GDP-GTP exchange processes of G{alpha}i1 protein are accelerated/decelerated depending on the type and the concentration of added detergents.

PubMed

Kubota, Makoto; Tanaka, Takeshi; Kohno, Toshiyuki; Wakamatsu, Kaori

2009-12-01

Although detergents have been widely used in G-protein studies to increase solubility and stability of the protein, we noticed that detergents modulate the nucleotide-binding properties of G-proteins. Hence, we analysed the effects of detergents on guanine nucleotide exchange reactions of Galpha(i1). Lubrol PX, a non-ionic detergent, which has been widely used in nucleotide dissociation/binding assays, was found to accelerate both GDP dissociation and GTPgammaS binding from/to Galpha in parallel at above its critical micelle concentration (cmc). Sodium cholate, an anionic detergent, which have been used to extract G-proteins from animal tissues, decelerated and accelerated GDP dissociation below and above its cmc, respectively. Surprisingly, micellar cholate decelerated GTPgammaS binding, and the binding rate constant was decreased by three orders of magnitude in the presence of 2% cholate. These results demonstrate that the guanine nucleotide exchange reactions of Galpha(i1) are drastically modulated by detergents differently depending on the type and the state (monomeric or micellar) of the detergents and that dissociation of GDP from Galpha(i1) does not necessarily lead to immediate binding of GTP to Galpha(i1) in some cases. These effects of detergents on G-proteins must be taken into account in G-protein experiments.

ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A/sub 1/

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gill, D.M.; Coburn, J.

1987-10-06

The authors have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S x GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S x GTP..gamma..S), forms over a period of 10-15 min at 37/sup 0/C. Both guanosine 5'-O-(2-thiodiphosphate)more » and GTP block this quasi-permanent activation. Some S x GTP..gamma..S forms in membranes that are exposed to CF alone and then to GTP..gamma..S, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S x GTP..gamma..S dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45/sup 0/C is decreased by GTP..gamma..S. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. They suggest that active S interacts directly with the enzymic A/sub 1/ fragments of cholera toxin and not with any toxin substrate. The activation and activity of S are independent of the state, or even the presence, of adenylate cyclase and seem to be involved with the cyclase system only via cholera toxin. S is apparently not related by function to certain other GTP binding proteins, including p21/sup ras/, and appears to be a new GTP binding protein whose physiologic role remains to be identified.« less

Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology

PubMed Central

Gebbink, Martijn F.B.G.; Kranenburg, Onno; Poland, Mieke; van Horck, Francis P.G.; Houssa, Brahim; Moolenaar, Wouter H.

1997-01-01

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth. PMID:9199174

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding.

PubMed

Goricanec, David; Stehle, Ralf; Egloff, Pascal; Grigoriu, Simina; Plückthun, Andreas; Wagner, Gerhard; Hagn, Franz

2016-06-28

Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

PubMed Central

Goricanec, David; Stehle, Ralf; Egloff, Pascal; Grigoriu, Simina; Wagner, Gerhard; Hagn, Franz

2016-01-01

Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein–coupled receptor (GPCR) activation. Agonist–receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape. PMID:27298341

Yrb4p, a yeast ran-GTP-binding protein involved in import of ribosomal protein L25 into the nucleus.

PubMed Central

Schlenstedt, G; Smirnova, E; Deane, R; Solsbacher, J; Kutay, U; Görlich, D; Ponstingl, H; Bischoff, F R

1997-01-01

Gsp1p, the essential yeast Ran homologue, is a key regulator of transport across the nuclear pore complex (NPC). We report the identification of Yrb4p, a novel Gsp1p binding protein. The 123 kDa protein was isolated from Saccharomyces cerevisiae cells and found to be related to importin-beta, the mediator of nuclear localization signal (NLS)-dependent import into the nucleus, and to Pse1p. Like importin-beta, Yrb4p and Pse1p specifically bind to Gsp1p-GTP, protecting it from GTP hydrolysis and nucleotide exchange. The GTPase block of Gsp1p complexed to Yrb4p or Pse1p is released by Yrb1p, which contains a Gsp1p binding domain distinct from that of Yrb4p. This might reflect an in vivo function for Yrb1p. Cells disrupted for YRB4 are defective in nuclear import of ribosomal protein L25, but show no defect in the import of proteins containing classical NLSs. Expression of a Yrb4p mutant deficient in Gsp1p-binding is dominant-lethal and blocks bidirectional traffic across the NPC in wild-type cells. L25 binds to Yrb4p and Pse1p and is released by Gsp1p-GTP. Consistent with its putative role as an import receptor for L25-like proteins, Yrb4p localizes to the cytoplasm, the nucleoplasm and the NPC. PMID:9321403

Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein*

PubMed Central

García-Márquez, Adrián; Gijsbers, Abril; de la Mora, Eugenio; Sánchez-Puig, Nuria

2015-01-01

Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1. PMID:25991726

Backbone resonance assignments for G protein α(i3) subunit in the GDP-bound state.

PubMed

Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

2014-10-01

Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gαi3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα(i3) in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα(i3)·GDP would be useful for the analyses of the dynamics of Gα(i3) and its interactions with various target molecules.

Discovery of G protein signaling.

PubMed

Selinger, Zvi

2008-01-01

The mechanism of transmembrane signaling by the receptor-activated adenylyl cyclase was an enigma. It was suggested that hydrolysis of GTP is a turn-off mechanism that resets the active adenylyl cyclase to the inactive state. To test this hypothesis, we developed a specific GTPase assay and found that the catecholamine adrenergic agonists stimulated the hydrolysis of GTP. To resolve the question of how the hormone concurrently stimulates GTP hydrolysis and activates the adenylyl cyclase, we suggested the regulatory GTPase cycle. Thus, because the hormone facilitates the binding of GTP, which is subsequently hydrolyzed, the regulatory cycle results in a hormone-stimulated GTPase activity. This model also predicts that two mechanisms could account for stimulation of adenylyl cyclase activity-either by the familiar hormone stimulation of the activation reaction or by an inhibition of the turn-off reaction. Indeed, we showed that cholera toxin enhances adenylyl cyclase activity by inhibition of GTP hydrolysis. Finally, we also showed that the hormone-activated receptor stimulates adenylyl cyclase activity by facilitating the exchange of bound GDP for free GTP. Thus, we presented, for the first time, an explicit mechanism for receptor action.

Dissociation of Rac1(GDP)·RhoGDI Complexes by the Cooperative Action of Anionic Liposomes Containing Phosphatidylinositol 3,4,5-Trisphosphate, Rac Guanine Nucleotide Exchange Factor, and GTP*

PubMed Central

Ugolev, Yelena; Berdichevsky, Yevgeny; Weinbaum, Carolyn; Pick, Edgar

2008-01-01

Rac plays a pivotal role in the assembly of the superoxide-generating NADPH oxidase of phagocytes. In resting cells, Rac is found in the cytosol in complex with Rho GDP dissociation inhibitor (RhoGDI). NADPH oxidase assembly involves dissociation of the Rac·RhoGDI complex and translocation of Rac to the membrane. We reported that liposomes containing high concentrations of monovalent anionic phospholipids cause Rac·RhoGDI complex dissociation (Ugolev, Y., Molshanski-Mor, S., Weinbaum, C., and Pick, E. (2006) J. Biol. Chem.281 ,19204 -1921916702219). We now designed an in vitro model mimicking membrane phospholipid remodeling during phagocyte stimulation in vivo. We showed that liposomes of “resting cell membrane” composition (less than 20 mol % monovalent anionic phospholipids), supplemented with 1 mol % of polyvalent anionic phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in conjunction with constitutively active forms of the guanine nucleotide exchange factors (GEFs) for Rac, Trio, or Tiam1 and a non-hydrolyzable GTP analogue, cause dissociation of Rac1(GDP)·RhoGDI complexes, GDP to GTP exchange on Rac1, and binding of Rac1(GTP) to the liposomes. Complexes were not dissociated in the absence of GEF and GTP, and optimal dissociation required the presence of PtdIns(3,4,5)P3 in the liposomes. Dissociation of Rac1(GDP)·RhoGDI complexes was correlated with the affinity of particular GEF constructs, via the N-terminal pleckstrin homology domain, for PtdIns(3,4,5)P3 and involved GEF-mediated GDP to GTP exchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P3 responded by NADPH oxidase activation upon exposure in vitro to Rac1(GDP)·RhoGDI complexes, p67phox, GTP, and Rac GEF constructs with affinity for PtdIns(3,4,5)P3 at a level superior to that of native membranes. PMID:18505730

Structural studies of the nudix hydrolase DR1025 from deinococcus radiodurans and its ligand complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ranatunga, Wasantha; Hill, Emma E.; Mooster, Jana L.

We have determined the crystal structure, at 1.4, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans. The protein forms an intertwined homodimer by exchanging N-terminal segments between chains. We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked b-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket. Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap4A (both at 1.6 resolution). Inmore » the Ap4Aco-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme. The GTP analog bound structure showed that GTP was bound almost identically as ATP. Neither nucleoside triphosphate was further cleaved.« less

Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

PubMed

Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

2015-04-03

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

PubMed Central

Sandri, Chiara; Caccavari, Francesca; Valdembri, Donatella; Camillo, Chiara; Veltel, Stefan; Santambrogio, Martina; Lanzetti, Letizia; Bussolino, Federico; Ivaska, Johanna; Serini, Guido

2012-01-01

During developmental and tumor angiogenesis, semaphorins regulate blood vessel navigation by signaling through plexin receptors that inhibit the R-Ras subfamily of small GTPases. R-Ras is mainly expressed in vascular cells, where it induces adhesion to the extracellular matrix (ECM) through unknown mechanisms. We identify the Ras and Rab5 interacting protein RIN2 as a key effector that in endothelial cells interacts with and mediates the pro-adhesive and -angiogenic activity of R-Ras. Both R-Ras-GTP and RIN2 localize at nascent ECM adhesion sites associated with lamellipodia. Upon binding, GTP-loaded R-Ras converts RIN2 from a Rab5 guanine nucleotide exchange factor (GEF) to an adaptor that first interacts at high affinity with Rab5-GTP to promote the selective endocytosis of ligand-bound/active β1 integrins and then causes the translocation of R-Ras to early endosomes. Here, the R-Ras/RIN2/Rab5 signaling module activates Rac1-dependent cell adhesion via TIAM1, a Rac GEF that localizes on early endosomes and is stimulated by the interaction with both Ras proteins and the vesicular lipid phosphatidylinositol 3-monophosphate. In conclusion, the ability of R-Ras-GTP to convert RIN2 from a GEF to an adaptor that preferentially binds Rab5-GTP allows the triggering of the endocytosis of ECM-bound/active β1 integrins and the ensuing funneling of R-Ras-GTP toward early endosomes to elicit the pro-adhesive and TIAM1-mediated activation of Rac1. PMID:22825554

IκB Kinase γ/Nuclear Factor-κB-Essential Modulator (IKKγ/NEMO) Facilitates RhoA GTPase Activation, which, in Turn, Activates Rho-associated Kinase (ROCK) to Phosphorylate IKKβ in Response to Transforming Growth Factor (TGF)-β1*

PubMed Central

Kim, Hee-Jun; Kim, Jae-Gyu; Moon, Mi-Young; Park, Seol-Hye; Park, Jae-Bong

2014-01-01

Transforming growth factor (TGF)-β1 plays several roles in a variety of cellular functions. TGF-β1 transmits its signal through Smad transcription factor-dependent and -independent pathways. It was reported that TGF-β1 activates NF-κB and RhoA, and RhoA activates NF-κB in several kinds of cells in a Smad-independent pathway. However, the activation molecular mechanism of NF-κB by RhoA upon TGF-β1 has not been clearly elucidated. We observed that RhoA-GTP level was increased by TGF-β1 in RAW264.7 cells. RhoA-GDP and RhoGDI were bound to N- and C-terminal domains of IKKγ, respectively. Purified IKKγ facilitated GTP binding to RhoA complexed with RhoGDI. Furthermore, Dbs, a guanine nucletotide exchange factor of RhoA much more enhanced GTP binding to RhoA complexed with RhoGDI in the presence of IKKγ. Indeed, si-IKKγ abolished RhoA activation in response to TGF-β1 in cells. However, TGF-β1 stimulated the release of RhoA-GTP from IKKγ and Rho-associated kinase (ROCK), an active RhoA effector protein, directly phosphorylated IKKβ in vitro, whereas TGF-β1-activated kinase 1 activated RhoA upon TGF-β1 stimulation. Taken together, our data indicate that IKKγ facilitates RhoA activation via a guanine nucletotide exchange factor, which in turn activates ROCK to phosphorylate IKKβ, leading to NF-κB activation that induced the chemokine expression and cell migration upon TGF-β1. PMID:24240172

A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction.

PubMed

Kopra, Kari; Ligabue, Alessio; Wang, Qi; Syrjänpää, Markku; Blaževitš, Olga; Veltel, Stefan; van Adrichem, Arjan J; Hänninen, Pekka; Abankwa, Daniel; Härmä, Harri

2014-07-01

A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.

GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oberdisse, E.; Lapetina, E.G.

1987-05-14

Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with (/sup 3/H)inositol. Ca/sup 2 +/ has opposite effects on the formation of (/sup 3/H)inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of (/sup 3/H)inositol phosphates is inhibited by Ca/sup 2 +/, action of thrombin is stimulated by Ca/sup 2 +/. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma Smore » on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca/sup 2 +/ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca/sup 2 +/ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.« less

Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

NASA Technical Reports Server (NTRS)

Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

1993-01-01

Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2*

PubMed Central

Hosokawa, Hiroyuki; Dip, Phat Vinh; Merkulova, Maria; Bakulina, Anastasia; Zhuang, Zhenjie; Khatri, Ashok; Jian, Xiaoying; Keating, Shawn M.; Bueler, Stephanie A.; Rubinstein, John L.; Randazzo, Paul A.; Ausiello, Dennis A.; Grüber, Gerhard; Marshansky, Vladimir

2013-01-01

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H+-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1–17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1–17) and its amino acids Phe5, Met10, and Gln14 involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1–17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1–a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor. PMID:23288846

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khatoon, S.; Slevin, J.T.; Haley, B.E.

A decrease occurs (80-100%) in the (/sup 32/P)8N/sub 3/GTP photoinsertion into a cytosolic protein (55K M/sub r/) of Alzheimer's (AD) brain, tentatively identified as the ..beta..-subunit of tubulin (co-migration with purified tubulin, concentration dependence of interaction with GTP, ATP and their 8-azido photoprobes, and similar effects of Ca/sup 2 +/ and EDTA on photoinsertion). This agrees with prior observations of (/sup 32/P)8N/sub 3/GTP interactions with brain tubulin and a recent report on faulty microtubular assembly in AD brain. The decrease in (/sup 32/P)8N/sub 3/GTP photoinsertion into the 55K M/sub r/ protein of AD brain was in contrast with other photolabeledmore » proteins, which remained at equal levels in AD and age-matched normal brain tissues. The 55K and 45K M/sub r/ were the two major (/sup 32/P)8N/sub 3/GTP photoinsertion species in non-AD brain. Of 5 AD brains, the photoinsertion of (/sup 32/P)8N/sub 3/GTP into the 55K M/sub r/ region was low or absent in 4 (55K/45K=0.1); one was 75% below normals (55K/45K=0.24). Total protein migrating at 55K M/sub r/ was similar in AD and controls. AD brain tubulin, while present, has its exchangeable GTP binding site on ..beta..-tubulin blocked/modified such that (/sup 32/P)8N/sub 3/GTP cannot interact normally with this site.« less

Blue News Update: BODIPY-GTP Binds to the Blue-Light Receptor YtvA While GTP Does Not

PubMed Central

Schmieder, Peter

2012-01-01

Light is an important environmental factor for almost all organisms. It is mainly used as an energy source but it is also a key factor for the regulation of multiple cellular functions. Light as the extracellular stimulus is thereby converted into an intracellular signal by photoreceptors that act as signal transducers. The blue-light receptor YtvA, a bacterial counterpart of plant phototropins, is involved in the stress response of Bacillus subtilis. The mechanism behind its activation, however, remains unknown. It was suggested based on fluorescence spectroscopic studies that YtvA function involves GTP binding and that this interaction is altered by absorption of light. We have investigated this interaction by several biophysical methods and show here using fluorescence spectroscopy, ITC titrations, and three NMR spectroscopic assays that while YtvA interacts with BODIPY-GTP as a fluorescent GTP analogue originally used for the detection of GTP binding, it does not bind GTP. PMID:22247770

Thermodynamics of the GTP-GDP-operated Conformational Switch of Selenocysteine-specific Translation Factor SelB*

PubMed Central

Paleskava, Alena; Konevega, Andrey L.; Rodnina, Marina V.

2012-01-01

SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNASec) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNASec to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5′-O-(γ-thio)triphosphate (GTPγS) and guanosine 5′-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (−621, −467, −235, and −275 cal × mol−1 × K−1, with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15–19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form. PMID:22740700

Thermodynamics of the GTP-GDP-operated conformational switch of selenocysteine-specific translation factor SelB.

PubMed

Paleskava, Alena; Konevega, Andrey L; Rodnina, Marina V

2012-08-10

SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNA(Sec)) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNA(Sec) to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5'-O-(γ-thio)triphosphate (GTPγS) and guanosine 5'-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (-621, -467, -235, and -275 cal × mol(-1) × K(-1), with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15-19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form.

Into the linker's DENN: A tyrosine's control of autophagy.

PubMed

Caplan, Steve

2017-04-28

The small GTP-binding protein Rab12 plays an important role in the initiation of starvation-induced macroautophagy (autophagy) and is activated by the guanine-nucleotide exchange factor DENND3. However, the molecular mechanism by which DENND3 becomes activated has remained elusive. Xu and McPherson now identify a novel mechanism of DENND3 intramolecular binding that is regulated by the phosphorylation of a single tyrosine residue. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

PubMed Central

Van Eps, Ned; Thomas, Celestine J.; Hubbell, Wayne L.; Sprang, Stephen R.

2015-01-01

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF. PMID:25605908

Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.

PubMed

Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

2002-10-01

Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His6-tagged ScRAB protein was expressed in E. coli, and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding.

The pretranslocation ribosome is targeted by GTP-bound EF-G in partially activated form

PubMed Central

Hauryliuk, Vasili; Mitkevich, Vladimir A.; Eliseeva, Natalia A.; Petrushanko, Irina Yu.; Ehrenberg, Måns; Makarov, Alexander A.

2008-01-01

Translocation of the tRNA·mRNA complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor G (EF-G). We have used isothermal titration calorimetry to characterize the binding of GDP and GTP to free EF-G at 4°C, 20°C, and 37°C. The binding affinity of EF-G is higher to GDP than to GTP at 4°C, but lower at 37°C. The binding enthalpy and entropy change little with temperature in the case of GDP binding but change greatly in the case of GTP binding. These observations are compatible with a large decrease in the solvent-accessible hydrophobic surface area of EF-G on GTP, but not GDP, binding. The explanation we propose is the locking of the switch 1 and switch 2 peptide loops in the G domain of EF-G to the γ-phosphate of GTP. From these data, in conjunction with previously reported structural data on guanine nucleotide-bound EF-G, we suggest that EF-G enters the pretranslocation ribosome as an “activity chimera,” with the G domain activated by the presence of GTP but the overall factor conformation in the inactive form typical of a GDP-bound multidomain guanosine triphosphatase. We propose that the active overall conformation of EF-G is attained only in complex with the ribosome in its “ratcheted state,” with hybrid tRNA binding sites. PMID:18836081

Cloning and characterization of EF-Tu and EF-Ts from Pseudomonas aeruginosa.

PubMed

Palmer, Stephanie O; Rangel, Edna Y; Montalvo, Alberto E; Tran, Alexis T; Ferguson, Kate C; Bullard, James M

2013-01-01

We have cloned genes encoding elongation factors EF-Tu and EF-Ts from Pseudomonas aeruginosa and expressed and purified the proteins to greater than 95% homogeneity. Sequence analysis indicated that P. aeruginosa EF-Tu and EF-Ts are 84% and 55% identical to E. coli counterparts, respectively. P. aeruginosa EF-Tu was active when assayed in GDP exchange assays. Kinetic parameters for the interaction of EF-Tu with GDP in the absence of EF-Ts were observed to be K M = 33 μM, k cat (obs) = 0.003 s(-1), and the specificity constant k cat (obs)/K M was 0.1 × 10(-3) s(-1) μM(-1). In the presence of EF-Ts, these values were shifted to K M = 2 μM, k cat (obs) = 0.005 s(-1), and the specificity constant k(cat)(obs)/K M was 2.5 × 10(-3) s(-1) μM(-1). The equilibrium dissociation constants governing the binding of EF-Tu to GDP (K GDP) were 30-75 nM and to GTP (K GTP) were 125-200 nM. EF-Ts stimulated the exchange of GDP by EF-Tu 10-fold. P. aeruginosa EF-Tu was active in forming a ternary complex with GTP and aminoacylated tRNA and was functional in poly(U)-dependent binding of Phe-tRNA(Phe) at the A-site of P. aeruginosa ribosomes. P. aeruginosa EF-Tu was active in poly(U)-programmed polyphenylalanine protein synthesis system composed of all P. aeruginosa components.

Nucleotide-dependent bisANS binding to tubulin.

PubMed

Chakraborty, S; Sarkar, N; Bhattacharyya, B

1999-07-13

Non-covalent hydrophobic probes such as 5, 5'-bis(8-anilino-1-naphthalenesulfonate) (bisANS) have become increasingly popular to gain information about protein structure and conformation. However, there are limitations as bisANS binds non-specifically at multiple sites of many proteins. Successful use of this probe depends upon the development of binding conditions where only specific dye-protein interaction will occur. In this report, we have shown that the binding of bisANS to tubulin occurs instantaneously, specifically at one high affinity site when 1 mM guanosine 5'-triphosphate (GTP) is included in the reaction medium. Substantial portions of protein secondary structure and colchicine binding activity of tubulin are lost upon bisANS binding in absence of GTP. BisANS binding increases with time and occurs at multiple sites in the absence of GTP. Like GTP, other analogs, guanosine 5'-diphosphate, guanosine 5'-monophosphate and adenosine 5'-triphosphate, also displace bisANS from the lower affinity sites of tubulin. We believe that these multiple binding sites are generated due to the bisANS-induced structural changes on tubulin and the presence of GTP and other nucleotides protect those structural changes.

Activation of Elongation Factor G by Phosphate Analogues

PubMed Central

Salsi, Enea; Farah, Elie

2016-01-01

EF-G is a universally conserved translational GTPase that promotes the translocation of tRNA and mRNA through the ribosome. EF-G binds to the ribosome in a GTP-bound form and subsequently catalyzes GTP hydrolysis. The contribution of the ribosome-stimulated GTP hydrolysis by EF-G to tRNA/mRNA translocation remains debated. Here, we show that while EF-G•GDP does not stably bind to the ribosome and induce translocation, EFG• GDP in complex with phosphate group analogues BeF3− and AlF4− promotes the translocation of tRNA and mRNA. Furthermore, the rates of mRNA translocation induced by EF-G in the presence of GTP and a non-hydrolysable analogue of GTP, GDP•BeF3−are similar. Our results are consistent with the model suggesting that GTP hydrolysis is not directly coupled to mRNA/tRNA translocation. Hence, GTP binding is required to induce the activated, translocation-competent conformation of EF-G while GTP hydrolysis triggers EF-G release from the ribosome. PMID:27063503

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini

NASA Technical Reports Server (NTRS)

Perdue, D. O.; Lomax, T. L.

1992-01-01

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

Characterization of GTP binding and hydrolysis in plasma membranes of zucchini.

PubMed

Perdue, D O; Lomax, T L

1992-01-01

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras.

PubMed

Xu, Shenyuan; Long, Brian N; Boris, Gabriel H; Chen, Anqi; Ni, Shuisong; Kennedy, Michael A

2017-12-01

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras-GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

Structural insight into the rearrangement of the switch I region in GTP-bound G12A K-Ras

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Shenyuan; Long, Brian N.; Boris, Gabriel H.

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras–GTP complex, the switch I region undergoes amore » significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.« less

Molecular modeling study for interaction between Bacillus subtilis Obg and Nucleotides.

PubMed

Lee, Yuno; Bang, Woo Young; Kim, Songmi; Lazar, Prettina; Kim, Chul Wook; Bahk, Jeong Dong; Lee, Keun Woo

2010-09-07

The bacterial Obg proteins (Spo0B-associated GTP-binding protein) belong to the subfamily of P-loop GTPase proteins that contain two equally and highly conserved domains, a C-terminal GTP binding domain and an N-terminal glycine-rich domain which is referred as the "Obg fold" and now it is considered as one of the new targets for antibacterial drug. When the Obg protein is associated with GTP, it becomes activated, because conformation of Obg fold changes due to the structural changes of GTPase switch elements in GTP binding site. In order to investigate the effects and structural changes in GTP bound to Obg and GTPase switch elements for activation, four different molecular dynamics (MD) simulations were performed with/without the three different nucleotides (GTP, GDP, and GDP + Pi) using the Bacillus subtilis Obg (BsObg) structure. The protein structures generated from the four different systems were compared using their representative structures. The pattern of C(alpha)-C(alpha) distance plot and angle between the two Obg fold domains of simulated apo form and each system (GTP, GDP, and GDP+Pi) were significantly different in the GTP-bound system from the others. The switch 2 element was significantly changed in GTP-bound system. Also root-mean-square fluctuation (RMSF) analysis revealed that the flexibility of the switch 2 element region was much higher than the others. This was caused by the characteristic binding mode of the nucleotides. When GTP was bound to Obg, its gamma-phosphate oxygen was found to interact with the key residue (D212) of the switch 2 element, on the contrary there was no such interaction found in other systems. Based on the results, we were able to predict the possible binding conformation of the activated form of Obg with L13, which is essential for the assembly with ribosome.

Germline Missense Mutations Affecting KRAS Isoform B Are Associated with a Severe Noonan Syndrome Phenotype

PubMed Central

Carta, Claudio; Pantaleoni, Francesca; Bocchinfuso, Gianfranco; Stella, Lorenzo; Vasta, Isabella; Sarkozy, Anna; Digilio, Cristina; Palleschi, Antonio; Pizzuti, Antonio; Grammatico, Paola; Zampino, Giuseppe; Dallapiccola, Bruno; Gelb, Bruce D.; Tartaglia, Marco

2006-01-01

Noonan syndrome (NS) is a developmental disorder characterized by short stature, facial dysmorphia, congenital heart disease, and multiple skeletal and hematologic defects. NS is an autosomal dominant trait and is genetically heterogeneous. Gain of function of SHP-2, a protein tyrosine phosphatase that positively modulates RAS signaling, is observed in nearly 50% of affected individuals. Here, we report the identification of heterozygous KRAS gene mutations in two subjects exhibiting a severe NS phenotype with features overlapping those of cardiofaciocutaneous and Costello syndromes. Both mutations were de novo and affected exon 6, which encodes the C-terminal portion of KRAS isoform B but does not contribute to KRAS isoform A. Structural analysis indicated that both substitutions (Val152Gly and Asp153Val) perturb the conformation of the guanine ring–binding pocket of the protein, predicting an increase in the guanine diphosphate/guanine triphosphate (GTP) dissociation rate that would favor GTP binding to the KRASB isoform and bypass the requirement for a guanine nucleotide exchange factor. PMID:16773572

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTP gamma S to brain membranes.

PubMed

Moni, R W; Romero, F S; Daly, J W

1995-08-01

1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog, Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes. 2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 microM) for A1-adenosine receptors, 30% (100 microM) for A2a-adenosine receptors, 20% (2 microM) for alpha 2-adrenergic receptors, and 30% (10 microM) for 5HT1A receptors. High affinity agonist binding for A1-, alpha 2-, and 5HT1A-receptors was virtually abolished by GTP gamma S in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin. 3. The effect of adenoregulin on binding of N6-cyclohexyladenosine ([3H]CHA) to A1-receptors was relatively slow and was irreversible. Adenoregulin increased the Bmax value for [3H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [3H]CHA dissociation. Binding of the A1-selective antagonist, [3H]DPCPX, was maximally enhanced by only 13% at 2 microM adenoregulin. Basal and A1-adenosine receptor-stimulated binding of [35S]GTP gamma S were maximally enhanced 45% and 23%, respectively, by 50 microM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [3H]CHA and [3H]DPCPX were enhanced by adenoregulin. Binding of [3H]CHA to membranes from DDT1 MF-2 cells was maximally enhanced 17% at 20 microM adenoregulin. In intact DDT1 MF-2 cells, 20 microM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediated via the adenosine A1 receptor. 4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

PubMed

Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

2006-07-01

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

How a low-fidelity DNA polymerase chooses non-Watson-Crick from Watson-Crick incorporation.

PubMed

Wu, Wen-Jin; Su, Mei-I; Wu, Jian-Li; Kumar, Sandeep; Lim, Liang-Hin; Wang, Chun-Wei Eric; Nelissen, Frank H T; Chen, Ming-Chuan Chad; Doreleijers, Jurgen F; Wijmenga, Sybren S; Tsai, Ming-Daw

2014-04-02

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

PubMed Central

Ostrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.

2014-01-01

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1–3. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP4 and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis5,6. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP7 and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner. PMID:24256730

Zampanolide Binding to Tubulin Indicates Cross-Talk of Taxane Site with Colchicine and Nucleotide Sites.

PubMed

Field, Jessica J; Pera, Benet; Gallego, Juan Estévez; Calvo, Enrique; Rodríguez-Salarichs, Javier; Sáez-Calvo, Gonzalo; Zuwerra, Didier; Jordi, Michel; Andreu, José M; Prota, Andrea E; Ménchon, Grégory; Miller, John H; Altmann, Karl-Heinz; Díaz, J Fernando

2018-03-23

The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 10 5 M -1 in the case of unmodified tubulin to 1.4 ± 0.3 × 10 5 M -1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.

GTP effects in rat brain slices support the non-interconvertability of M/sub 1/ and M/sub 2/ muscarinic acetylcholine receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spencer, D.G. Jr.; Horvath, E.; Traber, J.

GTP (guanosine-5'-triphosphate) markedly reduced high-affinity /sup 3/H-oxotremorine-M binding to M/sub 2/ receptors on brain slices in autoradiographic experiments while /sup 3/H-pirenzepine binding to M/sub 1/ receptors was largely unaffected. The distribution of M/sub 1/ receptors so labelled was also not altered by GTP to include former M/sub 2/-rich regions, thus indicating that GTP could not, by itself, interconvert high agonist-affinity M/sub 2/ receptors to M/sub 1/ receptors. 18 references, 1 figure.

An Arabidopsis Ran-binding protein, AtRanBP1c, is a co-activator of Ran GTPase-activating protein and requires the C-terminus for its cytoplasmic localization

NASA Technical Reports Server (NTRS)

Kim, Soo-Hwan; Roux, Stanley J.

2003-01-01

Ran-binding proteins (RanBPs) are a group of proteins that bind to Ran (Ras-related nuclear small GTP-binding protein), and thus either control the GTP/GDP-bound states of Ran or help couple the Ran GTPase cycle to a cellular process. AtRanBP1c is a Ran-binding protein from Arabidopsis thaliana (L.) Heynh. that was recently shown to be critically involved in the regulation of auxin-induced mitotic progression [S.-H. Kim et al. (2001) Plant Cell 13:2619-2630]. Here we report that AtRanBP1c inhibits the EDTA-induced release of GTP from Ran and serves as a co-activator of Ran-GTPase-activating protein (RanGAP) in vitro. Transient expression of AtRanBP1c fused to a beta-glucuronidase (GUS) reporter reveals that the protein localizes primarily to the cytosol. Neither the N- nor C-terminus of AtRanBP1c, which flank the Ran-binding domain (RanBD), is necessary for the binding of PsRan1-GTP to the protein, but both are needed for the cytosolic localization of GUS-fused AtRanBP1c. These findings, together with a previous report that AtRanBP1c is critically involved in root growth and development, imply that the promotion of GTP hydrolysis by the Ran/RanGAP/AtRanBP1c complex in the cytoplasm, and the resulting concentration gradient of Ran-GDP to Ran-GTP across the nuclear membrane could be important in the regulation of auxin-induced mitotic progression in root tips of A. thaliana.

High-throughput screening identifies small molecules that bind to the RAS:SOS:RAS complex and perturb RAS signaling.

PubMed

Burns, Michael C; Howes, Jennifer E; Sun, Qi; Little, Andrew J; Camper, DeMarco V; Abbott, Jason R; Phan, Jason; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2018-05-01

K-RAS is mutated in approximately 30% of human cancers, resulting in increased RAS signaling and tumor growth. Thus, RAS is a highly validated therapeutic target, especially in tumors of the pancreas, lung and colon. Although directly targeting RAS has proven to be challenging, it may be possible to target other proteins involved in RAS signaling, such as the guanine nucleotide exchange factor Son of Sevenless (SOS). We have previously reported on the discovery of small molecules that bind to SOS1, activate SOS-mediated nucleotide exchange on RAS, and paradoxically inhibit ERK phosphorylation (Burns et al., PNAS, 2014). Here, we describe the discovery of additional, structurally diverse small molecules that also bind to SOS1 in the same pocket and elicit similar biological effects. We tested >160,000 compounds in a fluorescence-based assay to assess their effects on SOS-mediated nucleotide exchange. X-Ray structures revealed that these small molecules bind to the CDC25 domain of SOS1. Compounds that elicited high levels of nucleotide exchange activity in vitro increased RAS-GTP levels in cells, and inhibited phospho ERK levels at higher treatment concentrations. The identification of structurally diverse SOS1 binding ligands may assist in the discovery of new molecules designed to target RAS-driven tumors. Copyright © 2018 Elsevier Inc. All rights reserved.

Role of the Rho GTPase Rac in the activation of the phagocyte NADPH oxidase

PubMed Central

Pick, Edgar

2014-01-01

The superoxide-generating NADPH oxidase of phagocytes consists of the membrane-associated cytochrome b558 (a heterodimer of Nox2 and p22phox) and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase, Rac, in complex with RhoGDI. Superoxide is produced by the NADPH-driven reduction of molecular oxygen, via a redox gradient located in Nox2. Electron flow in Nox2 is initiated by interaction with cytosolic components, which translocate to the membrane, p67phox playing the central role. The participation of Rac is expressed in the following sequence: (1) Translocation of the RacGDP-RhoGDI complex to the membrane; (2) Dissociation of RacGDP from RhoGDI; (3) GDP to GTP exchange on Rac, mediated by a guanine nucleotide exchange factor; (4) Binding of RacGTP to p67phox; (5) Induction of a conformational change in p67phox, promoting interaction with Nox2. The particular involvement of Rac in NADPH oxidase assembly serves as a paradigm for signaling by Rho GTPases, in general. PMID:24598074

The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

NASA Astrophysics Data System (ADS)

Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

2016-02-01

Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP-GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer.

ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses

PubMed Central

Cheung, Ming-Yan; Li, Xiaorong; Miao, Rui; Fong, Yu-Hang; Li, Kwan-Pok; Yung, Yuk-Lin; Yu, Mei-Hui; Wong, Kam-Bo; Lam, Hon-Ming

2016-01-01

G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein’s ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure–function relationships of the YchF subfamily of G proteins in all kingdoms of life. PMID:26912459

The microtubule-active antitumor compound TTI-237 has both paclitaxel-like and vincristine-like properties.

PubMed

Beyer, Carl F; Zhang, Nan; Hernandez, Richard; Vitale, Danielle; Nguyen, Thai; Ayral-Kaloustian, Semiramis; Gibbons, James J

2009-09-01

To compare TTI-237 (5-chloro-6-[2,6-difluoro-4-[3-(methylamino)propoxy]phenyl]-N-[(1S)-2,2,2-trifluoro-1-methylethyl]-[1, 2, 4]triazolo[1,5-a]pyrimidin-7-amine butanedioate) with paclitaxel and vincristine in order to better understand the properties of this new anti-microtubule agent. Tubulin polymerization and depolymerization were followed by turbidimetric assays. Effects of compounds on the binding of [(3)H]guanosine triphosphate ([(3)H]GTP) to tubulin were studied by competition binding assays. Effects of compounds on the phosphorylation of a panel of intracellular proteins were determined by flow cytometry using phosphoprotein-specific antibodies. At low molar ratios of TTI-237:tubulin heterodimer (about 1:30), TTI-237 enhanced depolymerization kinetics in response to low temperature, but stabilized the aggregates at higher ratios (about 1:4). Similarly, the aggregates induced in microtubule protein by TTI-237 were depolymerized by excess Ca(++) at low TTI-237:tubulin-heterodimer molar ratios, but were stable at higher ratios. TTI-237 inhibited the exchange of [(3)H]GTP at the exchangeable nucleotide site of the tubulin heterodimer, and was similar to vincristine in its effects on the phosphorylation of eight intracellular proteins in HeLa cells. TTI-237 has properties that distinguish it from typical vinca-site and taxoid-site ligands, and therefore it may exemplify a new class of microtubule-active compounds.

Influence of GTP/GDP and magnesium ion on the solvated structure of the protein FtsZ: a molecular dynamics study.

