Guanine Diphosphate Research Articles

Copper-doped Lanthanide Coordination Polymers as Luminescent Nanoenzyme for Ratiometric Sensing of H2O2 and Glutathione.

Design and fabrication of integrated multifunctional probes with intrinsic catalytic and detection abilities is of great importance to simplify the operation in biosensing application with high sensitivity. Herein, dual-emitting lanthanide coordination polymers (Ln-CPs) were facilely prepared by self-assembly of guanine diphosphate (GDP), terephthalic acid (TA), Tb3+ and Cu2+ designated as Tb/Cu-GDP/TA CPs. The doped Cu2+ endowed CPs with obviously enhanced peroxidase mimicking activity compared with free Cu2+. In the presence of H2O2, the probe catalyzed the oxidation of TA generating a new blue fluorescent product, while the fluorescence of Tb3+ decreased simultaneously. Therefore, a new sensitive ratiometric fluorescent sensor for H2O2 has been developed with a good linear range from 0.01 to 300μM and limit of 1.62nM. Moreover, the proposed platform could be extended to GSH ratiometric assay in the presence of H2O2, and interestingly, the detection performance could be easily adjusted by adding different concentration of H2O2. This work will facilitate the development of luminescent nanoenzymes based on Ln-CPs to construct the simple ratiomatric sensing platform.

Lanthanide coordination polymers used for fluorescent ratiometric sensing of H2O2 and glucose

Scheme 1. Schematic description of the preparation of GOx@Tb-ATA/GDP CPs and detection of glucose. • The dual-emission multi-functional probes based on Ln CPs were facilely prepared by self-assembly. • The ratiometric fluorescence method based on double ligand Ln 3+ coordination polymers for H 2 O 2 detection was realized. • By integrating GOx through the adaptive inclusion property of CPs in the self-assembly process, a simple, highly selective and sensitive one-pot retiometric glucose assay was developed. A dual-emission lanthanide coordination polymers (Ln CPs) were facilely prepared by self-assembly of guanine diphosphate (GDP) and 2-aminoterephthalic acid (ATA) with Tb 3+ , termed as Tb-ATA/GDP CPs. GDP played as the antenna ligand to sensitize the luminescence of Tb 3+ , and ATA served as a fluorescent auxiliary ligand for the ratiometric assay. The obtained Tb-ATA/GDP CPs exhibited both blue-emission of ATA at 430 nm and characteristic green-emission of Tb 3+ under a single excitation wavelength. Upon addition of H 2 O 2 , the fluorescence of Tb 3+ could be selectively quenched and that of ATA slightly enhanced. The fluorescence intensity ratio at 430 nm and 549 nm (I 430 /I 549 ) increased linearly with increasing the concentration of H 2 O 2 in the range of 0.005–20 μM and 120–500 μM, and the detection limit was 3.42 nM. Furthermore, by integrating the glucose oxidase (GOx) through the adaptive inclusion ability of Ln CPs, GOx@Tb-ATA/GDP CPs were facilely obtained. Based on their high affinity to oxidize glucose and the fluorescence response to the generated H 2 O 2 , a simple, highly selective and sensitive one-pot ratiometric glucose assay was developed. This work will facilitate the design and construct of multi-functional fluorescent probes based on the Ln CPs, and exploit their applications in bio-sensing and environmental analysis.

The structure–function analysis of Obg‐like GTPase proteins along the evolutionary tree from bacteria to humans

Obg proteins belong to P‐loop guanine triphosphatase (GTPase) that are conserved from bacteria to humans. Like other GTPases, Obg cycles between guanine triphosphate (GTP) bound “on” state and guanine diphosphate (GDP)‐bound “off” state, thereby controlling various cellular processes. Different members of this group have unique structural characteristics; a conserved glycine‐rich N‐terminal domain known as obg fold, a central conserved nucleotide binding domain, and a less conserved C‐terminal domain of other functions. Obg is a ribosome dependent GTPase helps in ribosome maturation by interacting with several proteins of the 50S subunit of the ribosome. Obg proteins have been widely considered as a regulator of cellular functions, helping in DNA replication, cell division. Apart from that, this protein also takes part in various stress adaptation pathways like a stringent response, sporulation, and general stress response. In this particular review, the structural features of ObgE have been highlighted and how the structure plays important role in interacting with regulators like GTP, ppGpp that are crucial for executing biological function has been orchestrated. In particular, we believe that Obg‐like proteins can provide a link between different global pathways that are necessary for fine‐tuning cellular processes to maintain the cellular energy status.

