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YqeK is a bacterial HD-domain metalloprotein that hydrolyzes the putative second messenger diadenosine tetraphosphate (Ap4A). Elevated Ap4A levels are primarily observed upon exposure of bacteria to factors such as heat or oxidative stress and cause pleiotropic effects, including antibiotic sensitivity and disrupted biofilm formation. Ap4A thus plays a central role in bacterial physiology and metabolism, and its hydrolysis by YqeK is intimately linked to the ability of these microbes to cope with stress. Although YqeK is reported to hydrolyze Ap4A under aerobic conditions, all four existing crystal structures reveal an active site that consists of a diiron center, portraying a cryptic chemical nature for the active metallocofactor. This study examines two YqeK proteins from two ecologically diverse parent organisms: the obligate anaerobe Clostridium acetobutylicum and the facultative aerobe Bacillus halodurans. Both enzymes utilize Fe-based cofactors for catalysis, while under ambient or oxidative conditions, Bh YqeK hydrolyzes Ap4A more efficiently compared to Ca YqeK. This redox-dependent activity difference stems from the following two molecular mechanisms: the incorporation of mixed-metal, Fe-based bimetallic cofactors, in which the second metal is redox inert (i.e., Fe–Zn) and the upshift of the Fe–Fe cofactor reduction potentials. In addition, three strictly conserved, positively charged residues vicinal to the active site are critical for tuning Ap4A hydrolysis. In conclusion, YqeK is an Fe-dependent phosphohydrolase that appears to have evolved to permit Ap4A hydrolysis under different environmental niches (aerobic vs. anaerobic) by expanding its cofactor configuration and O2 tolerance. [ABSTRACT FROM AUTHOR]

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) initially emerged as oral antidiabetic medication but were subsequently discovered to exhibit pleiotropic actions. Insomnia is a prevalent and debilitating sleep disorder. To date, the causality between SGLT2 inhibitors and insomnia remains unclear. This study aims to evaluate the causality between SGLT2 inhibitors and insomnia and identify potential plasma protein mediators. Using a two-sample Mendelian Randomization (MR) analysis, we estimated the causality of SGLT2 inhibition on insomnia and sleep duration. Additionally, employing a two-step and proteome-wide MR analysis, we evaluated the causal link of SGLT2 inhibition on 4907 circulating proteins and the causality of SGLT2 inhibition-driven plasma proteins on insomnia. We applied a false discovery rate (FDR) correction for multiple comparisons. Furthermore, mediation analyses were used to identify plasma proteins that mediate the effects of SGLT2 inhibition on insomnia. SGLT2 inhibition was negatively correlated with insomnia (odds ratio [OR] = 0.791, 95 % confidence interval [CI] [0.715, 0.876], P = 5.579*10^-6) and positively correlated with sleep duration (β = 0.186, 95 % CI [0.059, 0.314], P = 0.004). Among the 4907 circulating proteins, diadenosine tetraphosphatase (Ap4A) was identified as being linked to both SGLT2 inhibition and insomnia. Mediation analysis indicated that the effect of SGLT2 inhibition on insomnia partially operates through Ap4A (β = −0.018, 95 % CI [−0.036, −0.005], P = 0.023), with a mediation proportion of 7.7 %. The study indicated a causality between SGLT2 inhibition and insomnia, with plasma Ap4A potentially serving as a mediator. • Few previous studies have explored the relationship between SGLT2 inhibition and insomnia. • Mendelian randomization made us able to evaluate the causality of SGLT2 inhibition on insomnia. • Genetically-predicted SGLT2 inhibition negatively correlated to insomnia. • Plasma AP4A level mediates the inhibitory effect of SGLT2 inhibition on insomnia. [ABSTRACT FROM AUTHOR]

The recent expansion of the field of RNA chemical modifications has changed our understanding of post‐transcriptional gene regulation. Apart from internal nucleobase modifications, 7‐methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non‐canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non‐canonical RNA cap, diadenosine tetraphosphate (Ap4A), in human and rat cell lines. Ap4A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non‐canonical initiating nucleotide (NCIN). Using liquid chromatography‐mass spectrometry (LC–MS), we show that the amount of capped Ap4A‐RNA is independent of the cellular concentration of Ap4A. A decapping enzyme screen identifies two enzymes cleaving Ap4A‐RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4A‐RNA and show that although it is not translated, Ap4A‐RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation. [ABSTRACT FROM AUTHOR]