PubMed

Jamous, Carla; Basdevant, Nathalie; Ha-Duong, Tap

2014-01-01

We present here a structural analysis of ten extensive all-atom molecular dynamics simulations of the monomeric protein FtsZ in various binding states. Since the polymerization and GTPase activities of FtsZ depend on the nature of a bound nucleotide as well as on the presence of a magnesium ion, we studied the structural differences between the average conformations of the following five systems: FtsZ-Apo, FtsZ-GTP, FtsZ-GDP, FtsZ-GTP-Mg, and FtsZ-GDP-Mg. The in silico solvated average structure of FtsZ-Apo significantly differs from the crystallographic structure 1W59 of FtsZ which was crystallized in a dimeric form without nucleotide and magnesium. The simulated Apo form of the protein also clearly differs from the FtsZ structures when it is bound to its ligand, the most important discrepancies being located in the loops surrounding the nucleotide binding pocket. The three average structures of FtsZ-GTP, FtsZ-GDP, and FtsZ-GTP-Mg are overall similar, except for the loop T7 located at the opposite side of the binding pocket and whose conformation in FtsZ-GDP notably differs from the one in FtsZ-GTP and FtsZ-GTP-Mg. The presence of a magnesium ion in the binding pocket has no impact on the FtsZ conformation when it is bound to GTP. In contrast, when the protein is bound to GDP, the divalent cation causes a translation of the nucleotide outwards the pocket, inducing a significant conformational change of the loop H6-H7 and the top of helix H7.

A spatial focusing model for G protein signals. Regulator of G protein signaling (RGS) protien-mediated kinetic scaffolding.

PubMed

Zhong, Huailing; Wade, Susan M; Woolf, Peter J; Linderman, Jennifer J; Traynor, John R; Neubig, Richard R

2003-02-28

Regulators of G protein signaling (RGS) are GTPase-accelerating proteins (GAPs), which can inhibit heterotrimeric G protein pathways. In this study, we provide experimental and theoretical evidence that high concentrations of receptors (as at a synapse) can lead to saturation of GDP-GTP exchange making GTP hydrolysis rate-limiting. This results in local depletion of inactive heterotrimeric G-GDP, which is reversed by RGS GAP activity. Thus, RGS enhances receptor-mediated G protein activation even as it deactivates the G protein. Evidence supporting this model includes a GTP-dependent enhancement of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to G(i) by RGS. The RGS domain of RGS4 is sufficient for this, not requiring the NH(2)- or COOH-terminal extensions. Furthermore, a kinetic model including only the GAP activity of RGS replicates the GTP-dependent enhancement of GTPgammaS binding observed experimentally. Finally in a Monte Carlo model, this mechanism results in a dramatic "spatial focusing" of active G protein. Near the receptor, G protein activity is maintained even with RGS due to the ability of RGS to reduce depletion of local Galpha-GDP levels permitting rapid recoupling to receptor and maintained G protein activation near the receptor. In contrast, distant signals are suppressed by the RGS, since Galpha-GDP is not depleted there. Thus, a novel RGS-mediated "kinetic scaffolding" mechanism is proposed which narrows the spatial range of active G protein around a cluster of receptors limiting the spill-over of G protein signals to more distant effector molecules, thus enhancing the specificity of G(i) protein signals.

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

PubMed Central

Faurobert, E; Otto-Bruc, A; Chardin, P; Chabre, M

1993-01-01

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding. Images PMID:8223434

The N54-αs Mutant Has Decreased Affinity for βγ and Suggests a Mechanism for Coupling Heterotrimeric G Protein Nucleotide Exchange with Subunit Dissociation.

PubMed

Cleator, John H; Wells, Christopher A; Dingus, Jane; Kurtz, David T; Hildebrandt, John D

2018-05-01

Ser54 of G s α binds guanine nucleotide and Mg 2+ as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For α i/o , this results from persistent binding of α to βγ , whereas for small GTP binding proteins and α s this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of βγ interactions in mediating the properties of the Ser54-like mutants of G α subunits. Unexpectedly, WT- α s or N54- α s coexpressed with α 1B -adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT- α s was more effective than the mutant. One explanation for this result would be that α s , like Ser47 α i/o , blocks receptor activation by sequestering βγ ; implying that N54- α S has reduced affinity for βγ since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT- α s was more effective than the mutant in inhibiting βγ activation of phospholipase C β 2. Further, in vitro synthesized N54- α s bound biotinylated- βγ with lower apparent affinity than did WT- α s The Cys54 mutation also decreased βγ binding but less effectively than N54- α s Substitution of the conserved Ser in α o with Cys or Asn increased βγ binding, with the Cys mutant being more effective. This suggests that Ser54 of α s is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Synthesis of two fluorescent GTPγS molecules and their biological relevance.

PubMed

Trans, Denise J; Bai, Ruoli; Addison, J Bennet; Liu, Ruiwu; Hamel, Ernest; Coleman, Matthew A; Henderson, Paul T

2017-06-03

Fluorescent GTP analogues are utilized for an assortment of nucleic acid and protein characterization studies. Non-hydrolysable analogues such as GTPγS offer the advantage of keeping proteins in a GTP-bound conformation due to their resistance to hydrolysis into GDP. Two novel fluorescent GTPγS molecules were developed by linking fluorescein and tetramethylrhodamine to the γ-thiophosphate of GTPγS. Chemical and biological analysis of these two compounds revealed their successful synthesis and ability to bind to the nucleotide-binding site of tubulin. These two new fluorescent non-hydrolysable nucleotides offer new possibilities for biophysical and biochemical characterization of GTP-binding proteins.

Mechanistic Insights from Structural Analyses of Ran-GTPase-Driven Nuclear Export of Proteins and RNAs.

PubMed

Matsuura, Yoshiyuki

2016-05-22

Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes is a fundamental problem in biology. Exportins are Ran-GTPase-dependent nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP-dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic FG (phenylalanine-glycine) repeat motifs facilitate nuclear pore complex passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single-particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release and also begins to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza. Copyright © 2015 Elsevier Ltd. All rights reserved.

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.

PubMed

Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A; Iverson, T M; Meiler, Jens; Hamm, Heidi E

2016-09-09

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dissecting EB1-microtubule interactions from every direction: using single-molecule visualization and static and dynamic binding measurements

NASA Astrophysics Data System (ADS)

Lopez, Benjamin

2015-03-01

EB1 is an important microtubule associating protein (MAP) that acts as a master coordinator of protein activity at the growing plus-end of the microtubule. We can recapitulate the plus-end binding behavior of EB1 along the entire length of a static microtubule using microtubules polymerized in the presence of the nonhydrolyzable GTP analogs GMPCPP and GTP γS instead of GTP. Through the use of single-molecule TIRF imaging we find that EB1 is highly dynamic (with a sub-second characteristic binding lifetime) and continuously diffusive while bound to the microtubule. We measure the diffusion coefficient, D, through linear fitting to mean-squared displacement of individually labeled proteins, and the binding lifetime, τ, by fitting a single exponential decay to the probability distribution of trajectory lifetimes. In agreement with measurements of other diffusive MAPs, we find that D increases and τ decreases with increasing ionic strength. We also find that D is sensitive to the choice of GTP analog: EB1 proteins bound to GTP γS polymerized microtubules have a D half of that found with GMPCPP polymerized microtubules. To compare these single-molecule measurements to the bulk binding behavior of EB1, we use TIRF imaging to measure the intensity of microtubules coated with EB1-GFP as a function of EB1 concentration. We find that EB1 binding is cooperative and both the quantity of EB1 bound and the dissociation constant are sensitive to GTP analog and ionic concentration. The correlation between binding affinity and D and the cooperative nature of EB1-microtubule binding leads to a decrease in D with increasing EB1 concentration. Interestingly, we also find an increase in τ at high EB1 concentrations, consistent with attractive EB1-microtubule interactions driving the cooperativity. To further understand the nature of the cooperativity we estimate the interaction energy by measuring the association and dissociation rates (kon and koff respectively) at different concentrations of EB1.

Calculations of binding affinity between C8-substituted GTP analogs and the bacterial cell-division protein FtsZ

PubMed Central

Hritz, Jozef; Läppchen, Tilman

2010-01-01

The FtsZ protein is a self-polymerizing GTPase that plays a central role in bacterial cell division. Several C8-substituted GTP analogs are known to inhibit the polymerization of FtsZ by competing for the same binding site as its endogenous activating ligand GTP. Free energy calculations of the relative binding affinities to FtsZ for a set of five C8-substituted GTP analogs were performed. The calculated values agree well with the available experimental data, and the main contribution to the free energy differences is determined to be the conformational restriction of the ligands. The dihedral angle distributions around the glycosidic bond of these compounds in water are known to vary considerably depending on the physicochemical properties of the substituent at C8. However, within the FtsZ protein, this substitution has a negligible influence on the dihedral angle distributions, which fall within the narrow range of −140° to −90° for all investigated compounds. The corresponding ensemble average of the coupling constants 3J(C4,H1′) is calculated to be 2.95 ± 0.1 Hz. The contribution of the conformational selection of the GTP analogs upon binding was quantified from the corresponding populations. The obtained restraining free energy values follow the same trend as the relative binding affinities to FtsZ, indicating their dominant contribution. PMID:20559630

Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins.

PubMed

Maehama, T; Takahashi, K; Ohoka, Y; Ohtsuka, T; Ui, M; Katada, T

1991-06-05

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guaninemore » binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.« less

Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding, G proteins, phosphoinositide breakdown, cyclic AMP generation, and calcium influx.

PubMed

Shin, Y; Moni, R W; Lueders, J E; Daly, J W

1994-04-01

1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A1-adenosine receptors. 2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTP gamma S to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins. 3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin. N-Ethylmaleimide inhibits the response to both peptides. 4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTP gamma S-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects. 5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered by N-ethylmaleimide. 6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.

Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivkees, S.A.; Carlson, L.L.; Reppert, S.M.

Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using {sup 125}I-labeled melatonin ({sup 125}I-Mel), a potent melatonin agonist. {sup 125}I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K{sub d} of 2.3 {plus minus} 1.0 {times} 10{sup {minus}11} M and 2.06 {plus minus} 0.43 {times} 10{sup {minus}10} M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)), significantly reduced the number of high-affinity receptors and increasedmore » the dissociation rate of {sup 125}I-Mel from its receptor. Furthermore, GTP({gamma}S) treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of {sup 125}I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M{sub r} > 400,000 and M{sub r} ca. 110,000. This elution profile was markedly altered by pretreatment with GTP({gamma}S) before solubilization; only the M{sub r} 110,000 peak was present in GTP({gamma}S)-pretreated membranes. The results strongly suggest that {sup 125}I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.« less

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PubMed

Vashisht, Kapil; Verma, Sonia; Gupta, Sunita; Lynn, Andrew M; Dixit, Rajnikant; Mishra, Neelima; Valecha, Neena; Hamblin, Karleigh A; Maytum, Robin; Pandey, Kailash C; van der Giezen, Mark

2017-01-24

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Computational Simulation of the Activation Cycle of Gα Subunit in the G Protein Cycle Using an Elastic Network Model

PubMed Central

Kim, Min Hyeok; Kim, Young Jin; Kim, Hee Ryung; Jeon, Tae-Joon; Choi, Jae Boong; Chung, Ka Young; Kim, Moon Ki

2016-01-01

Agonist-activated G protein-coupled receptors (GPCRs) interact with GDP-bound G protein heterotrimers (Gαβγ) promoting GDP/GTP exchange, which results in dissociation of Gα from the receptor and Gβγ. The GTPase activity of Gα hydrolyzes GTP to GDP, and the GDP-bound Gα interacts with Gβγ, forming a GDP-bound G protein heterotrimer. The G protein cycle is allosterically modulated by conformational changes of the Gα subunit. Although biochemical and biophysical methods have elucidated the structure and dynamics of Gα, the precise conformational mechanisms underlying the G protein cycle are not fully understood yet. Simulation methods could help to provide additional details to gain further insight into G protein signal transduction mechanisms. In this study, using the available X-ray crystal structures of Gα, we simulated the entire G protein cycle and described not only the steric features of the Gα structure, but also conformational changes at each step. Each reference structure in the G protein cycle was modeled as an elastic network model and subjected to normal mode analysis. Our simulation data suggests that activated receptors trigger conformational changes of the Gα subunit that are thermodynamically favorable for opening of the nucleotide-binding pocket and GDP release. Furthermore, the effects of GTP binding and hydrolysis on mobility changes of the C and N termini and switch regions are elucidated. In summary, our simulation results enabled us to provide detailed descriptions of the structural and dynamic features of the G protein cycle. PMID:27483005

Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP γ-Phosphate*

PubMed Central

Knihtila, Ryan; Holzapfel, Genevieve; Weiss, Kevin; Meilleur, Flora; Mattos, Carla

2015-01-01

RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated γ-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the start of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. The neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases. PMID:26515069

Neutron crystal structure of RAS GTPase puts in question the protonation state of the GTP γ-phosphate

DOE PAGES

Knihtila, Ryan; Holzapfel, Genevieve; Weiss, Kevin; ...

2015-10-29

RAS GTPase is a prototype for nucleotide-binding proteins that function by cycling between GTP and GDP, with hydrogen atoms playing an important role in the GTP hydrolysis mechanism. It is one of the most well studied proteins in the superfamily of small GTPases, which has representatives in a wide range of cellular functions. These proteins share a GTP-binding pocket with highly conserved motifs that promote hydrolysis to GDP. The neutron crystal structure of RAS presented here strongly supports a protonated gamma-phosphate at physiological pH. This counters the notion that the phosphate groups of GTP are fully deprotonated at the startmore » of the hydrolysis reaction, which has colored the interpretation of experimental and computational data in studies of the hydrolysis mechanism. As a result, the neutron crystal structure presented here puts in question our understanding of the pre-catalytic state associated with the hydrolysis reaction central to the function of RAS and other GTPases.« less

Effects of guanyl nucleotides on CCKB receptor binding in brain tissue and continuous cell lines: a comparative study.

PubMed

Kaufmann, R; Schöneberg, T; Henklein, P; Meyer, R; Martin, H; Ott, T

1995-07-01

The effects of non-hydrolyzable guanyl nucleotide analogue GTP-gamma S on CCKB receptor binding in human and guinea-pig cortex, Jurkat T-cells, rat pituitary GH3 cells, rat glioma C6 cells and human small cell lung cancer NCI-H69 cells were investigated by using [3H]CCK-8S saturation and competition binding studies. GTP-gamma S caused inhibition of specific [3H]CCK-8S binding in a concentration dependent manner with a plateau at 10-25 microM. 25 microM GTP-gamma S resulted in a small but significant increase in Kd and IC50 values with amount very similar in all CCKB receptor models tested. However, the maximal number of specific [3H]CCK-8S binding sites (Bmax) was unaffected. Results suggest that CCKB receptors are G-protein coupled in a similar way to human and guinea-pig cortex, Jurkat cells, GH3 cells, C6 cells and NCI-H69 cells.

The mechanism of GTP hydrolysis by dynamin II: a transient kinetic study.

PubMed

Binns, D D; Helms, M K; Barylko, B; Davis, C T; Jameson, D M; Albanesi, J P; Eccleston, J F

2000-06-20

Dynamin II is a 98 kDa protein (870 amino acids) required for the late stages of clathrin-mediated endocytosis. The GTPase activity of dynamin is required for its function in the budding stages of receptor-mediated endocytosis and synaptic vesicle recycling. This activity is stimulated when dynamin self-associates on multivalent binding surfaces, such as microtubules and anionic liposomes. We first investigated the oligomeric state of dynamin II by analytical ultracentrifuge sedimentation equilibrium measurements at high ionic strength and found that it was best described by a monomer-tetramer equilibrium. We then studied the intrinsic dynamin GTPase mechanism by using a combination of fluorescence stopped-flow and HPLC methods using the fluorescent analogue of GTP, mantdGTP (2'-deoxy-3'-O-(N-methylanthraniloyl) guanosine-5'-triphosphate), under the same ionic strength conditions. The results are interpreted as showing that mantdGTP binds to dynamin in a two-step mechanism. The dissociation constant of mantdGTP binding to dynamin, calculated from the ratio of the off-rate to the on-rate (k(off)/k(on)), was 0.5 microM. Cleavage of mantdGTP then occurs to mantdGDP and P(i) followed by the rapid release of mantdGDP and P(i). No evidence of reversibility of hydrolysis was observed. The cleavage step itself is the rate-limiting step in the mechanism. This mechanism more closely resembles that of the Ras family of proteins involved in cell signaling than the myosin ATPase involved in cellular motility.

Genetic and biochemical analysis of the interaction of Bacillus subtilis CodY with branched-chain amino acids.

PubMed

Villapakkam, Anuradha C; Handke, Luke D; Belitsky, Boris R; Levdikov, Vladimir M; Wilkinson, Anthony J; Sonenshein, Abraham L

2009-11-01

Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

Biphasic association of T7 RNA polymerase and a nucleotide analogue, cibacron blue as a model to understand the role of initiating nucleotide in the mechanism of enzyme action.

PubMed

Pai, Sudipta; Das, Mili; Banerjee, Rahul; Dasgupta, Dipak

2011-08-01

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. Here we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, its effect on the function of the enzyme and the probable mode of binding of the dye. We have used difference absorption spectroscopy and isothermal titration calorimetry to show that the dye binds T7 RNAP in a biphasic manner. The first phase of the binding is characterized by inactivation of the enzyme. The second binding site overlaps with the common substrate-binding site of the enzyme. We have carried out docking experiment to map the binding site of the dye in the promoter bound protein. Competitive displacement of the dye from the high affinity site by labeled GTP and isothermal titration calorimetry of high affinity GTP bound enzyme with the dye suggests a strong correlation between the high affinity dye binding and the high affinity GTP binding in T7 RNAP reported earlier from our laboratory.

Structural basis for nuclear import complex dissociation by RanGTP.

PubMed

Lee, Soo Jae; Matsuura, Yoshiyuki; Liu, Sai Man; Stewart, Murray

2005-06-02

Nuclear protein import is mediated mainly by the transport factor importin-beta that binds cytoplasmic cargo, most often via the importin-alpha adaptor, and then transports it through nuclear pore complexes. This active transport is driven by disassembly of the import complex by nuclear RanGTP. The switch I and II loops of Ran change conformation with nucleotide state, and regulate its interactions with nuclear trafficking components. Importin-beta consists of 19 HEAT repeats that are based on a pair of antiparallel alpha-helices (referred to as the A- and B-helices). The HEAT repeats stack to yield two C-shaped arches, linked together to form a helicoidal molecule that has considerable conformational flexibility. Here we present the structure of full-length yeast importin-beta (Kap95p or karyopherin-beta) complexed with RanGTP, which provides a basis for understanding the crucial cargo-release step of nuclear import. We identify a key interaction site where the RanGTP switch I loop binds to the carboxy-terminal arch of Kap95p. This interaction produces a change in helicoidal pitch that locks Kap95p in a conformation that cannot bind importin-alpha or cargo. We suggest an allosteric mechanism for nuclear import complex disassembly by RanGTP.

Spontaneous nucleotide exchange in low molecular weight GTPases by fluorescently labeled γ-phosphate-linked GTP analogs

PubMed Central

Korlach, Jonas; Baird, Daniel W.; Heikal, Ahmed A.; Gee, Kyle R.; Hoffman, Gregory R.; Webb, Watt W.

2004-01-01

Regulated guanosine nucleotide exchange and hydrolysis constitute the fundamental activities of low molecular weight GTPases. We show that three guanosine 5′-triphosphate analogs with BODIPY fluorophores coupled via the gamma phosphate bind to the GTPases Cdc42, Rac1, RhoA, and Ras and displace guanosine 5′-diphosphate with high intrinsic exchange rates in the presence of Mg2+ ions, thereby acting as synthetic, low molecular weight guanine nucleotide exchange factors. The accompanying large fluorescence enhancements (as high as 12-fold), caused by a reduction in guanine quenching of the environmentally sensitive BODIPY dye fluorescence on protein binding, allow for real-time monitoring of this spontaneous nucleotide exchange in the visible spectrum with high signal-to-noise ratios. Binding affinities increased with longer aliphatic linkers connecting the nucleotide and BODIPY fluorophore and were in the 10–100 nM range. Steady-state and time-resolved fluorescence spectroscopy showed an inverse relationship between linker length and fluorescence enhancement factors and differences in protein-bound fluorophore mobilities, providing optimization criteria for future applications of such compounds as efficient elicitors and reporters of nucleotide exchange. EDTA markedly enhanced nucleotide exchange, enabling rapid loading of GTPases with these probes. Differences in active site geometries, in the absence of Mg2+, caused qualitatively different reporting of the bound state by the different analogs. The BODIPY analogs also prevented the interaction of Cdc42 with p21 activated kinase. Together, these results validate the use of these analogs as valuable tools for studying GTPase functions and for developing potent synthetic nucleotide exchange factors for this important class of signaling molecules. PMID:14973186

Kinetics of Interaction between ADP-ribosylation Factor-1 (Arf1) and the Sec7 Domain of Arno Guanine Nucleotide Exchange Factor, Modulation by Allosteric Factors, and the Uncompetitive Inhibitor Brefeldin A

PubMed Central

Rouhana, Jad; Padilla, André; Estaran, Sébastien; Bakari, Sana; Delbecq, Stephan; Boublik, Yvan; Chopineau, Joel; Pugnière, Martine; Chavanieu, Alain

2013-01-01

The GDP/GTP nucleotide exchange of Arf1 is catalyzed by nucleotide exchange factors (GEF), such as Arno, which act through their catalytic Sec7 domain. This exchange is a complex mechanism that undergoes conformational changes and intermediate complex species involving several allosteric partners such as nucleotides, Mg2+, and Sec7 domains. Using a surface plasmon resonance approach, we characterized the kinetic binding parameters for various intermediate complexes. We first confirmed that both GDP and GTP counteract equivalently to the free-nucleotide binary Arf1-Arno complex stability and revealed that Mg2+ potentiates by a factor of 2 the allosteric effect of GDP. Then we explored the uncompetitive inhibitory mechanism of brefeldin A (BFA) that conducts to an abortive pentameric Arf1-Mg2+-GDP-BFA-Sec7 complex. With BFA, the association rate of the abortive complex is drastically reduced by a factor of 42, and by contrast, the 15-fold decrease of the dissociation rate concurs to stabilize the pentameric complex. These specific kinetic signatures have allowed distinguishing the level and nature as well as the fate in real time of formed complexes according to experimental conditions. Thus, we showed that in the presence of GDP, the BFA-resistant Sec7 domain of Arno can also associate to form a pentameric complex, which suggests that the uncompetitive inhibition by BFA and the nucleotide allosteric effect combine to stabilize such abortive complex. PMID:23255605

A non-canonical mechanism for Crm1-export cargo complex assembly.

PubMed

Fischer, Ute; Schäuble, Nico; Schütz, Sabina; Altvater, Martin; Chang, Yiming; Faza, Marius Boulos; Panse, Vikram Govind

2015-04-21

The transport receptor Crm1 mediates the export of diverse cargos containing leucine-rich nuclear export signals (NESs) through complex formation with RanGTP. To ensure efficient cargo release in the cytoplasm, NESs have evolved to display low affinity for Crm1. However, mechanisms that overcome low affinity to assemble Crm1-export complexes in the nucleus remain poorly understood. In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2. In vitro, Slx9 binds Rio2 and RanGTP, forming a complex. This complex directly loads Crm1, unveiling a non-canonical stepwise mechanism to assemble a Crm1-export complex. A mutation in Slx9 that impairs Crm1-export complex assembly inhibits 40S pre-ribosome export. Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export. This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export.

Septins - active GTPases or just GTP-binding proteins?

PubMed

Abbey, Megha; Gaestel, Matthias; Menon, Manoj B

2018-05-10

Septins are conserved cytoskeletal proteins with unique filament forming capabilities and roles in cytokinesis and cell morphogenesis. Septins undergo hetero-oligomerization and assemble into higher order structures including filaments, rings and cages. Hetero- and homotypic interactions of septin isoforms involve alternating GTPase (G)-domain interfaces and those mediated by N- and C-terminal extensions. While most septins bind GTP, display weak GTP-hydrolysis activity and incorporate guanine nucleotides in their interaction interfaces, studies using GTPase-inactivating mutations have failed to conclusively establish a crucial role for GTPase activity in mediating septin functions. In this mini-review, we will critically assess the role of GTP-binding and -hydrolysis on septin assembly and function. The relevance of G-domain activity will also be discussed in the context of human septin mutations as well as the development of specific small-molecules targeting septin polymerization. As structural determinants of septin oligomer interfaces, G-domains are attractive targets for ligand-based inhibition of septin assembly. Whether such an intervention can predictably alter septin function is a major question for future research. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simonetti, Angelita; Marzi, Stefano; Fabbretti, Attilio

2013-06-01

The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2more » is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.« less

Structural basis for the binding of didemnins to human elongation factor eEF1A and rationale for the potent antitumor activity of these marine natural products.

PubMed

Marco, Esther; Martín-Santamaría, Sonsoles; Cuevas, Carmen; Gago, Federico

2004-08-26

Didemnins and tamandarins are closely related marine natural products with potent inhibitory effects on protein synthesis and cell viability. On the basis of available biochemical and structural evidence and results from molecular dynamics simulations, a model is proposed that accounts for the strong and selective binding of these compounds to human elongation factor eEF1A in the presence of GTP. We suggest that the p-methoxyphenyl ring of these cyclic depsipeptides is inserted into the same pocket in eEF1A that normally lodges either the 3' terminal adenine of aminoacylated tRNA, as inferred from two prokaryotic EF-Tu.GTP.tRNA complexes, or the aromatic side chain of Phe/Tyr-163 from the nucleotide exchange factor eEF1Balpha, as observed in several X-ray crystal structures of a yeast eEF1A:eEF1Balpha complex. This pocket, which has a strong hydrophobic character, is formed by two protruding loops on the surface of eEF1A domain 2. Further stabilization of the bound depsipeptide is brought about by additional crucial interactions involving eEF1A domain 1 in such a way that the molecule fits snugly at the interface between these two domains. In the GDP-bound form of eEF1A, this binding site exists only as two separate halves, which accounts for the much greater affinity of didemnins for the GTP-bound form of this elongation factor. This binding mode is entirely different from those seen in the complexes of the homologous prokaryotic EF-Tu with kirromycin-type antibiotics or the cyclic thiazolyl peptide antibiotic GE2270A. Interestingly, the set of interactions used by didemnins to bind to eEF1A is also distinct from that used by eEF1Balpha or eEF1Bbeta, thus establishing a competition for binding to a common site that goes beyond simple molecular mimicry. The model presented here is consistent with both available biochemical evidence and known structure-activity relationships for these two classes of natural compounds and synthetic analogues and provides fertile ground for future research.

Synthetic inhibitors of bacterial cell division targeting the GTP-binding site of FtsZ.

PubMed

Ruiz-Avila, Laura B; Huecas, Sonia; Artola, Marta; Vergoñós, Albert; Ramírez-Aportela, Erney; Cercenado, Emilia; Barasoain, Isabel; Vázquez-Villa, Henar; Martín-Fontecha, Mar; Chacón, Pablo; López-Rodríguez, María L; Andreu, José M

2013-09-20

Cell division protein FtsZ is the organizer of the cytokinetic Z-ring in most bacteria and a target for new antibiotics. FtsZ assembles with GTP into filaments that hydrolyze the nucleotide at the association interface between monomers and then disassemble. We have replaced FtsZ's GTP with non-nucleotide synthetic inhibitors of bacterial division. We searched for these small molecules among compounds from the literature, from virtual screening (VS), and from our in-house synthetic library (UCM), employing a fluorescence anisotropy primary assay. From these screens we have identified the polyhydroxy aromatic compound UCM05 and its simplified analogue UCM44 that specifically bind to Bacillus subtilis FtsZ monomers with micromolar affinities and perturb normal assembly, as examined with light scattering, polymer sedimentation, and negative stain electron microscopy. On the other hand, these ligands induce the cooperative assembly of nucleotide-devoid archaeal FtsZ into distinct well-ordered polymers, different from GTP-induced filaments. These FtsZ inhibitors impair localization of FtsZ into the Z-ring and inhibit bacterial cell division. The chlorinated analogue UCM53 inhibits the growth of clinical isolates of antibiotic-resistant Staphylococcus aureus and Enterococcus faecalis. We suggest that these interfacial inhibitors recapitulate binding and some assembly-inducing effects of GTP but impair the correct structural dynamics of FtsZ filaments and thus inhibit bacterial division, possibly by binding to a small fraction of the FtsZ molecules in a bacterial cell, which opens a new approach to FtsZ-based antibacterial drug discovery.

Solubilization and purification of melatonin receptors from lizard brain.

PubMed

Rivkees, S A; Conron, R W; Reppert, S M

1990-09-01

Melatonin receptors in lizard brain were identified and characterized using 125I-labeled melatonin ([125I]MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resulted in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.

The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells

PubMed Central

Cerny, Alexander C.; Altendorfer, André; Schopf, Krystina; Baltner, Karla; Maag, Nathalie; Sehn, Elisabeth; Wolfrum, Uwe; Huber, Armin

2015-01-01

Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14 P75L mutant. The ttd14 P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14 P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane. PMID:26509977

Domain structure, GTP-hydrolyzing activity and 7S RNA binding of Acidianus ambivalens ffh-homologous protein suggest an SRP-like complex in archaea.

PubMed

Moll, R; Schmidtke, S; Schäfer, G

1999-01-01

In this study we provide, for the first time, experimental evidence that a protein homologous to bacterial Ffh is part of an SRP-like ribonucleoprotein complex in hyperthermophilic archaea. The gene encoding the Ffh homologue in the hyperthermophilic archaeote Acidianus ambivalens has been cloned and sequenced. Recombinant Ffh protein was expressed in E. coli and subjected to biochemical and functional studies. A. ambivalens Ffh encodes a 50.4-kDa protein that is structured by three distinct regions: the N-terminal hydrophilic N-region (N), the GTP/GDP-binding domain (G) and a C-terminal located C-domain (C). The A. ambivalens Ffh sequence shares 44-46% sequence similarity with Ffh of methanogenic archaea, 34-36% similarity with eukaryal SRP54 and 30-34% similarity with bacterial Ffh. A polyclonal antiserum raised against the first two domains of A. ambivalens Ffh reacts specifically with a single protein (apparent molecular mass: 46 kDa, termed p46) present in cytosolic and in plasmamembrane cell fractions of A. ambivalens. Recombinant Ffh has a melting point of tm = 89 degreesC. Its intrinsic GTPase activity obviously depends on neutral pH and low ionic strength with a preference for chloride and acetate salts. Highest rates of GTP hydrolysis have been achieved at 81 degreesC in presence of 0.1-1 mm Mg2+. GTP hydrolysis is significantly inhibited by high glycerol concentrations, and the GTP hydrolysis rate also markedly decreases by addition of detergents. The Km for GTP is 13.7 microm at 70 degreesC and GTP hydrolysis is strongly inhibited by GDP (Ki = 8 microm). A. ambivalens Ffh, which includes an RNA-binding motif in the C-terminal domain, is shown to bind specifically to 7S RNA of the related crenarchaeote Sulfolobus solfataricus. Comparative sequence analysis reveals the presence of typical signal sequences in plasma membrane as well as extracellular proteins of hyperthermophilic crenarchaea which strongly supposes recognition events by an Ffh containing SRP-like particle in these organisms.

What makes Ras an efficient molecular switch: a computational, biophysical, and structural study of Ras-GDP interactions with mutants of Raf.

PubMed

Filchtinski, Daniel; Sharabi, Oz; Rüppel, Alma; Vetter, Ingrid R; Herrmann, Christian; Shifman, Julia M

2010-06-11

Ras is a small GTP-binding protein that is an essential molecular switch for a wide variety of signaling pathways including the control of cell proliferation, cell cycle progression and apoptosis. In the GTP-bound state, Ras can interact with its effectors, triggering various signaling cascades in the cell. In the GDP-bound state, Ras looses its ability to bind to known effectors. The interaction of the GTP-bound Ras (Ras(GTP)) with its effectors has been studied intensively. However, very little is known about the much weaker interaction between the GDP-bound Ras (Ras(GDP)) and Ras effectors. We investigated the factors underlying the nucleotide-dependent differences in Ras interactions with one of its effectors, Raf kinase. Using computational protein design, we generated mutants of the Ras-binding domain of Raf kinase (Raf) that stabilize the complex with Ras(GDP). Most of our designed mutations narrow the gap between the affinity of Raf for Ras(GTP) and Ras(GDP), producing the desired shift in binding specificity towards Ras(GDP). A combination of our best designed mutation, N71R, with another mutation, A85K, yielded a Raf mutant with a 100-fold improvement in affinity towards Ras(GDP). The Raf A85K and Raf N71R/A85K mutants were used to obtain the first high-resolution structures of Ras(GDP) bound to its effector. Surprisingly, these structures reveal that the loop on Ras previously termed the switch I region in the Ras(GDP).Raf mutant complex is found in a conformation similar to that of Ras(GTP) and not Ras(GDP). Moreover, the structures indicate an increased mobility of the switch I region. This greater flexibility compared to the same loop in Ras(GTP) is likely to explain the natural low affinity of Raf and other Ras effectors to Ras(GDP). Our findings demonstrate that an accurate balance between a rigid, high-affinity conformation and conformational flexibility is required to create an efficient and stringent molecular switch. Copyright 2010 Elsevier Ltd. All rights reserved.

Elastic Network Model of a Nuclear Transport Complex

NASA Astrophysics Data System (ADS)

Ryan, Patrick; Liu, Wing K.; Lee, Dockjin; Seo, Sangjae; Kim, Young-Jin; Kim, Moon K.

2010-05-01

The structure of Kap95p was obtained from the Protein Data Bank (www.pdb.org) and analyzed RanGTP plays an important role in both nuclear protein import and export cycles. In the nucleus, RanGTP releases macromolecular cargoes from importins and conversely facilitates cargo binding to exportins. Although the crystal structure of the nuclear import complex formed by importin Kap95p and RanGTP was recently identified, its molecular mechanism still remains unclear. To understand the relationship between structure and function of a nuclear transport complex, a structure-based mechanical model of Kap95p:RanGTP complex is introduced. In this model, a protein structure is simply modeled as an elastic network in which a set of coarse-grained point masses are connected by linear springs representing biochemical interactions at atomic level. Harmonic normal mode analysis (NMA) and anharmonic elastic network interpolation (ENI) are performed to predict the modes of vibrations and a feasible pathway between locked and unlocked conformations of Kap95p, respectively. Simulation results imply that the binding of RanGTP to Kap95p induces the release of the cargo in the nucleus as well as prevents any new cargo from attaching to the Kap95p:RanGTP complex.

Solution structure, mutagenesis, and NH exchange studies of the MutT enzyme-Mg 2+-8-oxo-dGMP complex

NASA Astrophysics Data System (ADS)

Massiah, M. A.; Saraswat, V.; Azurmendi, H. F.; Mildvan, A. S.

2004-08-01

The MutT pyrophosphohydrolase from E. coli (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP), including 8-oxo-dGTP, by substitution at Pβ, to yield NMP and pyrophosphate. The product, 8-oxo-dGMP is an unusually tight binding, slowly exchanging inhibitor with a KD=52 nM, (Δ G°=-9.8 kcal/mol) which is 6.1 kcal/mol tighter than the binding of dGMP (Δ G°=-3.7 kcal/mol). The higher affinity for 8-oxo-dGMP results from a more favorable Δ Hbinding (-32 kcal/mol) despite an unfavorable - TΔ S° binding (+22 kcal/mol). The solution structure of the MutT-Mg 2+-8-oxo-dGMP complex shows a narrowed, hydrophobic nucleotide-binding cleft with Asn-119 and Arg-78 among the few polar residues. The N119A, N119D, R78K and R78A single mutations, and the R78K+N119A double mutant all showed largely intact active sites, on the basis of small changes in the kinetic parameters of dGTP hydrolysis and in 1H- 15N HSQC spectra. However, the N119A mutation profoundly weakened the active site binding of 8-oxo-dGMP by 4.3 kcal/mol (1650-fold). The N119D mutation also weakened 8-oxo-dGMP binding but only by 2.1 kcal/mol (37-fold), suggesting that Asn-119 functioned both as a hydrogen bond donor to C8O, and a hydrogen bond acceptor from N7H of 8-oxo-dGMP, while aspartate at position -119 functioned as an acceptor of a single hydrogen bond. Much smaller weakening effects (0.3-0.4 kcal/mol) on the binding of dGMP and dAMP were found, indicating specific hydrogen bonding of Asn-119 to 8-oxo-dGMP. While formation of the wild type MutT-Mg 2+-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in conformational flexibility of the N119A mutant. The R78K and R78A mutations weakened the binding of 8-oxo-dGMP by 1.7 and 1.1 kcal/mol, respectively, indicating a lesser role of Arg-78 than of Asn-119 in the selective binding of 8-oxo-dGMP, likely donating a single hydrogen bond to its C6O. The R78K+N119A double mutant weakened the binding of 8-oxo-dGMP ( KIslope=3.1 mM) by 6.5±0.2 kcal/mol which overlaps, within error with the sum of the effects of the two single mutants (6.0±0.3 kcal/mol). Such additive effects of the two single mutants in the double mutant are most simply explained by the independent functioning of Asn-119 and Arg-78 in the binding of 8-oxo-dGMP. Independent functioning of these two residues in nucleotide binding is consistent with their locations in the MutT-Mg 2+-8-oxo-dGMP complex, on opposite sides of the active site cleft, with a distance of 8.4±0.5 Å between their side chain nitrogens.

GTP requirement for inositol-1,4,5-trisphosphate-induced Ca2+ release from sarcoplasmic reticulum in smooth muscle.

PubMed

Saida, K; van Breemen, C

1987-05-14

We have examined inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from the sarcoplasmic reticulum (SR) in the skinned vascular smooth muscle. The amount of Ca2+ in the SR was estimated indirectly by caffeine-induced contraction of the skinned preparation. The Ca2+ release from the SR by IP3 required GTP. A non-hydrolyzable analogue of GTP, guanosine 5'-(beta gamma-imido) triphosphate (GppNHp) could substitute for GTP in the IP3-induced Ca2+ release. These results suggest an involvement of GTP-binding protein in the mechanism of Ca2+ release from the SR by IP3 in smooth muscle.