Identification and Characterization of Small‐Molecule Ras Modulators and their Impact in Cancer Drug Discovery

Ras is a small GTPase enzyme that binds guanine diphosphate (GDP), a nucleotide substrate, to its guanine‐nucleotide binding pocket. Ras activation occurs by the coordination of SOS, a guanine nucleotide exchange factor, which catalyzes the substitution of GDP for guanine triphosphate (GTP), rendering the Ras protein in an active state. Oncogenic Ras mutants are constitutively active, propagating proliferative cell signaling that leads to aberrant cell growth and metastatic tumors that are present in 30% of all human cancers. Discovery of a potent, small molecule Ras inhibitor has been challenging. We have characterized a novel class of Ras modulators that exhibit both Ras activation. These small molecules were discovered using Ras‐SOS molecular modeling and computational docking studies focused on identifying potential compound binding to the Ras‐SOS complex interface. Several in‐vitro biochemical assays were performed to elucidate the structural significance, binding kinetics, and nucleotide exchange effects of these small molecule binders. A fluorescence nucleotide exchange assay was optimized to discover compounds that had an atypical nucleotide exchange rate (off‐state to on‐state) in a kinetic assay. Compounds discovered in the assay were then analyzed by Surface Plasmon Resonance (SPR) to further elucidate details on the binding kinetics between Ras and compound as well as between Ras and SOS in the presence of the compound. Of the examined compounds, several exhibited activation of the Ras‐SOS catalytic reaction. An ongoing structure activity relationship (SAR) study has identified essential components that facilitate binding to the Ras:SOS interface. This presumably allosteric interaction may have uncovered an unprobed regulatory site that can be manipulated to create Ras inhibitors. This discovery of activator compounds provides a promising new direction for the development of potent Ras modulators.Support or Funding InformationThe Center for Healthcare Innovation (CHI), Stevens Institute of Technology

Elongation Factor Tu Switch I Element is a Gate for Aminoacyl-tRNA Selection

Selection of correct aminoacyl (aa)-tRNA at the ribosomal A site is fundamental to maintaining translational fidelity. Aa-tRNA selection is a multistep process facilitated by the guanosine triphosphatase elongation factor (EF)-Tu. EF-Tu delivers aa-tRNA to the ribosomal A site and participates in tRNA selection. The structural mechanism of how EF-Tu is involved in proofreading remains to be fully resolved. Here, we provide evidence that switch I of EF-Tu facilitates EF-Tu's involvement during aa-tRNA selection. Using structure-based and explicit solvent molecular dynamics simulations based on recent cryo-electron microscopy reconstructions, we studied the conformational change of EF-Tu from the guanosine triphosphate to guanine diphosphate conformation during aa-tRNA accommodation. Switch I of EF-Tu rapidly converts from an α-helix into a β-hairpin and moves to interact with the acceptor stem of the aa-tRNA. In doing so, switch I gates the movement of the aa-tRNA during accommodation through steric interactions with the acceptor stem. Pharmacological inhibition of the aa-tRNA accommodation pathway prevents the proper positioning of switch I with the aa-tRNA acceptor stem, suggesting that the observed interactions are specific for cognate aa-tRNA substrates, and thus capable of contributing to the fidelity mechanism.

Mechanistic Insights into the Differential Catalysis by RheB and Its Mutants: Y35A and Y35A-D65A.

