Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5'-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5'-diphosphates (5'pp). Genomes from mammalian reoviruses with 5'pp termini, 5'pp-RNA isolated from yeast L-A virus, and base-paired 5'pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5'pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5'pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5'pp-RNA, like that of 5'ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (trans,trans)-farnesyl diphosphate (FPP) (1a) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (cis,trans)-FPP (1b) as an alternative substrate. Strikingly, TEAS was catalytically robust in the enzymatic conversion of (cis,trans)-FPP (1b) to exclusively (>/=99.5%) cisoid products. Further, crystallographic characterization of wild-type TEAS and a catalytically promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-trans FPP (1a) and (cis,trans)-FPP (1b) revealed binding modes consistent with preorganization of the farnesyl chain. These results provide a structural glimpse into both cisoid and transoid cyclization pathways efficiently templated by a single enzyme active site, consistent with the recently elucidated stereochemistry of the cisoid products. Further, computational studies using density functional theory calculations reveal concerted, highly asynchronous cyclization pathways leading to the major cisoid cyclization products. The implications of these discoveries for expanded sesquiterpene diversity in nature are discussed.

We report on the successful synthesis of diammonium magnesium dihydrogendiphosphate (V) dihydrate compound (NH4)2Mg(H2P2O7)2•2H2O using a wet chemical route. Single crystal X-ray diffraction analysis and micro Raman spectroscopy are employed to characterize the compound. We demonstrate, using a multidisciplinary approach, that this compound is ideal for carbon dioxide (CO2) capture in addition to other anthropogenic gasses. We show here -from both an experimental as well as from a density functional theory (DFT) calculations routes- the potential for adopting this compound into domestic air-conditioning units (ACUs). From these experiments, the resistance to bacterial growth is also investigated, which is critical for the adoption of this compound in ACUs. Our  compound exhibits a higher methane (CH4) sorptivity as compared to CO2 at 25 °C and 45 °C under pressures up to 50 bars. Furthermore, DFT electronic structure calculations are used to compute the main structural and electronic properties of the compound, taking into consideration the characteristics of the identified pores as a function of the progressive CO2 vs. CH4 loadings. Finally, the antibacterial assay reveals a strong antibacterial activity against the tested Gram-positive and Gram-negative bacteria, with a large zone of inhibition against the tested E. Coli, S. Aureus and K. Pneumonia.

Ribonucleotide reductase catalyzes the conversion of ribonucleotide diphosphates to deoxyribonucleotide diphosphates. The functional enzyme consists of two subunits - one large (RRM1) and one small (RRM2 or RRM2b) subunit. Expression levels of each subunit have been implicated in prognostic outcomes in several different types of cancers.

Analysis of the updated reference tomato genome found 34 full-length TPS genes and 18 TPS pseudogenes. Biochemical analysis has now identified the catalytic activities of all enzymes encoded by the 34 TPS genes: one isoprene synthase, 10 exclusively or predominantly monoterpene synthases, 17 sesquiterpene synthases and six diterpene synthases. Among the monoterpene and sesquiterpene and diterpene synthases, some use trans-prenyl diphosphates, some use cis-prenyl diphosphates and some use both. The isoprene synthase is cytosolic; six monoterpene synthases are plastidic, and four are cytosolic; the sesquiterpene synthases are almost all cytosolic, with the exception of one found in the mitochondria; and three diterpene synthases are found in the plastids, one in the cytosol and two in the mitochondria. New trans-prenyltransferases (TPTs) were characterised; together with previously characterised TPTs and cis-prenyltransferases (CPTs), tomato plants can make all cis and trans C10 , C15 and C20 prenyl diphosphates. Every type of plant tissue examined expresses some TPS genes and some TPTs and CPTs. Phylogenetic comparison of the TPS genes from tomato and Arabidopsis shows expansions in each clade of the TPS gene family in each lineage (and inferred losses), accompanied by changes in subcellular localisations and substrate specificities.