Negative inotropic effect of carbachol and interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein in mouse isolated atrium--a novel methodological trial.

PubMed

Okada, Muneyoshi; Noma, Chihiro; Yamawaki, Hideyuki; Hara, Yukio

2013-01-01

Interaction between acetylcholine receptor-operated potassium channel (K.ACh channel) and GTP binding protein was examined by an immunoprecipitation-Western blotting system in mouse isolated atrium. The carbachol-induced negative inotropic action in indomethacin-pretreated mouse atrium was significantly inhibited by a K.ACh channel blocker, tertiapin or atropine. Kir3.1 K.ACh channel (Kir3.1) was immunoprecipitated with a mouse anti-Kir3.1 antibody. Coprecipitating Gβ with Kir3.1, detected by Western blotting, was significantly augmented by carbachol. Atropine, but not tertiapin, significantly inhibited the carbachol-induced coprecipitating Gβ with Kir3.1. The data indicate that immunoprecipitation with Kir3.1 and Western blotting of Gβ system is a useful method for assessing interaction between K.ACh channel and GTP binding protein in mouse atrium.

Pertussis toxin modifies the characteristics of both the inhibitory GTP binding proteins and the somatostatin receptor in anterior pituitary tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahy, N.; Woolkalis, M.; Thermos, K.

1988-08-01

The effects of pertussis toxin treatment on the characteristics of somatostatin receptors in the anterior pituitary tumor cell line AtT-20 were examined. Pertussis toxin selectively catalyzed the ADP ribosylation of the alpha subunits of the inhibitory GTP binding proteins in AtT-20 cells. Toxin treatment abolished somatostatin inhibition of forskolin-stimulated adenylyl cyclase activity and somatostatin stimulation of GTPase activity. To examine the effects of pertussis toxin treatment on the characteristics of the somatostatin receptor, the receptor was labeled by the somatostatin analog (125I)CGP 23996. (125I)CGP 23996 binding to AtT-20 cell membranes was saturable and within a limited concentration range was tomore » a single high affinity site. Pertussis toxin treatment reduced the apparent density of the high affinity (125I)CGP 23996 binding sites in AtT-20 cell membranes. Inhibition of (125I)CGP 23996 binding by a wide concentration range of CGP 23996 revealed the presence of two binding sites. GTP predominantly reduced the level of high affinity sites in control membranes. Pertussis toxin treatment also diminished the amount of high affinity sites. GTP did not affect (125I)CGP 23996 binding in the pertussis toxin-treated membranes. The high affinity somatostatin receptors were covalently labeled with (125I) CGP 23996 and the photoactivated crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. No high affinity somatostatin receptors, covalently bound to (125I)CGP 23996, were detected in the pertussis toxin-treated membranes. These results are most consistent with pertussis toxin uncoupling the inhibitory G proteins from the somatostatin receptor thereby converting the receptor from a mixed population of high and low affinity sites to only low affinity receptors.« less

A non-canonical mechanism for Crm1-export cargo complex assembly

PubMed Central

Fischer, Ute; Schäuble, Nico; Schütz, Sabina; Altvater, Martin; Chang, Yiming; Boulos Faza, Marius; Panse, Vikram Govind

2015-01-01

The transport receptor Crm1 mediates the export of diverse cargos containing leucine-rich nuclear export signals (NESs) through complex formation with RanGTP. To ensure efficient cargo release in the cytoplasm, NESs have evolved to display low affinity for Crm1. However, mechanisms that overcome low affinity to assemble Crm1-export complexes in the nucleus remain poorly understood. In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2. In vitro, Slx9 binds Rio2 and RanGTP, forming a complex. This complex directly loads Crm1, unveiling a non-canonical stepwise mechanism to assemble a Crm1-export complex. A mutation in Slx9 that impairs Crm1-export complex assembly inhibits 40S pre-ribosome export. Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export. This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export. DOI: http://dx.doi.org/10.7554/eLife.05745.001 PMID:25895666

Role of guanine nucleotides in the vinblastine-induced self-association of tubulin: effects of guanosine alpha,beta-methylenetriphosphate and guanosine alpha,beta-methylenediphosphate.

PubMed

Vulevic, B; Lobert, S; Correia, J J

1997-10-21

It is now well established that guanine nucleotides are allosteric effectors of the vinca alkaloid-induced self-association of tubulin. GDP enhances self-association for vinblastine-, vincristine- and vinorelbine-induced spiral assembly relative to GTP by 0.90 +/- 0.17 kcal/mol [Lobert et al. (1996) Biochemistry 35, 6806-6814]. Since chemical modifications of the vinca alkaloid structure are known to modulate the overall affinity of drug binding, it is very likely that, by Wyman linkage, chemical modifications of guanine nucleotide allosteric effectors also modulate drug binding. Here we compare the effects of the GTP and GDP alpha,beta-methylene analogues GMPCPP and GMPCP on vinblastine-induced tubulin association in 10 and 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), 1 mM MgSO4, and 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), pH 6. 9, at different temperatures. We found that GMPCPP perfectly mimics GTP in its effect on spiral assembly under all ionic strength and temperature conditions. However, GMPCP in 10 mM Pipes behaves not as a GDP analogue, but as a GTP analogue. In 100 mM Pipes, GMPCP has characteristics that are intermediate between GDP and GTP. These data suggest that the alpha,beta methylene group in GMPCP and GMPCPP is sufficient to produce a GTP-like effect on vinblastine-induced tubulin self-assembly. This is consistent with previous observations that GMPCP-tubulin will assemble into microtubules in a 2 M glycerol and 100 mM Pipes buffer [Vulevic & Correia (1997) Biophys. J. 72, 1357-1375]. Our results demonstrate that an alpha,beta methylene modification of the guanine nucleotide phosphate moiety can induce a salt-dependent conformational change in the tubulin heterodimer that favors the GTP-tubulin structure. This has important implications for understanding allosteric interactions that occur in the binding of guanine nucleotides to tubulin.

Deletion mutants of Harvey ras p21 protein reveal the absolute requirement of at least two distant regions for GTP-binding and transforming activities.

PubMed Central

Lacal, J C; Anderson, P S; Aaronson, S A

1986-01-01

Deletions of small sequences from the viral Harvey ras gene have been generated, and resulting ras p21 mutants have been expressed in Escherichia coli. Purification of each deleted protein allowed the in vitro characterization of GTP-binding, GTPase and autokinase activity of the proteins. Microinjection of the highly purified proteins into quiescent NIH/3T3 cells, as well as transfection experiments utilizing a long terminal repeat (LTR)-containing vector, were utilized to analyze the biological activity of the deleted proteins. Two small regions located at 6-23 and 152-165 residues are shown to be absolutely required for in vitro and in vivo activities of the ras product. By contrast, the variable region comprising amino acids 165-184 was shown not to be necessary for either in vitro or in vivo activities. Thus, we demonstrate that: (i) amino acid sequences at positions 5-23 and 152-165 of ras p21 protein are probably directly involved in the GTP-binding activity; (ii) GTP-binding is required for the transforming activity of ras p21 and by extension for the normal function of the proto-oncogene product; and (iii) the variable region at the C-terminal end of the ras p21 molecule from amino acids 165 to 184 is not required for transformation. Images Fig.2. Fig.4. PMID:3011420

Elevated guanosine 5'-diphosphate 3'-diphosphate level inhibits bacterial growth and interferes with FtsZ assembly.

PubMed

Yamaguchi, Takayoshi; Iida, Ken-Ichiro; Shiota, Susumu; Nakayama, Hiroaki; Yoshida, Shin-Ichi

2015-12-01

FtsZ, a protein essential for prokaryotic cell division, forms a ring structure known as the Z-ring at the division site. FtsZ has a GTP binding site and is assembled into linear structures in a GTP-dependent manner in vitro. We assessed whether guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a global regulator of gene expression in starved bacteria, affects cell division in Salmonella Paratyphi A. Elevation of intracellular ppGpp levels by using the relA expression vector induced repression of bacterial growth and incorrect FtsZ assembly. We found that FtsZ forms helical structures in the presence of ppGpp by using the GTP binding site; however, ppGpp levels required to form helical structures were at least 20-fold higher than the required GTP levels in vitro. Furthermore, once formed, helical structures did not change to the straight form even after GTP addition. Our data indicate that elevation of the ppGpp level leads to inhibition of bacterial growth and interferes with FtsZ assembly. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A distinct class of dominant negative Ras mutants: cytosolic GTP-bound Ras effector domain mutants that inhibit Ras signaling and transformation and enhance cell adhesion.

PubMed

Fiordalisi, James J; Holly, Stephen P; Johnson, Ronald L; Parise, Leslie V; Cox, Adrienne D

2002-03-29

Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a beta(1)-subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Ras-induced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF- or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.

Solubilization and purification of melatonin receptors from lizard brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rivkees, S.A.; Conron, R.W. Jr.; Reppert, S.M.

Melatonin receptors in lizard brain were identified and characterized using {sup 125}I-labeled melatonin (({sup 125}I)MEL) after solubilization with the detergent digitonin. Saturation studies of solubilized material revealed a high affinity binding site, with an apparent equilibrium dissociation constant of 181 +/- 45 pM. Binding was reversible and inhibited by melatonin and closely related analogs, but not by serotonin or norepinephrine. Treatment of solubilized material with the non-hydrolyzable GTP analog, guanosine 5'-(3-O-thiotriphosphate) (GTP-gamma-S), significantly reduced receptor affinity. Gel filtration chromatography of solubilized melatonin receptors revealed a high affinity, large (Mr 400,000) peak of specific binding. Pretreatment with GTP-gamma-S before solubilization resultedmore » in elution of a lower affinity, smaller (Mr 150,000) peak of specific binding. To purify solubilized receptors, a novel affinity chromatography resin was developed by coupling 6-hydroxymelatonin with Epoxy-activated Sepharose 6B. Using this resin, melatonin receptors were purified approximately 10,000-fold. Purified material retained the pharmacologic specificity of melatonin receptors. These results show that melatonin receptors that bind ligand after detergent treatment can be solubilized and substantially purified by affinity chromatography.« less

A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme.

PubMed

Bullard-Feibelman, Kristen M; Fuller, Benjamin P; Geiss, Brian J

2016-01-01

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that causes severe and debilitating disease symptoms. Alarmingly, transmission rates of CHIKV have increased dramatically over the last decade resulting in 1.7 million suspected cases in the Western hemisphere alone. There are currently no antivirals for treatment of CHIKV infection and novel anti-alphaviral compounds are badly needed. nsP1 is the alphavirus protein responsible for the methyltransferase and guanylyltransferase activities necessary for formation of the 5' type 0 cap structure added to newly formed viral RNA. Formation of this cap depends on nsP1 binding GTP and transferring a methylated GMP to nascent viral RNA. We have developed a fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time. Determining the relative affinities of 15 GTP analogs for nsP1 GTP revealed important structural aspects of GTP that will inform identification of inhibitors able to outcompete GTP for the nsP1 binding site. Validation of the assay for HTS was completed and a secondary orthogonal assay that measures guanylation activity was developed in order to evaluate hits from future drug screens. This platform provides an avenue for identification of potent nsP1 inhibitors, which would potentially provide compounds capable of treating disease caused by CHIKV infection.

¹H, ¹³C and ¹⁵N resonance assignment for the human K-Ras at physiological pH.

PubMed

Vo, Uybach; Embrey, Kevin J; Breeze, Alexander L; Golovanov, Alexander P

2013-10-01

K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(β), as well as partial H(α), H(β) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Young, M.E.; Keegstra, K.; Froehlich, J.E.

Protein import into chloroplasts is an energy-requiring process mediated by a pertinacious import apparatus. Although previous work has shown that low levels of ATP or GTP can support precursor binding, the role of GTP during the import process remains unclear. Specifically, it is unknown whether GTP plays a separate role from ATP during the early stages of protein import and whether GTP has any role in the later stages of transport. The authors investigated the role of GTP during the various stages of protein import into chloroplasts by using purified GTP analogs and an in vitro import assay. GTP, GDP,more » the nonhydrolyzable analog GMP-PNP, and the slowly hydrolyzable analogs guanosine 5{prime}-O-(2-thiodiphosphate) and guanosine 5{prime}-O-(3-thiotriphosphate) were used in this study. Chromatographically purified 5{prime}-guanylyl-imido-diphosphate and guanosine 5{prime}-O-(3-thiotriphosphate) were found to inhibit the formation of early-import intermediates, even in the presence of ATP. The authors also observed that GTP does not play a role during the translocation of precursors from the intermediate state. They conclude that GTP hydrolysis influences events leading to the formation of early-import intermediates, but not subsequent steps such as precursor translocation.« less

Structural insights into cell cycle control by essential GTPase Era.

PubMed

Ji, Xinhua

Era (Escherichia coli Ras-like protein), essential for bacterial cell viability, is composed of an N-terminal GTPase domain and a C-terminal KH domain. In bacteria, it is required for the processing of 16S ribosomal RNA (rRNA) and maturation of 30S (small) ribosomal subunit. Era recognizes 10 nucleotides ( 1530 GAUCACCUCC 1539 ) near the 3' end of 16S rRNA and interacts with helix 45 (h45, nucleotides 1506-1529). GTP binding enables Era to bind RNA, RNA binding stimulates Era's GTP-hydrolyzing activity, and GTP hydrolysis releases Era from matured 30S ribosomal subunit. As such, Era controls cell growth rate via regulating the maturation of the 30S ribosomal subunit. Ribosomes manufacture proteins in all living organisms. The GAUCA sequence and h45 are highly conserved in all three kingdoms of life. Homologues of Era are present in eukaryotic cells. Hence, the mechanism of bacterial Era action also sheds light on the cell cycle control of eukaryotes.

Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, G.P.

1987-01-01

Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted inmore » a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.« less

GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

PubMed

Uejima, Tamami; Ihara, Kentaro; Goh, Tatsuaki; Ito, Emi; Sunada, Mariko; Ueda, Takashi; Nakano, Akihiko; Wakatsuki, Soichi

2010-11-19

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

In Situ Live Cell Sensing of Multiple Nucleotides Exploiting DNA/RNA Aptamers and Graphene Oxide Nanosheets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Ying; Li, Zhaohui; Weber, Thomas J.

2013-07-23

Adenosine-5’-triphosphate (ATP) and guanosine-5’-triphosphate (GTP) are primary energy resources and function coordinately for numerous reactions such as microtubule assembly, insulin secretion and ion channel regulation. We have developed a novel DNA/RNA aptamer- graphene oxide nanosheet (GO-nS) sensing platform that can selectively and simultaneously detect ATP and GTP in live cells. A fluorescent tag is covalently attached to aptamers and fluorescence is quenched upon binding of aptamer to the GO-nS. Fluorescently tagged aptamers that selectively bind ATP or GTP were isolated from an aptamer library and were adsorbed onto GO-nS. Upon incubation with targets (ATP and/or GTP), the aptamers readily dissociatedmore » from GO-nS and the fluorescent signal was recovered. By covalently attaching fluorophores, both ATP and GTP sensing aptamers could be exploited to simultaneously visualize aptamer dissociation in live cells. In addition, the GO-nS appear to be biocompatible and protect the adsorbed DNA/RNA aptamers from enzymatic cleavage. Our results support the application of aptamer/GO-nS as a sensing platform for nucleotides in living cells and have implications for the development of additional sensor platforms for other bio-molecules that show selective interactions with aptamers and other biomarkers.« less

Effective GTP-replacing FtsZ inhibitors and antibacterial mechanism of action.

PubMed

Artola, Marta; Ruiz-Avila, Laura B; Vergoñós, Albert; Huecas, Sonia; Araujo-Bazán, Lidia; Martín-Fontecha, Mar; Vázquez-Villa, Henar; Turrado, Carlos; Ramírez-Aportela, Erney; Hoegl, Annabelle; Nodwell, Matthew; Barasoain, Isabel; Chacón, Pablo; Sieber, Stephan A; Andreu, Jose M; López-Rodríguez, María L

2015-03-20

Essential cell division protein FtsZ is considered an attractive target in the search for antibacterials with novel mechanisms of action to overcome the resistance problem. FtsZ undergoes GTP-dependent assembly at midcell to form the Z-ring, a dynamic structure that evolves until final constriction of the cell. Therefore, molecules able to inhibit its activity will eventually disrupt bacterial viability. In this work, we report a new series of small molecules able to replace GTP and to specifically inhibit FtsZ, blocking the bacterial division process. These new synthesized inhibitors interact with the GTP-binding site of FtsZ (Kd = 0.4-0.8 μM), display antibacterial activity against Gram-positive pathogenic bacteria, and show selectivity against tubulin. Biphenyl derivative 28 stands out as a potent FtsZ inhibitor (Kd = 0.5 μM) with high antibacterial activity [MIC (MRSA) = 7 μM]. In-depth analysis of the mechanism of action of compounds 22, 28, 33, and 36 has revealed that they act as effective inhibitors of correct FtsZ assembly, blocking bacterial division and thus leading to filamentous undivided cells. These findings provide a compelling rationale for the development of compounds targeting the GTP-binding site as antibacterial agents and open the door to antibiotics with novel mechanisms of action.

The RanGTP Pathway: From Nucleo-Cytoplasmic Transport to Spindle Assembly and Beyond

PubMed Central

Cavazza, Tommaso; Vernos, Isabelle

2016-01-01

The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD) Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context. PMID:26793706

Kirromycin, an Inhibitor of Protein Biosynthesis that Acts on Elongation Factor Tu

PubMed Central

Wolf, Heinz; Chinali, Gianni; Parmeggiani, Andrea

1974-01-01

Kirromycin, a new inhibitor of protein synthesis, is shown to interfere with the peptide transfer reaction by acting on elongation factor Tu (EF-Tu). All the reactions associated with this elongation factor are affected. Formation of the EF-Tu·GTP complex is strongly stimulated. Peptide bond formation is prevented only when Phe-tRNAPhe is bound enzymatically to ribosomes, presumably because GTP hydrolysis associated with enzymatic binding of Phe-tRNAPhe is not followed by release of EF-Tu·GDP from the ribosome. This antibiotic also enables EF-Tu to catalyze the binding of Phe-tRNAPhe to the poly(U)·ribosome complex even in the absence of GTP. EF-Tu activity in the GTPase reaction is dramatically affected by kirromycin: GTP hydrolysis, which normally requires ribosomes and aminoacyl-tRNA, takes place with the elongation factor alone. This GTPase shows the same Km for GTP as the one dependent on Phe-tRNAPhe and ribosomes in the absence of the antibiotic. Ribosomes and Phe-tRNAPhe, but not tRNAPhe or Ac-Phe-tRNAPhe, stimulate the kirromycin-induced EF-Tu GTPase. These results indicate that the catalytic center of EF-Tu GTPase that is dependent upon aminoacyl-tRNA and ribosomes is primarily located on the elongation factor. In conclusion, kirromycin can substitute for GTP, aminoacyl-tRNA, or ribosomes in various reactions involving EF-Tu, apparently by affecting the allosteric controls between the sites on the EF-Tu molecule interacting with these components. PMID:4373734

Muscarinic-agonist and guanine nucleotide stimulation of myo-inositol trisphosphate formation in membranes isolated from bovine iris sphincter smooth muscle: effects of short-term cholinergic desensitization.

PubMed

Honkanen, R E; Abdel-Latif, A A

1989-01-01

The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [3H]myo-inositol or 32P from [gamma-32P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTP gamma S, or GTP gamma S plus carbachol (CCh), and the production of [3H]myo-inositol 1,4,5-trisphosphate (IP3) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (greater than or equal to 1 mM), GTP gamma S (greater than 10 microM) and GTP gamma S (1 microM) plus CCh (10 microM), but not GDP or GDP beta S, increased phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and IP3 production. GTP gamma S increased IP3 accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTP gamma S, potentiated the effects of GTP gamma S. The effect of CCh plus GTP gamma S on IP3 production was inhibited by atropine, had an absolute requirement for nM amounts of Ca2+ and was not affected by pertussis toxin. At higher concentrations (greater than 1 microM), Ca2+ alone induced PIP2 hydrolysis. Short-term exposure (less than 60 min) of the muscle to CCh (100 microM) did not affect the total number (Bmax) of mAChRs nor their affinity (KD) for [3H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTP gamma S; (2) a loss of the ability of GTP gamma S to affect CCh binding to the receptors; and (3) an attenuation of the GTP gamma S plus CCh-stimulated PIP2 hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization results in (1) the uncoupling of the receptor-G-protein complex and (2) the attenuation of mAChR-activation of phospholipase C.

Modification of opiate agonist binding by pertussis toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abood, M.E.; Lee, N.M.; Loh, H.H.

1986-03-05

Opiate agonist binding is decreased by GTP, suggesting the possible involvement of GTP binding proteins in regulation of opiate receptor binding. This possibility was addressed by asking whether pertussis toxin treatment, which results in ADP-ribosylation and modification of G proteins, would alter opiate agonist binding. The striatum was chosen for the initial brain area to be studied, since regulation of opiate action in this area had been shown to be modified by pertussis toxin. Treatment of striatal membranes with pertussis toxin results in up to a 55% decrease in /sup 3/(H)-DADLE binding as compared with membranes treated identically without toxin.more » This corresponds to a near complete ADP-ribosylation of both G proteins in the striatal membrane. The decrease in agonist binding appears to be due to an altered affinity of the receptor for agonist as opposed to a decrease in the number of sites. This effect of pertussis toxin on opiate agonist binding demonstrates the actual involvement of G proteins in regulation of opiate receptor binding.« less

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

PubMed

Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2014-03-04

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins.

PubMed

Aktories, K

1994-09-01

The actin cytoskeleton appears to be as the cellular target of various clostridial ADP-ribosyltransferases which have been described during recent years. Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium spiroforme toxin ADP-ribosylate actin monomers and inhibit actin polymerization. Clostridium botulinum exoenzyme C3 and Clostridium limosum exoenzyme ADP-ribosylate the low-molecular-mass GTP-binding proteins of the Rho family, which participate in the regulation of the actin cytoskeleton. ADP-ribosylation inactivates the regulatory Rho proteins and disturbs the organization of the actin cytoskeleton.

Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ott, S.; Costa, T.; Herz, A.

1988-07-25

The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex.more » The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa.« less

Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum.

PubMed Central

Witter, K; Cahill, D J; Werner, T; Ziegler, I; Rödl, W; Bacher, A; Gütlich, M

1996-01-01

The GTP cyclohydrolase I (GTP-CH) gene of the cellular slime mould Dictyostelium discoideum has been cloned and sequenced. The 855 bp cDNA of this gene contains the open reading frame (ORF) encoding 232 amino acids with a predicted molecular mass of approx. 26 kDa. Southern blot analysis indicated the presence of a single gene for GTP-CH in Dictyostelium. PCR amplification of the ORF from chromosomal DNA and sequencing showed the existence of a 101 bp intron in the GTP-CH gene of Dictyostelium discoideum. The amino acid sequence has 47% and 49% positional identity to those of the human and yeast enzymes respectively. Most of the sequence variation between species is located in the N-terminal part of the protein. The overall identity with the E. coli protein is markedly lower. The enzyme was expressed in E. coli and purified as a 68 kDa fusion protein with the maltose-binding protein of E. coli. GTP-CH of Dictyostelium is heat-stable and showed maximal activity at 60 degrees C. The Km value for GTP is 50 microM. PMID:8870645

Activation of the Lbc Rho Exchange Factor Proto-Oncogene by Truncation of an Extended C Terminus That Regulates Transformation and Targeting

PubMed Central

Sterpetti, Paola; Hack, Andrew A.; Bashar, Mariam P.; Park, Brian; Cheng, Sou-De; Knoll, Joan H. M.; Urano, Takeshi; Feig, Larry A.; Toksoz, Deniz

1999-01-01

The human lbc oncogene product is a guanine nucleotide exchange factor that specifically activates the Rho small GTP binding protein, thus resulting in biologically active, GTP-bound Rho, which in turn mediates actin cytoskeletal reorganization, gene transcription, and entry into the mitotic S phase. In order to elucidate the mechanism of onco-Lbc transformation, here we report that while proto- and onco-lbc cDNAs encode identical N-terminal dbl oncogene homology (DH) and pleckstrin homology (PH) domains, proto-Lbc encodes a novel C terminus absent in the oncoprotein that includes a predicted α-helical region homologous to cyto-matrix proteins, followed by a proline-rich region. The lbc proto-oncogene maps to chromosome 15, and onco-lbc represents a fusion of the lbc proto-oncogene N terminus with a short, unrelated C-terminal sequence from chromosome 7. Both onco- and proto-Lbc can promote formation of GTP-bound Rho in vivo. Proto-Lbc transforming activity is much reduced compared to that of onco-Lbc, and a significant increase in transforming activity requires truncation of both the α-helical and proline-rich regions in the proto-Lbc C terminus. Deletion of the chromosome 7-derived C terminus of onco-Lbc does not destroy transforming activity, demonstrating that it is loss of the proto-Lbc C terminus, rather than gain of an unrelated C-terminus by onco-Lbc, that confers transforming activity. Mutations of onco-Lbc DH and PH domains demonstrate that both domains are necessary for full transforming activity. The proto-Lbc product localizes to the particulate (membrane) fraction, while the majority of the onco-Lbc product is cytosolic, and mutations of the PH domain do not affect this localization. The proto-Lbc C-terminus alone localizes predominantly to the particulate fraction, indicating that the C terminus may play a major role in the correct subcellular localization of proto-Lbc, thus providing a mechanism for regulating Lbc oncogenic potential. PMID:9891067

TCDD causes stimulation of c-ras expression in the hepatic plasma membranes in vivo and in vitro.

PubMed

Tullis, K; Olsen, H; Bombick, D W; Matsumura, F; Jankun, J

1992-01-01

A series of in vivo and in vitro experiments were conducted to determine the effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) administered on the expression of c-ras. Differences in c-ras expression between control and TCDD treated groups were determined by immunoassay of p21ras protein, or indirectly measured by the specific binding of 3H-GTP to hepatic plasma membrane preparations. Intraperitoneal injection of sublethal doses of TCDD significantly elevated (P less than 0.05, Student t test) levels of hepatic p21ras protein in Sprague-Dawley rats and TCDD sensitive C57BL/6J mice. Such an increase occurred at an early stage of poisoning in the C57BL/6J mice. The earliest increase was detectable 6 hr after dosing, and the difference became statistically significant by 12 and 24 hr after dosing. In contrast, TCDD tolerant DBA/2J mice had only a marginal increase in hepatic p21ras protein which did not become statistically significant even at 24 hr host-dosing. TCDD evoked increases in hepatic p21ras protein of C57BL/6J mice were accompanied by the increase in the specific binding of GTP to hepatic plasma membranes. Column chromatography of solubilized rat hepatic membrane proteins on sephadex G-50 showed TCDD administration increased levels of a 3H-GTP binding protein with MW of approximately 21 Kd. 3H-GTP binding in total hepatic membranes was also elevated (P less than 0.05, Fisher PLSD multiple comparison test) 6 hr and 24 hr after dosing of C57BL/6J mice, but as expected the effect of TCDD was not as conspicuous as that found in the plasma membrane. TCDD treatment increased levels of a 21 Kd protein found in the in vitro translation products of RNA purified from guinea pig liver. This protein was identified as a c-ras protein based upon its ability to bind GTP, precipitation by a polyclonal antibody against the rasHa and Ki proteins and subsequent SDS-PAGE which showed a single protein band of approximately 21 Kd.

The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

DOE PAGES

Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

2015-02-27

Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNAmore » transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.« less

The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNAmore » transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.« less

Vibrational studies of phosphoryl transfer enzymes: ras- p21(*)magnesium-GTP and Myosin S1(*)magnesium-ADP- vanadate

NASA Astrophysics Data System (ADS)

Wang, Jianghua

1999-07-01

We have measured the Raman spectra of monophosphate compounds in aqueous solution. The measured frequencies were correlated with P••O valence bond order by using a modification of the Hardcastle- Wachs procedure. The P••O bond order and bond length in phosphates can be determined from vibrational spectra by using the derived bond order/stretching frequency correlation and the bond length/bond order correlation of Brown and Wu. The Raman and infrared spectra of guanosine 5'-diphosphate (GDP) and guanosine 5'-triphosphate (GTP) in aqueous solution were also examined. Frequency shifts were observed as Mg2+ complexes with GDP and GTP in aqueous solution. These results suggested that Mg2+ binds to GDP in a bidentate manner to the α,β P••O bonds and in a tridentate manner to the α,β and γ P••O bonds of Mg•GTP . We have analyzed the previously obtained isotope edited Raman difference spectra of 1:1 complexes of Mg•GDP and Mg•GTP in ras-p21. Frequency changes of the phosphate groups were observed when Mg•GDP , Mg•GTP bind to the protein. Employing both the previous empirical relationships between bond orders/lengths and frequencies as well as vibrational analysis from ab initio calculations, the spectral changes can be explained by the change of the Mg2+ binding sites and hydrogen-bonding. Implications of these structural results for the reaction mechanism of GTP hydrolysis catalyzed by the GTPase are discussed. We have analyzed previously obtained isotope edited Raman difference spectra of the non-bridging V••O bonds of vanadates, both in solution, and when bound to the myosin S1•MgADP complex. By use of ab initio calculations on a model of the vanadate binding site in myosin, the angles between the non-bridging V••O bonds and between these bonds and the apical bonds in the myosin S1•MgADP -Vi complex were determined. The summed bond order of the two apical bonds between the attacking and leaving group oxygens with the central vanadium ion in the S1•MgADP -Vi complex was found to increase only slightly compared with the bond order of the ester V-O bond of a monoester vanadate model compound in solution, suggesting an SN2 like mechanism for the phosphoryl transfer reaction catalyzed by myosin.

Interspecies conservation of retinal guanosine 5'-triphosphatase. Characterization by photoaffinity labelling and tryptic-peptide mapping.

PubMed Central

McMurray, M M; Hansen, J S; Haley, B E; Takemoto, D J; Takemoto, L J

1985-01-01

Light-activated hydrolysis of cyclic GMP is achieved through the photoexcitation of rhodopsin, a process which then triggers the replacement of GDP for GTP by a retinal guanosine 5'-triphosphatase referred to as 'transducin'. The transducin-GTP complex then switches on the phosphodiesterase [Fung, Hurley & Stryer (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 152-156]. The bovine transducin consists of an alpha-subunit (39000 Mr), which is a GTP-binding component, together with a beta-(37000 Mr) and a gamma-subunit (10000 Mr). We have purified retinal transducin from cow, pig, chick and frog. The enzyme specific activities and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic profiles indicate that this enzyme is similar in all species except the frog. Whereas the bovine, pig and chick transducins consist of major 37000- and 39000-Mr components, that of the frog consists of a single 75000-Mr component. Labelling of the GTP-binding components with the photoaffinity label 8-azidoguanosine [gamma-32P]triphosphate demonstrated that the 37000-Mr components of the cow, pig and chick and the 75000-Mr component of the frog were major GTP-binding components. In addition, peptide maps of radioiodinated tryptic peptides indicate that the frog 75000-Mr protein is highly related to the pig transducin. These results demonstrate evolutionary conservation of retinal transducin and the presence of a higher-Mr, but nonetheless highly conserved form, of transducin in the frog. The relationship of this component to the recently reported rod-outer-segment inhibitor protein [Yamazaki, Stein, Chernoff & Bitensky (1983) J. Biol. Chem. 258, 8188-8194] is discussed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2983663

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast*

PubMed Central

Muñoz, Sofía; Manjón, Elvira; Sánchez, Yolanda

2014-01-01

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3+ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation. PMID:24947517

Structure-function studies of adenylosuccinate synthetase from Escherichia coli.

PubMed

Honzatko, R B; Fromm, H J

1999-10-01

Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, thermodynamically coupling the hydrolysis of GTP to the formation of adenylosuccinate from l-aspartate and IMP. The enzyme from Esherichia coli undergoes a ligand-induced dimerization, which leads to the assembly of a complete active site. The binding of IMP causes conformational changes over distances of 30 A, the end result of which is the activation of essential catalytic elements and the organization of the binding pocket for Mg(2+)-GTP. The enzyme promotes first a phosphoryl transfer from GTP to the 6-oxygen atom of IMP, by way of a transition state that has characteristics of both associative and dissociative reaction pathways. Following the formation of 6-phosphoryl-IMP, the enzyme then catalyzes the nucleophilic displacement of the 6-phosphoryl group by the alpha-amino group of l-aspartate in a transition state, which requires two metal cations. Copyright 1999 Academic Press.

Binding of ATP by pertussis toxin and isolated toxin subunits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hausman, S.Z.; Manclark, C.R.; Burns, D.L.

1990-07-03

The binding of ATP to pertussis toxin and its components, the A subunit and B oligomer, was investigated. Whereas, radiolabeled ATP bound to the B oligomer and pertussis toxin, no binding to the A subunit was observed. The binding of ({sup 3}H)ATP to pertussis toxin and the B oligomer was inhibited by nucleotides. The relative effectiveness of the nucleotides was shown to be ATP > GTP > CTP > TTP for pertussis toxin and ATP > GTP > TTP > CTP for the B oligomer. Phosphate ions inhibited the binding of ({sup 3}H)ATP to pertussis toxin in a competitive manner;more » however, the presence of phosphate ions was essential for binding of ATP to the B oligomer. The toxin substrate, NAD, did not affect the binding of ({sup 3}H)ATP to pertussis toxin, although the glycoprotein fetuin significantly decreased binding. These results suggest that the binding site for ATP is located on the B oligomer and is distinct from the enzymatically active site but may be located near the eukaryotic receptor binding site.« less

Adenylosuccinate synthetase: recent developments.

PubMed

Honzatko, R B; Stayton, M M; Fromm, H J

1999-01-01

By exerting strategic control on purine nucleotide biosynthesis, and by engaging GTP-dependent transphosphorylation of IMP to activate loss of an oxygen atom during catalysis, adenylosuccinate synthetase remains as enzyme that justifiably fascinates students of enzyme catalysis. This review describes how the balanced application of X-ray crystallography and enzyme kinetics has advanced the comprehension of the catalytic and regulatory properties of adenylosuccinate synthetase. Detailed analysis has demonstrated the formation of 6-phosphoryl-IMP, an intermediate originally postulated over 40 years ago on the basis of oxygen-18 exchange experiments showing that position-6 oxygen of IMP becomes incorporated into phosphate. Inferences about the participation of amino acid side-chains that stabilize 6-P-IMP during catalysis have also been confirmed by site-directed mutagenesis and examination of such mutations on various kinetic parameters. Moreover, the action of certain regulatory ligands have also been viewed at atomic level resolution. For example, magnesium ion and GDP can induce conformational changes linked to the stabilization of one of two known conformations of the so-called 40s loop. Another significant finding is that two magnesium ions play fundamental roles: one binding with high affinity to the substrate GTP, and a second binding with lower affinity to the co-substrate aspartate. These structural and kinetic studies have also formed the basis for clarifying the action of various inhibitors and potentially important pharmacologic agents with this key regulatory enzyme. Finally, this review explores the current status of investigations on gene structure and gene expression in a number of organisms.

Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor.

PubMed

Oesterlin, Lena K; Goody, Roger S; Itzen, Aymelt

2012-04-10

Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

PubMed Central

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Nucleolar Trafficking of Nucleostemin Family Proteins: Common versus Protein-Specific Mechanisms▿ §

PubMed Central

Meng, Lingjun; Zhu, Qubo; Tsai, Robert Y. L.

2007-01-01

The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins. PMID:17923687

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

The GTP binding proteins Gem and Rad are negative regulators of the Rho–Rho kinase pathway

PubMed Central

Ward, Yvona; Yap, Seow-Fong; Ravichandran, V.; Matsumura, Fumio; Ito, Masaaki; Spinelli, Beth; Kelly, Kathleen

2002-01-01

The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins are important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the Rad, Gem, and Kir family) for which biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) α and β. Gem binds ROKβ independently of RhoA in the ROKβ coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKβ-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKβ. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKβ- and Rad opposed ROKα-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKβ containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKβ is required for the effects observed. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK. PMID:11956230

Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.

PubMed Central

Lill, R; Robertson, J M; Wintermeyer, W

1989-01-01

A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA. PMID:2583120

Green tea polyphenols improve cardiac muscle mRNA and protein levels of signal pathways related to insulin and lipid metabolism and inflammation in insulin-resistant rats.

PubMed

Qin, Bolin; Polansky, Marilyn M; Harry, Dawson; Anderson, Richard A

2010-05-01

Epidemiological studies indicate that the consumption of green tea polyphenols (GTP) may reduce the risk of coronary artery disease. To explore the underlying mechanisms of action at the molecular level, we examined the effects of GTP on the cardiac mRNA and protein levels of genes involved in insulin and lipid metabolism and inflammation. In rats fed a high-fructose diet, supplementation with GTP (200 mg/kg BW daily dissolved in distilled water) for 6 wk, reduced systemic blood glucose, plasma insulin, retinol-binding protein 4, soluble CD36, cholesterol, triglycerides, free fatty acids and LDL-C levels, as well as the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and IL-6. GTP did not affect food intake, bodyweight and heart weight. In the myocardium, GTP also increased the insulin receptor (Ir), insulin receptor substrate 1 and 2 (Irs1 and Irs2), phosphoinositide-3-kinase (Pi3k), v-akt murine thymoma viral oncogene homolog 1 (Akt1), glucose transporter 1 and 4 (Glut1 and Glut4) and glycogen synthase 1 (Gys1) expression but inhibited phosphatase and tensin homolog deleted on chromosome ten (Pten) expression and decreased glycogen synthase kinase 3beta (Gsk3beta) mRNA expression. The sterol regulatory element-binding protein-1c (Srebp1c) mRNA, microsomal triglyceride transfer protein (Mttp) mRNA and protein, Cd36 mRNA and cluster of differentiation 36 protein levels were decreased and peroxisome proliferator-activated receptor (Ppar)gamma mRNA levels were increased. GTP also decreased the inflammatory factors: Tnf, Il1b and Il6 mRNA levels, and enhanced the anti-inflammatory protein, zinc-finger protein, protein and mRNA expression. In summary, consumption of GTP ameliorated the detrimental effects of high-fructose diet on insulin signaling, lipid metabolism and inflammation in the cardiac muscle of rats.

