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GTP cyciohydrolase I (GCYH-I) is an essential [Zn.sup.2+]-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ~25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under [Zn.sup.2+]-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during [Zn.sup.2+] starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, [Zn.sup.2+]-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development. doi: 10.1128/JB.00287-09

The previously 'orphan' G protein-coupled receptor GPR17 is structurally related to both P2Y nucleotide receptors and to receptors for cysteinyl leukotrienes. Genomic analysis revealed two putative open reading frames encoding for a 'short' and a 'long' receptor isoform of 339- and 367-amino acids, respectively, with the latter displaying a 28-amino acid longer N[H.sub.2] terminus. The short isoform has been recently 'deorphanized,' revealing dual responses to uracil nucleotides and cysteinyl leukotrienes. No information regarding the ligand specificity, tissue distribution, or pathophysiological roles of the long receptor isoform is available. In the present study, we cloned human long-GPR17, determined its tissue distribution, and characterized its pharmacological specificity in 1321N1 cells by [[sup.35]S]GTP[gamma]S binding (which measures the ability of G protein-coupled receptor agonists to increase GTP binding to G proteins) and whole cell patch-clamp recording measuring receptor coupling to [K.sup.+] channels. [[sup.35]S]GTP[gamma]/S binding in long-GPR 17-expressing 1321N 1 cells revealed concentration-dependent responses to uracil nucleotides (UDP-galactose = UDP > UDP-glucose) and cysteinyl leukotrienes (LT[C.sub.4] > LT[D.sub.4]), which were counteracted by a purinergic (cangrelor) and a cysteinyl leukotriene antagonist (montelukast), respectively. The nonhydrolyzable ATP analog ATP[gamma]S also acted as an antagonist. GPR17 coupled to Gi and, to a lesser extent, [G.sub.q] proteins. UDP-glucose and LT[D.sub.4] also induced increases in overall outward [K.sup.+] currents, which were antagonized by the pufinergic antagonists MRS2179 and cangrelor and by montelukast. We conclude that the previously uncharacterized long-GPR17 isoform is a functional receptor that is stimulated by both uracil nucleotides and cysteinyl leukotrienes. We also show that the signaling pathway of GPR17 involves the generation of outward [K.sup.+] currents, an important protective mechanism that, in brain, is specifically aimed at reducing neuronal hyperexcitability and resultant neuronal injury. uracil nucleotides; cysteinyl leukotrienes; G protein-coupled receptors; big-conductance [K.sup.+] channels; neuronal damage; guanosine 5'-O-(3-thiotriphosphate) doi: 10.1152/ajpcell.00658.2008

Natriuretic peptide receptors A (NPR-A) and B (NPR-B) mediate most effects of natriuretic peptides by synthesizing cGMP. ATP increases the activity of these receptors by an unknown mechanism. We recently reported that a nonhydrolyzable form of ATP, adenylyl imidodiphosphate (AMPPNP), stabilizes but is not required for the activation of NPR-A and NPR-B in membranes from highly overexpressing cells. Here, we repeated these studies on receptors expressed in endogenous settings. Kinetic analysis indicated that both AMPPNP and ATP dramatically decrease the apparent [K.sub.m] of both receptors for GTP but had little effect on the [V.sub.max]. The [EC.sub.50] for AMPPNP decreased as substrate concentration increased whereas the magnitude of the effect was greater at lower GTP concentrations. ATP increased the activity of a mutant receptor containing glutamates substituted for all known phosphorylation sites similarly to the wild-type receptor, consistent with a phosphorylation independent mechanism. Finally, the putative ATP binding sites were investigated. Mutation of the ATP modulatory domain region had no effect, but mutation of K535A dramatically diminished ANP-dependent cyclase activity in a manner that was unresponsive to ATP. Mutation of the highly conserved 630-KSS to AAA (all alanines) resulted in an expressed receptor that had no detectable guanylyl cyclase activity. We conclude that ATP is not required for the initial activation of NPRs but does increase activity over time by reducing the apparent [K.sub.m] for GTP. guanylyl cyclase; cyclic guanosine monophosphate; guanosine triphosphate; adenosine triphosphate; Michaelis-Menten constant; heart failure; hypertension; bone growth