K(ATP) channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K(ATP) channel. In contrast, the R301C and R314C mutants, which are also "PIP2-insensitive," remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the K(ATP) channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since K(ATP) channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.

We have isolated and expressed a cDNA from the parasitic nematode Trichinella spiralis encoding a novel secreted nucleotidase which catalyses the hydrolysis of nucleoside 5'-diphosphates and 5'-monophosphates, but not 5'-triphosphates. The full length cDNA encodes a protein of 550 amino acids with an N-terminal signal peptide, but lacking a C-terminal signature sequence for addition of a glycosyl phosphatidylinositol (GPI) anchor. Expression in Pichia pastoris resulted in the secretion of an active enzyme with the catalytic properties of both a Mg2+-dependent diphosphohydrolase/apyrase and a 5'-nucleotidase. The protein sequence is homologous to 5'-nucleotidases from a wide variety of organisms but contains no sequences specifically conserved in apyrases, suggesting that it is a representative of a new class of secreted nucleotidase. The enzyme was essentially monospecific for AMP among the nucleoside 5'-monophosphates and catalysed the hydrolysis of nucleoside 5'-diphosphates in the order of UDP >> ADP. The diphosphatase activity was dependent on the presence of magnesium ions and a reducing agent, while the 5'-nucleotidase activity was enhanced by these additions. Kinetic analyses indicated that the enzyme exhibits allosteric behaviour. Determination of the number of active sites suggested that catalysis of the two different reactions occurs at the same active site. The data are discussed in terms of regulation of host purinergic signalling during infection.

The activity of ribonucleotide reductase (RNR), which catalyses the transformation of four ribonucleoside diphosphates (NDPs) to their corresponding deoxyribonucleoside diphosphates (dNDPs), is the main determiner of the cellular concentration of dNTP pools and should be tightly coordinated with DNA synthesis and cell-cycle progression. Constitutively increased or decreased RNR activity interferes with DNA replication and leads to arrested cell cycle progression; however, the mechanisms underlying these disruptive effects in higher plants remain to be uncovered. In this study, we identified a RNR large subunit mutant, sistl1, in Setaria italica (foxtail millet), which exhibited growth retardation as well as striped leaf phenotype, i.e. irregularly reduced leaf vein distances and decreased chloroplast biogenesis. We determined that a Gly737 to Glu substitution occurring in the C-terminus of the SiSTL1 protein slightly affected its optimal function, leading in turn to the reduced expression of genes variously involved in the assembly and activation of the DNA pre-replicative complex, elongation of replication forks and S phase entry. Our study provides new insights into how SiSTL1 regulates plant growth, chloroplast biogenesis, and cell cycle progression in Poaceae crops.

Vγ9Vδ2 T cells is the dominant γδ T cell subset in human blood. They are cytotoxic and activated by phosphoantigens whose concentrations are increased in cancer cells, making the cancer cells targets for Vγ9Vδ2 T cell immunotherapy. For successful immunotherapy, it is important both to characterise Vγ9Vδ2 T cell proliferation and optimise the assessment of their cytotoxic potential, which is the aim of this study. We found that supplementation with freshly thawed human serum potentiated Vγ9Vδ2 T cell proliferation from peripheral mononuclear cells (PBMCs) stimulated with (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and consistently enabled Vγ9Vδ2 T cell proliferation from cryopreserved PBMCs. In cryopreserved PBMCs the proliferation was higher than in freshly prepared PBMCs. In a panel of short-chain prenyl alcohols, monophosphates and diphosphates, most diphosphates and also dimethylallyl monophosphate stimulated Vγ9Vδ2 T cell proliferation. We developed a method where the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells is assessed at the single cell level using flow cytometry, which gives more clear-cut results than the traditional bulk release assays. Moreover, we found that HMBPP enhances the Vγ9Vδ2 T cell cytotoxicity towards colon cancer cells. In summary, we have developed an easily interpretable method to assess the cytotoxicity of Vγ9Vδ2 T cells towards adherent cells, found that Vγ9Vδ2 T cell proliferation can be potentiated by media-supplementation and how misclassification of non-responders may be avoided. Our findings will be useful in the further development of Vγ9Vδ2 T cell immunotherapy.