The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins.

PubMed

Kawada, Daiki; Kobayashi, Hiromu; Tomita, Tsuyoshi; Nakata, Eisuke; Nagano, Makoto; Siekhaus, Daria Elisabeth; Toshima, Junko Y; Toshima, Jiro

2015-01-01

Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Signaling through G protein coupled receptors.

PubMed

Tuteja, Narendra

2009-10-01

Heterotrimeric G proteins (Galpha, Gbeta/Ggamma subunits) constitute one of the most important components of cell signaling cascade. G Protein Coupled Receptors (GPCRs) perceive many extracellular signals and transduce them to heterotrimeric G proteins, which further transduce these signals intracellular to appropriate downstream effectors and thereby play an important role in various signaling pathways. GPCRs exist as a superfamily of integral membrane protein receptors that contain seven transmembrane alpha-helical regions, which bind to a wide range of ligands. Upon activation by a ligand, the GPCR undergoes a conformational change and then activate the G proteins by promoting the exchange of GDP/GTP associated with the Galpha subunit. This leads to the dissociation of Gbeta/Ggamma dimer from Galpha. Both these moieties then become free to act upon their downstream effectors and thereby initiate unique intracellular signaling responses. After the signal propagation, the GTP of Galpha-GTP is hydrolyzed to GDP and Galpha becomes inactive (Galpha-GDP), which leads to its re-association with the Gbeta/Ggamma dimer to form the inactive heterotrimeric complex. The GPCR can also transduce the signal through G protein independent pathway. GPCRs also regulate cell cycle progression. Till to date thousands of GPCRs are known from animal kingdom with little homology among them, but only single GPCR has been identified in plant system. The Arabidopsis GPCR was reported to be cell cycle regulated and also involved in ABA and in stress signaling. Here I have described a general mechanism of signal transduction through GPCR/G proteins, structure of GPCRs, family of GPCRs and plant GPCR and its role.

IbeA and OmpA of Escherichia coli K1 Exploit Rac1 Activation for Invasion of Human Brain Microvascular Endothelial Cells

PubMed Central

Maruvada, Ravi

2012-01-01

Meningitis-causing Escherichia coli K1 internalization of the blood-brain barrier is required for penetration into the brain, but the host-microbial interactions involved in E. coli entry of the blood-brain barrier remain incompletely understood. We show here that a meningitis-causing E. coli K1 strain RS218 activates Rac1 (GTP-Rac1) of human brain microvascular endothelial cells (HBMEC) in a time-dependent manner. Both activation and bacterial invasion were significantly inhibited in the presence of a Rac1 inhibitor. We further showed that the guanine nucleotide exchange factor Vav2, not β-Pix, was involved in E. coli K1-mediated Rac1 activation. Since activated STAT3 is known to bind GTP-Rac1, the relationship between STAT3 and Rac1 was examined in E. coli K1 invasion of HBMEC. Downregulation of STAT3 resulted in significantly decreased E. coli invasion compared to control HBMEC, as well as a corresponding decrease in GTP-Rac1, suggesting that Rac1 activation in response to E. coli is under the control of STAT3. More importantly, two E. coli determinants contributing to HBMEC invasion, IbeA and OmpA, were shown to affect both Rac1 activation and their association with STAT3. These findings demonstrate for the first time that specific E. coli determinants regulate a novel mechanism of STAT3 cross talk with Rac1 in E. coli K1 invasion of HBMEC. PMID:22451524

IbeA and OmpA of Escherichia coli K1 exploit Rac1 activation for invasion of human brain microvascular endothelial cells.

PubMed

Maruvada, Ravi; Kim, Kwang Sik

2012-06-01

Meningitis-causing Escherichia coli K1 internalization of the blood-brain barrier is required for penetration into the brain, but the host-microbial interactions involved in E. coli entry of the blood-brain barrier remain incompletely understood. We show here that a meningitis-causing E. coli K1 strain RS218 activates Rac1 (GTP-Rac1) of human brain microvascular endothelial cells (HBMEC) in a time-dependent manner. Both activation and bacterial invasion were significantly inhibited in the presence of a Rac1 inhibitor. We further showed that the guanine nucleotide exchange factor Vav2, not β-Pix, was involved in E. coli K1-mediated Rac1 activation. Since activated STAT3 is known to bind GTP-Rac1, the relationship between STAT3 and Rac1 was examined in E. coli K1 invasion of HBMEC. Downregulation of STAT3 resulted in significantly decreased E. coli invasion compared to control HBMEC, as well as a corresponding decrease in GTP-Rac1, suggesting that Rac1 activation in response to E. coli is under the control of STAT3. More importantly, two E. coli determinants contributing to HBMEC invasion, IbeA and OmpA, were shown to affect both Rac1 activation and their association with STAT3. These findings demonstrate for the first time that specific E. coli determinants regulate a novel mechanism of STAT3 cross talk with Rac1 in E. coli K1 invasion of HBMEC.

Structure of Escherichia coli dGTP Triphosphohydrolase: A Hexameric Enzyme with DNA Effector Molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Deepa; Gawel, Damian; Itsko, Mark

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present paper, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNAmore » with high affinity (K d ~ 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent K m for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Finally, our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.« less

Structure of Escherichia coli dGTP Triphosphohydrolase: A Hexameric Enzyme with DNA Effector Molecules

DOE PAGES

Singh, Deepa; Gawel, Damian; Itsko, Mark; ...

2015-02-18

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present paper, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNAmore » with high affinity (K d ~ 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent K m for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Finally, our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.« less

CLASPs are required for proper microtubule localization of End-binding proteins

PubMed Central

Grimaldi, Ashley D.; Maki, Takahisa; Fitton, Benjamin P.; Roth, Daniel; Yampolsky, Dmitry; Davidson, Michael W.; Svitkina, Tatyana; Straube, Anne; Hayashi, Ikuko; Kaverina, Irina

2014-01-01

Summary Microtubule (MT) plus-end tracking proteins (+TIPs) preferentially localize to MT plus-ends. End-binding proteins (EBs) are master regulators of the +TIP complex; however, it is unknown whether EBs are regulated by other +TIPs. Here, we show that Cytoplasmic linker associated proteins (CLASPs) modulate EB localization at MTs. In CLASP-depleted cells, EBs localized along the MT lattice in addition to plus-ends. The MT-binding region of CLASP was sufficient for restoring normal EB localization, while neither EB-CLASP interactions nor EB tail-binding proteins are involved. In vitro assays revealed that CLASP directly functions to remove EB from MTs. Importantly, this effect occurs specifically during MT polymerization, but not at pre-formed MTs. Increased GTP-tubulin content within MTs in CLASP-depleted cells suggests that CLASPs facilitate GTP-hydrolysis to reduce EB lattice binding. Together, these findings suggest that CLASPs influence the MT lattice itself to regulate EB and determine exclusive plus-end localization of EBs in cells. PMID:25117684

Determination of the kinetics of guanine nucleotide exchange on EF-Tu and EF-Ts: continuing uncertainties.

PubMed

Manchester, Keith L

2004-01-30

An analysis is made of the rate constants for the reactions involving the interactions of EF-Tu, EF-Ts, GDP, and GTP recently derived by Gromadski et al. [Biochemistry 41 (2002) 162]. Though their measured values appear to allow a reasonable rate of nucleotide exchange sufficient to support rates of protein synthesis in vivo, their data underestimate the thermodynamic barrier involved in nucleotide exchange and therefore cannot be considered definitive. A kinetic scheme consistent with the thermodynamic barrier can be achieved by modification of various rate constants, particularly of those involving the release of EF-Ts from EF-Tu.GTP.EF-Ts, but such constants are markedly different from what are experimentally observed. It thus remains impossible at present satisfactorily to model guanine nucleotide exchange on EF-Tu, catalysed by EF-Ts by a double displacement mechanism, with experimentally derived rate constants. Metabolic control analysis has been applied to determine the degree of flux control of the different steps in the pathway.

Valerian extract and valerenic acid are partial agonists of the 5-HT5a receptor in vitro.

PubMed

Dietz, Birgit M; Mahady, Gail B; Pauli, Guido F; Farnsworth, Norman R

2005-08-18

Insomnia is the most frequently encountered sleep complaint worldwide. While many prescription drugs are used to treat insomnia, extracts of valerian (Valeriana officinalis L., Valerianaceae) are also used for the treatment of insomnia and restlessness. To determine novel mechanisms of action, radioligand binding studies were performed with valerian extracts (100% methanol, 50% methanol, dichloromethane [DCM], and petroleum ether [PE]) at the melatonin, glutamate, and GABA(A) receptors, and 8 serotonin receptor subtypes. Both DCM and PE extracts had strong binding affinity to the 5-HT(5a) receptor, but only weak binding affinity to the 5-HT(2b) and the serotonin transporter. Subsequent binding studies focused on the 5-HT(5a) receptor due to the distribution of this receptor in the suprachiasmatic nucleus of the brain, which is implicated in the sleep-wake cycle. The PE extract inhibited [(3)H]lysergic acid diethylamide (LSD) binding to the human 5-HT(5a) receptor (86% at 50 microg/ml) and the DCM extract inhibited LSD binding by 51%. Generation of an IC(50) curve for the PE extract produced a biphasic curve, thus GTP shift experiments were also performed. In the absence of GTP, the competition curve was biphasic (two affinity sites) with an IC(50) of 15.7 ng/ml for the high-affinity state and 27.7 microg/ml for the low-affinity state. The addition of GTP (100 microM) resulted in a right-hand shift of the binding curve with an IC(50) of 11.4 microg/ml. Valerenic acid, the active constituent of both extracts, had an IC(50) of 17.2 microM. These results indicate that valerian and valerenic acid are new partial agonists of the 5-HT(5a) receptor.

Valerian extract and valerenic acid are partial agonists of the 5-HT5a receptor in vitro

PubMed Central

Dietz, Birgit M.; Mahady, Gail B.; Pauli, Guido F.; Farnsworth, Norman R.

2018-01-01

Insomnia is the most frequently encountered sleep complaint worldwide. While many prescription drugs are used to treat insomnia, extracts of valerian (Valeriana officinalis L., Valerianaceae) are also used for the treatment of insomnia and restlessness. To determine novel mechanisms of action, radioligand binding studies were performed with valerian extracts (100% methanol, 50% methanol, dichloromethane [DCM], and petroleum ether [PE]) at the melatonin, glutamate, and GABAA receptors, and 8 serotonin receptor subtypes. Both DCM and PE extracts had strong binding affinity to the 5-HT5a receptor, but only weak binding affinity to the 5-HT2b and the serotonin transporter. Subsequent binding studies focused on the 5-HT5a receptor due to the distribution of this receptor in the suprachiasmatic nucleus of the brain, which is implicated in the sleep–wake cycle. The PE extract inhibited [3H]lysergic acid diethylamide (LSD) binding to the human 5-HT5a receptor (86% at 50 μg/ml) and the DCM extract inhibited LSD binding by 51%. Generation of an IC50 curve for the PE extract produced a biphasic curve, thus GTP shift experiments were also performed. In the absence of GTP, the competition curve was biphasic (two affinity sites) with an IC50 of 15.7 ng/ml for the high-affinity state and 27.7 μg/ml for the low-affinity state. The addition of GTP (100 AM) resulted in a right-hand shift of the binding curve with an IC50 of 11.4 μg/ml. Valerenic acid, the active constituent of both extracts, had an IC50 of 17.2 AM. These results indicate that valerian and valerenic acid are new partial agonists of the 5-HT5a receptor. PMID:15921820

Transportin acts to regulate mitotic assembly events by target binding rather than Ran sequestration

PubMed Central

Bernis, Cyril; Swift-Taylor, Beth; Nord, Matthew; Carmona, Sarah; Chook, Yuh Min; Forbes, Douglass J.

2014-01-01

The nuclear import receptors importin β and transportin play a different role in mitosis: both act phenotypically as spatial regulators to ensure that mitotic spindle, nuclear membrane, and nuclear pore assembly occur exclusively around chromatin. Importin β is known to act by repressing assembly factors in regions distant from chromatin, whereas RanGTP produced on chromatin frees factors from importin β for localized assembly. The mechanism of transportin regulation was unknown. Diametrically opposed models for transportin action are as follows: 1) indirect action by RanGTP sequestration, thus down-regulating release of assembly factors from importin β, and 2) direct action by transportin binding and inhibiting assembly factors. Experiments in Xenopus assembly extracts with M9M, a superaffinity nuclear localization sequence that displaces cargoes bound by transportin, or TLB, a mutant transportin that can bind cargo and RanGTP simultaneously, support direct inhibition. Consistently, simple addition of M9M to mitotic cytosol induces microtubule aster assembly. ELYS and the nucleoporin 107–160 complex, components of mitotic kinetochores and nuclear pores, are blocked from binding to kinetochores in vitro by transportin, a block reversible by M9M. In vivo, 30% of M9M-transfected cells have spindle/cytokinesis defects. We conclude that the cell contains importin β and transportin “global positioning system”or “GPS” pathways that are mechanistically parallel. PMID:24478460

Identification of small molecule inhibitors of the Chikungunya virus nsP1 RNA capping enzyme.

PubMed

Feibelman, Kristen M; Fuller, Benjamin P; Li, Linfeng; LaBarbera, Daniel V; Geiss, Brian J

2018-06-01

Chikungunya virus (CHIKV) is an arthropod-borne alphavirus. Alphaviruses are positive strand RNA viruses that require a 5' cap structure to direct translation of the viral polyprotein and prevent degradation of the viral RNA genome by host cell nucleases. Formation of the 5' RNA cap is orchestrated by the viral protein nsP1, which binds GTP and provides the N-7 methyltransferase and guanylyltransferase activities that are necessary for cap formation. Viruses with aberrant nsP1 activity are unable to replicate effectively suggesting that nsP1 is a promising target for antiviral drug discovery. Given the absence of commercially available antiviral therapies for CHIKV, it is imperative to identify compounds that could be developed as potential therapeutics. This study details a high-throughput screen of 3051 compounds from libraries containing FDA-approved drugs, natural products, and known bioactives against CHIKV nsP1 using a fluorescence polarization-based GTP competition assay. Several small molecule hits from this screen were able to compete with GTP for the CHIKV nsP1 GTP binding site at low molar concentrations. Compounds were also evaluated with an orthogonal assay that measured the ability of nsP1 to perform the guanylation step of the capping reaction in the presence of inhibitor. In addition, live virus assays with CHIKV and closely related alphavirus, Sindbis virus, were used in conjunction with cell toxicity assays to determine the antiviral activity of compounds in cell culture. The naturally derived compound lobaric acid was found to inhibit CHIKV nsP1 GTP binding and guanylation as well as attenuate viral growth in vitro at both 24 hpi and 48 hpi in hamster BHK21 and human Huh 7 cell lines. These data indicate that development of lobaric acid and further exploration of CHIKV nsP1 as a drug target may aid in the progress of anti-alphaviral drug development strategies. Copyright © 2018. Published by Elsevier B.V.

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia.

PubMed

Smith, Clyde A; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B

2014-06-01

The bifunctional acetyltransferase(6')-Ie-phosphotransferase(2'')-Ia [AAC(6')-Ie-APH(2'')-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2'')-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2'')-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2'')-IIa and APH(2'')-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2'')-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2'')-IIIa enzyme. In APH(2'')-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2'')-Ia into a dual-specificity enzyme.

Elongation factor Ts directly facilitates the formation and disassembly of the Escherichia coli elongation factor Tu·GTP·aminoacyl-tRNA ternary complex.

PubMed

Burnett, Benjamin J; Altman, Roger B; Ferrao, Ryan; Alejo, Jose L; Kaur, Navdep; Kanji, Joshua; Blanchard, Scott C

2013-05-10

Aminoacyl-tRNA (aa-tRNA) enters the ribosome in a ternary complex with the G-protein elongation factor Tu (EF-Tu) and GTP. EF-Tu·GTP·aa-tRNA ternary complex formation and decay rates are accelerated in the presence of the nucleotide exchange factor elongation factor Ts (EF-Ts). EF-Ts directly facilitates the formation and disassociation of ternary complex. This system demonstrates a novel function of EF-Ts. Aminoacyl-tRNA enters the translating ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here, we describe bulk steady state and pre-steady state fluorescence methods that enabled us to quantitatively explore the kinetic features of Escherichia coli ternary complex formation and decay. The data obtained suggest that both processes are controlled by a nucleotide-dependent, rate-determining conformational change in EF-Tu. Unexpectedly, we found that this conformational change is accelerated by elongation factor Ts (EF-Ts), the guanosine nucleotide exchange factor for EF-Tu. Notably, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. These results suggest that EF-Ts serves an unanticipated role in the cell of actively regulating the abundance and stability of ternary complex in a manner that contributes to rapid and faithful protein synthesis.

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

PubMed

Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate

PubMed Central

Lee, Young Kwang; Low-Nam, Shalini T.; Chung, Jean K.; Hansen, Scott D.; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T.

2017-01-01

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification. PMID:28452363

Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

PubMed

Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

2017-04-28

The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

An improved procedure for reconstitution of the uncoupling protein and in-depth analysis of H+/OH- transport.

PubMed

Winkler, E; Klingenberg, M

1992-07-01

An improved procedure for reincorporation of isolated uncoupling protein (UCP) from brown adipose tissue into phospholipid vesicles is reported and H+ uptake in K(+)-driven exchange diffusion quantitatively analyzed. UCP is isolated and reconstituted with medium-length linear-chain alkyl polyoxyethylene. In the critical step of vesicle formation, the stepwise removal of the detergent by polystyrene beads is applied. Vesicles are generated in the presence of solutes and buffers to be internalized which are then removed by gel filtration. The internal volume is about 4 microliters/mg phospholipid with a vesicle diameter of 100 nm. One vesicle contains, on average, six molecules UCP. The best results are obtained with purified egg yolk phosphatidylcholine. Addition of PtdEtn, PtdSer decreases the vesicle size and, still more, H(+)-transport activity by UCP. Asolectin completely inactivates UCP. K(+)-gradient-driven H+ uptake is 80% inhibited by external GTP and 95% by internal plus external GTP. When H+ transport is recorded externally by a pH electrode and internally by pyranine, the kinetics show no delay resulting from intervening membrane-bound H+ pools. Total H+ uptake after addition of carbonylcyanine m-chlorophenylhydrazone (CCCP) and valinomycin corresponds to the diffusion between H+ and K+ and is unchanged by GTP. The linear correlation of H(+)-transport inhibition to GTP binding demonstrates that all UCP molecules incorporated are equally active. The exchange diffusion between H+ uptake and K+ efflux is demonstrated using a K+ electrode and 86Rb measurements. Recording delta psi using 3,3'-diispropylthiadicarbocyanine shows a rapid generation of delta psi on valinomycin addition, which decreases only slightly with H+ uptake, even after addition of CCCP or gramicidin. The delta psi collapses only after addition of external K+. By demonstrating that valinomycin-induced K+ and H+ fluxes reflect relaxation into the diffusion equilibrium state, the transport rate of UCP can be evaluated as a first-order rate, VH+/CH+, in which the rate, VH+, is related to H(+)-uptake capacity, CH+. This allows quantitative comparison of transport rates independently of the variable CH+. The dependence on delta psi of H+ transport is measured by varying external K+ concentration. A virtually linear relation of the rate to the K(+)-diffusion potential is observed, although the capacity is only slightly changed. The linear VH+/delta psi relationship resembles an open-channel type of transport, but is discussed in terms of a low-activation-barrier type of carrier mechanism, in contrast to the log (VH+/delta psi) relation found for the ADP/ATP carrier with high activation barriers.

Disease-Causing Mutations in the G Protein Gαs Subvert the Roles of GDP and GTP.

PubMed

Hu, Qi; Shokat, Kevan M

2018-05-17

The single most frequent cancer-causing mutation across all heterotrimeric G proteins is R201C in Gαs. The current model explaining the gain-of-function activity of the R201 mutations is through the loss of GTPase activity and resulting inability to switch off to the GDP state. Here, we find that the R201C mutation can bypass the need for GTP binding by directly activating GDP-bound Gαs through stabilization of an intramolecular hydrogen bond network. Having found that a gain-of-function mutation can convert GDP into an activator, we postulated that a reciprocal mutation might disrupt the normal role of GTP. Indeed, we found R228C, a loss-of-function mutation in Gαs that causes pseudohypoparathyroidism type 1a (PHP-Ia), compromised the adenylyl cyclase-activating activity of Gαs bound to a non-hydrolyzable GTP analog. These findings show that disease-causing mutations in Gαs can subvert the canonical roles of GDP and GTP, providing new insights into the regulation mechanism of G proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

14-3-3 Proteins Interact with a Hybrid Prenyl-Phosphorylation Motif to Inhibit G Proteins

PubMed Central

Riou, Philippe; Kjær, Svend; Garg, Ritu; Purkiss, Andrew; George, Roger; Cain, Robert J.; Bineva, Ganka; Reymond, Nicolas; McColl, Brad; Thompson, Andrew J.; O’Reilly, Nicola; McDonald, Neil Q.; Parker, Peter J.; Ridley, Anne J.

2013-01-01

Summary Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins. PMID:23622247

“Gate-keeper” Residues and Active-Site Rearrangements in DNA Polymerase μ Help Discriminate Non-cognate Nucleotides

PubMed Central

Li, Yunlang; Schlick, Tamar

2013-01-01

Incorporating the cognate instead of non-cognate substrates is crucial for DNA polymerase function. Here we analyze molecular dynamics simulations of DNA polymerase μ (pol μ) bound to different non-cognate incoming nucleotides including A:dCTP, A:dGTP, A(syn):dGTP, A:dATP, A(syn):dATP, T:dCTP, and T:dGTP to study the structure-function relationships involved with aberrant base pairs in the conformational pathway; while a pol μ complex with the A:dTTP base pair is available, no solved non-cognate structures are available. We observe distinct differences of the non-cognate systems compared to the cognate system. Specifically, the motions of active-site residue His329 and Asp330 distort the active site, and Trp436, Gln440, Glu443 and Arg444 tend to tighten the nucleotide-binding pocket when non-cognate nucleotides are bound; the latter effect may further lead to an altered electrostatic potential within the active site. That most of these “gate-keeper” residues are located farther apart from the upstream primer in pol μ, compared to other X family members, also suggests an interesting relation to pol μ's ability to incorporate nucleotides when the upstream primer is not paired. By examining the correlated motions within pol μ complexes, we also observe different patterns of correlations between non-cognate systems and the cognate system, especially decreased interactions between the incoming nucleotides and the nucleotide-binding pocket. Altered correlated motions in non-cognate systems agree with our recently proposed hybrid conformational selection/induced-fit models. Taken together, our studies propose the following order for difficulty of non-cognate system insertions by pol μ: T:dGTP

Green tea polyphenols ameliorate non-alcoholic fatty liver disease through upregulating AMPK activation in high fat fed Zucker fatty rats.

PubMed

Tan, Yi; Kim, Jane; Cheng, Jing; Ong, Madeleine; Lao, Wei-Guo; Jin, Xing-Liang; Lin, Yi-Guang; Xiao, Linda; Zhu, Xue-Qiong; Qu, Xian-Qin

2017-06-07

To investigate protective effects and molecular mechanisms of green tea polyphenols (GTP) on non-alcoholic fatty liver disease (NAFLD) in Zucker fatty (ZF) rats. Male ZF rats were fed a high-fat diet (HFD) for 2 wk then treated with GTP (200 mg/kg) or saline (5 mL/kg) for 8 wk, with Zucker lean rat as their control. At the end of experiment, serum and liver tissue were collected for measurement of metabolic parameters, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), inflammatory cytokines and hepatic triglyceride and liver histology. Immunoblotting was used to detect phosphorylation of AMP-activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC), and sterol regulatory element-binding protein 1c (SREBP1c). Genetically obese ZF rats on a HFD presented with metabolic features of hepatic pathological changes comparable to human with NAFLD. GTP intervention decreased weight gain (10.1%, P = 0.052) and significantly lowered visceral fat (31.0%, P < 0.01). Compared with ZF-controls, GTP treatment significantly reduced fasting serum insulin, glucose and lipids levels. Reduction in serum ALT and AST levels (both P < 0.01) were observed in GTP-treated ZF rats. GTP treatment also attenuated the elevated TNFα and IL-6 in the circulation. The increased hepatic TG accumulation and cytoplasmic lipid droplet were attenuated by GTP treatment, associated with significantly increased expression of AMPK-Thr172 ( P < 0.05) and phosphorylated ACC and SREBP1c (both P < 0.05), indicating diminished hepatic lipogenesis and triglycerides out flux from liver in GTP treated rats. The protective effects of GTP against HFD-induced NAFLD in genetically obese ZF rats are positively correlated to reduction in hepatic lipogenesis through upregulating the AMPK pathway.

Photoregeneration of bovine rhodopsin from its signaling state.

PubMed

Arnis, S; Hofmann, K P

1995-07-25

In rhodopsin, 11-cis-retinal is bound by a protonated Schiff base and acts as a strong antagonist, which holds the receptor in its inactive ground state conformation. Light induces cis-/trans-retinal isomerization and a sequence of thermal transitions through intermediates. The active conformation that catalyzes GDP/GTP exchange in the G-protein (Gt) is generated from the metarhodopsin II intermediate (MII) and mediated by Schiff base proton translocation and proton uptake from the aqueous phase. In the stable nucleotide-free MII-Gt complex, any thermal transition of MII into other forms of rhodopsin is blocked. We have now studied how Gt affects flash-induced photochemical conversions of MII. Difference spectra from measured absorption changes show that MII photolyzes through two parallel pathways, with fast (1 ms) and slow (50 ms) kinetics (12 degrees C, pH 6). The slow pathway regenerates rhodopsin (9- or 11-cis) via Schiff base reprotonation and proton release. We infer a cis-isomerized early photoproduct (reverted meta, RM) preceding these thermal transitions. When MII is photolyzed in the MII-Gt complex, the slow absorption change is abolished, indicating that Gt blocks the completion of the regeneration process. This is due to the formation of a stable RM-Gt complex, as shown by successive photolysis of MII, RM, and ground state rhodopsin, and the application of GTP gamma S at different stages. The complex dissociates with GTP gamma S, and rhodopsin relaxes to the ground state. The results indicate that cis-retinal and Gt can bind to the receptor at the same time. We discuss the result that the protonations in the meta II state uncouple retinal geometry from Gt interaction.

Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

DOE PAGES

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; ...

2015-06-16

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas G12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas G12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

PubMed Central

Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

2015-01-01

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia

PubMed Central

Smith, Clyde A.; Toth, Marta; Bhattacharya, Monolekha; Frase, Hilary; Vakulenko, Sergei B.

2014-01-01

The bifunctional acetyltransferase(6′)-Ie-phosphotransfer­ase(2′′)-Ia [AAC(6′)-Ie-APH(2′′)-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2′′)-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2′′)-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2′′)-IIa and APH(2′′)-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2′′)-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2′′)-IIIa enzyme. In APH(2′′)-Ia this GTP selectivity is governed by the presence of a ‘gatekeeper’ residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2′′)-Ia into a dual-specificity enzyme. PMID:24914967

GTP analogues promote release of the alpha subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells.

PubMed Central

Milligan, G; Mullaney, I; Unson, C G; Marshall, L; Spiegel, A M; McArdle, H

1988-01-01

The major pertussis-toxin-sensitive guanine nucleotide-binding protein of rat glioma C6 BU1 cells corresponded immunologically to Gi2. Antibodies which recognize the alpha subunit of this protein indicated that it has an apparent molecular mass of 40 kDa and a pI of 5.7. Incubation of membranes of these cells with guanosine 5'-[beta gamma-imido]triphosphate, or other analogues of GTP, caused release of this polypeptide from the membrane in a time-dependent manner. Analogues of GDP or of ATP did not mimic this effect. The GTP analogues similarly caused release of the alpha subunit of Gi2 from membranes of C6 cells in which this G-protein had been inactivated by pretreatment with pertussis toxin. The beta subunit was not released from the membrane under any of these conditions, indicating that the release process was a specific response to the dissociation of the G-protein after binding of the GTP analogue. Similar nucleotide profiles for release of the alpha subunits of forms of Gi were noted for membranes of both the neuroblastoma x glioma hybrid cell line NG108-15 and of human platelets. These data provide evidence that: (1) pertussis-toxin-sensitive G-proteins, in native membranes, do indeed dissociate into alpha and beta gamma subunits upon activation; (2) the alpha subunit of 'Gi-like' proteins need not always remain in intimate association with the plasma membrane; and (3) the alpha subunit of Gi2 can still dissociate from the beta/gamma subunits after pertussis-toxin-catalysed ADP-ribosylation. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3140801

Structures of rat cytosolic PEPCK: insight into the mechanism of phosphorylation and decarboxylation of oxaloacetic acid.

PubMed

Sullivan, Sarah M; Holyoak, Todd

2007-09-04

The structures of the rat cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK) reported in the PEPCK-Mn2+, -Mn2+-oxaloacetic acid (OAA), -Mn2+-OAA-Mn2+-guanosine-5'-diphosphate (GDP), and -Mn2+-Mn2+-guanosine-5'-tri-phosphate (GTP) complexes provide insight into the mechanism of phosphoryl transfer and decarboxylation mediated by this enzyme. OAA is observed to bind in a number of different orientations coordinating directly to the active site metal. The Mn2+-OAA and Mn2+-OAA-Mn2+GDP structures illustrate inner-sphere coordination of OAA to the manganese ion through the displacement of two of the three water molecules coordinated to the metal in the holo-enzyme by the C3 and C4 carbonyl oxygens. In the PEPCK-Mn2+-OAA complex, an alternate bound conformation of OAA is present. In this conformation, in addition to the previous interactions, the C1 carboxylate is directly coordinated to the active site Mn2+, displacing all of the waters coordinated to the metal in the holo-enzyme. In the PEPCK-Mn2+-GTP structure, the same water molecule displaced by the C1 carboxylate of OAA is displaced by one of the gamma-phosphate oxygens of the triphosphate nucleotide. The structures are consistent with a mechanism of direct in-line phosphoryl transfer, supported by the observed stereochemistry of the reaction. In the catalytically competent binding mode, the C1 carboxylate of OAA is sandwiched between R87 and R405 in an environment that would serve to facilitate decarboxylation. In the reverse reaction, these two arginines would form the CO2 binding site. Comparison of the Mn2+-OAA-Mn2+GDP and Mn2+-Mn2+GTP structures illustrates a marked difference in the bound conformations of the nucleotide substrates in which the GTP nucleotide is bound in a high-energy state resulting from the eclipsing of all three of the phosphoryl groups along the triphosphate chain. This contrasts a previously determined structure of PEPCK in complex with a triphosphate nucleotide analogue in which the analogue mirrors the conformation of GDP as opposed to GTP. Last, the structures illustrate a correlation between conformational changes in the P-loop, the nucleotide binding site, and the active site lid that are important for catalysis.

Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

NASA Astrophysics Data System (ADS)

Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

2015-11-01

Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

Structural insight into TPX2-stimulated microtubule assembly

PubMed Central

2017-01-01

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements (’ridge’ and ‘wedge’) in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation. PMID:29120325

A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes

PubMed Central

Yu, Xinchao; Breitman, Marianna; Goldberg, Jonathan

2012-01-01

Summary Budding of COPI-coated vesicles from Golgi membranes requires an Arf-family G protein and the coatomer complex recruited from cytosol. Arf is also required with coatomer-related clathrin adaptor complexes to bud vesicles from the trans-Golgi network and endosomal compartments. To understand the structural basis for Arf-dependent recruitment of a vesicular coat to the membrane, we determined the structure of Arf1 bound to the γζ-COP subcomplex of coatomer. Structure-guided biochemical analysis reveals that a second Arf1-GTP molecule binds to βδ-COP at a site common to the γ- and β-COP subunits. The Arf1-binding sites on coatomer are spatially related to PtdIns4,5P2-binding sites on the endocytic AP2 complex, providing evidence that the orientation of membrane binding is general for this class of vesicular coat proteins. A bivalent GTP-dependent binding mode has implications for the dynamics of coatomer interaction with the Golgi and for the selection of cargo molecules. PMID:22304919

Brefeldin A Inhibits Cell-Free, De Novo Synthesis of Poliovirus

PubMed Central

Cuconati, Andrea; Molla, Akhteruzzaman; Wimmer, Eckard

1998-01-01

Brefeldin A (BFA), an inhibitor of intracellular vesicle-dependent secretory transport, is a potent inhibitor of poliovirus RNA replication in infected cells. We have determined that the unknown mechanism of BFA inhibition of replication is reproduced in the cell-free poliovirus translation, replication, and encapsidation system. Furthermore, we provide evidence suggesting that the cellular mechanism targeted by BFA, the GTP-dependent synthesis of secretory transport vesicles, may be involved in viral RNA replication in the system via a soluble cellular GTP-binding and -hydrolyzing activity. This activity is related to the ARF (ADP-ribosylation factor) family of GTP-binding proteins. ARFs are required for the formation of several classes of secretory vesicles, and some family members are indirectly inactivated by BFA. Peptides that function as competitive inhibitors of ARF activity in cell-free transport systems also inhibit poliovirus RNA replication, and this inhibitory effect can be countered by the addition of exogenous ARF. We suggest that BFA inhibition of replication is diagnostic of a requirement for ARF activity in the cell-free system. PMID:9658088

Structural model of FeoB, the iron transporter from Pseudomonas aeruginosa, predicts a cysteine lined, GTP-gated pore

PubMed Central

Seyedmohammad, Saeed; Fuentealba, Natalia Alveal; Marriott, Robert A.J.; Goetze, Tom A.; Edwardson, J. Michael; Barrera, Nelson P.; Venter, Henrietta

2016-01-01

Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an N-terminal GTP-binding domain and a C-terminal membrane domain. Despite the availability of X-ray crystal structures of the N-terminal domain, many aspects of the structure and function of FeoB remain unclear, such as the structure of the membrane domain, the oligomeric state of the protein, the molecular mechanism of iron transport, and how this is coupled to GTP hydrolysis at the N-terminal domain. In the present study, we describe the first homology model of FeoB. Due to the lack of sequence homology between FeoB and other transporters, the structures of four different proteins were used as templates to generate the homology model of full-length FeoB, which predicts a trimeric structure. We confirmed this trimeric structure by both blue-native-PAGE (BN-PAGE) and AFM. According to our model, the membrane domain of the trimeric protein forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) lined by aspartate residues. Biochemical analysis of FeoB from Pseudomonas aeruginosa further reveals a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. These results indicate that FeoB might not act as a transporter, but rather as a GTP-gated channel. PMID:26934982

Fusel Alcohols Regulate Translation Initiation by Inhibiting eIF2B to Reduce Ternary Complex in a Mechanism That May Involve Altering the Integrity and Dynamics of the eIF2B Body

PubMed Central

Taylor, Eleanor J.; Campbell, Susan G.; Griffiths, Christian D.; Reid, Peter J.; Slaven, John W.; Harrison, Richard J.; Sims, Paul F.G.; Pavitt, Graham D.; Delneri, Daniela

2010-01-01

Recycling of eIF2-GDP to the GTP-bound form constitutes a core essential, regulated step in eukaryotic translation. This reaction is mediated by eIF2B, a heteropentameric factor with important links to human disease. eIF2 in the GTP-bound form binds to methionyl initiator tRNA to form a ternary complex, and the levels of this ternary complex can be a critical determinant of the rate of protein synthesis. Here we show that eIF2B serves as the target for translation inhibition by various fusel alcohols in yeast. Fusel alcohols are endpoint metabolites from amino acid catabolism, which signal nitrogen scarcity. We show that the inhibition of eIF2B leads to reduced ternary complex levels and that different eIF2B subunit mutants alter fusel alcohol sensitivity. A DNA tiling array strategy was developed that overcame difficulties in the identification of these mutants where the phenotypic distinctions were too subtle for classical complementation cloning. Fusel alcohols also lead to eIF2α dephosphorylation in a Sit4p-dependent manner. In yeast, eIF2B occupies a large cytoplasmic body where guanine nucleotide exchange on eIF2 can occur and be regulated. Fusel alcohols impact on both the movement and dynamics of this 2B body. Overall, these results confirm that the guanine nucleotide exchange factor, eIF2B, is targeted by fusel alcohols. Moreover, they highlight a potential connection between the movement or integrity of the 2B body and eIF2B regulation. PMID:20444979

Mixed Inhibition of cPEPCK by Genistein, Using an Extended Binding Site Located Adjacent to Its Catalytic Cleft

PubMed Central

Dhanjal, Jaspreet Kaur; Sundar, Durai

2015-01-01

Cytosolic phosphoenolpyruvate carboxykinase (cPEPCK) is a critical enzyme involved in gluconeogenesis, glyceroneogenesis and cataplerosis. cPEPCK converts oxaloacetic acid (OAA) into phosphoenol pyruvate (PEP) in the presence of GTP. cPEPCK is known to be associated with type 2 diabetes. Genistein is an isoflavone compound that shows anti-diabetic and anti-obesitic properties. Experimental studies have shown a decrease in the blood glucose level in the presence of genistein by lowering the functional activity of cPEPCK, an enzyme of gluconeogenesis. Using computational techniques such as molecular modeling, molecular docking, molecular dynamics simulation and binding free energy calculations, we identified cPEPCK as a direct target of genistein. We studied the molecular interactions of genistein with three possible conformations of cPEPCK—unbound cPEPCK (u_cPEPCK), GTP bound cPEPCK (GTP_cPEPCK) and GDP bound cPEPCK (GDP_cPEPCK). Binding of genistein was also compared with an already known cPEPCK inhibitor. We analyzed the interactions of genistein with cPEPCK enzyme and compared them with its natural substrate (OAA), product (PEP) and known inhibitor (3-MPA). Our results demonstrate that genistein uses the mechanism of mixed inhibition to block the functional activity of cPEPCK and thus can serve as a potential anti-diabetic and anti-obesity drug candidate. We also identified an extended binding site in the catalytic cleft of cPEPCK which is used by 3-MPA to inhibit cPEPCK non-competitively. We demonstrate that extended binding site of cPEPCK can further be exploited for designing new drugs against cPEPCK. PMID:26528723

A Small GTP-Binding Host Protein Is Required for Entry of Powdery Mildew Fungus into Epidermal Cells of Barley1

PubMed Central

Schultheiss, Holger; Dechert, Cornelia; Kogel, Karl-Heinz; Hückelhoven, Ralph

2002-01-01

Small GTP-binding proteins such as those from the RAC family are cytosolic signal transduction proteins that often are involved in processing of extracellular stimuli. Plant RAC proteins are implicated in regulation of plant cell architecture, secondary wall formation, meristem signaling, and defense against pathogens. We isolated a RacB homolog from barley (Hordeum vulgare) to study its role in resistance to the barley powdery mildew fungus (Blumeria graminis f.sp. hordei). RacB was constitutively expressed in the barley epidermis and its expression level was not strongly influenced by inoculation with B. graminis. However, after biolistic bombardment of barley leaf segments with RacB-double-stranded RNA, sequence-specific RNA interference with RacB function inhibited fungal haustorium establishment in a cell-autonomous and genotype-specific manner. Mutants compromised in function of the Mlo wild-type gene and the Ror1 gene (genotype mlo5 ror1) that are moderately susceptible to B. graminis showed no alteration in powdery mildew resistance upon RacB-specific RNA interference. Thus, the phenotype, induced by RacB-specific RNA interference, was apparently dependent on the same processes as mlo5-mediated broad resistance, which is suppressed by ror1. We conclude that an RAC small GTP-binding protein is required for successful fungal haustorium establishment and that this function may be linked to MLO-associated functions. PMID:11950993

Green tea polyphenols causes cell cycle arrest and apoptosis in prostate cancer cells by suppressing class I histone deacetylases

PubMed Central

Thakur, Vijay S.; Gupta, Sanjay

2012-01-01

Green tea polyphenols (GTPs) reactivate epigenetically silenced genes in cancer cells and trigger cell cycle arrest and apoptosis; however, the mechanisms whereby these effects occur are not well understood. We investigated the molecular mechanisms underlying the antiproliferative effects of GTP, which may be similar to those of histone deacetylase (HDAC) inhibitors. Exposure of human prostate cancer LNCaP cells (harboring wild-type p53) and PC-3 cells (lacking p53) with 10–80 μg/ml of GTP for 24 h resulted in dose-dependent inhibition of class I HDAC enzyme activity and its protein expression. GTP treatment causes an accumulation of acetylated histone H3 in total cellular chromatin, resulting in increased accessibility to bind with the promoter sequences of p21/waf1 and Bax, consistent with the effects elicited by an HDAC inhibitor, trichostatin A. GTP treatment also resulted in increased expression of p21/waf1 and Bax at the protein and message levels in these cells. Furthermore, treatment of cells with proteasome inhibitor, MG132 together with GTP prevented degradation of class I HDACs, compared with cells treated with GTP alone, indicating increased proteasomal degradation of class I HDACs by GTP. These alterations were consistent with G0–G1 phase cell cycle arrest and induction of apoptosis in both cell lines. Our findings provide new insight into the mechanisms of GTP action in human prostate cancer cells irrespective of their p53 status and suggest a novel approach to prevention and/or therapy of prostate cancer achieved via HDAC inhibition. PMID:22114073

Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics

PubMed Central

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M.; Abel, Steven M.; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S.; Hansen, Scott D.; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K.; Kuriyan, John; Groves, Jay T.

