Search

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

The Bacillus anthracis BA2291 gene codes for a sensor histidine kinase involved in the induction of sporulation. Genes for orthologs of the sensor domain of the BA2291 kinase exist in virulence plasmids in this organism, and these proteins, when expressed, inhibit sporulation by converting BA2291 to an apparent phosphatase of the sporulation phosphorelay. Evidence suggests that the sensor domains inhibit BA2291 by titrating its activating signal ligand. Studies with purified BA2291 revealed that this kinase is uniquely specific for GTP in the forward reaction and GDP in the reverse reaction. The G1 motif of BA2291 is highly modified from ATP-specific histidine kinases, and modeling this motif in the structure of the kinase catalytic domain suggested how guanine binds to the region. A mutation in the putative coiled-coil linker between the sensor domain and the catalytic domains was found to decrease the rate of the forward autophosphorylation reaction and not affect the reverse reaction from phosphorylated Spo0F. The results suggest that the activating ligand for BA2291 is a critical signal for sporulation and in a limited concentration in the cell Decreasing the response to it either by slowing the forward reaction through mutation or by titration of the ligand by expressing the plasmid-encoded sensor domains switches BA2291 from an inducer to an inhibitor of the phosphorelay and sporulation.

Many of the adaptive mechanisms that allow Bacillus subtilis to adjust to changes in nutrient availability are controiled by CodY. Binding of CodY to its target genes is stimulated by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon nutrient limitation, intracellular pools of these effectors are depleted and CodY can no Ionger repress genes required for adaptation. In vitro studies reported here explored in more detail the interaction of CodY with GTP. DNase I footprinting experiments indicated that CodY has an atfinity for GTP in the millimolar range. Further, CodY was shown to interact specifically with GTP and dGTP; no other naturally occurring nucleotides that were tested, including ppGpp and pppGpp, resulted in DNA protection. Two nonhydrolyzable anaiogs of GTP were fully able to activate CodY binding to target DNA, demonslrating that GTP hydrolysis is not necessary for CodY-dependent regulation. GTP and the BCAAs were shown to act additively to increase the affinity of CodY for DNA; increased protection was observed in DNase I footprinting experiments when both effectors were present, compared to either effector alone, and in in vitro transcription reactions, transcriptional repression by CodY was stronger in the presence of both GTP and BCAAs than of BCAAs alone. Thus, interaction of CodY with GTP is specific and results in increased attinity for its target genes. This increase in aflinity is independent of GTP hydrolysis and is augmented in the presence of BCAAs.

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.

The targeting of proteins into and across biological membranes to their correct cellular locations is mediated by a variety of transport pathways. These systems must couple the thermodynamically unfavorable processes of substrate translocation and integration with the expenditure of metabolic energy, using the free energy of ATP and GTP hydrolysis and/or a transmembrane protonmotive force. Several recent advances in our knowledge of the structure and function of these transport systems have provided insights into the mechanisms of energy transduction, force generation and energy use by different protein transport pathways.

The role of GTP in the activation of human neutrophil NADPH oxidase was studied. To eliminate the possibility of nucleotide contamination, experiments were conducted in a cell-free system wherein nucleotide levels were reduced by > 99.5%. The results showed that activation of NADPH oxidase is dependent on GTP. Furthermore, since analogs were as effective or more effective than GTP to activate NADPH oxidase, GTP-binding proteins may be involved in the in vivo activation. ATP was not required for activation, however, in the presence of GMP and GDP, ATP could indirectly activate the enzyme through its role in GTP synthesis via GMP and GTP.

Early screens for conditional lethal mutations that affected rRNA expression in Escherichia coli identified temperature-sensitive fda mutants (fda encodes the glycolytic enzyme fructose-l,6-diphosphate aldolase). It was shown that these fda(Ts) mutants were severely impaired in rRNA synthesis upon shift to the restrictive temperature, although the mechanism of inhibition was never determined. Here, we bring resolution to this long-standing question by showing that changes in the concentrations of guanosine 5'-diphosphate 3'-diphosphate and initiating nucleoside triphosphates can account for the previously observed effects of fda mutations on rRNA transcription.

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.