Dinucleoside polyphosphates, a class of nucleotides found amongst all the Trees of Life, have been gathering a lot of attention in the past decades due to their putative role as cellular alarmones. In particular, diadenosine tetraphosphate (AP4A) has been widely studied in bacteria facing various environmental challenges and has been proposed to be important for ensuring cellular survivability through harsh conditions. Here, we discuss the current understanding of AP4A synthesis and degradation, protein targets, their molecular structure where possible, and insights into the molecular mechanisms of AP4A action and its physiological consequences. Lastly, we will briefly touch on what is known with regards to AP4A beyond the bacterial kingdom, given its increasing appearance in the eukaryotic world. Altogether, the notion that AP4A is a conserved second messenger in organisms ranging from bacteria to humans and is able to signal and modulate cellular stress regulation seems promising. [ABSTRACT FROM AUTHOR]

Dinucleoside tetraphosphates, often described as alarmones because their cellular concentration increases in response to stress, have recently been shown to function in bacteria as precursors to nucleoside tetraphosphate (Np4) RNA caps. Removal of this cap is critical for initiating 5' end-dependent degradation of those RNAs, potentially affecting bacterial adaptability to stress; however, the predominant Np4 decapping enzyme in proteobacteria, ApaH, is inactivated by the very conditions of disulfide stress that enable Np4-capped RNAs to accumulate to high levels. Here, we show that, in Escherichia coli cells experiencing such stress, the RNA pyrophosphohydrolase RppH assumes a leading role in decapping those transcripts, preferring them as substrates over their triphosphorylated and diphosphorylated counterparts. Unexpectedly, this enzyme recognizes Np4-capped 5' ends by a mechanism distinct from the one it uses to recognize other 5' termini, resulting in a one-nucleotide shift in substrate specificity. The unique manner in which capped substrates of this kind bind to the active site of RppH positions the δ-phosphate, rather than the β-phosphate, for hydrolytic attack, generating triphosphorylated RNA as the primary product of decapping. Consequently, a second RppH-catalyzed deprotection step is required to produce the monophosphorylated 5' terminus needed to stimulate rapid RNA decay. The unconventional manner in which RppH recognizes Np4-capped 5' ends and its differential impact on the rates at which such termini are deprotected as a prelude to RNA degradation could have major consequences for reprogramming gene expression during disulfide stress. [ABSTRACT FROM AUTHOR]

Cellular homeostasis requires adaption to environmental stress. In response to various environmental and genotoxic stresses, all cells produce dinucleoside polyphosphates (NpnNs), the best studied of which is diadenosine tetraphosphate (Ap4A). Despite intensive investigation, the precise biological roles of these molecules have remained elusive. However, recent studies have elucidated distinct and specific signaling mechanisms for these nucleotides in prokaryotes and eukaryotes. This review summarizes these key discoveries and describes the mechanisms of Ap4A and Ap4N synthesis, the mediators of the cellular responses to increased intracellular levels of these molecules and the hydrolytic mechanisms required to maintain low levels in the absence of stress. The intracellular responses to dinucleotide accumulation are evaluated in the context of the “friend” and “foe” scenarios. The “friend (or alarmone) hypothesis” suggests that ApnN act as bona fide secondary messengers mediating responses to stress. In contrast, the “foe” hypothesis proposes that ApnN and other NpnN are produced by non-canonical enzymatic synthesis as a result of physiological and environmental stress in critically damaged cells but do not actively regulate mitigating signaling pathways. In addition, we will discuss potential target proteins, and critically assess new evidence supporting roles for ApnN in the regulation of gene expression, immune responses, DNA replication and DNA repair. The recent advances in the field have generated great interest as they have for the first time revealed some of the molecular mechanisms that mediate cellular responses to ApnN. Finally, areas for future research are discussed with possible but unproven roles for intracellular ApnN to encourage further research into the signaling networks that are regulated by these nucleotides.