2014-01-01

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras–guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. PMID:24994643

Characterisation of Translation Elongation Factor eEF1B Subunit Expression in Mammalian Cells and Tissues and Co-Localisation with eEF1A2

PubMed Central

Janikiewicz, Justyna; Doig, Jennifer; Abbott, Catherine M.

2014-01-01

Translation elongation is the stage of protein synthesis in which the translation factor eEF1A plays a pivotal role that is dependent on GTP exchange. In vertebrates, eEF1A can exist as two separately encoded tissue-specific isoforms, eEF1A1, which is almost ubiquitously expressed, and eEF1A2, which is confined to neurons and muscle. The GTP exchange factor for eEF1A1 is a complex called eEF1B made up of subunits eEF1Bα, eEF1Bδ and eEF1Bγ. Previous studies have cast doubt on the ability of eEF1B to interact with eEF1A2, suggesting that this isoform might use a different GTP exchange factor. We show that eEF1B subunits are all widely expressed to varying degrees in different cell lines and tissues, and at different stages of development. We show that ablation of any of the subunits in human cell lines has a small but significant impact on cell viability and cycling. Finally, we show that both eEF1A1 and eEF1A2 colocalise with all eEF1B subunits, in such close proximity that they are highly likely to be in a complex. PMID:25436608

Characterization of the Deoxynucleotide Triphosphate Triphosphohydrolase (dNTPase) Activity of the EF1143 Protein from Enterococcus faecalis and Crystal Structure of the Activator-Substrate Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vorontsov, Ivan I.; Minasov, George; Kiryukhina, Olga

2012-06-19

The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn{sup 2+}. In contrast, with Mg{sup 2+} hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed amore » tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the {alpha}-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the {alpha}3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.« less

A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. Proofreading of the codon-anticodon interaction by ribosomes.

PubMed

Thompson, R C; Dix, D B; Gerson, R B; Karim, A M

1981-01-10

The characteristics of a GTPase reaction between poly(U)-programmed ribosomes, EFTu . GTP, and the near-cognate aminoacyl (aa)-tRNA, Leu-tRNA Leu 2, have been studied to assess the role of this reaction in proofreading of the codon-anticodon interaction. The reaction resembles the GTPase reaction with cognate aa-tRNAs and EFTu . GTP in its substrate requirements, in its involving EFTu . GTP . aa-tRNA ternary complexes, and in its requiring a free ribosomal A-site. The noncognate reaction differs from the cognate one in that aa-tRNA becomes stably bound to the ribosomes only 5% of the time; it therefore seems best characterized as an abortive enzymatic binding reaction. The rate of reaction is a significant fraction (4%) of that of the cognate aa-tRNA, indicating that recognition of ternary complexes by ribosomes involves a level of error greater than that of translation as a whole. The rejection of the noncognate aa-tRNA following GTP hydrolysis is therefore a vital step in the translation process and fulfills the criteria set for a proofreading reaction. Leu-tRNA Leu 2 which escapes rejection through proofreading, forms a stable complex with the ribosomal A-site, so it appears that the Leu-tRNA2 which was rejected never reached the A-site and that proofreading precedes full A-site binding.

Heat-Responsive Photosynthetic and Signaling Pathways in Plants: Insight from Proteomics.

PubMed

Wang, Xiaoli; Xu, Chenxi; Cai, Xiaofeng; Wang, Quanhua; Dai, Shaojun

2017-10-20

Heat stress is a major abiotic stress posing a serious threat to plants. Heat-responsive mechanisms in plants are complicated and fine-tuned. Heat signaling transduction and photosynthesis are highly sensitive. Therefore, a thorough understanding of the molecular mechanism in heat stressed-signaling transduction and photosynthesis is necessary to protect crop yield. Current high-throughput proteomics investigations provide more useful information for underlying heat-responsive signaling pathways and photosynthesis modulation in plants. Several signaling components, such as guanosine triphosphate (GTP)-binding protein, nucleoside diphosphate kinase, annexin, and brassinosteroid-insensitive I-kinase domain interacting protein 114, were proposed to be important in heat signaling transduction. Moreover, diverse protein patterns of photosynthetic proteins imply that the modulations of stomatal CO₂ exchange, photosystem II, Calvin cycle, ATP synthesis, and chlorophyll biosynthesis are crucial for plant heat tolerance.

Mechanism of pyranopterin ring formation in molybdenum cofactor biosynthesis

DOE PAGES

Hover, Bradley M.; Tonthat, Nam K.; Schumacher, Maria A.; ...

2015-05-04

The molybdenum cofactor (Moco) is essential for all kingdoms of life, plays central roles in various biological processes, and must be biosynthesized de novo. During Moco biosynthesis, the characteristic pyranopterin ring is constructed by a complex rearrangement of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin (cPMP) through the action of two enzymes, MoaA and MoaC (molybdenum cofactor biosynthesis protein A and C, respectively). Conventionally, MoaA was considered to catalyze the majority of this transformation, with MoaC playing little or no role in the pyranopterin formation. Recently, this view was challenged by the isolation of 3',8-cyclo-7,8-dihydro-guanosine 5'-triphosphate (3',8-cH 2GTP) as the productmore » of in vitro MoaA reactions. To elucidate the mechanism of formation of Moco pyranopterin backbone, in this paper we performed biochemical characterization of 3',8-cH 2GTP and functional and X-ray crystallographic characterizations of MoaC. These studies revealed that 3',8-cH 2GTP is the only product of MoaA that can be converted to cPMP by MoaC. Our structural studies captured the specific binding of 3',8-cH 2GTP in the active site of MoaC. These observations provided strong evidence that the physiological function of MoaA is the conversion of GTP to 3',8-cH 2GTP (GTP 3',8-cyclase), and that of MoaC is to catalyze the rearrangement of 3',8-cH 2GTP into cPMP (cPMP synthase). Furthermore, our structure-guided studies suggest that MoaC catalysis involves the dynamic motions of enzyme active-site loops as a way to control the timing of interaction between the reaction intermediates and catalytically essential amino acid residues. In conclusion, these results reveal the previously unidentified mechanism behind Moco biosynthesis and provide mechanistic and structural insights into how enzymes catalyze complex rearrangement reactions.« less

A novel mechanism for the Ca(2+)-sensitizing effect of protein kinase C on vascular smooth muscle: inhibition of myosin light chain phosphatase

PubMed Central

1994-01-01

Mechanisms of Ca2+ sensitization of both myosin light chain (MLC) phosphorylation and force development by protein kinase C (PKC) were studied in permeabilized tonic smooth muscle obtained from the rabbit femoral artery. For comparison, the Ca2+ sensitizing effect of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) was examined, which had been previously shown to inhibit MLC phosphatase in phasic vascular smooth muscle. We now report that PKC activators (phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor) and GTP gamma S significantly increase both MLC phosphorylation and force development at constant [Ca2+]. Major phosphorylation site occurring in the presence of phorbol-12,13- dibutyrate (PDBu) or GTP gamma S at constant [Ca2+] is the same serine residue (Ser-19) as that phosphorylated by MLC kinase in response to increased Ca2+ concentrations. In an ATP- and Ca(2+)-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4- diazepine (ML-9), to avoid the kinase activity, both PDBu and GTP gamma S significantly decreased the rate of MLC dephosphorylation to half its control value. However, PDBu inhibited the relaxation rate more than did GTP gamma S. In the presence of microcystin-LR to inhibit the phosphatase activity, neither PDBu nor GTP gamma S affected MLC phosphorylation and force development. These results indicate that PKC, like activation of GTP binding protein, increases Ca2+ sensitivity of both MLC phosphorylation and force production through inhibition of MLC phosphatase. PMID:7807049

Differential regulation of serotonin-1A receptor-stimulated [35S]GTP gamma S binding in the dorsal raphe nucleus by citalopram and escitalopram.

PubMed

Rossi, Dania V; Burke, Teresa F; Hensler, Julie G

2008-03-31

The effect of chronic citalopram or escitalopram administration on 5-HT1A receptor function in the dorsal raphe nucleus was determined by measuring [35S]GTP gamma S binding stimulated by the 5-HT1A receptor agonist (R)-(+)-8-OH-DPAT (1nM-10 microM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT1A receptors to activate G proteins, whereas citalopram treatment did not. The binding of [3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram.

K-Ras protein as a drug target.

PubMed

McCormick, Frank

2016-03-01

K-Ras proteins are major drivers of human cancers, playing a direct causal role in about one million cancer cases/year. In cancers driven by mutant K-Ras, the protein is locked in the active, GTP-bound state constitutively, through a defect in the off-switch mechanism. As such, the mutant protein resembles the normal K-Ras protein from a structural perspective, making therapeutic attack extremely challenging. K-Ras is a member of a large family of related proteins, which share very similar GDP/GTP-binding domains, making specific therapies more difficult. Furthermore, Ras proteins lack pockets to which small molecules can bind with high affinity, with a few interesting exceptions. However, new insights into the structure and function of K-Ras proteins reveal opportunities for intervention that were not appreciated many years ago, when efforts were launched to develop K-Ras therapies. Furthermore, K-Ras undergoes post-translational modification and interactions with cellular signaling proteins that present additional therapeutic opportunities, such as specific binding to calmodulin and regulation of non-canonical Wnt signaling.

Yeast Ran-Binding Protein 1 (Yrb1) Shuttles between the Nucleus and Cytoplasm and Is Exported from the Nucleus via a CRM1 (XPO1)-Dependent Pathway

PubMed Central

Künzler, Markus; Gerstberger, Thomas; Stutz, Françoise; Bischoff, F. Ralf; Hurt, Ed

2000-01-01

The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo. PMID:10825193

Rho proteins of plants--functional cycle and regulation of cytoskeletal dynamics.

PubMed

Mucha, Elena; Fricke, Inka; Schaefer, Antje; Wittinghofer, Alfred; Berken, Antje

2011-11-01

Rho-related ROP proteins are molecular switches that essentially regulate a wide variety of processes. Of central interest is their influence on the plant cytoskeleton by which they affect vital processes like cell division, growth, morphogenesis, and pathogen defense. ROPs switch between GTP- and GDP-bound conformations by strictly regulated nucleotide exchange and GTP-hydrolysis, and only the active GTP-form interacts with downstream effectors to ultimately provoke a biological response. However, the mode of action of the engaged regulators and effectors as well as their upstream and downstream interaction partners have long been largely unknown. As opposed to analogous systems in animals and fungi, plants use specific GTPase activating proteins (RopGAPs) with a unique domain composition and novel guanine nucleotide exchange factors (RopGEFs) with a probable link to cell surface receptors. Moreover, plants comprise novel effector molecules and adapters connecting ROPs to mostly unknown downstream targets on the route to the cytoskeleton. This review aims to summarize recent knowledge on the molecular mechanisms and reaction cascades involved in ROP dependent cytoskeletal rearrangements, addressing the structure and function of the unusual RopGAPs, RopGEFs and effectors, and the upstream and downstream pathways linking ROPs to cell receptor-like kinases, actin filaments, and microtubules. Copyright © 2010 Elsevier GmbH. All rights reserved.

Molecular kinetics. Ras activation by SOS: allosteric regulation by altered fluctuation dynamics.

PubMed

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen; Christensen, Sune M; Abel, Steven M; Iwig, Jeff; Wu, Hung-Jen; Gureasko, Jodi; Rhodes, Christopher; Petit, Rebecca S; Hansen, Scott D; Thill, Peter; Yu, Cheng-Han; Stamou, Dimitrios; Chakraborty, Arup K; Kuriyan, John; Groves, Jay T

2014-07-04

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. Copyright © 2014, American Association for the Advancement of Science.

Nucleotide Dependent Switching in Rho GTPase: Conformational Heterogeneity and Competing Molecular Interactions

PubMed Central

Kumawat, Amit; Chakrabarty, Suman; Kulkarni, Kiran

2017-01-01

Ras superfamily of GTPases regulate myriad cellular processes through a conserved nucleotide (GTP/GDP) dependent switching mechanism. Unlike Ras family of GTPases, for the Rho GTPases, there is no clear evidence for the existence of “sub-states” such as state 1 & state 2 in the GTP bound form. To explore the nucleotide dependent conformational space of the Switch I loop and also to look for existence of state 1 like conformations in Rho GTPases, atomistic molecular dynamics and metadynamics simulations on RhoA were performed. These studies demonstrate that both the nucleotide-free state and the GDP bound “OFF” state have very similar conformations, whereas the GTP bound “ON” state has unique conformations with signatures of two intermediate states. The conformational free energy landscape for these systems suggests the presence of multiple intermediate states. Interestingly, the energetic penalty of exposing the non-polar residues in the GTP bound form is counter balanced by the favourable hydrogen bonded interactions between the γ-phosphate group of GTP with the highly conserved Tyr34 and Thr37 residues. These competing molecular interactions lead to a tuneable energy landscape of the Switch I conformation, which can undergo significant changes based on the local environment including changes upon binding to effectors. PMID:28374773

Nucleotide Dependent Switching in Rho GTPase: Conformational Heterogeneity and Competing Molecular Interactions

NASA Astrophysics Data System (ADS)

Kumawat, Amit; Chakrabarty, Suman; Kulkarni, Kiran

2017-04-01

Ras superfamily of GTPases regulate myriad cellular processes through a conserved nucleotide (GTP/GDP) dependent switching mechanism. Unlike Ras family of GTPases, for the Rho GTPases, there is no clear evidence for the existence of “sub-states” such as state 1 & state 2 in the GTP bound form. To explore the nucleotide dependent conformational space of the Switch I loop and also to look for existence of state 1 like conformations in Rho GTPases, atomistic molecular dynamics and metadynamics simulations on RhoA were performed. These studies demonstrate that both the nucleotide-free state and the GDP bound “OFF” state have very similar conformations, whereas the GTP bound “ON” state has unique conformations with signatures of two intermediate states. The conformational free energy landscape for these systems suggests the presence of multiple intermediate states. Interestingly, the energetic penalty of exposing the non-polar residues in the GTP bound form is counter balanced by the favourable hydrogen bonded interactions between the γ-phosphate group of GTP with the highly conserved Tyr34 and Thr37 residues. These competing molecular interactions lead to a tuneable energy landscape of the Switch I conformation, which can undergo significant changes based on the local environment including changes upon binding to effectors.

Determination of Rab5 activity in the cell by effector pull-down assay.

PubMed

Qi, Yaoyao; Liang, Zhimin; Wang, Zonghua; Lu, Guodong; Li, Guangpu

2015-01-01

Rab5 targets to early endosomes and is a master regulator of early endosome fusion and endocytosis in all eukaryotic cells. Like other GTPases, Rab5 functions as a molecular switch by alternating between GTP-bound and GDP-bound forms, with the former being biologically active via interactions with multiple effector proteins. Thus the Rab5-GTP level in the cell reflects Rab5 activity in promoting endosome fusion and endocytosis and is indicative of cellular endocytic activity. In this chapter, we describe a Rab5 activity assay by using GST fusion proteins with the Rab5 effectors such as Rabaptin-5, Rabenosyn-5, and EEA1 that specifically bind to GTP-bound Rab5. We compare the efficiencies of the three GST fusion proteins in the pull-down of mammalian and fungal Rab5 proteins.

Ran-dependent nuclear export mediators: a structural perspective

PubMed Central

Güttler, Thomas; Görlich, Dirk

2011-01-01

Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP–exportin–cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions. PMID:21878989

The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

PubMed Central

Romero, Lisa C; Nguyen, Thanh V; Deville, Benoit; Ogunjumo, Oluwasanmi; James, Anthony A

2004-01-01

Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development. PMID:15222903

The step-wise pathway of septin hetero-octamer assembly in budding yeast.

PubMed

Weems, Andrew; McMurray, Michael

2017-05-25

Septin proteins bind guanine nucleotides and form rod-shaped hetero-oligomers. Cells choose from a variety of available septins to assemble distinct hetero-oligomers, but the underlying mechanism was unknown. Using a new in vivo assay, we find that a stepwise assembly pathway produces the two species of budding yeast septin hetero-octamers: Cdc11/Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11/Shs1. Rapid GTP hydrolysis by monomeric Cdc10 drives assembly of the core Cdc10 homodimer. The extended Cdc3 N terminus autoinhibits Cdc3 association with Cdc10 homodimers until prior Cdc3-Cdc12 interaction. Slow hydrolysis by monomeric Cdc12 and specific affinity of Cdc11 for transient Cdc12•GTP drive assembly of distinct trimers, Cdc11-Cdc12-Cdc3 or Shs1-Cdc12-Cdc3. Decreasing the cytosolic GTP:GDP ratio increases the incorporation of Shs1 vs Cdc11, which alters the curvature of filamentous septin rings. Our findings explain how GTP hydrolysis controls septin assembly, and uncover mechanisms by which cells construct defined septin complexes.

Structural insights into translational fidelity.

PubMed

Ogle, James M; Ramakrishnan, V

2005-01-01

The underlying basis for the accuracy of protein synthesis has been the subject of over four decades of investigation. Recent biochemical and structural data make it possible to understand at least in outline the structural basis for tRNA selection, in which codon recognition by cognate tRNA results in the hydrolysis of GTP by EF-Tu over 75 A away. The ribosome recognizes the geometry of codon-anticodon base pairing at the first two positions but monitors the third, or wobble position, less stringently. Part of the additional binding energy of cognate tRNA is used to induce conformational changes in the ribosome that stabilize a transition state for GTP hydrolysis by EF-Tu and subsequently result in accelerated accommodation of tRNA into the peptidyl transferase center. The transition state for GTP hydrolysis is characterized, among other things, by a distorted tRNA. This picture explains a large body of data on the effect of antibiotics and mutations on translational fidelity. However, many fundamental questions remain, such as the mechanism of activation of GTP hydrolysis by EF-Tu, and the relationship between decoding and frameshifting.

Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.

PubMed Central

Gong, M C; Iizuka, K; Nixon, G; Browne, J P; Hall, A; Eccleston, J F; Sugai, M; Kobayashi, S; Somlyo, A V; Somlyo, A P

1996-01-01

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase. Images Fig. 3 Fig. 4 PMID:8577766

A potential link between insulin signaling and GLUT4 translocation: Association of Rab10-GTP with the exocyst subunit Exoc6/6b

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sano, Hiroyuki; Peck, Grantley R.; Blachon, Stephanie

Insulin increases glucose transport in fat and muscle cells by stimulating the exocytosis of specialized vesicles containing the glucose transporter GLUT4. This process, which is referred to as GLUT4 translocation, increases the amount of GLUT4 at the cell surface. Previous studies have provided evidence that insulin signaling increases the amount of Rab10-GTP in the GLUT4 vesicles and that GLUT4 translocation requires the exocyst, a complex that functions in the tethering of vesicles to the plasma membrane, leading to exocytosis. In the present study we show that Rab10 in its GTP form binds to Exoc6 and Exoc6b, which are the twomore » highly homologous isotypes of an exocyst subunit, that both isotypes are found in 3T3-L1 adipocytes, and that knockdown of Exoc6, Exoc6b, or both inhibits GLUT4 translocation in 3T3-L1 adipocytes. These results suggest that the association of Rab10-GTP with Exoc6/6b is a molecular link between insulin signaling and the exocytic machinery in GLUT4 translocation. - Highlights: • Insulin stimulates the fusion of vesicles containing GLUT4 with the plasma membrane. • This requires vesicular Rab10-GTP and the exocyst plasma membrane tethering complex. • We find that Rab10-GTP associates with the Exoc6 subunit of the exocyst. • We find that knockdown of Exoc6 inhibits fusion of GLUT4 vesicles with the membrane. • The interaction of Rab10-GTP with Exoc6 potentially links signaling to exocytosis.« less

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

PubMed

Shimada-Shimizu, Naoko; Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2013-03-01

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1. © 2013 The Authors Journal compilation © 2013 FEBS.

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

PubMed

Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

1993-05-06

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I.

PubMed

Rebelo, Jorge; Auerbach, Günter; Bader, Gerd; Bracher, Andreas; Nar, Herbert; Hösl, Cornelia; Schramek, Nicholas; Kaiser, Johannes; Bacher, Adelbert; Huber, Robert; Fischer, Markus

2003-02-14

GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.

OXALOACETATE DECARBOXYLATION AND OXALOACETATE-CARBON DIOXIDE EXCHANGE IN ACETOBACTER XYLINUM

PubMed Central

Benziman, Moshe; Heller, N.

1964-01-01

Benziman, Moshe (The Hebrew University of Jerusalem, Jerusalem, Israel), and N. Heller. Oxaloacetate decarboxylation and oxaloacetate-carbon dioxide exchange in Acetobacter xylinum. J. Bacteriol. 88:1678–1687. 1964.—Extracts of Acetobacter xylinum, prepared by sonic treatment, were shown to catalyze the decarboxylation of oxaloacetate (OAA) to pyruvate and CO2, and the exchange of C14-carbon dioxide into the β-carboxyl of OAA. Fractionation of the extracts with ammonium sulfate resulted in a 10-fold increase of the specific activity of the enzyme system catalyzing the CO2 exchange and OAA decarboxylation reactions. The purified preparation catalyzed the exchange of pyruvate-3-C14 into OAA. Similar pH curves with a pH optimum of 5.6 were obtained for the CO2 exchange and OAA decarboxylation reactions. Both reactions require the presence of Mn2+ or Mg2+ ions. OAA decarboxylation was more strongly inhibited than the exchange of CO2 by dialysis or metal-chelating agents. Avidin did not inhibit either reaction. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), pyrophosphate, or inorganic phosphate did not promote OAA decarboxylation and the CO2-exchange reaction catalyzed by the purified preparation. The purified preparation failed to catalyze the carboxylation of phosphoenolpyruvate in the presence of GDP, ADP, or inorganic phosphate, and that of pyruvate in the presence of ATP or GTP, even when supplemented with an OAA-trapping system. A scheme for OAA decarboxylation which could account for the observed exchange reactions and for the failure to obtain net fixation of CO2 is proposed. The relation between the exchange reaction and the synthesis of cellulose from pyruvate by A. xylinum is discussed. PMID:14240957

Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.

PubMed Central

van der Geer, P; Henkemeyer, M; Jacks, T; Pawson, T

1997-01-01

The Ras guanine nucleotide-binding protein functions as a molecular switch in signalling downstream of protein-tyrosine kinases. Ras is activated by exchange of GDP for GTP and is turned off by hydrolysis of bound GTP to GDP. Ras itself has a low intrinsic GTPase activity that can be stimulated by GTPase-activating proteins (GAPs), including p120-Gap and neurofibromin. These GAPs possess a common catalytic domain but contain distinct regulatory elements that may couple different external signals to control of the Ras pathway. p120-Gap, for example, has two N-terminal SH2 domains that directly recognize phosphotyrosine motifs on activated growth factor receptors and cytoplasmic phosphoproteins. To analyze the role of p120-Gap in Ras regulation in vivo, we have used fibroblasts derived from mouse embryos with a null mutation in the gene for p120-Gap (Gap). Platelet-derived growth factor stimulation of Gap-/- cells led to an abnormally large increase in the level of Ras-GTP and in the duration of mitogen-activated protein (MAP) kinase activation compared with wild-type cells, suggesting that p120-Gap is specifically activated following growth factor stimulation. Induction of DNA synthesis in response to platelet-derived growth factor and morphological transformation by the v-src and EJ-ras oncogenes were not significantly affected by the absence of p120-Gap. However, we found that normal tyrosine phosphorylation of p190-rhoGap, a cytoplasmic protein that associates with the p120-Gap SH2 domains, was dependent on the presence of p120-Gap. Our results suggest that p120-Gap has specific functions in downregulating the Ras/MAP kinase pathway following growth factor stimulation, and in modulating the phosphorylation of p190-rhoGap, but is not required for mitogenic signalling. PMID:9121432

Functional properties of LRRK2 mutations in Taiwanese Parkinson disease.

PubMed

Chang, Kuo-Hsuan; Chen, Chiung-Mei; Lin, Chih-Hsin; Chang, Wen-Teng; Jiang, Pei-Ru; Hsiao, Ya-Chin; Wu, Yih-Ru; Lee-Chen, Guey-Jen

2017-03-01

Leucine-rich repeat kinase 2 (LRRK2) is a large protein encoding multiple functional domains. Mutations within different LRRK2 domains have been considered to be involved in the development of Parkinson disease by different mechanisms. Our previous study found three LRRK2 mutations-p.R767H, p.S885N, and p.R1441H-in Taiwanese patients with Parkinson disease. We evaluated the functional properties of LRRK2 p.R767H, p.S885N, and p.R1441H mutations by overexpressing them in human embryonic kidney 293 and neuroblastoma SK-N-SH cells. The common p.G2019S mutation in the kinase domain was included for comparison. In 293 cells, overexpressed p.R1441H-but not p.R767H, p.S885N, or p.G2019-increased GTP binding affinity to prolong the active state. Overexpressed p.R1441H and p.G2019S generated inclusions in 293 cells. In SK-N-SH cells, the α-synuclein was coexpressed with wild type as well as mutated p.R767H, p.S885N, p.R1441H, and p.G2019 LRRK2 proteins. Part of the perinuclear inclusions formed by p.R1441H and p.G2019S were colocalized with α-synuclein. Additionally, p.S885N and p.R1441H mutations caused reduced interaction between LRRK2 and ARHGEF7, a putative guanine nucleotide exchange factor for LRRK2, whereas this interaction was well preserved in p.R767H and p.G2019S mutations. Our study suggests that p.R1441H protein facilitates the formation of intracellular inclusions, compromises GTP hydrolysis by increasing its affinity for GTP, and reduces its interaction with ARHGEF7. Copyright © 2016. Published by Elsevier B.V.

The RanBP2/RanGAP1*SUMO1/Ubc9 SUMO E3 ligase is a disassembly machine for Crm1-dependent nuclear export complexes

PubMed Central

Ritterhoff, Tobias; Das, Hrishikesh; Hofhaus, Götz; Schröder, Rasmus R.; Flotho, Annette; Melchior, Frauke

2016-01-01

Continuous cycles of nucleocytoplasmic transport require disassembly of transport receptor/Ran-GTP complexes in the cytoplasm. A basic disassembly mechanism in all eukaryotes depends on soluble RanGAP and RanBP1. In vertebrates, a significant fraction of RanGAP1 stably interacts with the nucleoporin RanBP2 at a binding site that is flanked by FG-repeats and Ran-binding domains, and overlaps with RanBP2's SUMO E3 ligase region. Here, we show that the RanBP2/RanGAP1*SUMO1/Ubc9 complex functions as an autonomous disassembly machine with a preference for the export receptor Crm1. We describe three in vitro reconstituted disassembly intermediates, which show binding of a Crm1 export complex via two FG-repeat patches, cargo-release by RanBP2's Ran-binding domains and retention of free Crm1 at RanBP2 after Ran-GTP hydrolysis. Intriguingly, all intermediates are compatible with SUMO E3 ligase activity, suggesting that the RanBP2/RanGAP1*SUMO1/Ubc9 complex may link Crm1- and SUMO-dependent functions. PMID:27160050

Multiple Controls Regulate Nucleostemin Partitioning Between Nucleolus and Nucleoplasm

PubMed Central

Meng, Lingjun; Yasumoto, Hiroaki; Tsai, Robert Y.L.

2010-01-01

Summary Nucleostemin plays an essential role in maintaining the continuous proliferation of stem cells and cancer cells. The movement of nucleostemin between the nucleolus and the nucleoplasm provides a dynamic way to partition the nucleostemin protein between these two compartments. Here, we showed that nucleostemin contained two nucleolus-targeting regions, the basic and the GTP-binding domains, which exhibited a short and a long nucleolar retention time, respectively. In a GTP-unbound state, the nucleolus-targeting activity of nucleostemin was blocked by a mechanism that trapped its intermediate domain in the nucleoplasm. A nucleostemin-interacting protein, RSL1D1, was identified that contained a ribosomal L1-domain, co-resided with nucleostemin in the same subnucleolar compartment non-identical to the B23 and fibrillarin distributions, and displayed a longer nucleolar residence time than nucleostemin. RSL1D1 interacted with both the basic and the GTP-binding domains of nucleostemin through a non-nucleolus-targeting region. Overexpression of the nucleolus-targeting domain of RSL1D1 alone dispersed the nucleolar nucleostemin. Loss of RSL1D1 expression reduced the compartmental size and amount of nucleostemin in the nucleolus. This work reveals that the partitioning of nucleostemin employs complex mechanisms involving both nucleolar and nucleoplasmic components, and provides insight into the post-translational regulation of its activity. PMID:17158916

The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4.

PubMed

Isabet, Tatiana; Montagnac, Guillaume; Regazzoni, Karine; Raynal, Bertrand; El Khadali, Fatima; England, Patrick; Franco, Michel; Chavrier, Philippe; Houdusse, Anne; Ménétrey, Julie

2009-09-16

The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 A resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6-JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6-mediated motor switch regulatory function.

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

PubMed

Srikant, C B; Dahan, A; Craig, C

1990-02-04

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2+, Mn2+, Ca2+ and Co2+ augmented the binding of both T* SS-14 and LTT* SS-28, while higher than 4 mM Co2+ inhibited binding of both ligands. LTT* SS-28 binding was reduced in the presence of high concentrations of Ba2+ and Mn2+ also. Interestingly Ca2+ at higher than 10 mM preferentially inhibited LTT* SS-28 binding and increased the affinity of SS-14 but not SS-28 for LTT* SS-28 binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Effector region of the translation elongation factor EF-Tu.GTP complex stabilizes an orthoester acid intermediate structure of aminoacyl-tRNA in a ternary complex.

PubMed Central

Förster, C; Limmer, S; Zeidler, W; Sprinzl, M

1994-01-01

tRNA(Val) from Escherichia coli was aminoacylated with [1-13C]valine and its complex with Thermus thermophilus elongation factor EF-Tu.GTP was analyzed by 13C NMR spectroscopy. The results suggest that the aminoacyl residue of the valyl-tRNA in ternary complex with bacterial EF-Tu and GTP is not attached to tRNA by a regular ester bond to either a 2'- or 3'-hydroxyl group; instead, an intermediate orthoester acid structure with covalent linkage to both vicinal hydroxyls of the terminal adenosine-76 is formed. Mutation of arginine-59 located in the effector region of EF-Tu, a conserved residue in protein elongation factors and the alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins), abolishes the stabilization of the orthoester acid structure of aminoacyl-tRNA. PMID:8183898

RCC1 regulates inner centromeric composition in a Ran-independent fashion.

PubMed

Zhang, Michael Shaofei; Furuta, Maiko; Arnaoutov, Alexei; Dasso, Mary

2018-01-01

RCC1 associates to chromatin dynamically within mitosis and catalyzes Ran-GTP production. Exogenous RCC1 disrupts kinetochore structure in Xenopus egg extracts (XEEs), but the molecular basis of this disruption remains unknown. We have investigated this question, utilizing replicated chromosomes that possess paired sister kinetochores. We find that exogenous RCC1 evicts a specific subset of inner KT proteins including Shugoshin-1 (Sgo1) and the chromosome passenger complex (CPC). We generated RCC1 mutants that separate its enzymatic activity and chromatin binding. Strikingly, Sgo1 and CPC eviction depended only on RCC1's chromatin affinity but not its capacity to produce Ran-GTP. RCC1 similarly released Sgo1 and CPC from synthetic kinetochores assembled on CENP-A nucleosome arrays. Together, our findings indicate RCC1 regulates kinetochores at the metaphase-anaphase transition through Ran-GTP-independent displacement of Sgo1 and CPC.

Assay for Arf GTP-binding Proteins | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute's Laboratory of Cellular and Molecular Biology is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize an antibody-based proteomics assay.

The Gem GTP-binding protein promotes morphological differentiation in neuroblastoma.

PubMed

Leone, A; Mitsiades, N; Ward, Y; Spinelli, B; Poulaki, V; Tsokos, M; Kelly, K

2001-05-31

Gem is a small GTP-binding protein within the Ras superfamily whose function has not been determined. We report here that ectopic Gem expression is sufficient to stimulate cell flattening and neurite extension in N1E-115 and SH-SY5Y neuroblastoma cells, suggesting a role for Gem in cytoskeletal rearrangement and/or morphological differentiation of neurons. Consistent with this potential function, in clinical samples of neuroblastoma, Gem protein was most highly expressed within cells which had differentiated to express ganglionic morphology. Gem was also observed in developing trigeminal nerve ganglia in 12.5 day mouse embryos, demonstrating that Gem expression is a property of normal ganglionic development. Although Gem expression is rare in epithelial and hematopoietic cancer cell lines, constitutive Gem levels were detected in several neuroblastoma cell lines and could be further induced as much as 10-fold following treatment with PMA or the acetylcholine muscarinic agonist, carbachol.

ARF1 and SAR1 GTPases in Endomembrane Trafficking in Plants

PubMed Central

Cevher-Keskin, Birsen

2013-01-01

Small GTPases largely control membrane traffic, which is essential for the survival of all eukaryotes. Among the small GTP-binding proteins, ARF1 (ADP-ribosylation factor 1) and SAR1 (Secretion-Associated RAS super family 1) are commonly conserved among all eukaryotes with respect to both their functional and sequential characteristics. The ARF1 and SAR1 GTP-binding proteins are involved in the formation and budding of vesicles throughout plant endomembrane systems. ARF1 has been shown to play a critical role in COPI (Coat Protein Complex I)-mediated retrograde trafficking in eukaryotic systems, whereas SAR1 GTPases are involved in intracellular COPII-mediated protein trafficking from the ER to the Golgi apparatus. This review offers a summary of vesicular trafficking with an emphasis on the ARF1 and SAR1 expression patterns at early growth stages and in the de-etiolation process. PMID:24013371

Ethylene Rapidly Up-Regulates the Activities of Both Monomeric GTP-Binding Proteins and Protein Kinase(s) in Epicotyls of Pea1

PubMed Central

Moshkov, Igor E.; Novikova, Galina V.; Mur, Luis A.J.; Smith, Aileen R.; Hall, Michael A.