1. The effects of mexiletine were evaluated on the ATP-sensitive K+ channel (K(ATP)) of rat skeletal muscle fibres using patch clamp techniques. The effects of mexiletine were studied on macropatch currents 20 s (maximally activated), 8 min (early stage of rundown) and 15 min (late stage of rundown) after excision in the absence or in the presence of internal ADP (50-100 microM) or UDP (500 microM). In addition, the effects of mexiletine were tested on single channel. 2. In the absence of ADP and UDP, mexiletine inhibited the current through maximally activated channels with an IC50 of -5.58+/-0.3 M. Nucleoside diphosphates shifted the current versus mexiletine concentration relationship to the right on the log concentration axis. UDP (500 microM) was more efficacious than ADP (50-100 microM) in this effect. 3. At the early stage of rundown, the sensitivity of the channel to mexiletine was reduced and nucleoside diphosphates, particularly UDP, antagonized the effect of mexiletine. At the late stage of rundown, mexiletine did not affect the currents. 4. At the single channel level, 1 microM mexiletine reduced the mean burst duration by 63% and prolonged the arithmetic mean closed time intervals between the bursts of openings without altering the open time and closed time distributions. Mexiletine did not affect the single channel conductance. 5. These results show that in skeletal muscle, mexiletine is a state-dependent K(ATP) channel inhibitor which either acts through the nucleotide binding site or a site allosterically coupled to it.

Although the existence of ATP-sensitive K+ (KATP) channels in vascular muscle is widely accepted, there appears to be little consensus as to what the primary regulator of these channels is under physiological or pathophysiological conditions. Recent evidence has suggested that nucleotide diphosphates (NDPs) may play a more important role than ATP. However, since the properties of vascular KATP channels are quite diverse, and the effects of these nucleotides are poorly understood, the aim of this study was to test the hypothesis that intracellular ATP can regulate whole-cell KATP current (IK,ATP) in the absence of NDPs.

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, the building blocks of DNA. RNRs are specific for either ribonucleoside diphosphates or triphosphates as substrates. As far as is known, oxygen-dependent class I RNRs (NrdAB) all reduce ribonucleoside diphosphates, and oxygen-sensitive class III RNRs (NrdD) are all ribonucleoside triphosphate reducers, whereas the adenosylcobalamin-dependent class II (NrdJ) contains both ribonucleoside diphosphate and triphosphate reducers. However, it is unknown how this specificity is conveyed by the active site of the enzymes and how this feature developed in RNR evolution. By structural comparison of the active sites in different RNRs, we identified the apical loop of the phosphate-binding site as a potential structural determinant of substrate specificity. Grafting two residues from this loop from a diphosphate- to a triphosphate-specific RNR caused a change in preference from ribonucleoside triphosphate to diphosphate substrates in a class II model enzyme, confirming them as the structural determinants of phosphate specificity. The investigation of the phylogenetic distribution of this motif in class II RNRs yielded a likely monophyletic clade with the diphosphate-defining motif. This indicates a single evolutionary-split event early in NrdJ evolution in which diphosphate specificity developed from the earlier triphosphate specificity. For those interesting cases where organisms contain more than one nrdJ gene, we observed a preference for encoding enzymes with diverse phosphate specificities, suggesting that this varying phosphate specificity confers a selective advantage.

Bitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves.