RheB GTPase is a Ras-related molecular switch, which regulates the mTOR signaling pathway by cycling between the active [guanosine triphosphate (GTP)] state and inactive [guanine diphosphate (GDP)] state. Impairment of GTPase activity because of mutations in several small GTPases is known to be associated with several cancers. The conventional GTPase mechanism such as in H-Ras requires a conserved glutamine (Q64) in the switch-II region of RheB to align the catalytic water molecule for efficient GTP hydrolysis. The conformation of this conserved glutamine is different in RheB, resulting in an altered conformation of the entire switch-II region. Studies on the atypical switch-II conformation in RheB revealed a distinct, noncanonical mode of GTP hydrolysis. An RheB mutant Y35A was previously shown to exclusively enhance the intrinsic GTPase activity of RheB, whereas the Y35A-D65A double mutant was shown to reduce the elevated GTPase activity. Here, we have used all-atom molecular dynamics (MD) simulations for comprehensive understanding of the conformational dynamics associated with the fast (Y35A) and slow (Y35A-D65A) hydrolyzing mutants of RheB. Using a combination of starting models from PDB structures and in-silico generated mutant structures, we discuss the observed conformational deviations in wild type (WT) versus mutants. Our results show that a number of interactions of RheB with phosphates of GTP as well as Mg2+ are destabilized in Y35A mutant in the switch-I region. We report distinct water dynamics at the active site of WT and mutants. Furthermore, principal component analysis showed significant differences in the conformational space sampled by the WT and mutants. Our observations provide improved understanding of the noncanonical GTP hydrolysis mechanism adopted by RheB and its modulation by Y35A and Y35A-D65A mutants.

Rac1: an emerging player in stretch-stimulated glucose transport.

Rac1: an emerging player in stretch-stimulated glucose transport.

The O-specific polysaccharide structure and gene cluster of serotype O:12 of the Yersinia pseudotuberculosis complex, and the identification of a novel L-quinovose biosynthesis gene

A major virulence factor for Yersinia pseudotuberculosis is lipopolysaccharide, including O-polysaccharide (OPS). Currently, the OPS based serotyping scheme for Y. pseudotuberculosis includes 21 known O-serotypes, with genetic and structural data available for 17 of them. The completion of the OPS structures and genetics of this species will enable the visualization of relationships between O-serotypes and allow for analysis of the evolutionary processes within the species that give rise to new serotypes. Here we present the OPS structure and gene cluster of serotype O:12, thus adding one more to the set of completed serotypes, and show that this serotype is present in both Y. pseudotuberculosis and the newly identified Y. similis species. The O:12 structure is shown to include two rares ugars: 4-C[(R)-1-hydroxyethyl]-3,6-dideoxy-D-xylo-hexose(D-yersiniose) and 6-deoxy-L-glucopyranose (L-quinovose).We have identified a novel putative guanine diphosphate(GDP)-L-fucose 4-epimerase gene and propose a pathway for the synthesis of GDP-L-quinovose, which extends the known GDP-L-fucose pathway.

Activation of Rho GTPases by DOCK Exchange Factors Is Mediated by a Nucleotide Sensor

Activation of Rho guanosine triphosphatases (GTPases) to the guanine triphosphate (GTP)-bound state is a critical event in their regulation of the cytoskeleton and cell signaling. Members of the DOCK family of guanine nucleotide exchange factors (GEFs) are important activators of Rho GTPases, but the mechanism of activation by their catalytic DHR2 domain is unknown. Through structural analysis of DOCK9-Cdc42 complexes, we identify a nucleotide sensor within the alpha10 helix of the DHR2 domain that contributes to release of guanine diphosphate (GDP) and then to discharge of the activated GTP-bound Cdc42. Magnesium exclusion, a critical factor in promoting GDP release, is mediated by a conserved valine residue within this sensor, whereas binding of GTP-Mg2+ to the nucleotide-free complex results in magnesium-inducing displacement of the sensor to stimulate discharge of Cdc42-GTP. These studies identify an unusual mechanism of GDP release and define the complete GEF catalytic cycle from GDP dissociation followed by GTP binding and discharge of the activated GTPase.