Human histidine triad nucleotide‐binding protein 2 (hHINT2) is an important player in human mitochondrial bioenergetics, but little is known about its catalytic capabilities or its nucleotide phosphoramidate prodrug (proTide)‐activating activity akin to the cytosolic isozyme hHINT1. Here, a similar substrate specificity profile (kcat/Km) for model phosphoramidate substrates was found for hHINT2 but with higher kcat and Km values when compared with hHINT1. A broader pH range for maximum catalytic activity was determined for hHINT2 (pK1 = 6.76 ± 0.16, pK2 = 8.41 ± 0.07). In addition, the known hHINT1‐microphthalmia‐inducing transcription factor‐regulating molecule Ap4A was found to have no detectable binding to HINT1 nor HINT2 by isothermal titration calorimetry. These results demonstrate that despite differences in their sequence and localization, HINT1 and HINT2 have similar nucleotide substrate specificities, which should be considered in future proTide design and in studies of their natural function. [ABSTRACT FROM AUTHOR]

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5' end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant. [ABSTRACT FROM AUTHOR]

The recent expansion of the field of RNA chemical modifications has changed our understanding of post-transcriptional gene regulation. Apart from internal nucleobase modifications, 7-methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non-canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non-canonical RNA cap, diadenosine tetraphosphate (Ap 4 A), in human and rat cell lines. Ap 4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non-canonical initiating nucleotide (NCIN). Using liquid chromatography-mass spectrometry (LC-MS), we show that the amount of capped Ap 4 A-RNA is independent of the cellular concentration of Ap 4 A. A decapping enzyme screen identifies two enzymes cleaving Ap 4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap 4 A-RNA and show that although it is not translated, Ap 4 A-RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The pathogenesis of glaucoma involves numerous intracellular mechanisms including the purinergic system contribution. Furthermore, the presence and release of nucleotides and dinucleotides during the glaucomatous damage and the maintenance of degradation machinery through ecto-nucleotidase activity are participating in the modulation of the suitable extracellular complex balance. The aim of this study was to investigate the levels of diadenosine tetraphosphate (Ap4A) and the pattern of ecto-nucleotidase activity expression in glaucomatous retinas during the progress the pathology. Ap4A levels were analyzed by HPLC in glaucomatous retinas from the DBA/2J mice at 3, 9, 15, and 23 months of age. For that, retinas were dissected as flattened whole-mounts and stimulated in Ringer buffer with or without 59 mM KCl. NPP1 expression was analyzed by RT-PCR and western blot and its distribution was assessed by immunohistochemistry studies examined under confocal microscopy. Glaucomatous mice exhibited Ap4A values, which changed in stimulated retinas as long as the pathology progressed varying from 0.73 ± 0.04 (3 months) to 0.170 ± 0.05 pmol/mg retina (23 months). Concomitantly, NPP1 expression was significantly increased (82.15%) in the DBA/2J mice at 15 months. Furthermore, immunohistochemical studies showed that NPP1 labeling was stronger in OPL and IPL labeling tangentially in the vitreal part of the retina and was upregulated at 15 months of age. Our findings demonstrate that Ap4A decreased levels may be related with exacerbated activity of NPP1 protein in glaucomatous degeneration and in this way contributing to elucidate different mechanisms involved in retinal impairment in glaucomatous degeneration. [ABSTRACT FROM AUTHOR]

Glaucoma is a neuropathology, often accompanied by an elevated intraocular pressure (IOP), which can lead to blindness. Since DBA/2J mice develop glaucoma, several studies of the physiopathology of glaucoma have been reported in this animal model. It is also known that purinergic receptors are involved in the pathology of glaucoma by controlling aqueous humor production and drainage and therefore controlling IOP. There are no studies on purinergic receptors in the DBA/2J model of glaucoma and their relation to the development of the pathology, so the aim of this study was to make an approach to the purinergic mechanisms involved in glaucoma. All the experiments were performed using DBA/2J and C57BL/6J mice and investigating P2Y1, P2Y2, and P2Y6 receptors. IOP measurements were made with a non-invasive rebound tonometer, and animals were instilled with diadenosine tetraphosphate (Ap4A) and the corresponding purinergic antagonists in order to see their effects on IOP. The expression of mRNA for P2Y1, P2Y2, and P2Y6 purinergic receptors was carried out by quantitative real-time PCR. Additionally, P2Y-receptor expression was performed by immunohistochemical techniques carried out on the ciliary processes. The results showed that IOP decreases when Ap4A was instilled and that the expressions of the analyzed purinergic receptors were stable throughout all the ages under study in the C57BL/6J mice (control mice). On the other hand, there were significant changes in the purinergic receptor expression in DBA/2J suggesting that elevated IOP in these animals could be related to an increase of P2Y2 expression and a decrease in P2Y1 receptors.

