g5Rp in mRNA degradation. Collectively, these results provide the structural basis of interaction between RNA and g5Rp and highlight the inhibitory mechanism of InsP6 on mRNA decapping by g5Rp. [ABSTRACT FROM AUTHOR]

Phytate (myo-inositol hexakisphosphate or InsP6) is the main phosphorus reservoir that is present in almost all wholegrains, legumes, and oilseeds. It is a major component of the Mediterranean and Dietary Approaches to Stop Hypertension (DASH) diets. Phytate is recognized as a nutraceutical and is classified by the Food and Drug Administration (FDA) as Generally Recognized As Safe (GRAS). Phytate has been shown to be effective in treating or preventing certain diseases. Phytate has been shown to inhibit calcium salt crystallization and, therefore, to reduce vascular calcifications, calcium renal calculi and soft tissue calcifications. Moreover, the adsorption of phytate to the crystal faces can inhibit hydroxyapatite dissolution and bone resorption, thereby playing a role in the treatment/prevention of bone mass loss. Phytate has a potent antioxidation and anti-inflammatory action. It is capable of inhibiting lipid peroxidation through iron chelation, reducing iron-related free radical generation. As this has the effect of mitigating neuronal damage and loss, phytate shows promise in the treatment/prevention of neurodegenerative disease. It is reported that phytate improves lipid and carbohydrate metabolism, increases adiponectin, decreases leptin and reduces protein glycation, which is linked with macrovascular and microvascular diabetes complications. In this review, we summarize the benefits of phytate intake as seen in in vitro, animal model, epidemiological and clinical trials, and we also identify questions to answer in the future.

Phytic acid (inositol hexakisphosphate, InsP6) is an important phosphate store and signal molecule necessary for maintenance of basal resistance to plant pathogens. Arabidopsis thaliana ('arabidopsis') has three genes encoding myo‐inositol phosphate synthases (IPS1–3), the enzymes that catalyse conversion of glucose‐6‐phosphate to InsP, the first step in InsP6 biosynthesis. There is one gene for inositol‐(1,3,4,5,6)‐pentakisphosphate 2‐kinase (IPK1), which catalyses the final step. Previously, we showed that mutation of IPS2 and IPK1 but not IPS1 increased susceptibility to pathogens. Our aim was to better understand the InsP6 biosynthesis pathway in plant defence. Here we found that the susceptibility of arabidopsis (Col‐0) to virulent and avirulent Pseudomonas syringae pv. tomato was also increased in ips3 and ips2/3 double mutants. Also, ipk1 plants had compromised expression of local acquired resistance induced by treatment with the pathogen‐derived molecular pattern (PAMP) molecule flg22, but were unaffected in other responses to flg22, including Ca2+ influx and the oxidative burst, seedling root growth inhibition, and transcriptional up‐regulation of the PAMP‐triggered genes MITOGEN‐ACTIVATED PROTEIN KINASE (MPK) 3, MPK11, CINNAMYL ALCOHOL DEHYDROGENASE 5, and FLG22‐INDUCED RECEPTOR‐LIKE KINASE 1. IPK1 mutation did not prevent the induction of systemic acquired resistance by avirulent P. syringae. Also, ips2 and ips2/3 double mutant plants, like ipk1, were hypersusceptible to P. syringae but were not compromised in flg22‐induced local acquired resistance. The results support the role of InsP6 biosynthesis enzymes in effective basal resistance and indicate that there is more than one basal resistance mechanism dependent upon InsP6 biosynthesis. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanisms of retroviral assembly has been a decades-long endeavor. With the recent discovery of inositol hexakisphosphate (IP6) acting as an assembly co-factor for human immunodeficiency virus (HIV), great strides have been made in retroviral research. In this review, the enzymatic pathways to synthesize and metabolize inositol phosphates (IPs) relevant to retroviral assembly are discussed. The functions of these enzymes and IPs are outlined in the context of the cellular biology important for retroviruses. Lastly, the recent advances in understanding the role of IPs in retroviral biology are surveyed.