RAS in cellular transformation and senescence

In 1964, a transforming retrovirus was described that produced tumours in mice [1]. The Harvey rat sarcoma virus, aptly named H-RAS after the discovering scientist, encoded an oncogene that had been hijacked from its host. A similar virus was isolated in 1970 and was named the Kirsten rat sarcoma virus or K-RAS [2]. In 1982, the human genes homologous to the viral genes were elucidated and were designated c-H-RAS and c-K-RAS [3], and subsequently the third and final RAS family member was isolated from human neuroblastoma samples and termed c-N-RAS. An examination of other human cancers also revealed RAS genes capable of transforming mammalian cells, and interestingly, these alleles were transforming due to point mutations that inactivated the proteins’ intrinsic guanine triphosphatase (GTPase) activity [4,5]. RAS functions downstream of mitogenic growth factor receptors. Following the ligation with growth factors, these receptor tyrosine kinases undergo autophosphorylation and thereby recruit adaptor proteins that bind guanine nucleotide exchange factors (GEFs) (for review, see [6]). GEFs catalyse the exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) on small GTP-binding proteins, including RAS. GTP-bound RAS subsequently interacts with a number of effectors to regulate cellular proliferation and survival, notably including the RAF family of proteins and type I phosphoinositol-3-kinase (PI3K). RAS proteins mutated at codons 12, 13, or 61 are rendered constitutively GTP bound and consequently have increased affinity to RAF proteins and PI3K, leading to activation of downstream pathways. The RAF family of serine/threonine kinases consists of three members: ARAF, BRAF, and CRAF [6]. Following activation by RAS, RAF kinases phosphorylate Mitogen-Activated Protein Kinase (MEK) kinases (MEK1 and MEK2), resulting in their activation. MEK proteins subsequently phosphorylate Extracellular Regulated MAP Kinase (ERK) (ERK1 and ERK2) in the cytoplasm, resulting in their activation and nuclear translocation. ERK kinases have a variety of cytoplasmic and nuclear targets, importantly including ETS transcription factors to regulate the expression of many pro-proliferative genes. RAS-GTP also interacts with and stimulates the activity of PI3K [6]. Upon activation, PI3K catalyses the conversion of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) to produce phosphatidylinositol-3,4,5trisphosphate (PtdIns(3,4,5)P3). Increased local concentrations of PtdIns(3,4,5)P3 result in recruitment of the kinase PDK1 via its pleckstrin homology domain, which phosphorylates and activates the kinase AKT. AKT in turn promotes cellular survival and activates mammalian target of rapamycin (mTOR) to increase ribosomal biogenesis and messenger RNA (mRNA) translation. Additionally, active PI3K also stimulates the GTP-binding protein RAC, a regulator of the actin cytoskeleton. Therefore, rather than relying upon a single pathway for cellular transformation, it is hypothesised that numerous alterations in multiple biochemical pathways collectively promote cellular transformation by oncogenic RAS. Oncogenic RAS mutations are the most common oncogenic events identified in human tumours, being found in 30% of human cancers. KRAS is the most commonly mutated member of the RAS family, present in over 90% of ductal pancreatic cancers, 40−50% colorectal cancers, and 30% of non-small cell lung cancers (6). A detailed characterisation of the cellular and molecular pathways altered by oncogenic RAS has been pursued in order to identify potential therapeutic targets for such cancers, but despite its expected role in promoting tumourigenesis, ectopic expression of oncogenic RAS in primary cell cultures induces a paradoxical irreversible growth arrest known as oncogene-induced senescence (OIS) [7]. OIS is characterised by a flattened cellular morphology, a large nucleus with a prominent nucleolus, chromatin reorganisation, and activation of the p53 and p16INK4a pathways [7]. Although ectopic expression of oncogenic RAS in primary cells induces OIS, the exact molecular mechanisms remain unclear. Studies have

Coactivation of G Protein Signaling by Cell-Surface Receptors and an Intracellular Exchange Factor

