Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the alpha subunits of Gs (stimulatory) (alpha 45 and alpha 52), a 41-kDa subunit of Gi (inhibitory) (alpha 41), a 40-kDa protein (alpha 40), and the 36-kDa beta subunit. No protein that comigrated with the alpha subunit of Go (unknown function) (alpha 39) was detected. In cells grown in the presence of [3H]myristic acid, alpha 41 and alpha 40 contained 3H label, while the beta subunit did not. Chemical analysis of lipids attached covalently to purified alpha 41 and alpha 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein beta and gamma subunits and of Gt (transducin) subunits (alpha, beta, and gamma) failed to reveal fatty acids. The fatty acid associated with alpha 41, alpha 40, and alpha 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.

G-quadruplexes (G4s) are four-stranded, guanine-rich nucleic acid structures that can influence a variety of biological processes such as the transcription and translation of genes and DNA replication. In many cases, a single G4-forming nucleic acid sequence can adopt multiple different folded conformations that interconvert on biologically relevant timescales, entropically stabilizing the folded state. The coexistence of different folded conformations also suggests that there are multiple pathways leading from the unfolded to the folded state ensembles, potentially modulating the folding rate and biological activity. We have developed an experimental method for quantifying the contributions of individual pathways to the folding of conformationally heterogeneous G4s that is based on mutagenesis, thermal hysteresis kinetic experiments and global analysis, and validated our results using photocaged kinetic NMR experiments. We studied the regulatory Pu22 G4 from the c-myc oncogene promoter, which adopts at least four distinct folded isomers. We found that the presence of four parallel pathways leads to a 2.5-fold acceleration in folding; that is, the effective folding rate from the unfolded to folded ensembles is 2.5 times as large as the rate constant for the fastest individual pathway. Since many G4 sequences can adopt many more than four isomers, folding accelerations of more than an order of magnitude are possible via this mechanism.

Stimuli-controlled structural transitions of nucleic acids have received growing attentions owing to their potential applications in the fields of chemical and synthetic biology. Here, we describe the development of reduction-responsive deoxyribonucleic acid (DNA) duplexes, in which guanine rings bearing a reduction-responsive cleavable nitrobenzyl (NB) group at the O 6 position (GNB) are introduced at defined positions. We demonstrate that the artificial NB group can be removed in response to reduction stimulus without the dissociation of the intermolecular duplex structure, which comprises a G-quadruplex forming nucleic acid strand with one GNB and its complementary sequence with one mismatch pair. Meanwhile, another duplex that comprised a G-quadruplex forming nucleic acid strand with two GNB and its complementary sequence with three mismatch pairs exhibited reduction-responsive structural transitions from intermolecular duplex to intramolecular quadruplex. These findings might be useful for the development of DNA architectures endowed with reduction-responsive functions.

A suite of crystal structures is reported for a cellular mRNA cap (guanine-N7) methyltransferase in complex with AdoMet, AdoHcy, and the cap guanylate. Superposition of ligand complexes suggests an in-line mechanism of methyl transfer, albeit without direct contacts between the enzyme and either the N7 atom of guanine (the attacking nucleophile), the methyl carbon of AdoMet, or the sulfur of AdoMet/AdoHcy (the leaving group). The structures indicate that catalysis of cap N7 methylation is accomplished by optimizing proximity and orientation of the substrates, assisted by a favorable electrostatic environment. The enzyme-ligand structures, together with new mutational data, fully account for the biochemical specificity of the cap guanine-N7 methylation reaction, an essential and defining step of eukaryotic mRNA synthesis.

By substitution of natural nucleotides by their abasic analogs (i.e., 1',2'-dideoxyribose phosphate residue) at critically chosen positions within 27-bp DNA constructs originating from the first intron of N-myc gene, we hindered hybridization within the guanine- and cytosine-rich central region and followed formation of non-canonical structures. The impeded hybridization between the complementary strands leads to time-dependent structural transformations of guanine-rich strand that are herein characterized with the use of solution-state NMR, CD spectroscopy, and native polyacrylamide gel electrophoresis. Moreover, the DNA structural changes involve transformation of intra- into inter-molecular G-quadruplex structures that are thermodynamically favored. Intriguingly, the transition occurs in the presence of complementary cytosine-rich strands highlighting the inability of Watson-Crick base-pairing to preclude the transformation between G-quadruplex structures that occurs via intertwining mechanism and corroborates a role of G-quadruplex structures in DNA recombination processes.