Glaucoma is a neuropathology, often accompanied by an elevated intraocular pressure (IOP), which can lead to blindness. Since DBA/2J mice develop glaucoma, several studies of the physiopathology of glaucoma have been reported in this animal model. It is also known that purinergic receptors are involved in the pathology of glaucoma by controlling aqueous humor production and drainage and therefore controlling IOP. There are no studies on purinergic receptors in the DBA/2J model of glaucoma and their relation to the development of the pathology, so the aim of this study was to make an approach to the purinergic mechanisms involved in glaucoma. All the experiments were performed using DBA/2J and C57BL/6J mice and investigating P2Y1, P2Y2, and P2Y6 receptors. IOP measurements were made with a non-invasive rebound tonometer, and animals were instilled with diadenosine tetraphosphate (Ap4A) and the corresponding purinergic antagonists in order to see their effects on IOP. The expression of mRNA for P2Y1, P2Y2, and P2Y6 purinergic receptors was carried out by quantitative real-time PCR. Additionally, P2Y-receptor expression was performed by immunohistochemical techniques carried out on the ciliary processes. The results showed that IOP decreases when Ap4A was instilled and that the expressions of the analyzed purinergic receptors were stable throughout all the ages under study in the C57BL/6J mice (control mice). On the other hand, there were significant changes in the purinergic receptor expression in DBA/2J suggesting that elevated IOP in these animals could be related to an increase of P2Y2 expression and a decrease in P2Y1 receptors. [ABSTRACT FROM AUTHOR]

Diadenosine tetraphosphate abbreviated Ap4A is a naturally occurring dinucleotide, which is present in most of the ocular fluids. Due to its intrinsic resistance to enzyme degradation compared to mononucleotides, this molecule can exhibit profound actions on ocular tissues, including the ocular surface, ciliary body, trabecular meshwork, and probably the retina. The actions of Ap4A are mostly carried out by P2Y2 receptors, but the participation of P2X2 and P2Y6 in processes such as the regulation of intraocular pressure (IOP), together with the P2Y2, is pivotal. Beyond the physiological role, this dinucleotide can present on the ocular surface keeping a right production of tear secretion or regulating IOP. It is important to note that exogenous application of Ap4A to cells or animal models can significantly modify pathophysiological conditions and thus is an attractive therapeutic molecule. The ocular location where Ap4A actions have not been fully elucidated is in the retina. Although some analogues show interesting actions on pathological situations such as retinal detachment, little is known about the real effect of this dinucleotide, this being one of the challenges that require pursuing in the near future. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the possible use of soft contact lenses (CL) to improve the secretagogue role of diadenosine tetraphosphate (Ap4A) promoting tear secretion. Methods: Two conventional hydrogel CL (Omafilcon A and Ocufilcon D) and two silicone hydrogel (SiH) CL (Comfilcon A and Balafilcon A) were used. Ap4A was loaded into the lenses by soaking in a 1 mM Ap4A solution during 12 h. In vitro experiments were performed by placing the lenses in multi-wells during 2 h containing 1 ml of ultrapure water. 100 μl aliquots were taken at time zero and every minute for the first 10 min, and then every 15 min. In vivo experiments were performed in New Zealand rabbits and both the dinucleotide release from SiH and tear secretion were measured by means of Schirmer strips and high-pressure liquid chromatography (HPLC) analysis. Results: Ap4A in vitro release experiments in hydrogel CL presented a release time 50 (RT50) of 3.9 ± 0.2 min and 3.1 ± 0.1 min for the non-ionic and the ionic CL, respectively. SiH CL released also Ap4A with RT50 values of 5.1 ± 0.1 min for the non-ionic and 2.7 ± 0.1 min for the ionic CL. In vivo experiments with SiH CL showed RT50 values of 9.3 ± 0.2 min and 8.5 ± 0.2 min for the non-ionic and the ionic respectively. The non-ionic lens Ap4A release was able to induce tear secretion above baseline tear levels for almost 360 min. Conclusion: The delivery of Ap4A is slower and the effect lasts longer with non-ionic lenses than ionic lenses.