AKT, a serine/threonine protein kinase and mammalian target of rapamycin (mTOR) plays a critical role in the proliferation and resistance to apoptosis that are essential to the development and progression of colon cancer. Therefore, AKT/mTOR signaling pathway has been recognized as an attractive target for anticancer therapy. Inositol hexaphosphate (InsP6), a natural occurring phytochemical, has been shown to have both preventive and therapeutic effects against various cancers, however, its exact molecular mechanisms of action are not fully understood. The aim of the in vitro study was to investigate the anticancer activity of InsP6 on colon cancer with the focus on inhibiting the AKT1 kinase and p70S6K1 as mTOR effector, in relation to proliferation and apoptosis of cells. The colon cancer Caco-2 cells were cultured using standard techniques and exposed to InsP6 at different concentrations (1 mM, 2.5 mM and 5 mM). Cellular proliferative activity was monitored by 5-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA. Flow cytometric analysis was performed for cell cycle progression and apoptosis studies. Real-time RT-qPCR was used to validate mRNA levels of CDNK1A, CDNK1B, CASP3, CASP9, AKT1 and S6K1 genes. The concentration of p21 protein as well as the activities of caspase 3, AKT1 and p70S6K1 were determined by the ELISA method. The results revealed that IP6 inhibited proliferation and stimulated apoptosis of colon cancer cells. This effect was mediated by an increase in the expression of genes encoding p21, p27, caspase 3, caspase 9 as well a decrease in transcription of AKT1 and S6K1. InsP6 suppressed phosphorylation of AKT1 and p70S6K1, downstream effector of mTOR. Based on these studies it may be concluded that InsP6 can reduce proliferation and induce apoptosis through inhibition of the AKT/mTOR pathway and mTOR effector followed by modulation of the expression and activity of several key components of these pathways in colon cancer cells. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Oral treatment with inositol hexaphosphate (InsP6) has shown to be efficient in decreasing adverse effects in patients with breast cancer under chemotherapy. This study was aimed at evaluating and comparing the efficacy of topical InsP6 in improving quality of life in women treated with anticancer drugs. PATIENTS AND METHODS: The study was a double-blind, randomized controlled trial (RCT) with allocation concealment of 20 patients in two groups, one (experimental) applied 4% topical formulation of InsP6 once a day, whereas the second one (control) a gel containing hyaluronic acid. InsP6 therapy started 6 weeks after lumpectomy. Blood tests were monitored in both groups and quality of life was assessed using standardized QLQ-C30 and QLQ-BR23. RESULTS: Patients who applied InsP6 on the breast significantly improved their quality of life and functional status reducing side effects compared to control group; moreover, after treatment, a significant difference between the two groups was observed in the white blood cells and platelets count values. CONCLUSIONS: Topical InsP6 treatment has demonstrated to be effective and safe in preventing and/or mitigating chemotherapy-induced side effects as well as the preserving quality of life in women with ductal breast cancer. [ABSTRACT FROM AUTHOR]

Background: Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. Results: In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag. The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. Conclusion: pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins. [ABSTRACT FROM AUTHOR]

Inositol pyrophosphates are unique cellular signaling molecules with recently discovered roles in energy sensing and metabolism. Studies in eukaryotes have revealed that these compounds have a rapid turnover, and thus only small amounts accumulate. Inositol pyrophosphates have not been the subject of investigation in plants even though seeds produce large amounts of their precursor, myo-inositol hexakisphosphate (InsP6). Here, we report that Arabidopsis and maize InsP6 transporter mutants have elevated levels of inositol pyrophosphates in their seed, providing unequivocal identification of their presence in plant tissues. We also show that plant seeds store a little over 1% of their inositol phosphate pool as InsP7 and InsP8. Many tissues, including, seed, seedlings, roots and leaves accumulate InsP7 and InsP8, thus synthesis is not confined to tissues with high InsP6. We have identified two highly similar Arabidopsis genes, AtVip1 and AtVip2, which are orthologous to the yeast and mammalian VIP kinases. Both AtVip1 and AtVip2 encode proteins capable of restoring InsP7 synthesis in yeast mutants, thus AtVip1 and AtVip2 can function as bonafide InsP6 kinases. AtVip1 and AtVip2 are differentially expressed in plant tissues, suggesting non-redundant or non-overlapping functions in plants. These results contribute to our knowledge of inositol phosphate metabolism and will lay a foundation for understanding the role of InsP7 and InsP8 in plants. [ABSTRACT FROM AUTHOR]