2003-01-01

It is demonstrated that, in etiolated pea (Pisum sativum) epicotyls, ethylene affects the activation of both monomeric GTP-binding proteins (monomeric G-proteins) and protein kinases. For monomeric G-proteins, the effect may be a rapid (2 min) and bimodal up-regulation, a transiently unimodal activation, or a transient down-regulation. Pretreatment with 1-methylcyclopropene abolishes the response to ethylene overall. Immunoprecipitation studies indicate that some of the monomeric G-proteins affected may be of the Rab class. Protein kinase activity is rapidly up-regulated by ethylene, the effect is inhibited by 1-methylcyclopropene, and the activation is bimodal. Immunoprecipitation indicates that the kinase(s) are of the MAP kinase ERK1 group. It is proposed that the data support the hypothesis that a transduction chain exists that is separate and antagonistic to that currently revealed by studies on Arabidopsis mutants. PMID:12692330

Role of Structural Dynamics at the Receptor G Protein Interface for Signal Transduction.

PubMed

Rose, Alexander S; Zachariae, Ulrich; Grubmüller, Helmut; Hofmann, Klaus Peter; Scheerer, Patrick; Hildebrand, Peter W

2015-01-01

GPCRs catalyze GDP/GTP exchange in the α-subunit of heterotrimeric G proteins (Gαßγ) through displacement of the Gα C-terminal α5 helix, which directly connects the interface of the active receptor (R*) to the nucleotide binding pocket of G. Hydrogen-deuterium exchange mass spectrometry and kinetic analysis of R* catalysed G protein activation have suggested that displacement of α5 starts from an intermediate GDP bound complex (R*•GGDP). To elucidate the structural basis of receptor-catalysed displacement of α5, we modelled the structure of R*•GGDP. A flexible docking protocol yielded an intermediate R*•GGDP complex, with a similar overall arrangement as in the X-ray structure of the nucleotide free complex (R*•Gempty), however with the α5 C-terminus (GαCT) forming different polar contacts with R*. Starting molecular dynamics simulations of GαCT bound to R* in the intermediate position, we observe a screw-like motion, which restores the specific interactions of α5 with R* in R*•Gempty. The observed rotation of α5 by 60° is in line with experimental data. Reformation of hydrogen bonds, water expulsion and formation of hydrophobic interactions are driving forces of the α5 displacement. We conclude that the identified interactions between R* and G protein define a structural framework in which the α5 displacement promotes direct transmission of the signal from R* to the GDP binding pocket.

A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

PubMed

Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

2015-12-01

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs. © 2015 Society for Laboratory Automation and Screening.

StarD13 is a tumor suppressor in breast cancer that regulates cell motility and invasion

PubMed Central

HANNA, SAMER; KHALIL, BASSEM; NASRALLAH, ANITA; SAYKALI, BECHARA A.; SOBH, RANIA; NASSER, SELIM; EL-SIBAI, MIRVAT

2014-01-01

Breast cancer is one of the most commonly diagnosed cancers in women around the world. In general, the more aggressive the tumor, the more rapidly it grows and the more likely it metastasizes. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in breast cancer cell motility and metastasis. The switch between active GTP-bound and inactive GDP-bound state is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine-nucleotide dissociation inhibitors (GDIs). We studied the role of StarD13, a recently identified Rho-GAP that specifically inhibits the function of RhoA and Cdc42. We aimed to investigate its role in breast cancer proliferation and metastasis. The levels of expression of this Rho-GAP in tumor tissues of different grades were assayed using immunohistochemistry. We observed that, while the level of StarD13 expression decreases in cancer tissues compared to normal tissues, it increases as the grade of the tumor increased. This was consistent with the fact that although StarD13 was indeed a tumor suppressor in our breast cancer cells, as seen by its effect on cell proliferation, it was needed for cancer cell motility. In fact, StarD13 knockdown resulted in an inhibition of cell motility and cells were not able to detach their tail and move forward. Our study describes, for the first time, a tumor suppressor that plays a positive role in cancer motility. PMID:24627003

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

PubMed Central

Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

Structure of Shigella IpgB2 in Complex with Human RhoA

PubMed Central

Klink, Björn U.; Barden, Stephan; Heidler, Thomas V.; Borchers, Christina; Ladwein, Markus; Stradal, Theresia E. B.; Rottner, Klemens; Heinz, Dirk W.

2010-01-01

A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP·Mg2+ complex is preceded by the displacement of the metal ion to the α-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family. PMID:20363740

Inhibitors of Ras-SOS Interactions.

PubMed

Lu, Shaoyong; Jang, Hyunbum; Zhang, Jian; Nussinov, Ruth

2016-04-19

Activating Ras mutations are found in about 30 % of human cancers. Ras activation is regulated by guanine nucleotide exchange factors, such as the son of sevenless (SOS), which form protein-protein interactions (PPIs) with Ras and catalyze the exchange of GDP by GTP. This is the rate-limiting step in Ras activation. However, Ras surfaces lack any evident suitable pockets where a molecule might bind tightly, rendering Ras proteins still 'undruggable' for over 30 years. Among the alternative approaches is the design of inhibitors that target the Ras-SOS PPI interface, a strategy that is gaining increasing recognition for treating Ras mutant cancers. Herein we focus on data that has accumulated over the past few years pertaining to the design of small-molecule modulators or peptide mimetics aimed at the interface of the Ras-SOS PPI. We emphasize, however, that even if such Ras-SOS therapeutics are potent, drug resistance may emerge. To counteract this development, we propose "pathway drug cocktails", that is, drug combinations aimed at parallel (or compensatory) pathways. A repertoire of classified cancer, cell/tissue, and pathway/protein combinations would be beneficial toward this goal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

PubMed

Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

2012-10-09

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

GTPase activity, structure, and mechanical properties of filaments assembled from bacterial cytoskeleton protein MreB.

PubMed

Esue, Osigwe; Wirtz, Denis; Tseng, Yiider

2006-02-01

MreB, a major component of the recently discovered bacterial cytoskeleton, displays a structure homologous to its eukaryotic counterpart actin. Here, we study the assembly and mechanical properties of Thermotoga maritima MreB in the presence of different nucleotides in vitro. We found that GTP, not ADP or GDP, can mediate MreB assembly into filamentous structures as effectively as ATP. Upon MreB assembly, both GTP and ATP release the gamma phosphate at similar rates. Therefore, MreB is an equally effective ATPase and GTPase. Electron microscopy and quantitative rheology suggest that the morphologies and micromechanical properties of filamentous ATP-MreB and GTP-MreB are similar. In contrast, mammalian actin assembly is favored in the presence of ATP over GTP. These results indicate that, despite high structural homology of their monomers, T. maritima MreB and actin filaments display different assembly, morphology, micromechanics, and nucleotide-binding specificity. Furthermore, the biophysical properties of T. maritima MreB filaments, including high rigidity and propensity to form bundles, suggest a mechanism by which MreB helical structure may be involved in imposing a cylindrical architecture on rod-shaped bacterial cells.

THE RHODOPSIN-TRANSDUCIN COMPLEX HOUSES TWO DISTINCT RHODOPSIN MOLECULES

PubMed Central

Jastrzebska, Beata; Ringler, Philipe; Palczewski, Krzysztof; Engel, Andreas

2013-01-01

Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho*, which binds the heterotrimeric G protein, transducin (Gt) causing GDP to GTP exchange and Gt dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1 mM n-dodecyl-β-D-maltoside (DDM) and Rho monomers 5 mM in DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho*•Gt and sConA-Rho*•Gt complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3-D envelope calculated from projections of negatively stained Rho*•Gt-LMNG complexes accommodated two Rho molecules, one Gt heterotrimer and a detergent belt. Visualization of triple sConA-Rho*•Gt complexes unequivocally demonstrated a pentameric assembly of the Rho*•Gt complex in which the photoactivated Rho* dimer serves as a platform for binding the Gt heterotrimer. Importantly, individual monomers of the Rho* dimer in the heteropentameric complex exhibited different capabilities to be regenerated with either 11-cis or 9-cis-retinal. PMID:23458690

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex*

PubMed Central

Burnett, Benjamin J.; Altman, Roger B.; Ferrao, Ryan; Alejo, Jose L.; Kaur, Navdep; Kanji, Joshua; Blanchard, Scott C.

2013-01-01

Aminoacyl-tRNA enters the translating ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here, we describe bulk steady state and pre-steady state fluorescence methods that enabled us to quantitatively explore the kinetic features of Escherichia coli ternary complex formation and decay. The data obtained suggest that both processes are controlled by a nucleotide-dependent, rate-determining conformational change in EF-Tu. Unexpectedly, we found that this conformational change is accelerated by elongation factor Ts (EF-Ts), the guanosine nucleotide exchange factor for EF-Tu. Notably, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. These results suggest that EF-Ts serves an unanticipated role in the cell of actively regulating the abundance and stability of ternary complex in a manner that contributes to rapid and faithful protein synthesis. PMID:23539628

Sensor potency of the moonlighting enzyme-decorated cytoskeleton: the cytoskeleton as a metabolic sensor

PubMed Central

2013-01-01

Background There is extensive evidence for the interaction of metabolic enzymes with the eukaryotic cytoskeleton. The significance of these interactions is far from clear. Presentation of the hypothesis In the cytoskeletal integrative sensor hypothesis presented here, the cytoskeleton senses and integrates the general metabolic activity of the cell. This activity depends on the binding to the cytoskeleton of enzymes and, depending on the nature of the enzyme, this binding may occur if the enzyme is either active or inactive but not both. This enzyme-binding is further proposed to stabilize microtubules and microfilaments and to alter rates of GTP and ATP hydrolysis and their levels. Testing the hypothesis Evidence consistent with the cytoskeletal integrative sensor hypothesis is presented in the case of glycolysis. Several testable predictions are made. There should be a relationship between post-translational modifications of tubulin and of actin and their interaction with metabolic enzymes. Different conditions of cytoskeletal dynamics and enzyme-cytoskeleton binding should reveal significant differences in local and perhaps global levels and ratios of ATP and GTP. The different functions of moonlighting enzymes should depend on cytoskeletal binding. Implications of the hypothesis The physical and chemical effects arising from metabolic sensing by the cytoskeleton would have major consequences on cell shape, dynamics and cell cycle progression. The hypothesis provides a framework that helps the significance of the enzyme-decorated cytoskeleton be determined. PMID:23398642

Inhibition by Siomycin and Thiostrepton of Both Aminoacyl-tRNA and Factor G Binding to Ribosomes

PubMed Central

Ll, Juan Modole; Cabrer, Bartolomé; Parmeggiani, Andrea; Azquez, David V

1971-01-01

Siomycin, a peptide antibiotic that interacts with the 50S ribosomal subunit and inhibits binding of factor G, is shown also to inhibit binding of aminoacyl-tRNA; however, it does not impair binding of fMet-tRNA and completion of the initiation complex. Moreover, unlike other inhibitors of aminoacyl-tRNA binding (tetracycline, sparsomycin, and streptogramin A), siomycin completely abolishes the GTPase activity associated with the binding of aminoacyl-tRNA catalyzed by factor Tu. A single-site interaction of siomycin appears to be responsible for its effect on both the binding of the aminoacyl-tRNA-Tu-GTP complex and that of factor G. PMID:4331558

An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

PubMed

Baumann, P; Jackson, S P

1996-06-25

Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya.

RHGF-2 Is an Essential Rho-1 Specific RhoGEF that binds to the Multi-PDZ Domain Scaffold Protein MPZ-1 in Caenorhabditis elegans

PubMed Central

Lin, Li; Tran, Thuy; Hu, Shuang; Cramer, Todd; Komuniecki, Richard; Steven, Robert M.

2012-01-01

RhoGEF proteins activate the Rho family of small GTPases and thus play a key role in regulating fundamental cellular processes such as cell morphology and polarity, cell cycle progression and gene transcription. We identified a Caenorhabditis elegans RhoGEF protein, RHGF-2, as a binding partner of the C. elegans multi-PDZ domain scaffold protein MPZ-1 (MUPP1 in mammals). RHGF-2 exhibits significant identity to the mammalian RhoGEFs PLEKHG5/Tech/Syx and contains a class I C-terminal PDZ binding motif (SDV) that interacts most strongly to MPZ-1 PDZ domain eight. RHGF-2 RhoGEF activity is specific to the C. elegans RhoA homolog RHO-1 as determined by direct binding, GDP/GTP exchange and serum response element-driven reporter activity. rhgf-2 is an essential gene since rhgf-2 deletion mutants do not elongate during embryogenesis and hatch as short immobile animals that arrest development. Interestingly, the expression of a functional rhgf-2::gfp transgene appears to be exclusively neuronal and rhgf-2 overexpression results in loopy movement with exaggerated body bends. Transient expression of RHGF-2 in N1E-115 neuroblastoma cells prevents neurite outgrowth similar to constitutive RhoA activation in these cells. Together, these observations indicate neuronally expressed RHGF-2 is an essential RHO-1 specific RhoGEF that binds most strongly to MPZ-1 PDZ domain eight and is required for wild-type C. elegans morphology and growth. PMID:22363657

Synthesis and Evolution of a Threose Nucleic Acid Aptamer Bearing 7-Deaza-7-Substituted Guanosine Residues.

PubMed

Mei, Hui; Liao, Jen-Yu; Jimenez, Randi M; Wang, Yajun; Bala, Saikat; McCloskey, Cailen; Switzer, Christopher; Chaput, John C

2018-05-02

In vitro selection experiments carried out on artificial genetic polymers require robust and faithful methods for copying genetic information back and forth between DNA and xeno-nucleic acids (XNA). Previously, we have shown that Kod-RI, an engineered polymerase developed to transcribe DNA templates into threose nucleic acid (TNA), can function with high fidelity in the absence of manganese ions. However, the transcriptional efficiency of this enzyme diminishes greatly when individual templates are replaced with libraries of DNA sequences, indicating that manganese ions are still required for in vitro selection. Unfortunately, the presence of manganese ions in the transcription mixture leads to the misincorporation of tGTP nucleotides opposite dG residues in the templating strand, which are detected as G-to-C transversions when the TNA is reverse transcribed back into DNA. Here we report the synthesis and fidelity of TNA replication using 7-deaza-7-modified guanosine base analogues in the DNA template and incoming TNA nucleoside triphosphate. Our findings reveal that tGTP misincorporation occurs via a Hoogsteen base pair in which the incoming tGTP residue adopts a syn conformation with respect to the sugar. Substitution of tGTP for 7-deaza-7-phenyl tGTP enabled the synthesis of TNA polymers with >99% overall fidelity. A TNA library containing the 7-deaza-7-phenyl guanine analogue was used to evolve a biologically stable TNA aptamer that binds to HIV reverse transcriptase with low nanomolar affinity.

The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

PubMed Central

2011-01-01

Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry. Conclusion Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion. PMID:22204307

Downregulation of cardiac guanosine 5'-triphosphate-binding proteins in right atrium and left ventricle in pacing-induced congestive heart failure.

PubMed Central

Roth, D A; Urasawa, K; Helmer, G A; Hammond, H K

1993-01-01

The extent to which congestive heart failure (CHF) is dependent upon increased levels of the cardiac inhibitory GTP-binding protein (Gi), and the impact of CHF on the cardiac stimulatory GTP-binding protein (Gs) and mechanisms by which Gs may change remain unexplored. We have addressed these unsettled issues using pacing-induced CHF in pigs to examine physiological, biochemical, and molecular features of the right atrium (RA) and left ventricle (LV). CHF was associated with an 85 +/- 20% decrease in LV segment shortening (P < 0.001) and a 3.5-fold increase (P = 0.006) in the ED50 for isoproterenol-stimulated heart rate responsiveness. Myocardial beta-adrenergic receptor number was decreased 54% in RA (P = 0.004) and 57% in LV (P < 0.001), and multiple measures of adenylyl cyclase activity were depressed 49 +/- 8% in RA (P < 0.005), and 44 +/- 9% in LV (P < 0.001). Quantitative immunoblotting established that Gi and Gs were decreased in RA (Gi: 59% reduction; P < 0.0001; Gs: 28% reduction; P < 0.007) and LV (Gi: 35% reduction; P < 0.008; Gs: 28% reduction; P < 0.01) after onset of CHF. Reduced levels of Gi and Gs were confirmed by ADP ribosylation studies, and diminished function of Gs was established in reconstitution studies. Steady state levels for Gs alpha mRNA were increased in RA and unchanged in LV, and significantly more GS alpha was found in the supernatant (presumably cytosolic) fraction in RA and LV membrane homogenates after CHF, suggesting that increased Gs degradation, rather than decreased Gs synthesis, is the mechanism by which Gs is downregulated. We conclude that cardiac Gi content poorly predicts adrenergic responsiveness or contractile function, that decreased Gs is caused by increased degradation rather than decreased synthesis, and that alterations in beta-adrenergic receptors, adenylyl cyclase, and GTP-binding proteins are uniform in RA and LV in this model of congestive heart failure. Images PMID:8383705

Regulation of cyclic AMP metabolism by prostaglandins in rabbit cortical collecting tubule cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sonnenburg, W.K.

1987-01-01

In the rabbit cortical collecting tubule (RCCT), prostaglandin E/sub 1/ (PGE/sub 1/) and prostaglandin E/sub 2/ (PGE/sub 2/) at 1 nM inhibit arginine-vasopressin (AVP)-induced water reabsorption, while 100 nM PGE/sub 1/ and PGE/sub 2/ alone stimulate water reabsorption. Reported here are studies designed to investigate the molecular basis for the biphasic physiological action of PGE/sub 1/ and PGE/sub 2/ in the collecting duct. In freshly isolated RCCT cells, PGE/sub 1/, PGE/sub 2/, and 16,16-dimethyl-PGE/sub 2/ (DM-PGE/sub 2/) stimulated cAMP synthesis at concentrations ranging from 0.1 to 10 M. Other prostaglandins including the synthetic PGE/sub 2/ analogue, sulprostone, failed to stimulatemore » cAMP synthesis. Moreover, sulprostone did not antagonize PGE/sub 2/-stimulated cAMP formation. In contrast, PGE/sub 2/ and sulprostone at concentrations ranging from 1 to 100 nM, inhibited AVP-induced cAMP accumulation in freshly isolated RCCT cells. PGE/sub 2/, PGE/sub 1/, DM-PGE/sub 2/ and sulprostone at 100 nM were equally effective in inhibiting AVP-induced cAMP formation. Moreover sulprostone inhibited AVP-stimulated adenylate cyclase activity. These results suggest that PGE derivatives mediate either inhibition or activation of adenylate cyclase by stimulating different PGE receptors. To further test this concept, PGE/sub 2/ binding to freshly isolated RCCT cell membranes was characterized. Two different classes of PGE/sub 2/ binding were detected. //sup 3/H/PGE/sub 2/ binding to the high affinity class of sites was increased by the GTP-analogue, GTP S, while pertussis toxin pretreatment blocked the stimulatory action. In contrast, //sup 3/H/ PGE/sub 2/ binding to the low affinity class of sites was decreased by GTP S; this inhibitory effect was not blocked by pertussis toxin pretreatment.« less

Tyrosine phosphorylation switching of a G protein.

PubMed

Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke; Jia, Haiyan; Werth, Emily G; Mowrey, David D; Hicks, Leslie M; Dokholyan, Nikolay V; Torres, Matthew P; Jones, Alan M

2018-03-30

Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr 166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr 166 -dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr 166 forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr 166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr 166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching." © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of a small GTP-binding protein gene TaRab18 from wheat involved in the stripe rust resistance.

PubMed

Jiang, Zhengning; Wang, Hui; Zhang, Guoqin; Zhao, Renhui; Bie, Tongde; Zhang, Ruiqi; Gao, Derong; Xing, Liping; Cao, Aizhong

2017-04-01

The stripe rust resistance gene, Yr26, is commonly used in wheat production. Identification of Yr26 resistance related genes is important for better understanding of the resistance mechanism. TaRab18, a putative small GTP-binding protein, was screened as a resistance regulated gene as it showed differential expression between the Yr26-containing resistant wheat and the susceptible wheat at different time points after Pst inoculation. TaRab18 contains four typical domains (GI to GIV) of the small GTP-binding proteins superfamily and five domains (RabF1 to RabF5) specific to the Rab subfamily. From the phylogenetic tree that TaRab18 was identified as belonging to the RABC1 subfamily. Chromosome location analysis indicated that TaRab18 and its homeoalles were on the homeologous group 7 chromosomes, and the Pst induced TaRab18 was on the 7 B chromosome. Functional analysis by virus induced gene silencing (VIGS) indicated that TaRab18 was positively involved in the stripe rust resistance through regulating the hypersensitive response, and Pst can develop on the leaves of TaRab18 silenced 92R137. However, over-expression of TaRab18 in susceptible Yangmai158 did not enhance its resistance dramatically, only from 9 grade in Yangmai158 to 8 grade in the transgenic plant. However, histological observation indicated that the transgenic plants with over-expressed TaRab18 showed a strong hypersensitive response at the early infection stage. The research herein, will improve our understanding of the roles of Rab in wheat resistance. Copyright © 2017. Published by Elsevier Masson SAS.

Shift in the equilibrium between on and off states of the allosteric switch in Ras-GppNHp affected by small molecules and bulk solvent composition.

PubMed

Holzapfel, Genevieve; Buhrman, Greg; Mattos, Carla

2012-08-07

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

PubMed Central

Fong, Yu Hang; Wong, Ho Chun; Yuen, Man Hon; Lau, Pak Ho; Chen, Yu Wai; Wong, Kam-Bo

2013-01-01

Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease. PMID:24115911

Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

PubMed

Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L; Southworth, Daniel R; Banerjee, Ruma

2017-10-27

G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Exquisite Modulation of the Active Site of Methanocaldococcus jannaschii Adenylosuccinate Synthetase in Forward Reaction Complexes.

PubMed

Karnawat, Vishakha; Mehrotra, Sonali; Balaram, Hemalatha; Puranik, Mrinalini

2016-05-03

In enzymes that conduct complex reactions involving several substrates and chemical transformations, the active site must reorganize at each step to complement the transition state of that chemical step. Adenylosuccinate synthetase (ADSS) utilizes a molecule each of guanosine 5'-monophosphate (GTP) and aspartate to convert inosine 5'-monophosphate (IMP) into succinyl adenosine 5'-monophosphate (sAMP) through several kinetic intermediates. Here we followed catalysis by ADSS through high-resolution vibrational spectral fingerprints of each substrate and intermediate involved in the forward reaction. Vibrational spectra show differential ligand distortion at each step of catalysis, and band positions of substrates are influenced by binding of cosubstrates. We found that the bound IMP is distorted toward its N1-deprotonated form even in the absence of any other ligands. Several specific interactions between GTP and active-site amino acid residues result in large Raman shifts and contribute substantially to intrinsic binding energy. When both IMP and GTP are simultaneously bound to ADSS, IMP is converted into an intermediate 6-phosphoryl inosine 5'-monophosphate (6-pIMP). The 6-pIMP·ADSS complex was found to be stable upon binding of the third ligand, hadacidin (HDA), an analogue of l-aspartate. We find that in the absence of HDA, 6-pIMP is quickly released from ADSS, is unstable in solution, and converts back into IMP. HDA allosterically stabilizes ADSS through local conformational rearrangements. We captured this complex and determined the spectra and structure of 6-pIMP in its enzyme-bound state. These results provide important insights into the exquisite tuning of active-site interactions with changing substrate at each kinetic step of catalysis.

Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Holzapfel, Genevieve; Buhrman, Greg; Mattos, Carla

2012-08-31

Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) canmore » selectively shift the equilibrium to the 'on' state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the 'ordered off' state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-{beta}. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.« less

Structural and functional characterization of the Mycobacterium tuberculosis uridine monophosphate kinase: insights into the allosteric regulation.

PubMed

Labesse, Gilles; Benkali, Khaled; Salard-Arnaud, Isabelle; Gilles, Anne-Marie; Munier-Lehmann, Hélène

2011-04-01

Nucleoside Monophosphate Kinases (NMPKs) family are key enzymes in nucleotide metabolism. Bacterial UMPKs depart from the main superfamily of NMPKs. Having no eukaryotic counterparts they represent attractive therapeutic targets. They are regulated by GTP and UTP, while showing different mechanisms in Gram(+), Gram(-) and archaeal bacteria. In this work, we have characterized the mycobacterial UMPK (UMPKmt) combining enzymatic and structural investigations with site-directed mutagenesis. UMPKmt exhibits cooperativity toward ATP and an allosteric regulation by GTP and UTP. The crystal structure of the complex of UMPKmt with GTP solved at 2.5 Å, was merely identical to the modelled apo-form, in agreement with SAXS experiments. Only a small stretch of residues was affected upon nucleotide binding, pointing out the role of macromolecular dynamics rather than major structural changes in the allosteric regulation of bacterial UMPKs. We further probe allosteric regulation by site-directed mutagenesis. In particular, a key residue involved in the allosteric regulation of this enzyme was identified.

Microtubule assembly governed by tubulin allosteric gain in flexibility and lattice induced fit

PubMed Central

2018-01-01

Microtubules (MTs) are key components of the cytoskeleton and play a central role in cell division and development. MT assembly is known to be associated with a structural change in αβ-tubulin dimers from kinked to straight conformations. How GTP binding renders individual dimers polymerization-competent, however, is still unclear. Here, we have characterized the conformational dynamics and energetics of unassembled tubulin using atomistic molecular dynamics and free energy calculations. Contrary to existing allosteric and lattice models, we find that GTP-tubulin favors a broad range of almost isoenergetic curvatures, whereas GDP-tubulin has a much lower bending flexibility. Moreover, irrespective of the bound nucleotide and curvature, two conformational states exist differing in location of the anchor point connecting the monomers that affects tubulin bending, with one state being strongly favored in solution. Our findings suggest a new combined model in which MTs incorporate and stabilize flexible GTP-dimers with a specific anchor point state. PMID:29652248

Dynamic clustering of dynamin-amphiphysin helices regulates membrane constriction and fission coupled with GTP hydrolysis

PubMed Central

Kozai, Toshiya; Yang, Huiran; Ishikuro, Daiki; Seyama, Kaho; Kumagai, Yusuke; Abe, Tadashi; Yamada, Hiroshi; Uchihashi, Takayuki

2018-01-01

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission. PMID:29357276

Influence of bacterial toxins on the GTPase activity of transducin from bovine retinal rod outer segments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rybin, V.O.; Gureeva, A.A.

1986-05-10

The action of cholera toxin, capable of ADP-ribosylation of the activator N/sub s/ protein, and pertussis toxin, capable of ADP-ribosylation of the inhibitor N/sub i/ protein of the adenylate cyclase complex, on transducin, the GTP-binding protein of the rod outer segments of the retina, was investigated. It was shown that under the action of pertussis and cholera toxins, the GTPase activity of transducin is inhibited. Pertussin toxin inhibits the GTPase of native retinal rod outer segments by 30-40%, while GTPase of homogeneous transducin produces a 70-80% inhibition. The action of toxins on transducin depends on the presence and nature ofmore » the guanylic nucleotide with which incubation is performed. On the basis of the data obtained it is suggested that pertussis toxin interacts with pretransducin and with the transducin-GDP complex, while cholera toxin ADP-ribosylates the transducin-GTP complex and does not act on transducin lacking GTP.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dissanayake, V.U.; Hughes, J.; Hunter, J.C.

The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE boundmore » with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.« less

Molecular basis for allosteric specificity regulation in class Ia ribonucleotide reductase from Escherichia coli

PubMed Central

Zimanyi, Christina M; Chen, Percival Yang-Ting; Kang, Gyunghoon; Funk, Michael A; Drennan, Catherine L

2016-01-01

Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell. DOI: http://dx.doi.org/10.7554/eLife.07141.001 PMID:26754917

Cytokine-induced (interleukins-3, -6 and -8 and tumour necrosis factor-beta) activation and deactivation of human neutrophils.

PubMed Central

Brom, J; König, W

1992-01-01

The effect of various cytokines [interleukin-3(IL-3), IL-6, IL-8, tumour necrosis factor-beta (TNF-beta)] on human neutrophils (PMN) was analysed with regard to the generation of leukotrienes and the involvement of guanosine triphosphate (GTP)-binding proteins (G proteins). Incubation of cytochalasin B-pretreated PMN with cytokines alone did not lead to a generation of leukotrienes. However, the cytokines affected the formyl-methionyl-leucyl-phenylalanine-(FMLP)-induced formation of leukotrienes in a time-dependent manner. Preincubation of the cells with the different cytokines for short periods (15 seconds at 37 degrees) enhanced the subsequent FMLP-induced leukotriene generation, whereas preincubation for prolonged times resulted in a reduced formation of leukotrienes. These results correlated with the respective G protein-associated guanosine triphosphatase (GTPase) activities within isolated membrane fractions. The present study indicates a modulation of the FMLP-induced leukotriene formation by diverse cytokines via interaction with the GTP-binding proteins. PMID:1312995

Molecular analysis of ARF1 expression profiles during development of physic nut (Jatropha curcas L.).

PubMed

Qin, Xiaobo; Lin, Fanrong; Lii, Yifan; Gou, Chunbao; Chen, Fang

2011-03-01

A cDNA clone designated arf1 was isolated from a physic nut (Jatropha curcas L.) endosperm cDNA library which encodes a small GTP-binding protein and has significant homology to ADP-ribosylation factors (ARF) in plants, animals and microbes. The cDNA contains an open reading frame that encodes a polypeptide of 181 amino acids with a calculated molecular mass of 20.7 kDa. The deduced amino acid sequence showed high homology to known ARFs from other organisms. The products of the arf1 obtained by overexpression in E. coli revealed the specific binding activity toward GTP. The expression of arf1 was observed in flowers, roots, stems and leaves as analyzed by RT-PCR, and its transcriptional level was highest in flowers. In particular, the accumulation of arf1 transcripts was different under various environmental stresses in seedlings. The results suggest that arf1 plays distinct physiological roles in Jatropha curcas cells.

Structural differences between yeast and mammalian microtubules revealed by cryo-EM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howes, Stuart C.; Geyer, Elisabeth A.; LaFrance, Benjamin

Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrationsmore » used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. In conclusion, our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.« less

Crystal structure of a Schistosoma mansoni septin reveals the phenomenon of strand slippage in septins dependent on the nature of the bound nucleotide.

PubMed

Zeraik, Ana E; Pereira, Humberto M; Santos, Yuri V; Brandão-Neto, José; Spoerner, Michael; Santos, Maiara S; Colnago, Luiz A; Garratt, Richard C; Araújo, Ana P U; DeMarco, Ricardo

2014-03-14

Septins are filament-forming GTP-binding proteins involved in important cellular events, such as cytokinesis, barrier formation, and membrane remodeling. Here, we present two crystal structures of the GTPase domain of a Schistosoma mansoni septin (SmSEPT10), one bound to GDP and the other to GTP. The structures have been solved at an unprecedented resolution for septins (1.93 and 2.1 Å, respectively), which has allowed for unambiguous structural assignment of regions previously poorly defined. Consequently, we provide a reliable model for functional interpretation and a solid foundation for future structural studies. Upon comparing the two complexes, we observe for the first time the phenomenon of a strand slippage in septins. Such slippage generates a front-back communication mechanism between the G and NC interfaces. These data provide a novel mechanistic framework for the influence of nucleotide binding to the GTPase domain, opening new possibilities for the study of the dynamics of septin filaments.

Mechanisms of inverse agonist action at D2 dopamine receptors

PubMed Central

Roberts, David J; Strange, Philip G

2005-01-01

Mechanisms of inverse agonist action at the D2(short) dopamine receptor have been examined. Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [3H]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. Competition of inverse agonists versus [3H]NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. Ki values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. Kcoupled and Kuncoupled were statistically different for the set of compounds tested (ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. These observations were supported by simulations of these competition experiments according to the extended ternary complex model. Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [35S]GTPγS binding to varying degrees in concentration–response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (−)-sulpiride was unaffected. These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism. PMID:15735658

Identification of five reptile egg whites protein using MALDI-TOF mass spectrometry and LC/MS-MS analysis.

PubMed

Prajanban, Bung-on; Shawsuan, Laoo; Daduang, Sakda; Kommanee, Jintana; Roytrakul, Sittiruk; Dhiravisit, Apisak; Thammasirirak, Sompong

2012-03-16

Proteomics of egg white proteins of five reptile species, namely Siamese crocodile (Crocodylus siamensis), soft-shelled turtle (Trionyx sinensis taiwanese), red-eared slider turtle (Trachemys scripta elegans), hawksbill turtle (Eretmochelys imbricate) and green turtle (Chelonia mydas) were studied by 2D-PAGE using IPG strip pH 4-7 size 7 cm and IPG strip pH 3-10 size 24 cm. The protein spots in the egg white of the five reptile species were identified by MALDI-TOF mass spectrometry and LC/MS-MS analysis. Sequence comparison with the database revealed that reptile egg white contained at least seven protein groups, such as serpine, transferrin precursor/iron binding protein, lysozyme C, teneurin-2 (fragment), interferon-induced GTP-binding protein Mx, succinate dehydrogenase iron-sulfur subunit and olfactory receptor 46. This report confirms that transferrin precursor/iron binding protein is the major component in reptile egg white. In egg white of Siamese crocodile, twenty isoforms of transferrin precursor were found. Iron binding protein was found in four species of turtle. In egg white of soft-shelled turtle, ten isoforms of lysozyme were found. Apart from well-known reptile egg white constituents, this study identified some reptile egg white proteins, such as the teneurin-2 (fragment), the interferon-induced GTP-binding protein Mx, the olfactory receptor 46 and the succinate dehydrogenase iron-sulfur subunit. Copyright © 2012 Elsevier B.V. All rights reserved.

Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

PubMed Central

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-01

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

PubMed

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-09

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1.

PubMed

Stewart, James J P

2016-11-01

A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol -1 to ±0.1 kcal mol -1 . This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates.

An inhibitor of eIF2 activity in the sRNA pool of eukaryotic cells.

PubMed

Centrella, Michael; Porter, David L; McCarthy, Thomas L

2011-08-15

Eukaryotic protein synthesis is a multi-step and highly controlled process that includes an early initiation complex containing eukaryotic initiation factor 2 (eIF2), GTP, and methionine-charged initiator methionyl-tRNA (met-tRNAi). During studies to reconstruct formation of the ternary complex containing these molecules, we detected a potent inhibitor in low molecular mass RNA (sRNA) preparations of eukaryotic tRNA. The ternary complex inhibitor (TCI) was retained in the total sRNA pool after met-tRNAi was charged by aminoacyl tRNA synthetase, co-eluted with sRNA by size exclusion chromatography, but resolved from met-tRNAi by ion exchange chromatography. The adverse effect of TCI was not overcome by high GTP or magnesium omission and was independent of GTP regeneration. Rather, TCI suppressed the rate of ternary complex formation, and disrupted protein synthesis and the accumulation of heavy polymeric ribosomes in reticulocyte lysates in vitro. Lastly, a component or components in ribosome depleted cell lysate significantly reversed TCI activity. Since assembly of the met-tRNAi/eIF2/GTP ternary complex is integral to protein synthesis, awareness of TCI is important to avoid confusion in studies of translation initiation. A clear definition of TCI may also allow a better appreciation of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo. Copyright © 2011 Elsevier B.V. All rights reserved.

An archaebacterial homologue of the essential eubacterial cell division protein FtsZ.

PubMed Central

Baumann, P; Jackson, S P

1996-01-01

Life falls into three fundamental domains--Archaea, Bacteria, and Eucarya (formerly archaebacteria, eubacteria, and eukaryotes,. respectively). Though Archaea lack nuclei and share many morphological features with Bacteria, molecular analyses, principally of the transcription and translation machineries, have suggested that Archaea are more related to Eucarya than to Bacteria. Currently, little is known about the archaeal cell division apparatus. In Bacteria, a crucial component of the cell division machinery is FtsZ, a GTPase that localizes to a ring at the site of septation. Interestingly, FtsZ is distantly related in sequence to eukaryotic tubulins, which also interact with GTP and are components of the eukaryotic cell cytoskeleton. By screening for the ability to bind radiolabeled nucleotides, we have identified a protein of the hyperthermophilic archaeon Pyrococcus woesei that interacts tightly and specifically with GTP. Furthermore, through screening an expression library of P. woesei genomic DNA, we have cloned the gene encoding this protein. Sequence comparisons reveal that the P. woesei GTP-binding protein is strikingly related in sequence to eubacterial FtsZ and is marginally more similar to eukaryotic tubulins than are bacterial FtsZ proteins. Phylogenetic analyses reinforce the notion that there is an evolutionary linkage between FtsZ and tubulins. These findings suggest that the archaeal cell division apparatus may be fundamentally similar to that of Bacteria and lead us to consider the evolutionary relationships between Archaea, Bacteria, and Eucarya. Images Fig. 1 Fig. 2 PMID:8692886

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing

PubMed Central

James, Victoria; Zhang, Yining; Foxler, Daniel E.; de Moor, Cornelia H.; Kong, Yi Wen; Webb, Thomas M.; Self, Tim J.; Feng, Yungfeng; Lagos, Dimitrios; Chu, Chia-Ying; Rana, Tariq M.; Morley, Simon J.; Longmore, Gregory D.; Bushell, Martin; Sharp, Tyson V.