The article describes the newly discovered [Fe.sup.2}-dependent archael-specific GTP cyclohydrolase, MptA, which is obtained from Methanocaldococcus jannaschii and catalyzes the first step in the biosynthesis of methanopterin. The analysis reveals that the mutation of conserved histidine residues, expected to be involved in such binding, reduces the enzymatic activity, but not the amount of bond iron.

Rho GTPases can be activated in subcellular zones that appear semi-stable, yet are dynamically maintained. The basic features of Rho GTPases activity zones are reviewed, and the implications of zone properties from the perspective of both their function and how they are likely to be controlled are discussed.

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent ([rel.sup.+]; wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits [tRNA.sup.set] aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

Translational initiation factor 2 (IF2) is a guanine nucleotide-binding protein that can bind guanosine 3',5'-(bis) diphosphate (ppGpp), an alarmone involved in stringent response in bacteria. In cells growing under optimal conditions, the GTP concentration is very high, and that of ppGpp very low. However, under stress conditions, the GTP concentration may decline by as much as 50%, and that of ppGpp can attain levels comparable to those of GTP. Here we show that IF2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. Thus, GTP and the alarmone ppGpp can be considered two alternative physiologically relevant IF2 ligands, ppGpp interferes with IF2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and blocks the initiation step of translation. Our data suggest that IF2 has the properties of a cellular metabolic sensor and regulator that oscillates between an active GTP-bound form under conditions allowing active protein syntheses and an inactive ppGpp-bound form when shortage of nutrients would be detrimental, if not accompanied by slackening of this synthesis. fast kinetics | GTP | translation regulation | nutritional stress

Genistein, the primary isoflavone component of soy, consumed in the diet during the prepubertal period only, and the combined prepubertal and adult periods, suppresses chemically induced mammary cancer in rats. Gestational or adult-only exposures do not provide protection. An inverse relation exists between cancer susceptibility and mammary gland differentiation. The current study used proteomic technology to investigate genistein mechanisms of action as related to programming against chemically induced mammary cancer. Rats were injected subcutaneously with 500 [micro]g genistein/g body weight on d 16, 18, and 20 postpartum. At d 21, mammary glands were subjected to 2-dimensional polyacrylamide gel electrophoresis. After gel scanning, image analysis, and MS, 6 proteins were determined to be differentially regulated and identified. One protein, GTP-cyclohydrolase 1 (GTP-CH1), was confirmed as being significantly upregulated at d 21 by immunoblot analysis. Investigation of downstream signaling from GTP-CH1 showed that tyrosine hydroxylase was upregulated and vascular endothelial growth factor receptor 2 (VEGFR2) was downregulated in the mammary glands of 50-d-old rats treated with genistein in the prepubertal period. This and previous work suggest that early prepubertal exposure to genistein enhances cell proliferation by upregulating GTP-CH1 and the epidermal growth factor (EGF)-signaling pathway, and hence cell differentiation and gland maturation. This unique developmental maturation leads to a new biochemical blueprint, whereby the cells have reduced EGF signaling and VEGFR2, which renders the mature mammary gland less proliferative and less susceptible to cancer. This study demonstrated the usefulness of proteomics for the discovery of novel pathways that may be involved in cancer prevention. KEY WORDS: * 2-D gel * genistein * GTP-CH1 * VEGFR2 * proteomics