In this study 97 soil samples from different soil ecosystems were collected. The initial screening was performed on modified glycerol arginine agar (MGAA) to isolate common actinomycetes and on modified MGA-SE (MMGA-SE) to isolate rare actinomycetes. Sixty-seven isolates potentially producing extracellular phytate-degrading activity were identified. The potential to dephosphorylate phytate was confirmed in liquid culture for 46.3 % of the isolates. 12 strains were selected for a direct determination of their phytate-degrading capacity. The results highlighted that the selected isolates produced extracellular phytate-degrading activity; however their capacity in InsP degradation was different. In addition the fermentation medium had an effect on the extent of phytate degradation. Some enzymatic properties of the phytases from isolate No. 43 and isolate No. 63 were determined after obtaining phytase-enriched samples. The enzymes had maximum phytate-degrading capability at 55 °C and pH 5 (isolate No. 43) and 37 °C and pH 7 (isolates No. 63), respectively. Due to their properties, the phytase of isolate No. 43 behaves like a histidine acid phytase, whereas the phytase of No. 63 showed similar enzymatic properties to the phytase of lily. To our knowledge, the results from this study demonstrated for the first time that actinomycetes produce extracellular phytate-degrading activity. By 16SrRNA sequencing, the more closely studied phytase producers were identified a s Streptomyces sp. Isolate No. 43 showed 98 % identity to Streptomyces alboniger and S. venezuelae, while isolate No. 63 exhibited 98 % sequence identity to S. ambofaciens and S. lienomycini. [ABSTRACT FROM AUTHOR]

Abstract: Nopp140, a highly phosphorylated nucleolar protein, negatively regulates CK2, a kinase essential for cell proliferation. We quantitatively analyzed the interaction between two subunits of CK2 and Nopp140 and characterized the mechanism by which InsP6 inhibits the interaction. Nopp140 specifically binds to the catalytic subunit of CK2 (CK2α) with a dissociation constant of (K d) of 4nM, which interferes with the catalytic activity of CK2. The C-terminal region of Nopp140 is determined as CK2α-binding region by a yeast two-hybrid method as well as a direct measurement of the interaction between CK2α and deletion mutants of Nopp140. InsP6 specifically binds to CK2α and disrupts the interaction between CK2α and Nopp140 with an IC50 value of 25μM, thereby attenuating the Nopp140-mediated repression of CK2 activity. [Copyright &y& Elsevier]

Abstract: We have cloned and characterized four Itpk genes from soybean. All four recombinant Itpk proteins showed canonical Ins(1,3,4)P3 5/6-kinase activity, but a kinetic analysis raised questions about its biological significance. Instead, we provide evidence that one alternative biological role for soybean Itpks is to interconvert the Cl− channel inhibitor, Ins(3,4,5,6)P4, and its metabolic precursor, Ins(1,3,4,5,6)P5, within a substrate cycle. The soybean Itpks also phosphorylated Ins(3,4,6)P3 to Ins(1,3,4,6)P4 which was further phosphorylated to Ins(1,3,4,5,6)P5 by soybean Ipk2. Thus, soybean Itpks may participate in an inositol lipid-independent pathway of InsP6 synthesis. [Copyright &y& Elsevier]