α- and β-tubulin form heterodimers, with GTPase activity, that assemble into microtubules. Like other GTPases, the nucleotide-bound state of tubulin heterodimers controls whether the molecules are in a biologically active or inactive state. While α-tubulin in the heterodimer is constitutively bound to GTP, β-tubulin can be bound to either GDP (GDP-tubulin) or GTP (GTP-tubulin). GTP-tubulin hydrolyzes its GTP to GDP following assembly into a microtubule and, upon disassembly, must exchange its bound GDP for GTP to participate in subsequent microtubule polymerization. Tubulin dimers have been shown to exhibit rapid intrinsic nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary in cells. Here, we use quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. We further show that BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells.

Lesch-Nyhan syndrome encompasses a host of neurological symptoms, caused by a deficiency of the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). How the absence of this enzymes activity affects development of the nervous system is unknown. In this study, we examined the ability of N2aTG, a HGPRT-deficient neuroblastoma and its HGPRT-positive counterpart to proliferate and differentiate at various densities. In summary, N2aTG cells proliferated less and differentiated more than N2a cells, with the former cells exhibiting enhanced sensitivity to the effects of low-density culture. Given the homogeneity of this neuroblastoma cell line and its use in studies of neuronal development, the present study indicates that N2aTG cells may prove a suitable in vitro model for the study of non-dopaminergic neuronal development in Lesch-Nyhan syndrome.

8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) are among the most common oxidative DNA lesions and are substrates for 8-oxoguanine DNA glycosylase (OGG1)-initiated DNA base excision repair (BER). Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity in mice. The mouse cells were analyzed for telomere integrity by telomere quantitative fluorescence in situ hybridization (telomere-FISH), by chromosome orientation-FISH (CO-FISH), and by indirect immunofluorescence in combination with telomere-FISH and for oxidative base lesions by Fpg-incision/Southern blot assay. In comparison to the wild type, telomere lengthening was observed in Ogg1 null (Ogg1(-/-)) mouse tissues and primary embryonic fibroblasts (MEFs) cultivated in hypoxia condition (3% oxygen), whereas telomere shortening was detected in Ogg1(-/-) mouse hematopoietic cells and primary MEFs cultivated in normoxia condition (20% oxygen) or in the presence of an oxidant. In addition, telomere length abnormalities were accompanied by altered telomere sister chromatid exchanges, increased telomere single- and double-strand breaks, and preferential telomere lagging- or G-strand losses in Ogg1(-/-) mouse cells. Oxidative guanine lesions were increased in telomeres in Ogg1(-/-) mice with aging and primary MEFs cultivated in 20% oxygen. Furthermore, oxidative guanine lesions persisted at high level in Ogg1(-/-) MEFs after acute exposure to hydrogen peroxide, while they rapidly returned to basal level in wild-type MEFs. These findings indicate that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. Our studies demonstrate that BER pathway is required in repairing oxidative guanine damage in telomeres and maintaining telomere integrity in mammals.

Guanine quadruplex (G-quadruplex) motifs in the 5' untranslated region (5'-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophysical characterization and the inhibitory activity on translation was shown by luciferase reporter assays. In experiments with whole cell extracts from different eukaryotic cell lines, G-quadruplex-binding proteins were isolated by pull-down assays and subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The binding partners of the RNA G-quadruplexes we discovered included several heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and splicing factors, as well as other proteins that have previously not been described to interact with nucleic acids. While most of the proteins were specific for either of the investigated G-quadruplexes, some of them bound to both motifs. Selected candidate proteins were subsequently produced by recombinant expression and dissociation constants for the interaction between the proteins and RNA G-quadruplexes in the low nanomolar range were determined by surface plasmon resonance spectroscopy. The present study may thus help to increase our understanding of the mechanisms by which G-quadruplexes regulate translation.

DNA guanine quadruplexes or G4s are non-canonical DNA secondary structures which affect genomic processes like replication, transcription and recombination. G4s are computationally identified by specific nucleotide motifs which are also called putative G4 (PG4) motifs. Despite the general relevance of these structures, there is currently no tool available that can allow batch queries and genome-wide analysis of these motifs in a user-friendly interface. QuadBase2 (quadbase.igib.res.in) presents a completely reinvented web server version of previously published QuadBase database. QuadBase2 enables users to mine PG4 motifs in up to 178 eukaryotes through the EuQuad module. This module interfaces with Ensembl Compara database, to allow users mine PG4 motifs in the orthologues of genes of interest across eukaryotes. PG4 motifs can be mined across genes and their promoter sequences in 1719 prokaryotes through ProQuad module. This module includes a feature that allows genome-wide mining of PG4 motifs and their visualization as circular histograms. TetraplexFinder, the module for mining PG4 motifs in user-provided sequences is now capable of handling up to 20 MB of data. QuadBase2 is a comprehensive PG4 motif mining tool that further expands the configurations and algorithms for mining PG4 motifs in a user-friendly way.