Nucleotide second messengers act as intracellular 'secondary' signals that represent environmental or cellular cues, i.e. the 'primary' signals. As such, they are linking sensory input with regulatory output in all living cells. The amazing physiological versatility, the mechanistic diversity of second messenger synthesis, degradation, and action as well as the high level of integration of second messenger pathways and networks in prokaryotes has only recently become apparent. In these networks, specific second messengers play conserved general roles. Thus, (p)ppGpp coordinates growth and survival in response to nutrient availability and various stresses, while c-di-GMP is the nucleotide signaling molecule to orchestrate bacterial adhesion and multicellularity. c-di-AMP links osmotic balance and metabolism and that it does so even in Archaea may suggest a very early evolutionary origin of second messenger signaling. Many of the enzymes that make or break second messengers show complex sensory domain architectures, which allow multisignal integration. The multiplicity of c-di-GMP-related enzymes in many species has led to the discovery that bacterial cells are even able to use the same freely diffusible second messenger in local signaling pathways that can act in parallel without cross-talking. On the other hand, signaling pathways operating with different nucleotides can intersect in elaborate signaling networks. Apart from the small number of common signaling nucleotides that bacteria use for controlling their cellular "business," diverse nucleotides were recently found to play very specific roles in phage defense. Furthermore, these systems represent the phylogenetic ancestors of cyclic nucleotide-activated immune signaling in eukaryotes., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

The insertion domain 1 (ID1) of class IIA dimeric glycyl-tRNA synthetase (α 2 GRS) is an appended domain in the core catalytic region of the enzyme. ID1 has been shown to play a role in tRNA aminoacylation, mediating interaction with the acceptor arm of tRNA and diadenosine tetraphosphate (Ap4A) synthesis. Mutations in α 2 GRS, including those in the ID1 region, have been implicated in distal hereditary motor neuropathy-V (dHMN-V) and Charcot–Marie–Tooth (CMT) disease. Through sequence and structure based evolutionary analysis, we show that ID1 of α 2 GRS is a rubredoxin-like zinc ribbon domain. The zinc-chelating cysteines of ID1 are well conserved in all archaeal versions of the enzyme and also in several eukaryotes, which most likely have acquired them via horizontal gene transfer from bacteria; but in all other eukaryotes, the zinc-chelating residues are not preserved. ID1 from bacteria display a selective preservation of zinc-binding residues, ranging from complete conservation to complete loss. The ID1 from different organisms harbor variable-sized non-conserved insertions between the two zinc-binding half-sites of the zinc ribbon. Three of the previously identified CMT-associated mutations in α 2 GRS, viz., human D146N, mouse C157R and human S211F, are located in the zinc ribbon region of ID1. Interestingly, human Asp146 which is implicated in the synthesis of Ap4A, a molecule known to act during neuronal transmission, has also been reported to be mutated in dHMN-V, suggesting a possible link between hereditary motor neuropathy and Ap4A synthesis. [ABSTRACT FROM AUTHOR]

Lysyl‐tRNA synthetase (KRS) interacts with the laminin receptor (LR/RPSA) and enhances laminin‐induced cell migration in cancer metastasis. In this nuclear magnetic resonance (NMR)‐based study, we show that the anticodon‐binding domain of KRS binds directly to the C‐terminal region of 37LRP, and the previously found inhibitors BC‐K‐01 and BC‐K‐YH16899 interfere with KRS–37LRP binding. In addition, the anticodon‐binding domain of KRS binds to laminin, observed by NMR and SPR. These results provide crucial insights into the structural characteristics of the KRS–LR interaction on the cell surface.Anticodon‐binding domain of KRS binds to the C‐terminal region of 37LRP. BC‐K‐01 and BC‐K‐YH16899 interfere with the 37LRP‐induced KRS signal perturbation. Anticodon‐binding domain of KRS binds to laminin. [ABSTRACT FROM AUTHOR]