Abstract: Contemporary phytase research is primarily concerned with ameliorating the problem of inadequate digestion of inositol hexakisphosphate (phytate; InsP6) in monogastric farm animal feed, so as to reduce the pollution that results from the high phosphate content of the manure. In the current study we pursue a new, safe and cost-effective solution. We demonstrate that the rate of hydrolysis of InsP6 by recombinant avian MINPP (0.7μmol/mg protein/min) defines it as by far the most active phytase found to date in any animal cell (the corresponding activity of recombinant mammalian MINPP is only 0.006μmol/mg protein/min). Although avian MINPP has less than 20% sequence identity with microbial phytases, we create a homology model of MINPP in which it is predicted that the structure of the phytase active site is well-conserved. This model is validated by site-directed mutagenesis and by use of a substrate analogue, scyllo-InsP6, which we demonstrate is only a weak MINPP substrate. In a model chicken cell line, we overexpressed a mutant form of MINPP that is secretion-competent. This version of the enzyme was actively secreted without affecting either cell viability or the cellular levels of any inositol phosphates. Our studies offer a genetic strategy for greatly improving dietary InsP6 digestion in poultry. [Copyright &y& Elsevier]

Abstract: The dose-dependent inhibitory effect of sodium phytate (myo-inositol-hexaphosphate) on absorption of zinc and retention of calcium was studied in man. No systematic study of this dose–response effect has been reported to this time. Forty subjects were served meals containing white wheat rolls without/with additions of phytate. Ten subjects were given test meals containing one or two of the studied levels of phytate and in addition all subjects were served meals to which no phytate was added. The zinc content was 3.1mg (47μmol) and the calcium content 266mg (6.6mmol). The rolls were labelled extrinsically with radioisotopes, 65Zn and 47Ca, and whole-body retention of both minerals was measured. Totally 105 meals were served, 36 meals in which no phytate was added and 9–10 meals on each level of phytate. The zinc absorption in meals to which either 0, 25, 50, 75, 100, 140, 175 or 250mg of phytate-P (0, 134, 269, 403, 538, 753, 941 or 1344μmol phytate) had been added was 22%, 16%, 14%, 11%, 7%, 7%, 7% and 6%, respectively (mean values). The addition of 50mg phytate-P or more significantly decreased zinc absorption as compared to absorption from the test meals with no added phytate. The calcium retention at day 7 in the same meals was 31%, 28%, 27%, 26%, 22%, 19%, 14% and 11% (mean values). The addition of 100mg phytate-P or more significantly decreased calcium retention compared to the test meals with no added phytate. It was concluded that the inhibitory effect of phytate on the absorption of zinc and the retention of calcium was dose dependent. [Copyright &y& Elsevier]

Abstract: Phytic acid is enzymatically catabolized to numerous inositol polyphosphates and subsequently to myo-inositol and inorganic phosphate. These conversions are important in energy metabolism, metabolic regulation, and signal transduction pathways. A concise synthesis of phosphatase-resistant analogues of phytic acid (InsP6) is described in which the P-1 phosphate has been replaced by phosphorothioate, phosphoroselenate, or methylphosphonate moieties. In addition, a new synthesis of P-1 tethered aminoalkyl InsP6 and the corresponding affinity resin are described. [Copyright &y& Elsevier]