The Rab GTPase family is a major regulator of membrane traffic in eukaryotic cells. The Rab11 subfamily plays important roles in specific trafficking events such as exocytosis, endosomal recycling, and cytokinesis. SH3BP5 and SH3BP5-like (SH3BP5L) proteins have recently been found to serve as guanine nucleotide exchange factors (GEF) for Rab11. Here, we report the crystal structures of the SH3BP5 GEF domain alone and its complex with Rab11a. SH3BP5 exhibits a V-shaped structure comprising two coiled coils. The coiled coil composed of α1, and α4 is solely responsible for the Rab11a binding and GEF activity. SH3BP5 pulls out and deforms switch I of Rab11a so as to facilitate the GDP release from Rab11a. SH3BP5 interacts with the N-terminal region, switch I, interswitch, and switch II of Rab11a. SH3BP5 and SH3BP5L localize to Rab11-positive recycling endosomes and show GEF activity for all of the Rab11 family but not for Rab14. Fluorescence-based GEF assays combined with site-directed mutagenesis reveal the essential interactions between SH3BP5 and Rab11 family proteins for the GEF reaction on recycling endosomes.

Three Sec7 guanine nucleotide exchange factors (GEFs) activate ADP-ribosylation factors (ARFs) to facilitate coating of transport vesicles within the secretory and endosomal pathways. GBF1 recruits COPI to pre-Golgi and Golgi compartments, whereas BIG1 and BIG2 recruit AP1 and GGA clathrin adaptors to the trans-Golgi network (TGN) and endosomes. Here, we report a functional cascade between these GEFs by showing that GBF1-activated ARFs (ARF4 and ARF5, but not ARF3) facilitate BIG1 and BIG2 recruitment to the TGN. We localize GBF1 ultrastructurally to the pre-Golgi, the Golgi, and also the TGN. Our findings suggest a model in which GBF1 localized within pre-Golgi and Golgi compartments mediates ARF activation to facilitate recruitment of COPI to membranes, whereas GBF1 localized at the TGN mediates ARF activation that leads to the recruitment of BIG1 and BIG2 to the TGN. Membrane-associated BIG1/2 then activates ARFs that recruit clathrin adaptors. In this cascade, an early acting GEF (GBF1) activates ARFs that mediate recruitment of late acting GEFs (BIG1/2) to coordinate coating events within the pre-Golgi/Golgi/TGN continuum. Such coordination may optimize the efficiency and/or selectivity of cargo trafficking through the compartments of the secretory pathway.

Small Rho-GTPases are enzymes that are bound to GDP or GTP, which determines their inactive or active state, respectively. The exchange of GDP for GTP is catalyzed by so-called Rho-guanine nucleotide exchange factors (GEFs). Rho-GEFs are characterized by a Dbl-homology (DH) and adjacent Pleckstrin-homology (PH) domain that serves as enzymatic unit for the GDP/GTP exchange. Rho-GEFs show different GTPase specificities, meaning that a particular GEF can activate either multiple GTPases or only one specific GTPase. We recently reported that the Rho-GEF Trio, known to be able to exchange GTP on Rac1, RhoG and RhoA, regulates lamellipodia formation to mediate cell spreading and migration in a Rac1-dependent manner. In this commentary, we review the current knowledge of Trio in several aspects of cell biology.

Damaged tissues and cells release intracellular purine nucleotides, which serve as intercellular signaling factors. We previously showed that exogenously added adenine nucleotide (250 μM ATP) suppressed the activation of murine splenic T lymphocytes. Here, we examined the effects of other purine nucleotides/nucleosides on mouse T cell activation. First, we found that pretreatment of mouse spleen T cells with 250 μM GTP, GDP, GMP, guanosine, ITP, IDP, IMP or inosine significantly reduced the release of stimulus-inducible cytokine IL-2. This suppression of IL-2 release was not caused by induction of cell death. Further studies with GTP, ITP, guanosine and inosine showed that pretreatment with these nucleotides/nucleosides also suppressed release of IL-6. However, these nucleotides/nucleosides did not suppress stimulus-induced phosphorylation of ERK1/2, suggesting that the suppression of the release of inflammatory cytokines does not involve inhibition of ERK1/2 signaling. In contrast to ATP pretreatment at the same concentration, guanine or inosine nucleotides/nucleosides did not attenuate the expression of CD25. Our findings indicate that exogenous guanine or inosine nucleotides/nucleosides can suppress inflammatory cytokine release from T cells, and may be promising candidates for use as supplementary agents in the treatment of T cell-mediated immune diseases.