G protein-coupled receptors (GPCRs) mediate responses to a broad range of chemical and environmental signals. In yeast, a pheromone-binding GPCR triggers events leading to the fusion of haploid cells. In general, GPCRs function as guanine-nucleotide exchange factors (GEFs); upon agonist binding, the receptor induces a conformational change in the G protein alpha subunit, resulting in exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP) and in signal initiation. Signaling is terminated when GTP is hydrolyzed to GDP [1]. This well-established paradigm has in recent years been revised to include new components that rates of GDP release, GTP binding [2-8], and GTP hydrolysis[9, 10]. Here we report the discovery of a nonreceptor GEF, Arr4. Like receptors, Arr4 binds directly to the G protein,accelerates guanine-nucleotide exchange, and stabilizes the nucleotide-free state of the a subunit. Moreover, Arr4 promotes G protein-dependent cellular responses, including mitogen-activated protein kinase (MAPK) phosphorylation,new-gene transcription, and mating. In contrast to knownGPCRs, however, Arr4 is not a transmembrane receptor,but rather a soluble intracellular protein. Our data suggest that intracellular proteins function in cooperation with mating pheromones to amplify G protein signaling, thereby leading to full pathway activation.

47] Expression, purification, and functional assays for self-association of dynamin-1

This chapter discusses the expression, purification, and functional assays for self-association of dynamin-1. High-level expression of recombinant dynamin in baculovirus-infected insect cells provides a reproducible technique for the production of biologically active dynamin-1. With this expression system, researchers are able to apply a relatively simple purification procedure, which includes batch anion-exchange and hydroxyapatite chromatography, that routinely yields >95% pure preparation of dynamin in >10-mg quantities. Dynamin has the propensity to self-assemble, in conditions of low ionic strength or under physiologic salt conditions in the presence of guanine diphosphate (GDP) and AlF 4 – .The resulting rings and helical stacks of rings are readily sedimentable by ultracentrifugation, providing a simple means for quantitatively assaying self-assembly by velocity sedimentation. The column chromatography purification yields unassembled dynamin tetramers in high-salt buffers. The velocity sedimentation assay can be used to rapidly determine buffer conditions under which both wild type and mutant forms of dynamin can assemble in vitro.

Molecular basis for interactions of G protein betagamma subunits with effectors.

Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.

Pyrophosphate formation as the most efficient condensation reaction of activated nucleotides

When an oligonucleotide primer pG10 is incubated with the nucleotide analogue 9-[3-hydroxy-2-(hydroxymethyl)prop-1-yl] guanine diphosphate (pGp, I) in the presence of poly(C), addition of the monomer occurs almost exclusively at the 5'-terminal phosphate rather than the 3'-terminal cis-glycol. The implications of this finding in the context of prebiotic condensation reactions are discussed.

Structural and dynamic differences between normal and transforming N-ras gene products: a 31P and isotope-edited 1H NMR study.

[15N]Glycine was biosynthetically incorporated into normal cellular N-ras p21 and a position 12 transforming mutant, in order to produce p21 proteins containing several site-specific NMR probes at or near activating positions in the guanine nucleotide binding domain. We have previously assigned all five glycine resonances located in loops directly involved in binding of guanosine diphosphate in the wild-type p21 protein [Campbell-Burk, S., Papastavros, M. Z., McCormick, F., & Redfield, A. G. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 817-820]. In this report, the corresponding glycine resonances in the p21 mutant have been assigned, and spectral differences between normal and mutant p21-guanosine diphosphate (p21.GDP) complexes have been investigated. Our combined 1H[15N] and 31P NMR results show that substitution of aspartate for glycine-12 produces perturbations in the phosphoryl binding domain, near the point of the mutation. Although many of the remaining glycines were unaffected, spectral differences were also observed outside the GDP binding domain. Two of the five active-site glycines in wild-type p21.GDP have very slow amide proton exchange rates with water (kappa less than 2.8 x 10(-5) s-1). The active-site glycines are located in solvent-exposed loops, so their apparent solvent inaccessibility may result from strong hydrogen bond formation between glycine amide protons and bound guanine diphosphate and/or other nearby groups in p21.