InsP6 is abundant in cereals and legumes. InsP6 and lower inositol phosphates, in particular InsP3, participate in important intracellular processes. In addition, InsP6 possess significant health benefits, such as anti-cancer effect, kidney stones prevention, lowering serum cholesterol. Because of the insensitivity of existing methods for determination of non-radiolabeled inositol phosphates, little is known about the natural occurrence, much less on the concentrations of InsP6 and InsP3 in biological samples. Using gas chromatography-mass detection analysis of HPLC chromatographic fractions, we report a measurement of unlabeled total InsP3 and InsP6 (a) as they occur within cells culture, tissues, and plasma, and (b) their changes depending on the presence of exogenous InsP6. When rats were fed on a purified diet in which InsP6 was undetectable (AIN-76A) the levels of InsP6 in brain were 3.35 ± 0.57 (SE) μmol·kg−1 and in plasma 0.023 ± 0.008 (SE) μmol·l−1. The presence of InsP6 in diet dramatically influenced its levels in brain and in plasma. When rats were given an InsP6-sufficient diet (AIN-76A + 1% InsP6), the levels of InsP6 were about 100-fold higher in brain tissues (36.8 ± 1.8 (SE)) than in plasma (0.29 ± 0.02 (SE)); InsP6 concentrations were 8.5-fold higher than total InsP3 concentrations in either plasma (0.033 ± 0.012 (SE)) and brain (4.21 ± 0.55 (SE)). When animals were given an InsP6-poor diet (AIN-76A only), there was a 90% decrease in InsP6 content in both brain tissue and plasma (p < 0.001); however, there was no change in the level of total InsP3. In non-stimulated malignant cells (MDA-MB 231 and K562) the InsP6 contents were 16.2 ± 9.1 (SE) μmol·kg−1 for MDA-MB 231 cells and 15.6 ± 2.7 (SE) for K 562 cells. These values were around 3-fold higher than those of InsP3 (4.8 ± 0.5 μmol·kg−1 and 6.9 ± 0.1 (SE) for MDA-MB 231 and K562 cells respectively). Treatment of malignant cells with InsP6 resulted in a 2-fold increase in the intracellular concentrations of total InsP3 (9.5 ± 1.3 (SE) and 10.8 ± 1.0 (SE) μmol·kg−1 for MDA-MB 231 and K562 cells respectively, p < 0.05), without changes in InsP6 levels. These results indicate that exogenous InsP6 directly affects its physiological levels in plasma and brain of normal rats without changes on the total InsP3 levels. Although a similar fluctuation of InsP6 concentration was not seen in human malignant cell lines following InsP6 treatment, an increased intracellular levels of total InsP3 was clearly observed. [Copyright &y& Elsevier]

Visual arrestin (Arr1) terminates rhodopsin signaling by blocking its interaction with transducin. To do this, Arr1 translocates from the inner to the outer segment of photoreceptors upon light stimulation. Mounting evidence indicates that inositol phosphates (InsPs) affect Arr1 activity, but the Arr1-InsP molecular interaction remains poorly defined. We report the structure of bovine Arr1 in a ligand-free state featuring a near-complete model of the previously unresolved C-tail, which plays a crucial role in regulating Arr1 activity. InsPs bind to the N-domain basic patch thus displacing the C-tail, suggesting that they prime Arr1 for interaction with rhodopsin and help direct Arr1 translocation. These structures exhibit intact polar cores, suggesting that C-tail removal by InsP binding is insufficient to activate Arr1. These results show how Arr1 activity can be controlled by endogenous InsPs in molecular detail. [Display omitted] • The regulatory C-tail/N-domain interaction of arrestin-1 is resolved • Arrestin-1 binds InsPs using many of the same residues that hold the C-tail • InsP binding does not activate arrestin-1, but partially removes the C-tail Sander et al. resolve how the C-tail regulatory domain and the N-domain of arrestin 1 interact and maintain correct cellular localization. They further show that InsPs, including InsP 3 , compete for N-domain contacts responsible for binding the C-tail, thereby priming the structure for translocation. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanisms of retroviral assembly has been a decades-long endeavor. With the recent discovery of inositol hexakisphosphate (IP6) acting as an assembly co-factor for human immunodeficiency virus (HIV), great strides have been made in retroviral research. In this review, the enzymatic pathways to synthesize and metabolize inositol phosphates (IPs) relevant to retroviral assembly are discussed. The functions of these enzymes and IPs are outlined in the context of the cellular biology important for retroviruses. Lastly, the recent advances in understanding the role of IPs in retroviral biology are surveyed. [ABSTRACT FROM AUTHOR]

Protein purification is the most basic and critical step for protein biophysical and biochemical studies to understand its function and structure. Various fusion tags and proteases have been developed and assembled in expression and purification system. However, it is one of the fields that continues to innovate to develop improved systems that are more efficient, simpler, and less expensive. An efficient self-cleavage C-terminal fusion system was developed using the inositol hexakisphosphate-inducible Vibrio cholerae MARTX Vc toxin cysteine protease domain (CPD). CPD fusion proteins are expressed from the T7 promoter and purified using a 6xHis-tag with immobilized-metal affinity chromatography. The C-terminal CPD-tag is removed by self-cleavage at the final purification stage. Here, we describe an efficient cloning method using Gibson assembly, followed by expression and purification of tagless recombinant proteins of interest using CPD self-cleavage.