Although the bacterium Symbiobacterium thermophilum has a genome with a high guanine-cytosine (GC) content (69%), it belongs to a low GC content bacterial group. We detected only 18 low GC content regions with 5 or more consecutive genes whose GC contents were below 65% in the genome of this organism. S. thermophilum has 66 transposase genes, which are markers of transposable genetic elements, and 38 (58%) of them were located in the low GC content regions, suggesting that Symbiobacterium has a similar gene silencing system as Salmonella. The top hit (best match) analyses for each Symbiobacterium protein showed that putative horizontally transferred genes and vertically inherited genes are scattered across the genome. Approximately 25% of the 3338 Symbiobacterium proteins have the highest similarity with the protein of a phylogenetically distant organism. The putative horizontally transferred genes also have a high GC content, suggesting that Symbiobacterium has gained many DNA fragments from phylogenetically distant organisms during the early stage of Firmicutes evolution. After acquiring genes, Symbiobacterium increased the GC content of the horizontally transferred genes and thereby maintained a genome with a high GC content.

Extracellular/environmental stimuli trigger cellular responses to allow Schistosoma sp. parasites adaptation and decide development and survival fate. In this context, signal transduction involving eukaryotic protein kinases (ePKs) has an essential role in regulatory mechanisms. Functional studies had shown the importance of MAPK pathway for Schistosoma mansoni development. In addition, early studies demonstrated that Smp38 MAPK regulates the expression of a large set of genes, among them the hypoxanthine-guanine phosphoribosyl transferase 1 (SmHGPRTase 1, Smp_103560), a key enzyme in the purine salvage pathway that is part of a family comprising five different proteins.

Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that when two of the ANPs bind, they induce an unusual conformation change to the loop where the reaction product, pyrophosphate, is expected to bind. This and other structural differences between the Tbr and human enzymes suggest selective inhibitors for the Tbr enzyme can be designed.

Therapeutic treatment of tuberculosis (TB) is becoming increasingly problematic due to the emergence of drug resistant Mycobacterium tuberculosis (Mt). Thus, new targets for anti-TB drug discovery need to be identified to combat and eradicate this disease. One such target is hypoxanthine-guanine phosphoribosyltransferase (HGPRT) which synthesises the 6-oxopurine nucleoside monophosphates essential for DNA/RNA production. [3R,4R]-4-Hypoxanthin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine and [3R,4R]-4-guanin-9-yl-3-((S)-2-hydroxy-2-phosphonoethyl)oxy-1-N-(phosphonopropionyl)pyrrolidine (compound 6) are the most potent inhibitors of MtHGPRT yet discovered having Ki values of 60 nM. The crystal structure of the MtHGPRT.6 complex was obtained and compared with that of human HGPRT in complex with the same inhibitor. These structures provide explanations for the 60-fold difference in the inhibition constants between these two enzymes and a foundation for the design of next generation inhibitors. In addition, crystal structures of MtHGPRT in complex with two pyrrolidine nucleoside phosphosphonate inhibitors plus pyrophosphate provide insights into the final stage of the catalytic reaction. As the first step in ascertaining if such compounds have the potential to be developed as anti-TB therapeutics, the tetra-(ethyl L-phenylalanine) tetraamide prodrug of 6 was tested in cell based assays. This compound arrested the growth of virulent Mt not only in its replicating phase (IC50 of 14 μΜ) but also in its latent phase (IC50 of 29 μΜ). Furthermore, it arrested the growth of Mt in infected macrophages (MIC50 of 85 μΜ) and has a low cytotoxicity in mammalian cells (CC50 of 132 ± 20 μM). These inhibitors are therefore viewed as forerunners of new anti-TB chemotherapeutics.

The genetic information encoded in genomes must be faithfully replicated and transmitted to daughter cells. The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity. Here, we have shown that although 5caC pairs with guanine during DNA replication in vitro, G·5caC pairs stimulated DNA polymerase exonuclease activity and were recognized by the mismatch repair (MMR) proteins. Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC. These results suggest the epigenetic modification products of 5caC behave as DNA lesions.

Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein). TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs), Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K), appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.