The protein family of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase family) contains multiple members that hydrolyze nucleoside 5'-triphosphates and nucleoside 5'-diphosphates with varying preference for the individual type of nucleotide. We report the cloning and functional expression of rat NTPDase3. The rat brain-derived cDNA has an open reading frame of 1590 bp encoding 529 amino acid residues, a calculated molecular mass of 59.1 kDa and predicted N- and C-terminal hydrophobic sequences. It shares 94.3% and 81.7% amino acid identity with the mouse and human NTPDase3, respectively, and is more closely related to cell surface-located than to the intracellularly located members of the enzyme family. The NTPDase3 gene is allocated to chromosome 8q32 and organized into 11 exons. Rat NTPDase3 expressed in CHO cells hydrolyzed both nucleoside triphosphates and nucleoside diphosphates with hydrolysis ratios of ATP:ADP of 5:1 and UTP:UDP of 8:1. After addition of ATP, ADP is formed as an intermediate product that is further hydrolyzed to AMP. The enzyme is preferentially activated by Ca(2+) over Mg(2+) and reveals an alkaline pH optimum. Immunocytochemistry confirmed expression of heterologously expressed NTPDase3 to the surface of CHO cells. PC12 cells express endogenous surface-located NTPDase3. An immunoblot analysis detects NTPDase3 in all rat brain regions investigated. An alignment of the secondary structure domains of actin conserved within the actin/HSP70/sugar kinase superfamily to those of all members of the NTPDase family reveals apparent similarity. It infers that NTPDases share the two-domain structure with members of this enzyme superfamily.

The large superfamily of labdane-related diterpenoids is defined by the cyclization of linear geranylgeranyl pyrophosphate (GGPP), catalyzed by copalyl diphosphate synthases (CPSs) to form the basic decalin core, the copalyl diphosphates (CPPs). Three stereochemically distinct CPPs have been found in plants, namely (+)-CPP, ent-CPP and syn-CPP. Here, we used X-ray crystallography and cryo-EM methods to describe different oligomeric structures of a syn-copalyl diphosphate synthase from Oryza sativa (OsCyc1), and provided a cryo-EM structure of OsCyc1D367A mutant in complex with the substrate GGPP. Further analysis showed that tetramers are the dominant form of OsCyc1 in solution and are not necessary for enzyme activity in vitro. Through rational design, we identified an OsCyc1 mutant that can generate ent-CPP in addition to syn-CPP. Our work provides a structural and mechanistic basis for comparing different CPSs and paves the way for further enzyme design to obtain diterpene derivatives with specific chirality.

Nucleoside diphosphate kinase (NDK), an enzyme encoded by the Drosophila abnormal wing discs (awd) or human nm23 tumor suppressor genes, generates nucleoside triphosphates from respective diphosphates. We demonstrate that NDK regulates synaptic vesicle internalization at the stage where function of the dynamin GTPase is required. awd mutations lower the temperature at which behavioral paralysis, synaptic failure, and blocked membrane internalization occur at dynamin-deficient, shi(ts), mutant nerve terminals. Hypomorphic awd alleles display shi(ts)-like defects. NDK is present at synapses and its enzymatic activity is essential for normal presynaptic function. We suggest a model in which dynamin activity in nerve terminals is highly dependent on NDK-mediated supply of GTP. This connection between NDK and membrane internalization further strengthens an emerging hypothesis that endocytosis, probably of activated growth factor receptors, is an important tumor suppressor activity in vivo.

Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest k(cat)/K(m) values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.