2010-01-01

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5′ m7GTP cap–protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m7GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m7GTP-5′cap and Ago1/2 within the miRISC complex attached to the 3′-UTR of mRNA, creating an inhibitory closed-loop complex. PMID:20616046

UreE-UreG Complex Facilitates Nickel Transfer and Preactivates GTPase of UreG in Helicobacter pylori*♦

PubMed Central

Yang, Xinming; Li, Hongyan; Lai, Tsz-Pui; Sun, Hongzhe

2015-01-01

The pathogenicity of Helicobacter pylori relies heavily on urease, which converts urea to ammonia to neutralize the stomach acid. Incorporation of Ni2+ into the active site of urease requires a battery of chaperones. Both metallochaperones UreE and UreG play important roles in the urease activation. In this study, we demonstrate that, in the presence of GTP and Mg2+, UreG binds Ni2+ with an affinity (Kd) of ∼0.36 μm. The GTPase activity of Ni2+-UreG is stimulated by both K+ (or NH4+) and HCO3− to a biologically relevant level, suggesting that K+/NH4+ and HCO3− might serve as GTPase elements of UreG. We show that complexation of UreE and UreG results in two protein complexes, i.e. 2E-2G and 2E-G, with the former being formed only in the presence of both GTP and Mg2+. Mutagenesis studies reveal that Arg-101 on UreE and Cys-66 on UreG are critical for stabilization of 2E-2G complex. Combined biophysical and bioassay studies show that the formation of 2E-2G complex not only facilitates nickel transfer from UreE to UreG, but also enhances the binding of GTP. This suggests that UreE might also serve as a structural scaffold for recruitment of GTP to UreG. Importantly, we demonstrate for the first time that UreE serves as a bridge to grasp Ni2+ from HypA, subsequently donating it to UreG. The study expands our horizons on the molecular details of nickel translocation among metallochaperones UreE, UreG, and HypA, which further extends our knowledge on the urease maturation process. PMID:25752610

Excitation-contraction coupling in skeletal muscle fibres of rat and toad in the presence of GTP gamma S.

PubMed Central

Lamb, G D; Stephenson, D G

1991-01-01

1. Rapid force responses were elicited in single mechanically skinned fibres from extensor digitorum longus (EDL) muscle of the rat when the fibres were depolarized by substituting K+ in the bathing solution with Na+. The properties of these depolarization-induced responses, the responses to lowered [Mg2+], and the characteristics of the slow prolonged response ('second component') produced in 'loaded' fibres by choline chloride (ChCl) substitution, were virtually identical to those observed previously in skinned fibres from toad muscle. 2. At physiological levels of [Mg2+] (1 mM) and Ca2+ loading, application of 50 microM- to 1 mM-GTP gamma S (guanosine-5'-O-(3-thiotriphosphate), a non-hydrolysable analogue of GTP) did not produce a response in any mammalian or amphibian fibre, even though the depolarization-induced coupling was totally functional. Furthermore, the presence of GTP gamma S had no apparent effect on the size, the threshold or the maximum number of responses which could be elicited by depolarization. 3. GTP gamma S did not elicit any response when excitation-contraction coupling was abolished by prolonged depolarization or by chemically skinning the fibre with saponin or by 24 h exposure to low [Ca2+] (5 mM-EGTA). 4. GDP beta S (guanosine-5'-O-(2-thiodiphosphate), 250 microM or 1 mM) neither evoked a response nor affected the responses to depolarization or caffeine. 5. When the [Mg2+] was lowered to 0.2 mM and the fibres were heavily loaded with Ca2+, addition of GTP gamma S (250 microM or 1 mM) induced a small response in about 50% of fibres, but depolarization-induced responses were not affected in any fibres. 6. Asymmetric charge movement recorded in EDL fibres with the vaseline-gap voltage clamp was not affected by the application of 1 mM-GTP gamma S to the cut ends of the fibres for up to 1 h. 7. These data imply that GTP-binding proteins (G-proteins) are not involved in coupling the voltage sensors to Ca2+ release in skeletal muscle. Furthermore, there was no evidence that G-proteins play any role in modulating the voltage sensors, though this possibility could not be totally excluded. PMID:1726598

Excitation-contraction coupling in skeletal muscle fibres of rat and toad in the presence of GTP gamma S.

PubMed

Lamb, G D; Stephenson, D G

1991-12-01

1. Rapid force responses were elicited in single mechanically skinned fibres from extensor digitorum longus (EDL) muscle of the rat when the fibres were depolarized by substituting K+ in the bathing solution with Na+. The properties of these depolarization-induced responses, the responses to lowered [Mg2+], and the characteristics of the slow prolonged response ('second component') produced in 'loaded' fibres by choline chloride (ChCl) substitution, were virtually identical to those observed previously in skinned fibres from toad muscle. 2. At physiological levels of [Mg2+] (1 mM) and Ca2+ loading, application of 50 microM- to 1 mM-GTP gamma S (guanosine-5'-O-(3-thiotriphosphate), a non-hydrolysable analogue of GTP) did not produce a response in any mammalian or amphibian fibre, even though the depolarization-induced coupling was totally functional. Furthermore, the presence of GTP gamma S had no apparent effect on the size, the threshold or the maximum number of responses which could be elicited by depolarization. 3. GTP gamma S did not elicit any response when excitation-contraction coupling was abolished by prolonged depolarization or by chemically skinning the fibre with saponin or by 24 h exposure to low [Ca2+] (5 mM-EGTA). 4. GDP beta S (guanosine-5'-O-(2-thiodiphosphate), 250 microM or 1 mM) neither evoked a response nor affected the responses to depolarization or caffeine. 5. When the [Mg2+] was lowered to 0.2 mM and the fibres were heavily loaded with Ca2+, addition of GTP gamma S (250 microM or 1 mM) induced a small response in about 50% of fibres, but depolarization-induced responses were not affected in any fibres. 6. Asymmetric charge movement recorded in EDL fibres with the vaseline-gap voltage clamp was not affected by the application of 1 mM-GTP gamma S to the cut ends of the fibres for up to 1 h. 7. These data imply that GTP-binding proteins (G-proteins) are not involved in coupling the voltage sensors to Ca2+ release in skeletal muscle. Furthermore, there was no evidence that G-proteins play any role in modulating the voltage sensors, though this possibility could not be totally excluded.

The rhodopsin-transducin complex houses two distinct rhodopsin molecules.

PubMed

Jastrzebska, Beata; Ringler, Philippe; Palczewski, Krzysztof; Engel, Andreas

2013-05-01

Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho(∗), which binds the heterotrimeric G protein, transducin (Gt) causing GDP to GTP exchange and Gt dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1mM n-dodecyl-β-d-maltoside (DDM) and Rho monomers in 5mM DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho(∗)·Gt and sConA-Rho(∗)·Gt complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3 D envelope calculated from projections of negatively stained Rho(∗)·Gt-LMNG complexes accommodated two Rho molecules, one Gt heterotrimer and a detergent belt. Visualization of triple sConA-Rho(∗)·Gt complexes unequivocally demonstrated a pentameric assembly of the Rho(∗)·Gt complex in which the photoactivated Rho(∗) dimer serves as a platform for binding the Gt heterotrimer. Importantly, individual monomers of the Rho(∗) dimer in the heteropentameric complex exhibited different capabilities for regeneration with either 11-cis or 9-cis-retinal. Copyright © 2013 Elsevier Inc. All rights reserved.

Green tea polyphenols-induced apoptosis in human osteosarcoma SAOS-2 cells involves a caspase-dependent mechanism with downregulation of nuclear factor-{kappa}B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bin Hafeez, Bilal; Ahmed, Salahuddin; Wang, Naizhen

2006-10-01

Development of chemotherapy resistance and evasion from apoptosis in osteosarcoma, a primary malignant bone tumor, is often correlated with constitutive nuclear factor-{kappa}B (NF-{kappa}B) activation. Here, we investigated the ability of a polyphenolic fraction of green tea (GTP) that has been shown to have antitumor effects on various malignant cell lines to inhibit growth and induce apoptosis in human osteosarcoma SAOS-2 cells. Treatment of SAOS-2 cells with GTP (20-60 {mu}g/ml) resulted in reduced cell proliferation and induction of apoptosis, which correlated with decreased nuclear DNA binding of NF-{kappa}B/p65 and lowering of NF-{kappa}B/p65 and p50 levels in the cytoplasm and nucleus. GTPmore » treatment of cells reduced I{kappa}B-{alpha} phosphorylation but had no effect on its protein expression. Furthermore, GTP treatment resulted in the inhibition of IKK-{alpha} and IKK-{beta}, the upstream kinases that phosphorylate I{kappa}B-{alpha}. The increase in apoptosis in SAOS-2 cells was accompanied with decrease in the protein expression of Bcl-2 and concomitant increase in the levels of Bax. GTP treatment of SAOS-2 cells also resulted in significant activation of caspases as was evident by increased levels of cleaved caspase-3 and caspase-8 in these cells. Treatment of SAOS-2 cells with a specific caspase-3 inhibitor Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) and general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) rescued SAOS-2 cells from GTP-induced apoptosis. Taken together, these results indicate that GTP is a candidate therapeutic for osteosarcoma that mediates its antiproliferative and apoptotic effects via activation of caspases and inhibition of NF-{kappa}B.« less

The tRNA-modifying function of MnmE is controlled by post-hydrolysis steps of its GTPase cycle

PubMed Central

Prado, Silvia; Villarroya, Magda; Medina, Milagros; Armengod, M.-Eugenia

2013-01-01

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and Pi release can be uncoupled and that G-domain dissociation is directly responsible for the ‘ON’ state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and Pi. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle. PMID:23630314

Interconversion of two GDP-bound conformations and their selection in an Arf-family small G protein.

PubMed

Okamura, Hideyasu; Nishikiori, Masaki; Xiang, Hongyu; Ishikawa, Masayuki; Katoh, Etsuko

2011-07-13

ADP-ribosylation factor (Arf) and other Arf-family small G proteins participate in many cellular functions via their characteristic GTP/GDP conformational cycles, during which a nucleotide(∗)Mg(2+)-binding site communicates with a remote N-terminal helix. However, the conformational interplay between the nucleotides, the helix, the protein core, and Mg(2+) has not been fully delineated. Herein, we report a study of the dynamics of an Arf-family protein, Arl8, under various conditions by means of NMR relaxation spectroscopy. The data indicated that, when GDP is bound, the protein core, which does not include the N-terminal helix, reversibly transition between an Arf-family GDP form and another conformation that resembles the Arf-family GTP form. Additionally, we found that the N-terminal helix and Mg(2+), respectively, stabilize the aforementioned former and latter conformations in a population-shift manner. Given the dynamics of the conformational changes, we can describe the Arl8 GTP/GDP cycle in terms of an energy diagram. Copyright © 2011 Elsevier Ltd. All rights reserved.

Missense-depleted regions in population exomes implicate ras superfamily nucleotide-binding protein alteration in patients with brain malformation

PubMed Central

Ge, Xiaoyan; Gong, Henry; Dumas, Kevin; Litwin, Jessica; Phillips, Joanna J; Waisfisz, Quinten; Weiss, Marjan M; Hendriks, Yvonne; Stuurman, Kyra E; Nelson, Stanley F; Grody, Wayne W; Lee, Hane; Kwok, Pui-Yan; Shieh, Joseph T C

2016-01-01

Genomic sequence interpretation can miss clinically relevant missense variants for several reasons. Rare missense variants are numerous in the exome and difficult to prioritise. Affected genes may also not have existing disease association. To improve variant prioritisation, we leverage population exome data to identify intragenic missense-depleted regions (MDRs) genome-wide that may be important in disease. We then use missense depletion analyses to help prioritise undiagnosed disease exome variants. We demonstrate application of this strategy to identify a novel gene association for human brain malformation. We identified de novo missense variants that affect the GDP/GTP-binding site of ARF1 in three unrelated patients. Corresponding functional analysis suggests ARF1 GDP/GTP-activation is affected by the specific missense mutations associated with heterotopia. These findings expand the genetic pathway underpinning neurologic disease that classically includes FLNA. ARF1 along with ARFGEF2 add further evidence implicating ARF/GEFs in the brain. Using functional ontology, top MDR-containing genes were highly enriched for nucleotide-binding function, suggesting these may be candidates for human disease. Routine consideration of MDR in the interpretation of exome data for rare diseases may help identify strong genetic factors for many severe conditions, infertility/reduction in reproductive capability, and embryonic conditions contributing to preterm loss. PMID:28868155

Enhanced accumulation of atropine in Atropa belladonna transformed by Rac GTPase gene isolated from Scoparia dulcis.

PubMed

Asano, Kyouhei; Lee, Jung-Bum; Yamamura, Yoshimi; Kurosaki, Fumiya

2013-12-01

Leaf tissues of Atropa belladonna were transformed by Sdrac2, a Rac GTPase gene, that is isolated from Scoparia dulcis, and the change in atropine concentration of the transformants was examined. Re-differentiated A. belladonna overexpressing Sdrac2 accumulated considerable concentration of atropine in the leaf tissues, whereas the leaves of plants transformed by an empty vector accumulated only a very low concentration of the compound. A. belladonna transformed by CASdrac2, a modified Sdrac2 of which translate was expected to bind guanosine triphosphate (GTP) permanently, accumulated very high concentrations of atropine (approximately 2.4-fold excess to those found in the wild-type plant in its natural habitat). In sharp contrast, the atropine concentration in transformed A. belladonna prepared with negatively modified Sdrac2, DNSdrac2, expected to bind guanosine diphosphate instead of GTP, was very low. These results suggested that Rac GTPases play an important role in the regulation of secondary metabolism in plant cells and that overexpression of the gene(s) may be capable of enhancing the production of natural products accumulated in higher plant cells.

The Nuclear Transport Factor Kap121 Is Required for Stability of the Dam1 Complex and Mitotic Kinetochore Bi-orientation.

PubMed

Cairo, Lucas V; Wozniak, Richard W

2016-03-15

The karyopherin (Kap) family of nuclear transport factors facilitates macromolecular transport through nuclear pore complexes (NPCs). The binding of Kaps to their cargos can also regulate, both temporally and spatially, the interactions of the cargo protein with interacting partners. Here, we show that the essential yeast Kap, Kap121, binds Dam1 and Duo1, components of the microtubule (MT)-associated Dam1 complex required for linking dynamic MT ends with kinetochores (KTs). Like mutations in the Dam1 complex, loss of Kap121 function compromises the formation of normal KT-MT attachments during mitosis. We show that the stability of the Dam1 complex in vivo is dependent on its association with Kap121. Furthermore, we show that the Kap121/Duo1 complex is maintained in the presence of RanGTP but Kap121 is released by the cooperative actions of RanGTP and tubulin. We propose that Kap121 stabilizes the Dam1 complex and participates in escorting it to spindle MTs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Revisiting the Roco G-protein cycle.

PubMed

Terheyden, Susanne; Ho, Franz Y; Gilsbach, Bernd K; Wittinghofer, Alfred; Kortholt, Arjan

2015-01-01

Mutations in leucine-rich-repeat kinase 2 (LRRK2) are the most frequent cause of late-onset Parkinson's disease (PD). LRRK2 belongs to the Roco family of proteins which share a conserved Ras-like G-domain (Roc) and a C-terminal of Roc (COR) domain tandem. The nucleotide state of small G-proteins is strictly controlled by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because of contradictory structural and biochemical data, the regulatory mechanism of the LRRK2 Roc G-domain and the RocCOR tandem is still under debate. In the present study, we solved the first nucleotide-bound Roc structure and used LRRK2 and bacterial Roco proteins to characterize the RocCOR function in more detail. Nucleotide binding induces a drastic structural change in the Roc/COR domain interface, a region strongly implicated in patients with an LRRK2 mutation. Our data confirm previous assumptions that the C-terminal subdomain of COR functions as a dimerization device. We show that the dimer formation is independent of nucleotide. The affinity for GDP/GTP is in the micromolar range, the result of which is high dissociation rates in the s-1 range. Thus Roco proteins are unlikely to need GEFs to achieve activation. Monomeric LRRK2 and Roco G-domains have a similar low GTPase activity to small G-proteins. We show that GTPase activity in bacterial Roco is stimulated by the nucleotide-dependent dimerization of the G-domain within the complex. We thus propose that the Roco proteins do not require GAPs to stimulate GTP hydrolysis but stimulate each other by one monomer completing the catalytic machinery of the other.

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-γ-inducible cofactor

PubMed Central

Modiano, Nir; Lu, Yanping E.; Cresswell, Peter

2005-01-01

Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-γ-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-γ. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result. PMID:15937107

Golgi targeting of human guanylate-binding protein-1 requires nucleotide binding, isoprenylation, and an IFN-gamma-inducible cofactor.

PubMed

Modiano, Nir; Lu, Yanping E; Cresswell, Peter

2005-06-14

Human guanylate-binding protein-1 (hGBP-1) is a large GTPase, similar in structure to the dynamins. Like many smaller GTPases of the Ras/Rab family, it is farnesylated, suggesting it may dock into membranes and perhaps play a role in intracellular trafficking. To date, however, hGBP-1 has never been associated with a specific intracellular compartment. Here we present evidence that hGBP-1 can associate with the Golgi apparatus. Redistribution from the cytosol to the Golgi was observed by immunofluorescence and subcellular fractionation after aluminum fluoride treatment, suggesting that it occurs when hGBP-1 is in its GTP-bound state. Relocalization was blocked by a farnesyl transferase inhibitor. The C589S mutant of hGBP-1, which cannot be farnesylated, and the previously uncharacterized R48P mutant, which cannot bind GTP, both failed to localize to the Golgi. These two mutants had a dominant-negative effect, preventing endogenous wild-type hGBP-1 from efficiently redistributing after aluminum fluoride treatment. Furthermore, hGBP-1 requires another IFN-gamma-induced factor to be targeted to the Golgi, because constitutively expressed hGBP-1 remained cytosolic in cells treated with aluminum fluoride unless the cells were preincubated with IFN-gamma. Finally, two nonhydrolyzing mutants of hGBP-1, corresponding to active mutants of Ras family proteins, failed to constitutively associate with the Golgi; we propose three possible explanations for this surprising result.

Mutations in Elongation Factor Ef-1α Affect the Frequency of Frameshifting and Amino Acid Misincorporation in Saccharomyces Cerevisiae

PubMed Central

Sandbaken, M. G.; Culbertson, M. R.

1988-01-01

A mutational analysis of the eukaryotic elongation factor EF-1α indicates that this protein functions to limit the frequency of errors during genetic code translation. We found that both amino acid misincorporation and reading frame errors are controlled by EF-1α. In order to examine the function of this protein, the TEF2 gene, which encodes EF-1α in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine. Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations. DNA sequence analysis identified eight different sites in the EF-1α protein that elevate the frequency of mistranslation when mutated. These sites are located in two different regions of the protein. Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations. These mutations may effect mistranslation by altering the binding or hydrolysis of GTP. Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations. These mutations may alter the binding of aminoacyl-tRNA by EF-1α. The identification of frameshift and nonsense suppressor mutations in EF-1α indicates a role for this protein in limiting amino acid misincorporation and reading frame errors. We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms. PMID:3066688

Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.

PubMed

Stogios, Peter J; Shakya, Tushar; Evdokimova, Elena; Savchenko, Alexei; Wright, Gerard D

2011-01-21

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stogios, Peter J.; Shakya, Tushar; Evdokimova, Elena

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 {angstrom} resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity,more » indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2{double_prime}) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.« less

Regulating the Regulator: Post-Translational Modification of Ras

PubMed Central

Ahearn, Ian M.; Haigis, Kevin; Bar-Sagi, Dafna; Philips, Mark R.

2013-01-01

Ras proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on Ras is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which regulate the activation state of Ras without covalently modifying it. In contrast, post-translational modifications (PTMs) of Ras proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important Ras PTMs include the constitutive and irreversible remodelling of its C-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications including phosphorylation, peptidyl-proly isomerisation, mono- and di-ubiquitination, nitrosylation, ADP ribosylation and glucosylation. PMID:22189424

Study Illuminates K-Ras4B Activation, Which May Help Predict Drug Resistance | Poster

Cancer.gov

Until recently, researchers studying RAS, a family of proteins involved in transmitting signals within cells, believed that the exchange of guanosine 5’-diphosphate (GDP) by guanosine triphosphate (GTP) was sufficient to activate the protein. Once activated, RAS can cause unintended and overactive signaling in cells, which can lead to cell division and, ultimately, cancer.

EF-1 alpha is a target site for an inhibitory effect of quercetin in the peptide elongation process.

PubMed

Marcinkiewicz, C; Gałasiński, W; Gindzieński, A

1995-01-01

The effect of quercetin (3,3',4',5,7-pentahydroxyflavone) on the polypeptide elongation system isolated from rat liver cells, was investigated. Quercetin inhibited [14C]leucine incorporation into proteins in vitro and the inhibitory effect is being directed towards the elongation factor eEF-1, but not to eEF-2 and ribosomes. Quercetin was found to form a complex with EF-1 alpha, which was inactive in GTP-dependent binding to ribosomes. It can be suggested that quercetin can block the total or the part of the domain of EF-1 alpha structure that is responsible for formation of the ternary complex EF-1 alpha-GTP-[14C]Phe-tRNA and therefore preventing formation of the quaternary complex with ribosomes.

Rab2 promotes autophagic and endocytic lysosomal degradation

PubMed Central

Boda, Attila; Glatz, Gábor; Zobel, Martina; Bisi, Sara; Hegedűs, Krisztina; Scita, Giorgio

2017-01-01

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster. We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes. PMID:28483915

Fragment-Based Approaches to Enhance GTP Competitive KRAS G12C Inhibitors

DTIC Science & Technology

During the current period we completed work on a series of guanine nucleotide mimetics and published results. As part of this we developed and...reported a novel method of measuring small molecule binding to KRAS G12C active site. We also published 2 additional manuscripts about KRAS G12C directed

The Rapid Detection of Single Bacterial Cells by Deep UV Micro Raman Spectroscopy.

DTIC Science & Technology

1992-04-01

Saltzman and C.T. Gregg, Appl. Environ. Microbiol. 44, 1081 (1982). 14. D.’. Mc Greggor, W.K. Grace and G.C. Salzman, in "Rapid Methods and...was used by Dr. Marcus Peter of the Dana -Farber Cancer Institute. During that period he came to our laboratories weekly to study GTP- binding

A molecular network for the transport of the TI-VAMP/VAMP7 vesicles from cell center to periphery.

PubMed

Burgo, Andrea; Proux-Gillardeaux, Véronique; Sotirakis, Emmanuel; Bun, Philippe; Casano, Alessandra; Verraes, Agathe; Liem, Ronald K H; Formstecher, Etienne; Coppey-Moisan, Maïté; Galli, Thierry

2012-07-17

The compartmental organization of eukaryotic cells is maintained dynamically by vesicular trafficking. SNARE proteins play a crucial role in intracellular membrane fusion and need to be targeted to their proper donor or acceptor membrane. The molecular mechanisms that allow for the secretory vesicles carrying the v-SNARE TI-VAMP/VAMP7 to leave the cell center, load onto microtubules, and reach the periphery to mediate exocytosis are largely unknown. Here, we show that the TI-VAMP/VAMP7 partner Varp, a Rab21 guanine nucleotide exchange factor, interacts with GolginA4 and the kinesin 1 Kif5A. Activated Rab21-GTP in turn binds to MACF1, an actin and microtubule regulator, which is itself a partner of GolginA4. These components are required for directed movement of TI-VAMP/VAMP7 vesicles from the cell center to the cell periphery. The molecular mechanisms uncovered here suggest an integrated view of the transport of vesicles carrying a specific v-SNARE toward the cell surface. Copyright © 2012 Elsevier Inc. All rights reserved.

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains.

PubMed

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS.

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

PubMed Central

Siu, Fai Y.; Spanggord, Richard J.; Doudna, Jennifer A.

2007-01-01

The signal recognition particle (SRP) cotranslationally targets proteins to cell membranes by coordinated binding and release of ribosome-associated nascent polypeptides and a membrane-associated SRP receptor. GTP uptake and hydrolysis by the SRP-receptor complex govern this targeting cycle. Because no GTPase-activating proteins (GAPs) are known for the SRP and SRP receptor GTPases, however, it has been unclear whether and how GTP hydrolysis is stimulated during protein trafficking in vivo. Using both biochemical and genetic experiments, we show here that SRP RNA enhances GTPase activity of the SRP–receptor complex above a critical threshold required for cell viability. Furthermore, this stimulation is a property of the SRP RNA tetraloop. SRP RNA tetraloop mutants that confer defective growth phenotypes can assemble into SRP–receptor complexes, but fail to stimulate GTP hydrolysis in these complexes in vitro. Tethered hydroxyl radical probing data reveal that specific positioning of the RNA tetraloop within the SRP–receptor complex is required to stimulate GTPase activity to a level sufficient to support cell growth. These results explain why no external GAP is needed and why the phylogenetically conserved SRP RNA tetraloop is required in vivo. PMID:17164479

Characterization of a plasma membrane-associated prenylcysteine-directed alpha carboxyl methyltransferase in human neutrophils.

PubMed

Pillinger, M H; Volker, C; Stock, J B; Weissmann, G; Philips, M R

1994-01-14

Signal transduction in human neutrophils requires prenylcysteine-directed carboxyl methylation of ras-related low molecular weight GTP-binding proteins. We now report the subcellular localization and characterization of a neutrophil prenylcysteine alpha carboxyl methyltransferase. The highest carboxyl methyltransferase activity copurified with biotinylated neutrophil surface membranes, supporting a plasma membrane localization of the enzyme. Neutrophil nuclear fractions contained little or no methyltransferase activity. Methyltransferase activity was detergent-sensitive but could be reconstituted by removal of detergent in the presence of phosphatidyl choline and an anionic phospholipid. N-Acetyl-S-trans,trans-farnesyl-L-cysteine (AFC) and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine (AGGC) were effective substrates for neutrophil prenylcysteine-directed methyltransferase; Vmax values for AFC and AGGC (16.4 and 22.1 pmol of methylated/mg protein/min, respectively) are among the highest yet reported. Although both GTP gamma S and the chemoattractant fMet-Leu-Phe stimulated methylation of ras-related proteins, neither affected methylation of AFC. These data suggest that neutrophil plasma membranes contain a phospholipid-dependent, prenylcysteine-directed carboxyl methyltransferase of relatively high specific activity that modifies ras-related protein substrates in the GTP-bound, activated state.

Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.

PubMed

Duan, Zhiqiang; Xu, Haixu; Ji, Xinqin; Zhao, Jiafu; Xu, Houqiang; Hu, Yan; Deng, Shanshan; Hu, Shunlin; Liu, Xiufan

2018-12-31

The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336-433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

Kinesin-1 and mitochondrial motility control by discrimination of structurally equivalent but distinct subdomains in Ran-GTP-binding domains of Ran-binding protein 2.

PubMed

Patil, Hemangi; Cho, Kyoung-in; Lee, James; Yang, Yi; Orry, Andrew; Ferreira, Paulo A

2013-03-27

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein-protein and protein-phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD(n = 1-4)) with close structural homology to the PH domain of Bruton's tyrosine kinase. The RBD2, kinesin-binding domain (KBD) and RBD3 comprise a tripartite domain (R2KR3) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R2KR3 of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure-function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260,000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD2 and RBD3 towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD2 and RBD3 exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R2KR3 stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R2KR3 is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD2 and RBD3, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation*

PubMed Central

Kistler, Samantha; George, Samuel D.; Kuhlmann, Nora; Garvey, Leslie; Huynh, Minh; Bagni, Rachel K.; Lammers, Michael; Der, Channing J.; Campbell, Sharon L.

2017-01-01

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity. PMID:28154176

DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting regions and the active site.

PubMed

Kearney, Bradley M; Johnson, Christian W; Roberts, Daniel M; Swartz, Paul; Mattos, Carla

2014-02-06

Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras. Copyright © 2013 Elsevier Ltd. All rights reserved.

Assessment of the Activation of Rho Family GTP-Binding Proteins in Breast Cancer Cells and Specimens

DTIC Science & Technology

2001-08-01

lymphopenia Vav2 GEF for Rho, Rac, and Cdc42 proto-oncogene product; NM009500 Vav3 GEF for Rho and Rac proto-oncogene product; NM020505 hPEM -2 GEF for...junctions, and desmosomes play a fundamental role in maintaining the polarized phenotype and vectorial transport functions of epithelial cells. The tight

A molecular dynamics simulation study decodes the Zika virus NS5 methyltransferase bound to SAH and RNA analogue.

PubMed

Chuang, Chih-Hung; Chiou, Shean-Jaw; Cheng, Tian-Lu; Wang, Yeng-Tseng

2018-04-20

Since 2015, widespread Zika virus outbreaks in Central and South America have caused increases in microcephaly cases, and this acute problem requires urgent attention. We employed molecular dynamics and Gaussian accelerated molecular dynamics techniques to investigate the structure of Zika NS5 protein with S-adenosyl-L-homocysteine (SAH) and an RNA analogue, namely 7-methylguanosine 5'-triphosphate (m7GTP). For the binding motif of Zika virus NS5 protein and SAH, we suggest that the four Zika NS5 substructures (residue orders: 101-112, 54-86, 127-136 and 146-161) and the residues (Ser56, Gly81, Arg84, Trp87, Thr104, Gly106, Gly107, His110, Asp146, Ile147, and Gly148) might be responsible for the selectivity of the new Zika virus drugs. For the binding motif of Zika NS5 protein and m7GTP, we suggest that the three Zika NS5 substructures (residue orders: 11-31, 146-161 and 207-218) and the residues (Asn17, Phe24, Lys28, Lys29, Ser150, Arg213, and Ser215) might be responsible for the selectivity of the new Zika virus drugs.

Characterization of a RacGTPase up-regulated in the large yellow croaker Pseudosciaena crocea immunity.

PubMed

Han, Fang; Wang, Xiaoqing; Yang, Qilian; Cai, Mingyi; Wang, Zhi Yong

2011-02-01

The Rac proteins are members of the Rho family of small G proteins and are implicated in the regulation of several pathways, including those leading to cytoskeleton reorganization, gene expression, cell proliferation, cell adhesion and cell migration and survival. In this investigation, a Rac gene (named as LycRac gene) was obtained from the large yellow croaker and it was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified fusion protein (GST-LycRac). Moreover, the GTP-binding assay showed that the LycRac protein had GTP-binding activity. The LycRac gene was ubiquitously transcribed and expressed in 9 tissues. Quantitative real-time RT-PCR and Western blot analysis revealed the highest expression in gill and the weakest expression in spleen. Time-course analysis revealed that LycRac expression was obviously up-regulated in blood, spleen and liver after immunization with polyinosinic polycytidynic acid (poly I:C), formalin-inactive Gram-negative bacterium Vibrio parahemolyticus and bacterial lipopolysaccharides (LPS). These results suggested that LycRac protein might play an important role in the immune response against microorganisms in large yellow croaker. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

A fraction of Crm1 locates at centrosomes by its CRIME domain and regulates the centrosomal localization of pericentrin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Qinying; Jiang, Qing; Zhang, Chuanmao, E-mail: zhangcm@pku.edu.cn

Crm1 plays a role in exporting proteins containing nuclear export signals (NESs) from the nucleus to the cytoplasm. Some proteins that are capable of interacting with Ran/Crm1 were reported to be localized at centrosomes and to function as centrosome checkpoints. But it remains unclear how Crm1 locates at centrosomes. In this study, we found that a fraction of Crm1 is located at centrosomes through its N-terminal CRM1, importin {beta} etc. (CRIME) domain, which is responsible for interacting with RanGTP, suggesting that Crm1 might target to centrosomes through binding centrosomal RanGTP. Moreover, overexpression of the CRIME domain, which is free ofmore » NES binding domain, resulted in the dissociation of pericentrin and {gamma}-tubulin complex from centrosomes and the disruption of microtubule nucleation. Deficiency of Crm1 provoked by RNAi also decreased the spindle poles localization of pericentrin and {gamma}-tubulin complex, coupled with mitotic defects. Since pericentrin was sensitive to Crm1 specific inhibitor leptomycin B, we propose that the centrosomal Crm1 might interact with pericentrin and regulate the localization and function of pericentrin at centrosomes.« less

Complete kinetic mechanism for recycling of the bacterial ribosome

PubMed Central

Borg, Anneli; Pavlov, Michael

2016-01-01

How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec−1, is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec−1, are discussed in the perspective of rapidly growing bacterial cells. PMID:26527791

GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

NASA Astrophysics Data System (ADS)

Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

1992-11-01

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[β-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[γ-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[β-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

Carbon-Ion Irradiation Suppresses Migration and Invasiveness of Human Pancreatic Carcinoma Cells MIAPaCa-2 via Rac1 and RhoA Degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujita, Mayumi; Imadome, Kaori; Shoji, Yoshimi

2015-09-01

Purpose: To investigate the mechanisms underlying the inhibition of cancer cell migration and invasion by carbon (C)-ion irradiation. Methods and Materials: Human pancreatic cancer cells MIAPaCa-2, AsPC-1, and BxPC-3 were treated by x-ray (4 Gy) or C-ion (0.5, 1, 2, or 4 Gy) irradiation, and their migration and invasion were assessed 2 days later. The levels of guanosine triphosphate (GTP)-bound Rac1 and RhoA were determined by the active GTPase pull-down assay with or without a proteasome inhibitor, and the binding of E3 ubiquitin ligase to GTP-bound Rac1 was examined by immunoprecipitation. Results: Carbon-ion irradiation reduced the levels of GTP-bound Rac1 and RhoA, 2more » major regulators of cell motility, in MIAPaCa-2 cells and GTP-bound Rac1 in AsPC-1 and BxPC-3 cells. Proteasome inhibition reversed the effect, indicating that C-ion irradiation induced Rac1 and RhoA degradation via the ubiquitin (Ub)-proteasome pathway. E3 Ub ligase X-linked inhibitor of apoptosis protein (XIAP), which directly targets Rac1, was selectively induced in C-ion–irradiated MIAPaCa-2 cells and coprecipitated with GTP-bound Rac1 in C-ion–irradiated cells, which was associated with Rac1 ubiquitination. Cell migration and invasion reduced by C-ion radiation were restored by short interfering RNA–mediated XIAP knockdown, indicating that XIAP is involved in C-ion–induced inhibition of cell motility. Conclusion: In contrast to x-ray irradiation, C-ion treatment inhibited the activity of Rac1 and RhoA in MIAPaCa-2 cells and Rac1 in AsPC-1 and BxPC-3 cells via Ub-mediated proteasomal degradation, thereby blocking the motility of these pancreatic cancer cells.« less

Direct evidence of an elongation factor-Tu/Ts·GTP·Aminoacyl-tRNA quaternary complex.

PubMed

Burnett, Benjamin J; Altman, Roger B; Ferguson, Angelica; Wasserman, Michael R; Zhou, Zhou; Blanchard, Scott C

2014-08-22

During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNA(Phe) ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917-13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNA(aa). Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Rho-guanine nucleotide exchange factors during development

PubMed Central

Mulinari, Shai

2010-01-01

The development of multicellular organisms is associated with extensive rearrangements of tissues and cell sheets. The driving force for these rearrangements is generated mostly by the actin cytoskeleton. In order to permit the reproducible development of a specific body plan, dynamic reorganization of the actin cytoskeleton must be precisely coordinated in space and time. GTP-exchange factors that activate small GTPases of the Rho family play an important role in this process. Here we review the role of this class of cytoskeletal regulators during important developmental processes such as epithelial morphogenesis, cytokinesis, cell migration, cell polarity, neuronal growth cone extension and phagocytosis in different model systems. PMID:21686118

Assessment of the Activation State of Rho Family GTP-Binding Proteins in Breast Cancer Cells and Specimens

DTIC Science & Technology

2004-08-01

AF038388 Vav3 GEF for Rho and Rac Proto-oncogene product NM020505 hPEM -2 GEF for Cdc42 Predominantly expressed in brain AB007884 GEF-H1 GEF for Rac and Rho...fence tions, and desmosomes play a fundamental role in maintain- function). A functional tight junction is crucial to maintain ing the polarized phenotype

TIF-IA and Ebp1 regulate RNA synthesis in T cells.

PubMed

Saudemont, Aurore

2015-04-16

In this issue of Blood, Nguyen et al show that mycophenolic acid (MPA) induces GTP depletion, which inhibits the function of transcription initiation factor I (TIF-IA) and impacts the interaction of TIF-IA with ErbB3-binding protein 1 (Ebp1), a key in regulating proliferating cell nuclear antigen (PCNA) expression and ribosomal RNA (rRNA) synthesis in T cells during activation.

EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

PubMed Central

Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

2013-01-01

The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

Effects of nucleotides on [3H]bradykinin binding in guinea pig: further evidence for multiple B2 receptor subtypes.

PubMed

Seguin, L; Widdowson, P S

1993-02-01

We have suggested recently the existence of three subtypes of B2 bradykinin receptors in tissues of guinea pigs. We have classified these B2 bradykinin receptors into B2a, B2b, and B2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [3H]bradykinin to the B2 subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B2a and B2b subtypes, specific [3H]bradykinin binding was reduced with GDP (100 microM) and the nonmetabolized analogue of GTP, guanyl-5'-yl-imidodiphosphate (GppNHp; 100 microM). Competition studies with bradykinin and with [Hyp3]bradykinin, which shows approximately 20-fold greater selectivity for the B2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 microM). The competition experiments demonstrated that binding to the B2a subtype, which has higher affinity for [Hyp3]bradykinin and bradykinin than the B2b subtype, was lost in the presence of GppNHp, whereas binding to the B2b subtype was unaffected. In contrast, GppNHp (100 microM) and GDP (100 microM) failed to alter specific [3H]bradykinin binding to B2b and B2c subtypes in lung. [3H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 microM each). Based on this evidence, we suggest that the B2a bradykinin subtype is coupled to G proteins. The B2b and B2c subtypes are either not coupled to G proteins, or may be coupled to the Go-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and biochemical analysis of Escherichia coli ObgE, a central regulator of bacterial persistence.