Guanine triphosphate-binding site regulation by follicle-stimulating hormone and guanine diphosphate in membranes from immature rat Sertoli cells.

GTP binding to Sertoli cell membranes has been investigated using [3H]5'-guanylyl-beta gamma-imidodiphosphate [[3H]Gpp(NH)p], a nonhydrolyzable analog of GTP. Binding of [3H]Gpp(NH)p Gpp(NH)p to membranes prepared from Sertoli cells in serum-free culture was proportional to membrane protein concentration in the range of 5-50 micrograms. Competitive displacement studies using adenine (ATP, ADP, and AMP) and guanine nucleotides [GTP, GDP, GMP, and Gpp(NH)p] indicated that only GTP, its analog Gpp(NH)p, and GDP were effective ligands. The relative potencies were Gpp(NH)p much greater than GTP greater than GDP, as characterized by ED50 values of 0.8, 2.5, and 4 microM, respectively. Competitive inhibition by GTP, however, was similar to that by Gpp(NH)p in the presence of a nucleoside triphosphate-regenerating system, suggesting the involvement of an active GTPase. Equilibrium binding studies indicated a single high affinity site for GTP with a Ka of 3.3 +/- 0.2 X 10(7) M-1. This value was supported by other studies in which an association rate constant of 1.8 X 10(6) M-1 min-1 and a dissociation rate constant of 2.4 X 10(-2) min-1 were estimated. Maximal binding of [3H]Gpp(NH)p to Sertoli cell membranes ranged from 30-55 pmol/mg protein. FSH enhanced [3H]Gpp(NH)p binding by about 50% (P less than 0.05), reflecting an increase in the number of available binding sites rather than an effect on Ka. When GDP was preincubated with membranes in the absence of FSH, the number of available binding sites for [3H]Gpp(NH)p was decreased. This reduction in available binding sites by pretreatment with GDP could be reversed by adding FSH during the equilibrium binding analysis. These studies have demonstrated specific high affinity binding of Gpp(NH)p to Sertoli cell membranes with an affinity comparable to that required for activation of FSH-sensitive adenylate cyclase. Furthermore, a potent GTPase activity associated with the Sertoli cell membrane is responsible for rapid hydrolysis of GTP to GDP and may participate in inactivation of GTP-dependent adenylate cyclase activity. The role of FSH in the regulation of nucleoside binding appears to be in facilitating exchange of GTP for GDP by enhancing the release of bound GDP.

Induction of capsular polysaccharide synthesis by rho-fluorophenylalanine in Escherichia coli wild type and strains with altered phenylalanyl soluble ribonucleic acid synthetase.

Escherichia coli K-12 strain AB259 can be induced to form capsular polysaccharide (mucoid clones) by dl-p-fluorophenylalanine (FPA; 5 x 10(-6)m on agar plates at 37 C or 8 x 10(-5)m in liquid medium at 30 C). The change was shown to be phenotypic. An increase in enzymes probably involved in capsular polysaccharide synthesis [phosphomannose isomerase (3.3-fold), uridine diphosphate-d-galactose-4-epimerase (2.5-fold), and guanine diphosphate-l-fucose synthetase] was demonstrated as a result of growth in FPA. These increases appear sufficient to account for the increased synthesis of capsular polysaccharide due to growth in FPA. FPA-resistant derivatives of strain AB259 were obtained by selecting mutants on FPA-containing agar or by transducing in an altered phenylalanyl soluble ribonucleic acid synthetase that activates FPA poorly. Mucoid clones were formed by these strains only in the presence of 30 to 1,000 times as much FPA. Among these strains, there was a close correlation between incorporation of FPA-C(14) and induction of capsular polysaccharide synthesis. The results are thus consistent with the following model: FPA is incorporated into the protein product of the R(1) gene (repressor) and alters it sufficiently to allow derepression of several enzymes.