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The heat-inducible lysyl-tRNA synthetase from Escherichia coli (LysU; EC6/1/1/6.html) converts ATP to diadenosine tri- and tetraphosphates (Ap3A/Ap4A) in the presence of l-lysine/Mg2+/Zn2+. To understand LysU in more detail, 26 mutants were prepared: six of E264, four of R269 and sixteen mutants by alanine-scanning of the inner shell/motif 2 loop. In the presence of glycerol and absence of exogenously added Zn2+/ l-lysine, we unexpectedly found that E264K catalysed the production of glycerol-3-phosphate, powered by ATP turnover to ADP. E264Q and E264N are also capable of this activity, but all three show little formation of Ap4A/Ap3A under normal conditions (additional Zn2+/ l-lysine/Mg2+). By contrast, wild-type LysU has a weaker glycerol kinase-like capability in the absence of Zn2+ and is dominated by Ap4A/Ap3A synthesis in its presence. Kinetic and isothermal titration calorimetry results suggest that E264 is a crucial residue for Zn2+ promotion of Ap4A/Ap3A synthesis. This is consistent with the hypothesis that E264 provides an anchor point for a Zn2+ ion complexed to the active site, with simultaneous coordination to the enzyme bound lysyl-adenylate intermediate and secondary substrate ATP/ADP. The glycerol kinase-like activity is uncovered on disruption of this specific coordination. [ABSTRACT FROM AUTHOR]

Abstract: We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap4A)-‘molecular hook’ attached to magnetic beads enabling the isolation of Ap4A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap4A-binding partners, and we have expectations that others can be found with further technical improvements in our methods. [Copyright &y& Elsevier]

Purpose: We have previously demonstrated the importance of P2Y2 receptors in the corneal re-epithelialization effect triggered by diadenosine tetraphosphate (Ap4A). In addition, we have also shown the ERK1/2 and ROCK-I activation in Ap4A-wound repair response. Phospholipase C/Protein Kinase C (PLC/PKC) pathway activation has been suggested as a molecular mechanism of growth factors-modulated corneal cell migration and P2Y2 agonists. Hence, the aim of this study is to investigate the role of PLC/PKC cascade in the modification of re-epithelialization rate triggered by Ap4A in an established corneal epithelial cell line (Statens Seruminstitut rabbit cornea [SIRC] cells). Methods: In wounded confluent SIRC cell monolayers and in the presence or absence of Ap4A 100 µM, a group of PLC/PKC inhibitors (U73122 3 µM, Staurosporine 1 nM and Bisindolylmaleimide-I 10 µM) and activator (PDBU 1 µM) were assayed and the migration rate was evaluated. Also, the activation of ERK1/2 and ROCK-I was examined by Western blot assay after treatment with or without Ap4A, U73122, Staurosporine, Bisindolylmaleimide-I and PDBU. Results: Pre-treatment of wounded SIRC cells with PLC/PKC inhibitors significantly diminished the Ap4A-stimulated cell migration rate. Furthermore, PLC/PKC inhibitors also reduced ERK1/2 phosphorylation and ROCK-I activation triggered by Ap4A. Conclusions: The present study shows the involvement of PLC/PKC pathway in the activation of ERK1/2 and ROCK-I downstream signal transduction pathways stimulated by Ap4A/P2Y2 receptor during corneal epithelial wound repair. [ABSTRACT FROM AUTHOR]

Abstract: Background: Mono and dinucleoside polyphosphates (pnNs and NpnNs) exist in living organisms and induce diverse biological effects through interaction with intracellular and cytoplasmic membrane proteins. The source of these compounds is associated with secondary activities of a diverse group of enzymes. Scope of review: Here we discuss the mechanisms that can promote their synthesis at a molecular level. Although all the enzymes described in this review are able to catalyse the in vitro synthesis of NpnNs (and/or pnN), it is not clear which ones are responsible for their in vivo accumulation. Major conclusions: Despite the large amount of knowledge already available, important questions remain to be answered and a more complete understanding of pnNs and NpnNs synthesis mechanisms is required. With the possible exception of (GTP:GTP guanylyltransferase of Artemia), all enzymes able to catalyse the synthesis of pnNs and NpnNs are unspecific and the factors that can promote their synthesis relative to the canonical enzyme activities are unclear. General significance: The fact that pnNs and NpnNs syntheses are promiscuous activities of housekeeping enzymes does not reduce its physiological or pathological importance. Here we resume the current knowledge regarding their enzymatic synthesis and point the open questions on the field. [Copyright &y& Elsevier]

The article discusses a study which examined the expression and functions of nucleoside diphosphate linked moiety X (Nudix)-type motif 2 (NUDT2) in human breast carcinoma. The results of the study suggested that NUDT2 functions as an estrogen-repressed gene. NUDT2 was found to be induced by HER2 pathways in the breast cancer cells. The study concluded that NUDT2 plays a role in promoting the proliferation of cancer cells in the breast.