The general stress regulon of Bacillus subtilis is induced by activation of the [[sigma].sup.B] transcription factor. [[sigma].sup.B] activation occurs when one of two phosphatases responds to physical or nutritional stress to activate a positive [[sigma].sup.B] regulator by dephosphorylation. The signal that triggers the nutritional stress phosphatase (RsbP) is unknown; however, RsbP activation occurs under culture conditions (glucose/phosphate starvation, azide or decoyinine treatment) that reduce the cell's levels of ATP and/or GTP. Variances in nucleotide levels in these instances may be coincidental rather than causal. RsbP carries a domain (PAS) that in some regulatory systems can respond directly to changes in electron transport, proton motive force, or redox potential, changes that typically precede shifts in high-energy nucleotide levels. The current work uses Bacillus subtilis with mutations in the oxidative phosphorylation and purine nucleotide biosynthetic pathways in conjunction with metabolic inhibitors to better define the inducing signal for RsbP activation. The data argue that a drop in ATP, rather than changes in GTP, proton motive force, or redox state, is the key to triggering [[sigma].sup.B] activation.

ATP sulfurylase, from Escherichia coli K-12, conformationally couples the rates and chemical potentials of the two reactions that it catalyzes, GTP hydrolysis and activated sulfate synthesis. Two chemistry is linked at multiple points in the reaction coordinate.

Amino acids that are required, or partially required to support the formation of GDP-beta-L-fucose are identified using a combination of site-directed mutagenesis and UV photoaffinity cross-linking. The identification of the critical residues highlights how distinct GTP-L-fucose pyrophosphorylase is from other nucleotide-sugar pyrophosphorylases.

A recent study identified genuine tubulin proteins, BtubA and BtubB, in the bacterial genus Prosthecobacter. We have expressed BtubA and BtubB in Escherichia coli and studied their in vitro assembly. BtubB by itself formed rings with an outer diameter of 35-36 nm in the presence of GTP or GDP. Mixtures of BtubB and BtubA formed long protofilament bundles, 4-7 protofilaments wide (20-30 protofilaments in the three-dimensional bundle). Regardless of the starting stoichiometry, the polymers always contained equal concentrations of BtubA and BtubB, suggesting that BtubA and B alternate along the protofilament. BtubA showed negligible GTP hydrolysis, whereas BtubB hydrolyzed 0.40 mol GTP per min per mol BtubB. This GTPase activity increased to 1.37 per min when mixed 1:1 with BtubA. A critical concentration of 0.4-1.0 [micro]M was indicated by light scattering experiments and extrapolation of GTPase versus concentration, thus suggesting a cooperative assembly mechanism.

Nucleostemin (NS) was identified as a stem cell--and cancer cell-enriched nucleolar protein that controls the proliferation of these cells. Here, we report the mechanism that regulates its dynamic shuttling between the nucleolus and nucleoplasm. The nucleolar residence of nucleostemin involves a transient and a long-term binding by the basic and GTP-binding domains, and a dissociation mechanism mediated by the COOH-terminal region. This cycle is propelled by the GTP binding state of nucleostemin. We propose that a rapid nucleostemin cycle is designed to translate extra-and intra-cellular signals into the amount of nucleostemin in the nucleolus in a bidirectional and fast manner.

It is demonstrated that human pre-miRNA nuclear export, and miRNA function, are dependent on Exportin-5. The findings show that Exportin-5 can bind pre-miRNAs specifically in vitro, but only in the presence of the Ran-GTP cofactor.

Light and electron microscopic studies have revealed the binding of Xenopus laevis egg nuclear membrane vesicles to demembranated sperm chromatin and its subsequent fusion and nuclear envelope formation in the presence of cytosol. However, the addition of a hydrolysis-resistant gunaosine triphosphate analogueGTPgammaS inhibits this nuclear assembly process in direct proportion to the concentration of GTPgammaS added. These observations suggest a GTP-dependent soluble factor requiring GTP hydrolysis for activity.