Prenyldiphosphate synthases catalyze the reaction of allylic diphosphates with one or more isopentenyl diphosphate molecules to form compounds such as farnesyl diphosphate, used in, e.g., sterol biosynthesis and protein prenylation, as well as longer "polyprenyl" diphosphates, used in ubiquinone and menaquinone biosynthesis. Quinones play an essential role in electron transport and are associated with the inner mitochondrial membrane due to the presence of the polyprenyl group. In this work, we investigated the synthesis of the polyprenyl diphosphate that alkylates the ubiquinone ring precursor in Toxoplasma gondii, an opportunistic pathogen that causes serious disease in immunocompromised patients and the unborn fetus. The enzyme that catalyzes this early step of the ubiquinone synthesis is Coq1 (TgCoq1), and we show that it produces the C35 species heptaprenyl diphosphate. TgCoq1 localizes to the mitochondrion and is essential for in vitro T. gondii growth. We demonstrate that the growth defect of a T. gondii TgCoq1 mutant is rescued by complementation with a homologous TgCoq1 gene or with a (C45) solanesyl diphosphate synthase from Trypanosoma cruzi (TcSPPS). We find that a lipophilic bisphosphonate (BPH-1218) inhibits T. gondii growth at low-nanomolar concentrations, while overexpression of the TgCoq1 enzyme dramatically reduced growth inhibition by the bisphosphonate. Both the severe growth defect of the mutant and the inhibition by BPH-1218 were rescued by supplementation with a long-chain (C30) ubiquinone (UQ6). Importantly, BPH-1218 also protected mice against a lethal T. gondii infection. TgCoq1 thus represents a potential drug target that could be exploited for improved chemotherapy of toxoplasmosis. IMPORTANCE Millions of people are infected with Toxoplasma gondii, and the available treatment for toxoplasmosis is not ideal. Most of the drugs currently used are only effective for the acute infection, and treatment can trigger serious side effects requiring changes in the therapeutic approach. There is, therefore, a compelling need for safe and effective treatments for toxoplasmosis. In this work, we characterize an enzyme of the mitochondrion of T. gondii that can be inhibited by an isoprenoid pathway inhibitor. We present evidence that demonstrates that inhibition of the enzyme is linked to parasite death. In addition, the inhibitor can protect mice against a lethal dose of T. gondii. Our results thus reveal a promising chemotherapeutic target for the development of new medicines for toxoplasmosis.

The widespread utility of isoprenoids has recently sparked interest in efficient synthesis of isoprene-diphosphate precursors. Current efforts have focused on evaluating two-step "isoprenol pathways," which phosphorylate prenyl alcohols using promiscuous kinases/phosphatases. The convergence on isopentenyl phosphate kinases (IPKs) in these schemes has prompted further speculation about the class's utility in synthesizing non-natural isoprenoids. However, the substrate promiscuity of IPKs in general has been largely unexplored. Towards this goal, we report the biochemical characterization of five novel IPKs from Archaea and the assessment of their substrate specificity using 58 alkyl-monophosphates. This study reveals the IPK-catalyzed synthesis of 38 alkyl-diphosphate analogs and discloses broad substrate specificity of IPKs. Further, to demonstrate the biocatalytic utility of IPK-generated alkyl-diphosphates, we also highlight the synthesis of alkyl-l-tryptophan derivatives using coupled IPK-prenyltransferase reactions. These results reveal IPK-catalyzed reactions are compatible with downstream isoprenoid enzymes and further support their development as biocatalytic tools for the synthesis of non-natural isoprenoids.

Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleoside diphosphates (dNDPs) to provide dNTP precursors for DNA synthesis. Here, we report that acetylation and deacetylation of the RRM2 subunit of RNR acts as a molecular switch that impacts RNR activity, dNTP synthesis, and DNA replication fork progression. Acetylation of RRM2 at K95 abrogates RNR activity by disrupting its homodimer assembly. RRM2 is directly acetylated by KAT7, and deacetylated by Sirt2, respectively. Sirt2, which level peak in S phase, sustains RNR activity at or above a threshold level required for dNTPs synthesis. We also find that radiation or camptothecin-induced DNA damage promotes RRM2 deacetylation by enhancing Sirt2-RRM2 interaction. Acetylation of RRM2 at K95 results in the reduction of the dNTP pool, DNA replication fork stalling, and the suppression of tumor cell growth in vitro and in vivo. This study therefore identifies acetylation as a regulatory mechanism governing RNR activity.