Abstract: Previous studies have shown the presence of diadenosine tetraphosphate (Ap4A) and pentaphosphate (Ap5A) in the aqueous humour (AH) of different species. When topically applied to the rabbit cornea, Ap4A decreased IOP while Ap5A increased it. Here we study the presence of dinucleoside polyphosphates in the AH from human patients with or without glaucoma. AH was obtained at the time of cataract surgery from patients with (n =16) or without (n =10) primary open-angle glaucoma. AH (0.1–0.2ml) was collected at the beginning of surgery through a corneal paracentesis and immediately cooled in liquid nitrogen, kept frozen and protected from light. AH aliquots were analyzed by HPLC for the presence of Ap4A and Ap5A. Both, Ap4A and Ap5A were detected in the AH of both experimental groups. No significant differences were found for Ap5A. In contrast, Ap4A levels were increased by ∼15-fold in the AH from glaucomatous eyes ranging from 19.5±9.2nM in normal individuals to 286.03±30.9nM in glaucomatous patients. In conclusion, both Ap4A and Ap5A were detected for the first time in human AH. Interestingly, glaucomatous eyes presented elevated concentrations of Ap4A compared to controls. The role of Ap4A needs to be elucidated but it may help to protect the autonomic innervation in the ciliary body/trabecular meshwork. Also, because of its higher levels in glaucoma patients it may be considered as a possible glaucoma biomarker. [Copyright &y& Elsevier]

Abstract: The distribution of binding sites for [35S]5′-O-(2-thiodiphosphate) ([35S]ADPβS), a radioligand of P2Y1,12,13 receptors, and of ecto-nucleotide pyrophosphatase phosphodiesterase activity were analyzed in the rat forebrain. Binding sites for the radilogand are widespreadly distributed in the rat forebrain, showing the highest density in hypothalamus. K d values were in the range 1–2nM. Diadenosine tetraphosphate (Ap4A) and diethenoadenosine tetraphosphate, ɛ-(Ap4A), displaced the radioligand, indicating dinucleotide binding to ADPβS-recognizing P2Y receptors. Activity ecto-nucleotide pyrophosphatase phosphodiesterase 1 (NPP1), able to hydrolyze Ap4A and other diadenosine polyphosphates, is also widely distributed through the rat forebrain, with the highest activity in hypothalamus. These results suggests that Ap4A signalling mediated by P2Y1,12,13 receptors and enzymatically regulated by NPP1 activity may be particularly important in hypothalamus and add new support for neurotransmitter/neuromodulatory functions of diadenosine polyphosphates in brain. [Copyright &y& Elsevier]

Abstract: Diadenosine 5′,5″-P1P4 tetraphosphate (Ap4A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap4A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap4A binds Zn(II) weakly, with an apparent binding constant of ca. 104 at neutral pH. Such a low stability of Zn(II) complexes with Ap4A excludes a possibility for interactions between these two agents in vivo. [Copyright &y& Elsevier]

Abstract: Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA-amido conjugates, the most important of which appears to be JA-Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono- and dinucleoside polyphosphates, which are side-reaction products of many enzymes forming acyl∼adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile-conjugates was observed for (±)-JA and 9,10-dihydro-JA, while the rate of conjugation with 12-hydroxy-JA and OPC-4 (3-oxo-2-(2Z-pentenyl)cyclopentane-1-butyric acid) was only about 1–2% that for (±)-JA. Of the two stereoisomers of JA, (−)-JA and (+)-JA, rate of synthesis of the former was about 100-fold faster than for (+)-JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg2+, (−)-JA and tripolyphosphate the ligase produces adenosine 5′-tetraphosphate (p4A); (2) addition of isoleucine to that mixture halts the p4A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap3A) nor diadenosine tetraphosphate (Ap4A) and (4) Ap4A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA-Ile. [Copyright &y& Elsevier]