A family, or group, of proteins has been identified and given the name the GTPases. These proteins bind and utilize GTP (guanosine triphosphate), and are regulated by the binding of GTP. The structure of the GTPases, and the regulation of the turning on or off of the protein by binding of GTP, has been conserved throughout evolution. It is conserved in simple organisms such as bacteria and yeast, as well as in more complex animals such as flies and vertebrates, including man. The GTPases are coupled to and regulate other proteins that are involved in many cellular functions including: the synthesis of proteins; the transmission of signals by hormones or light into the cell; the passage of proteins through organelles in the cell so they can eventually be utilized by the body; the control of cell maturation or differentiation and cell growth; and the transport of vesicles that are necessary for secretion of proteins outside of the cells. Some of these GTPases, such as the ras and NF1 gene, if mutated, (genetically changed), are oncogenes, genes that are involved in the abnormal growth of cells in cancer. It is thought that further research will identify additional GTPases and their involvement in additional cellular functions. (Consumer Summary produced by Reliance Medical Information, Inc.)

RanGTP has a central role in spindle assembly, but the Ran-regulated factors required to initiate spindle bipolarity and stabilize MT growth toward the chromosomes remain unknown. However, three recent papers (Koffa et al., 2006; Sillje et al., 2006; Wong and Fang, 2006) have identified a single factor, HURP, that may encompass both of these properties.

Three aspects of FtsZ filament dynamics, namely, rates of GTP hydrolysis, subunit exchange between protofilaments, and disassembly induced by dilution or excess GDP measured at steady state are reported. The exchange of subunits between protofilaments at steady state involved two separate mechanisms, fragmentation or dissociation of subunits from protofilament which terminated following GTP hydrolysis and reversible association and dissociation of subunits from protofilament that terminated independent of hydrolysis.

In a large percentage of B-cell lymphomas, tumors of the lymphatic system, a translocation (in which certain regions of chromosomes are moved to another chromosome) occurs between chromosome 14 and 18, which moves a portion of the oncogene (tumor-causing gene) Bcl-2. Because of the change in chromosomal region, the Bcl-2 gene is no longer regulated and the protein the gene encodes, Bcl-2, is produced in large amounts. The Bcl-2 protein has been shown to have a function in the survival of cells, in the enhancement of the growth of cells, and in uncontrolled growth, i.e. cancer. It was thought that Bcl-2 binds guanidine triphosphate nucleotide (GTP) and this binding is thought to mediate the biological effects of Bcl-2. The levels of GTP binding proteins (G proteins) were examined in lymphoid cells that expressed Bcl-2. Several small G proteins, which were the same size as Bcl-2, were found to be expressed, but the level of expression did not change in cancerous cells, as does Bcl-2. Using protein chemistry and cell biological techniques, the Bcl-2 protein was separated from the small G proteins. It was shown that Bcl-2 did not bind GTP directly. Thus, the biochemical role of Bcl-2 is not known and the mechanisms behind its role in uncontrolled growth remain unknown as well. (Consumer Summary produced by Reliance Medical Information, Inc.)

The research focuses on discovering various inhibitors of the tissue transglutaminase (TGase) enzyme that play a very important role in neurodegenerative diseases by promoting the aggregation of disease-specific proteins. The kinetic analysis of the reaction between the enzyme and a slow-binding inhibitor shows that the potency and the kinetics of the inhibition are highly dependant on the structure of the substrate and also leads to several multiple conformational stages of the enzyme.

Protein-DNA complexes formed during the initial stages of P-element transposition is visualized using atomic force microscopy (AFM). The results revealed that guanosine triphosphate (GTP) acts to promote assembly of the first detectable noncovalent precleavage synaptic complex.

Proteins are directed to cellular compartments by specific localization signals. A GTP-driven cycle has now been identified as a mechanism for protein targeting to the nucleolus. The involvement of a GTP switch suggests that nucleolar localization can be regulated and may be responsive to extracellular stimuli via signaling pathways. The uncovered mechanism also implies that localization is determined by increased retention rather than directed targeting.