PubMed

Gkekas, Sotirios; Singh, Ranjan Kumar; Shkumatov, Alexander V; Messens, Joris; Fauvart, Maarten; Verstraeten, Natalie; Michiels, Jan; Versées, Wim

2017-04-07

The Obg protein family belongs to the TRAFAC (translation factor) class of P-loop GTPases and is conserved from bacteria to eukaryotes. Essential roles in many different cellular processes have been suggested for the Obg protein from Escherichia coli (ObgE), and we recently showed that it is a central regulator of bacterial persistence. Here, we report the first crystal structure of ObgE at 1.85-Å resolution in the GDP-bound state, showing the characteristic N-terminal domain and a central G domain that are common to all Obg proteins. ObgE also contains an intrinsically disordered C-terminal domain, and we show here that this domain specifically contributed to GTP binding, whereas it did not influence GDP binding or GTP hydrolysis. Biophysical analysis, using small angle X-ray scattering and multi-angle light scattering experiments, revealed that ObgE is a monomer in solution, regardless of the bound nucleotide. In contrast to recent suggestions, our biochemical analyses further indicate that ObgE is neither activated by K + ions nor by homodimerization. However, the ObgE GTPase activity was stimulated upon binding to the ribosome, confirming the ribosome-dependent GTPase activity of the Obg family. Combined, our data represent an important step toward further unraveling the detailed molecular mechanism of ObgE, which might pave the way to further studies into how this GTPase regulates bacterial physiology, including persistence. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Isolation and properties of the subunit form EF-1C of elongation factor 1 from Guerin epithelioma cells.

PubMed

Marcinkiewicz, C; Gałasiński, W

1993-01-01

EF-1C is a component of the aggregate EF-1B, consisting of the subunit forms EF-1A.EF-1C; it was isolated by dissociation of this aggregate in the presence of GTP. The subunit form EF-1C stimulates binding of aminoacyl-tRNA to ribosomes, catalysed by EF-1A, similarly as EF-1 beta gamma which stimulates the activity of EF-1 in other eukaryotic cells. EF-1C in the presence of 6 M urea was separated into two polypeptides. Polypeptide of molecular mass 32,000 Da is responsible for regeneration of the EF-1A.GTP active complex. Thermal sensitivity of EF-1A was much higher than that of EF-1B, thus a protective role of EF-1C in the EF-1A.EF-1C complex is suggested.

Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence*

PubMed Central

Tsujii, Akira; Miyamoto, Yoichi; Moriyama, Tetsuji; Tsuchiya, Yuko; Obuse, Chikashi; Mizuguchi, Kenji; Oka, Masahiro; Yoneda, Yoshihiro

2015-01-01

Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/β-mediated nuclear import. RBBP4 accelerates the release of importin β1 from importin α via competitive binding to the importin β-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/β-mediated nuclear import. We showed that the importin α/β pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin β1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence. PMID:26491019

Multiple sup 3 H-oxytocin binding sites in rat myometrial plasma membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crankshaw, D.; Gaspar, V.; Pliska, V.

1990-01-01

The affinity spectrum method has been used to analyse binding isotherms for {sup 3}H-oxytocin to rat myometrial plasma membranes. Three populations of binding sites with dissociation constants (Kd) of 0.6-1.5 x 10(-9), 0.4-1.0 x 10(-7) and 7 x 10(-6) mol/l were identified and their existence verified by cluster analysis based on similarities between Kd, binding capacity and Hill coefficient. When experimental values were compared to theoretical curves constructed using the estimated binding parameters, good fits were obtained. Binding parameters obtained by this method were not influenced by the presence of GTP gamma S (guanosine-5'-O-3-thiotriphosphate) in the incubation medium. The bindingmore » parameters agree reasonably well with those found in uterine cells, they support the existence of a medium affinity site and may allow for an explanation of some of the discrepancies between binding and response in this system.« less

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation.

PubMed

Yin, Guowei; Kistler, Samantha; George, Samuel D; Kuhlmann, Nora; Garvey, Leslie; Huynh, Minh; Bagni, Rachel K; Lammers, Michael; Der, Channing J; Campbell, Sharon L

2017-03-17

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteins involved in neuronal differentiation of neuroblastoma cell line N1E-115.

PubMed

Oh, Ji-Eun; Freilinger, Angelika; Gelpi, Ellen; Pollak, Arnold; Hengstschläger, Markus; Lubec, Gert

2007-06-01

Neuronal differentiation (ND) represents a well-defined phenomenon in biological terms but proteins involved have not been studied systematically. We therefore aimed to study ND by retinoic acid (RA) in a widely used neuroblastoma cell line by comparative proteomics. The ND was induced in the N1E-115 cell line by serum deprivation and RA treatment. Undifferentiated cells and cells undergoing serum deprivation served as controls. Protein extracts were run on 2-DE followed by MALDI-TOF or MALDI-TOF-TOF analysis. Quantification was carried out using specific software and stringent statistical analysis was performed. Tubulin beta 5, cat eye syndrome critical region protein 5 homolog, putative GTP-binding protein PTD004 homolog, and the metabolic proteins glyceraldehyde-3-phosphate dehydrogenase and transketolase were differentially regulated. Differential protein levels of cytoskeleton proteins including tubulins and metabolic proteins have been reported to be regulated by ND. Herein, specific signaling differences as reflected by putative GTP-binding protein PTD004 changes in differentiated cells are shown and a possible role for the Cat eye syndrome critical region protein 5 homolog is proposed. The protein disulfide isomerase associated 3 protein fits the already proposed findings of chaperon regulation by ND. The study forms the molecular basis for further evaluation of the functional roles of the differentially expressed proteins in ND.

Yeast Los1p Has Properties of an Exportin-Like Nucleocytoplasmic Transport Factor for tRNA

PubMed Central

Hellmuth, Klaus; Lau, Denise M.; Bischoff, F. Ralf; Künzler, Markus; Hurt, Ed; Simos, George

1998-01-01

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-β-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast. PMID:9774653

Tumours with class 3 BRAF mutants are sensitive to the inhibition of activated RAS.

PubMed

Yao, Zhan; Yaeger, Rona; Rodrik-Outmezguine, Vanessa S; Tao, Anthony; Torres, Neilawattie M; Chang, Matthew T; Drosten, Matthias; Zhao, Huiyong; Cecchi, Fabiola; Hembrough, Todd; Michels, Judith; Baumert, Hervé; Miles, Linde; Campbell, Naomi M; de Stanchina, Elisa; Solit, David B; Barbacid, Mariano; Taylor, Barry S; Rosen, Neal

2017-08-10

Approximately 200 BRAF mutant alleles have been identified in human tumours. Activating BRAF mutants cause feedback inhibition of GTP-bound RAS, are RAS-independent and signal either as active monomers (class 1) or constitutively active dimers (class 2). Here we characterize a third class of BRAF mutants-those that have impaired kinase activity or are kinase-dead. These mutants are sensitive to ERK-mediated feedback and their activation of signalling is RAS-dependent. The mutants bind more tightly than wild-type BRAF to RAS-GTP, and their binding to and activation of wild-type CRAF is enhanced, leading to increased ERK signalling. The model suggests that dysregulation of signalling by these mutants in tumours requires coexistent mechanisms for maintaining RAS activation despite ERK-dependent feedback. Consistent with this hypothesis, melanomas with these class 3 BRAF mutations also harbour RAS mutations or NF1 deletions. By contrast, in lung and colorectal cancers with class 3 BRAF mutants, RAS is typically activated by receptor tyrosine kinase signalling. These tumours are sensitive to the inhibition of RAS activation by inhibitors of receptor tyrosine kinases. We have thus defined three distinct functional classes of BRAF mutants in human tumours. The mutants activate ERK signalling by different mechanisms that dictate their sensitivity to therapeutic inhibitors of the pathway.

Developmentally regulated GTP-binding protein 2 depletion leads to mitochondrial dysfunction through downregulation of dynamin-related protein 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vo, Mai-Tram; Ko, Myoung Seok; Lee, Unn Hwa

Mitochondrial dynamics, including constant fusion and fission, play critical roles in maintaining mitochondrial morphology and function. Here, we report that developmentally regulated GTP-binding protein 2 (DRG2) regulates mitochondrial morphology by modulating the expression of the mitochondrial fission gene dynamin-related protein 1 (Drp1). shRNA-mediated silencing of DRG2 induced mitochondrial swelling, whereas expression of an shRNA-resistant version of DRG2 decreased mitochondrial swelling in DRG2-depleted cells. Analysis of the expression levels of genes involved in mitochondrial fusion and fission revealed that DRG2 depletion significantly decreased the level of Drp1. Overexpression of Drp1 rescued the defect in mitochondrial morphology induced by DRG2 depletion. DRG2more » depletion reduced the mitochondrial membrane potential, oxygen consumption rate (OCR), and amount of mitochondrial DNA (mtDNA), whereas it increased reactive oxygen species (ROS) production and apoptosis. Taken together, our data demonstrate that DRG2 acts as a regulator of mitochondrial fission by controlling the expression of Drp1. - Highlights: • DRG2 depletion increased mitochondrial swelling. • DRG2 depletion inhibited the expression of Drp1. • Overexpression of DRG2 or Drp1 rescued mitochondrial shape in DRG2 depleted cells. • DRG2 depletion induced mitochondrial dysfunction.« less

Transfer RNA mimicry among tymoviral genomic RNAs ranges from highly efficient to vestigial.

PubMed

Dreher, T W; Goodwin, J B

1998-10-01

Three tRNA-associated properties of a representative set of tymoviral RNAs have been quantitatively assessed using higher plant (wheat germ) proteins: aminoacylation, EF-1alpha*GTP binding, and 3'-adenylation of 3'-CC forms of the RNAs by CTP, ATP:tRNA nucleotidyltransferase. The RNAs fall into three classes differing in the extent of tRNA mimicry. Turnip yellow mosaic (TYMV) and kennedya yellow mosaic virus RNAs had activities in all three properties similar to those of a higher plant tRNAValtranscript, and thus are remarkable tRNA mimics. Although the isolated approximately 83 nt long tRNA-like structures showed high activity in these assays, in the case of TYMV, the 6318 nt long TYMV RNA was an even better substrate for valylation. Eggplant mosaic virus RNA, which has a differently constructed acceptor stem pseudoknot, differed from the above tymoviral RNAs in binding more weakly to EF-1alpha*GTP. Erysimum latent virus RNA, which lacks an identifiable anticodon domain, could not be valylated and had very low 3'-adenylation activity. The range of tRNA mimicry within the tymovirus genus thus ranges from extremely highly developed to minimal. The implications on the role of the tRNA mimicry in viral biology are discussed.

ADP-Ribosylation Factor 6 Regulates a Novel Plasma Membrane Recycling Pathway

PubMed Central

Radhakrishna, Harish; Donaldson, Julie G.

1997-01-01

ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor α subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton. PMID:9314528

On the mechanism of aluminum ion-induced neurotoxicity: The effects of aluminum species on G-protein-mediated processes and on drug interactions with the N-methyl-D-aspartate modulated ionophore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hubbard, C.M.

1989-01-01

To establish what effects Al{sup 3+} may have on G-protein mediate signal transduction, the effects of Al{sup 3+} on the signal-coupling G-protein from retinal rod outer segments (G{sub t} or transducin) have been investigated as a model for the effects of Al{sup 3+} on signal transduction by G-proteins in general. In this investigation, we have studied the effects of Al{sup 3+} on the isolated, light-dependent rhodopsin catalyzed GTP-GDP exchange on G{sub t}; the light-dependent GTPase activity of G{sub t}; the light-independent cGMP hydrolysis by PDE; and the light activated, rhodopsin catalyzed, cGMP hydrolysis by PDE in vitro. To determine themore » effects of two defined species of aluminum on N-methyl-D-aspartic acid (NMDA) receptor-channel modulation we utilized a specific radioligand binding assay. This allowed us to compare the effects of aluminum to other metal ions on specific ({sup 3}H)MK-801 binding to the NMDA receptor-channel complex. This complex is involved in long-term potentiation, which is currently being investigated as the mechanism by which learning and memory occur and has been implicated in the pathology of Alzheimer's disease. We have investigated the effects of two different species of aluminum, as well as Ca{sup 2+}, Zn{sup 2+}, Mg{sup 2+}, and Li{sup +} on the specific binding of ({sup 3}H)MK-801 to the NMDA receptor-channel complex under depolarized conditions.« less

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains

PubMed Central

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS. PMID:27924253

Acting on Actin: Rac and Rho Played by Yersinia.

PubMed

Aepfelbacher, Martin; Wolters, Manuel

2017-01-01

Pathogenic bacteria of the genus Yersinia include Y. pestis-the agent of plaque-and two enteropathogens, Y. enterocolitica, and Y. pseudotuberculosis. These pathogens have developed an array of virulence factors aimed at manipulating Rho GTP-binding proteins and the actin cytoskeleton in host cells to cross the intestinal barrier and suppress the immune system. Yersinia virulence factors include outer membrane proteins triggering cell invasion by binding to integrins, effector proteins injected into host cells to manipulate Rho protein functions and a Rho protein-activating exotoxin. Here, we present an overview of how Yersinia and host factors are integrated in a regulatory network that orchestrates the subversion of host defense.

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Negishi, M.; Ito, S.; Yokohama, H.

1988-05-15

Prostaglandin E/sub 2/ (PEG/sub 2/) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its ..cap alpha.. subunit from two known pertussismore » toxin substrate G-proteins (G/sub i/ and G/sub 0/) purified from bovine brain. The molecular weight of the ..cap alpha.. subunit was 40,000, which is between those of G/sub i/ and G/sub 0/. The purified protein was also distinguished immunologically from G/sub i/ and G/sub 0/ and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles resulted in a remarkable restoration of (/sup 3/H)PGE/sub 2/ binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G/sub 0/ in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.« less

Characterization of a ( sub 3 H)-5-hydroxtyryptamine binding site in rabbit caudate nucleus that differs from the 5-HT sub 1A , 5-HT sub 1B , 5-HT sub 1C and 5-HT sub 1D subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Wencheng; Nelson, D.L.

1989-01-01

({sup 3}H)5-HT binding sites were analyzed in membranes prepared from the rabbit caudate nucleus (CN). ({sup 3}H)5-HT labeled both 5-HT{sub 1A} and 5-HT{sub 1C} recognition sites, defined by nanomolar affinity for 8-OH-DPAT and mesulergine respectively; however, these represented only a fraction of total specific ({sup 3}H)5-HT binding. Saturation experiments of ({sup 3}H)5-HT binding in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine to block 5-HT{sub 1A} and 5-HT{sub 1C} sites revealed that non-5-HT{sub 1A}/non-5-HT{sub 1C} sites represented about 60% of the total 5-HT{sub 1} sites and that they exhibited saturable, high affinity, and homogeneous binding. The pharmacological profilemore » of the non-5-HT{sub 1A}/non-5-HT{sub 1C} sites (designated 5-HT{sub 1R}) also differed from that of 5-HT{sub 1B} and 5-HT{sub 2} sites, but was similar to that of the 5-HT{sub 1D} site. However, significant differences existed between the 5-HT{sub 1D} and 5-HT{sub 1B} sites for their K{sub i} values for spiperone, spirilene, metergoline, and methiothepin. The study of modulatory agents also showed differences between the 5-HT{sub 1R} and 5-HT{sub 1D} sites. In addition, calcium enhanced the effects of GTP on the 5-HT{sub 1R} sites, whereas calcium inhibited the GTP effect on the 5-HT{sub 1D} sites.« less

Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, from Bacillus subtilis.

PubMed

Levdikov, Vladimir M; Blagova, Elena; Young, Vicki L; Belitsky, Boris R; Lebedev, Andrey; Sonenshein, Abraham L; Wilkinson, Anthony J

2017-02-17

CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (c G MP-stimulated phosphodiesterases, a denylate cyclases, F hlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn α-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens

PubMed Central

Priya, Rashmi; Verma, Suzie; Kovacs, Eva M.; Jiang, Kai; Brown, Nicholas H.; Akhmanova, Anna; Stehbens, Samantha J.; Yap, Alpha S.

2014-01-01

Summary The biological impact of Rho depends critically on the precise subcellular localization of its active, GTP-loaded form. The spatio-temporal balance between molecules that promote nucleotide exchange or GTP hydrolysis can potentially determine the sites of Rho signalling. But how these activities may be coordinated is poorly understood. We now report a molecular pathway that achieves exactly this coordination at the epithelial zonula adherens. We identify an extramitotic activity of the centralspindlin complex, better understood as a cytokinetic regulator, which localises to the zonula adherens during interphase by interacting with the cadherin-associated protein, α-catenin. Centralspindlin recruits the Rho GEF, Ect2, to the zonula adherens to activate Rho and support junctional integrity through myosin IIA. Centralspindlin also inhibits the junctional localisation of p190RhoGAP B, which can inactivate Rho. Thus, a conserved molecular ensemble that governs Rho activation during cytokinesis is utilized in interphase cells to control the Rho GTPase cycle at the zonula adherens. PMID:22750944

Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination*

PubMed Central

Baker, Rachael; Wilkerson, Emily M.; Sumita, Kazutaka; Isom, Daniel G.; Sasaki, Atsuo T.; Dohlman, Henrik G.; Campbell, Sharon L.

2013-01-01

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation. PMID:24247240

A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTP.

PubMed

Koga, Hirohisa; Misawa, Satoru; Shibui, Tatsuro

2009-01-01

Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 microM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B(1-320)) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26 degrees C. 2009 American Institute of Chemical Engineers Biotechnol.

(1)H, (13)C, (15)N backbone and side-chain resonance assignment of Nostoc sp. C139A variant of the heme-nitric oxide/oxygen binding (H-NOX) domain.

PubMed

Alexandropoulos, Ioannis I; Argyriou, Aikaterini I; Marousis, Kostas D; Topouzis, Stavros; Papapetropoulos, Andreas; Spyroulias, Georgios A

2016-10-01

The H-NOX (Heme-nitric oxide/oxygen binding) domain is conserved across eukaryotes and bacteria. In human soluble guanylyl cyclase (sGC) the H-NOX domain functions as a sensor for the gaseous signaling agent nitric oxide (NO). sGC contains the heme-binding H-NOX domain at its N-terminus, which regulates the catalytic site contained within the C-terminal end of the enzyme catalyzing the conversion of GTP (guanosine 5'-triphosphate) to GMP (guanylyl monophosphate). Here, we present the backbone and side-chain assignments of the (1)H, (13)C and (15)N resonances of the 183-residue H-NOX domain from Nostoc sp. through solution NMR.

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

PubMed

Feriotto, G; Calza, R; Bergamini, C M; Griffin, M; Wang, Z; Beninati, S; Ferretti, V; Marzola, E; Guerrini, R; Pagnoni, A; Cavazzini, A; Casciano, F; Mischiati, C

2017-03-01

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Insight into Temperature Dependence of GTPase Activity in Human Guanylate Binding Protein-1

PubMed Central

Rahman, Safikur; Deep, Shashank; Sau, Apurba Kumar

2012-01-01

Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1. PMID:22859948

Construction of plasmid, bacterial expression, purification, and assay of dengue virus type 2 NS5 methyltransferase.

PubMed

Boonyasuppayakorn, Siwaporn; Padmanabhan, Radhakrishnan

2014-01-01

Dengue virus (DENV), a member of mosquito-borne flavivirus, causes self-limiting dengue fever as well as life-threatening dengue hemorrhagic fever and dengue shock syndrome. Its positive sense RNA genome has a cap at the 5'-end and no poly(A) tail at the 3'-end. The viral RNA encodes a single polyprotein, C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5. The polyprotein is processed into 3 structural proteins (C, prM, and E) and 7 nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). NS3 and NS5 are multifunctional enzymes performing various tasks in viral life cycle. The N-terminal domain of NS5 has distinct GTP and S-adenosylmethionine (SAM) binding sites. The role of GTP binding site is implicated in guanylyltransferase (GTase) activity of NS5. The SAM binding site is involved in both N-7 and 2'-O-methyltransferase (MTase) activities involved in formation of type I cap. The C-terminal domain of NS5 catalyzes RNA-dependent RNA polymerase (RdRp) activity involved in RNA synthesis. We describe the construction of the MTase domain of NS5 in an E. coli expression vector, purification of the enzyme, and conditions for enzymatic assays of N7- and 2'O-methyltransferase activities that yield the final type I 5'-capped RNA ((7Me)GpppA2'OMe-RNA).

Global temperature change potential of nitrogen use in agriculture: A 50-year assessment

PubMed Central

Fagodiya, R. K.; Pathak, H.; Kumar, A.; Bhatia, A.; Jain, N.

2017-01-01

Nitrogen (N) use in agriculture substantially alters global N cycle with the short- and long-term effects on global warming and climate change. It increases emission of nitrous oxide, which contributes 6.2%, while carbon dioxide and methane contribute 76% and 16%, respectively of the global warming. However, N causes cooling due to emission of NOx, which alters concentrations of tropospheric ozone and methane. NOx and NH3 also form aerosols with considerable cooling effects. We studied global temperature change potential (GTP) of N use in agriculture. The GTP due to N2O was 396.67 and 1168.32 Tg CO2e on a 20-year (GTP20) and 439.94 and 1295.78 Tg CO2e on 100-year scale (GTP100) during years 1961 and 2010, respectively. Cooling effects due to N use were 92.14 and 271.39 Tg CO2e (GTP20) and 15.21 and 44.80 Tg CO2e (GTP100) during 1961 and 2010, respectively. Net GTP20 was 369.44 and 1088.15 Tg CO2e and net GTP100 was 429.17 and 1264.06 Tg CO2e during 1961 and 2010, respectively. Thus net GTP20 is lower by 6.9% and GTP100 by 2.4% compared to the GTP considering N2O emission alone. The study shows that both warming and cooling effects should be considered to estimate the GTP of N use. PMID:28322322

Evidence for a role for the phosphotyrosine-binding domain of Shc in interleukin 2 signaling.

PubMed Central

Ravichandran, K S; Igras, V; Shoelson, S E; Fesik, S W; Burakoff, S J

1996-01-01

Stimulation via the T-cell growth factor interleukin 2 (IL-2) leads to tyrosine phosphorylation of Shc, the interaction of Shc with Grb2, and the Ras GTP/GDP exchange factor, mSOS. Shc also coprecipitates with the IL-2 receptor (IL-2R), and therefore, may link IL-2R to Ras activation. We have further characterized the Shc-IL-2R interaction and have made the following observations. (i) Among the two phosphotyrosine-interaction domains present in Shc, the phosphotyrosine-binding (PTB) domain, rather than its SH2 domain, interacts with the tyrosine-phosphorylated IL-2R beta chain. Moreover, the Shc-PTB domain binds a phosphopeptide derived from the IL-2R beta chain (corresponding to residues surrounding Y338, SCFTNQGpYFF) with high affinity. (ii) In vivo, mutant IL-2R beta chains lacking the acidic region of IL-2Rbeta (which contains Y338) fail to phosphorylate Shc. Furthermore, when wild type or mutant Shc proteins that lack the PTB domain were expressed in the IL-2-dependent CTLL-20 cell line, an intact Shc-PTB domain was required for Shc phosphorylation by the IL-2R, which provides further support for a Shc-PTB-IL-2R interaction in vivo. (iii) PTB and SH2 domains of Shc associate with different proteins in IL-2- and T-cell-receptor-stimulated lysates, suggesting that Shc, through the concurrent use of its two different phosphotyrosine-binding domains, could assemble multiple protein complexes. Taken together, our in vivo and in vitro observations suggest that the PTB domain of Shc interacts with Y338 of the IL-2R and provide evidence for a functional role for the Shc-PTB domain in IL-2 signaling. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8643566

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking

PubMed Central

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M.; Baehr, Wolfgang

2012-01-01

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d−/− mouse. PMID:22960045

Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering

PubMed Central

Blaževitš, Olga; Mideksa, Yonatan G.; Šolman, Maja; Ligabue, Alessio; Ariotti, Nicholas; Nakhaeizadeh, Hossein; Fansa, Eyad K.; Papageorgiou, Anastassios C.; Wittinghofer, Alfred; Ahmadian, Mohammad R.; Abankwa, Daniel

2016-01-01

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters. The latter activity may present an alternative mechanism for how overexpressed Gal-1 stimulates tumourigenesis. Here we revise the current model for the interaction of Gal-1 with H-ras. We show that it indirectly forms a complex with GTP-H-ras via a high-affinity interaction with the Ras binding domain (RBD) of Ras effectors. A computationally generated model of the Gal-1/C-Raf-RBD complex is validated by mutational analysis. Both cellular FRET as well as proximity ligation assay experiments confirm interaction of Gal-1 with Raf proteins in mammalian cells. Consistently, interference with H-rasG12V-effector interactions basically abolishes H-ras nanoclustering. In addition, an intact dimer interface of Gal-1 is required for it to positively regulate H-rasG12V nanoclustering, but negatively K-rasG12V nanoclustering. Our findings suggest stacked dimers of H-ras, Raf and Gal-1 as building blocks of GTP-H-ras-nanocluster at high Gal-1 levels. Based on our results the Gal-1/effector interface represents a potential drug target site in diseases with aberrant Ras signalling. PMID:27087647

Effect of green tea and Tai Chi on bone health in postmenopausal osteopenic women: a 6-month randomized placebo-controlled trial.

PubMed

Shen, C-L; Chyu, M-C; Yeh, J K; Zhang, Y; Pence, B C; Felton, C K; Brismée, J-M; Arjmandi, B H; Doctolero, S; Wang, J-S

2012-05-01

Postmenopausal women with osteopenia received green tea polyphenols (GTP) supplement and/or Tai Chi exercise for 6 months. Bone turnover biomarkers, calcium metabolism, and muscle strength were measured. This study showed that GTP supplementation and Tai Chi exercise increased bone formation biomarkers and improved bone turnover rate. Tai Chi exercise increased serum parathyroid hormone. GTP supplementation, Tai Chi exercise, and the combination of the two all improved muscle strength in postmenopausal women with osteopenia. This study evaluated the effect of GTP supplementation and Tai Chi (TC) exercise on serum markers of bone turnover (bone-specific alkaline phosphatase, BAP, and tartrate-resistant acid phosphatase, TRAP), calcium metabolism, and muscle strength in postmenopausal osteopenic women. One hundred and seventy-one postmenopausal osteopenic women were randomly assigned to four groups: (1) placebo (500 mg starch/day), (2) GTP (500 mg GTP/day), (3) placebo + TC (placebo plus TC training at 60 min/session, three sessions/week), and (4) GTP + TC (GTP plus TC training). Overnight fasting blood and urine samples were collected at baseline, 1, 3, and 6 months for biomarker analyses. Muscle strength was evaluated at baseline, 3, and 6 months. One hundred and fifty subjects completed the 6-month study. Significant increases in BAP level due to GTP intake (at 1 month) and TC (at 3 months) were observed. Significant increases in the change of BAP/TRAP ratio due to GTP (at 3 months) and TC (at 6 months) were also observed. Significant main effect of TC on the elevation in serum parathyroid hormone level was observed at 1 and 3 months. At 6 months, muscle strength significantly improved due to GTP, TC, and GTP + TC interventions. Neither GTP nor TC affected serum TRAP, serum and urinary calcium, and inorganic phosphate. In summary, GTP supplementation and TC exercise increased BAP and improved BAP/TRAP ratio. TC exercise increased serum parathyroid hormone. GTP supplementation, TC exercise, and the combination of the two all improved muscle strength in postmenopausal women with osteopenia.

Allosteric binding sites in Rab11 for potential drug candidates

PubMed Central

2018-01-01

Rab11 is an important protein subfamily in the RabGTPase family. These proteins physiologically function as key regulators of intracellular membrane trafficking processes. Pathologically, Rab11 proteins are implicated in many diseases including cancers, neurodegenerative diseases and type 2 diabetes. Although they are medically important, no previous study has found Rab11 allosteric binding sites where potential drug candidates can bind to. In this study, by employing multiple clustering approaches integrating principal component analysis, independent component analysis and locally linear embedding, we performed structural analyses of Rab11 and identified eight representative structures. Using these representatives to perform binding site mapping and virtual screening, we identified two novel binding sites in Rab11 and small molecules that can preferentially bind to different conformations of these sites with high affinities. After identifying the binding sites and the residue interaction networks in the representatives, we computationally showed that these binding sites may allosterically regulate Rab11, as these sites communicate with switch 2 region that binds to GTP/GDP. These two allosteric binding sites in Rab11 are also similar to two allosteric pockets in Ras that we discovered previously. PMID:29874286

Computational Analysis of KRAS Mutations: Implications for Different Effects on the KRAS p.G12D and p.G13D Mutations

PubMed Central

Liu, Yen-Yi; Hwang, Jenn-Kang; Barrio, Maria Jesus; Rodrigo, Maximiliano; Garcia-Toro, Enrique; Herreros-Villanueva, Marta

2013-01-01

Background The issue of whether patients diagnosed with metastatic colorectal cancer who harbor KRAS codon 13 mutations could benefit from the addition of anti-epidermal growth factor receptor therapy remains under debate. The aim of the current study was to perform computational analysis to investigate the structural implications of the underlying mutations caused by c.38G>A (p.G13D) on protein conformation. Methods Molecular dynamics (MD) simulations were performed to understand the plausible structural and dynamical implications caused by c.35G>A (p.G12D) and c.38G>A (p.G13D). The potential of mean force (PMF) simulations were carried out to determine the free energy profiles of the binding processes of GTP interacting with wild-type (WT) KRAS and its mutants (MT). Results Using MD simulations, we observed that the root mean square deviation (RMSD) increased as a function of time for the MT c.35G>A (p.G12D) and MT c.38G>A (p.G13D) when compared with the WT. We also observed that the GTP-binding pocket in the c.35G>A (p.G12D) mutant is more open than that of the WT and the c.38G>A (p.G13D) proteins. Intriguingly, the analysis of atomic fluctuations and free energy profiles revealed that the mutation of c.35G>A (p.G12D) may induce additional fluctuations in the sensitive sites (P-loop, switch I and II regions). Such fluctuations may promote instability in these protein regions and hamper GTP binding. Conclusions Taken together with the results obtained from MD and PMF simulations, the present findings implicate fluctuations at the sensitive sites (P-loop, switch I and II regions). Our findings revealed that KRAS mutations in codon 13 have similar behavior as KRAS WT. To gain a better insight into why patients with metastatic colorectal cancer (mCRC) and the KRAS c.38G>A (p.G13D) mutation appear to benefit from anti-EGFR therapy, the role of the KRAS c.38G>A (p.G13D) mutation in mCRC needs to be further investigated. PMID:23437064

Mechanisms of kinetic stabilization by the drugs paclitaxel and vinblastine

PubMed Central

Castle, Brian T.; McCubbin, Seth; Prahl, Louis S.; Bernens, Jordan N.; Sept, David; Odde, David J.

2017-01-01

Microtubule-targeting agents (MTAs), widely used as biological probes and chemotherapeutic drugs, bind directly to tubulin subunits and “kinetically stabilize” microtubules, suppressing the characteristic self-assembly process of dynamic instability. However, the molecular-level mechanisms of kinetic stabilization are unclear, and the fundamental thermodynamic and kinetic requirements for dynamic instability and its elimination by MTAs have yet to be defined. Here we integrate a computational model for microtubule assembly with nanometer-scale fluorescence microscopy measurements to identify the kinetic and thermodynamic basis of kinetic stabilization by the MTAs paclitaxel, an assembly promoter, and vinblastine, a disassembly promoter. We identify two distinct modes of kinetic stabilization in live cells, one that truly suppresses on-off kinetics, characteristic of vinblastine, and the other a “pseudo” kinetic stabilization, characteristic of paclitaxel, that nearly eliminates the energy difference between the GTP- and GDP-tubulin thermodynamic states. By either mechanism, the main effect of both MTAs is to effectively stabilize the microtubule against disassembly in the absence of a robust GTP cap. PMID:28298489

Effects of a foot bath containing green tea polyphenols on interdigital tinea pedis.

PubMed

Ikeda, Sugako; Kanoya, Yuka; Nagata, Shigeki

2013-01-01

It has been shown that green tea polyphenols (GTP) can directly kill Trichophyton in vitro; however, there are no published clinical studies that show anti-fungal activity of GTP. To identify the effects of GTP on interdigital tinea pedis in elderly patients. Ninety-four patients with interdigital tinea pedis were enrolled and were either given a lukewarm water foot bath containing GTP or placebo treatment. Effects of GTP were assessed based on changes in the size of the affected area, the rate of recurrence, microscopy findings, and overall assessment of skin changes. After 12 weeks of treatment with either GTP or placebo, a significant reduction in the size of the affected area was observed (p<0.001). There were no significant differences between the GTP or placebo groups in the size of the affected area (p=0.638), the recurrence rate (p=0.172), or the microscopy findings (p=1.000). However, the overall assessment demonstrated significant improvements (p=0.010) in the GTP group. These results show that GTP was effective in improving the symptoms of tinea pedis in comparison to only lukewarm water. Our results suggest that GTP could have anti-fungal activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

DHHC3 Contributions to Breast Cancer

DTIC Science & Technology

2014-11-01

oligosaccharide 1,2-alpha-mannosidase MAN1B1 0.021 0.572 IPI00015954 GTP-binding protein SAR1a SAR1A 0.546 0.573 IPI00011937 Peroxiredoxin-4 PRDX4...previous report that DHHC3 itself is an integral membrane protein (10). In addition, the most significant biological processes regulated by DHHC3 are cell...motion and vesicle-mediated transport (Fig. 8), two processes important for cancer cell invasion and metastasis. Figure 7. Gene ontology

The Conformational Change in Elongation Factor Tu Involves Separation of Its Domains

DOE PAGES

Lai, Jonathan; Ghaemi, Zhaleh; Luthey-Schulten, Zaida

2017-10-18

Elongation factor Tu (EF-Tu) is a highly conserved GTPase that is responsible for supplying the aminoacylated tRNA to the ribosome. Upon binding to the ribosome, EF-Tu undergoes GTP hydrolysis, which drives a major conformational change, triggering the release of aminoacylated tRNA to the ribosome. Using a combination of molecular simulation techniques, we studied the transition between the pre- and post-hydrolysis structures through two distinct pathways. Here, we show that the transition free energy is minimal along a non-intuitive pathway that involves “separation” of the GTP binding domain (domain 1) from the OB folds (domains 2 and 3), followed by domainmore » 1 rotation, and, eventually, locking the EF-Tu conformation in the post-hydrolysis state. The domain separation also leads to a slight extension of the linker connecting domain 1 to domain 2. Using docking tools and correlation-based analysis, we identified and characterized the EF-Tu conformations that release the tRNA. These calculations suggest that EF-Tu can release the tRNA before the domains separate and after domain 1 rotates by 25°. Lastly, we also examined the EF-Tu conformations in the context of the ribosome. Given the high degrees of sequence similarity with other translational GTPases, we predict a similar separation mechanism is followed.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jope, R.S.; Casebolt, T.L.; Johnson, G.V.

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with (/sup 3/H)inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of (/sup 3/H)inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly (/sup 3/H)inositol-1-phosphate. Incubation of slices with N-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with (/sup 3/H)inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slicesmore » and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4, 5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.« less

The Conformational Change in Elongation Factor Tu Involves Separation of Its Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Jonathan; Ghaemi, Zhaleh; Luthey-Schulten, Zaida

Elongation factor Tu (EF-Tu) is a highly conserved GTPase that is responsible for supplying the aminoacylated tRNA to the ribosome. Upon binding to the ribosome, EF-Tu undergoes GTP hydrolysis, which drives a major conformational change, triggering the release of aminoacylated tRNA to the ribosome. Using a combination of molecular simulation techniques, we studied the transition between the pre- and post-hydrolysis structures through two distinct pathways. Here, we show that the transition free energy is minimal along a non-intuitive pathway that involves “separation” of the GTP binding domain (domain 1) from the OB folds (domains 2 and 3), followed by domainmore » 1 rotation, and, eventually, locking the EF-Tu conformation in the post-hydrolysis state. The domain separation also leads to a slight extension of the linker connecting domain 1 to domain 2. Using docking tools and correlation-based analysis, we identified and characterized the EF-Tu conformations that release the tRNA. These calculations suggest that EF-Tu can release the tRNA before the domains separate and after domain 1 rotates by 25°. Lastly, we also examined the EF-Tu conformations in the context of the ribosome. Given the high degrees of sequence similarity with other translational GTPases, we predict a similar separation mechanism is followed.« less

The Expression Level of Septin12 Is Critical for Spermiogenesis

PubMed Central

Lin, Ying-Hung; Lin, Yung-Ming; Wang, Ya-Yun; Yu, I-Shing; Lin, Yi-Wen; Wang, Yun-Han; Wu, Ching-Ming; Pan, Hsien-An; Chao, Shin-Chih; Yen, Pauline H.; Lin, Shu-Wha; Kuo, Pao-Lin

2009-01-01

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis. PMID:19359518

Synergistic effects of green tea polyphenols and alphacalcidol on chronic inflammation-induced bone loss in female rats

PubMed Central

Yeh, J. K.; Cao, J. J.; Tatum, O. L.; Dagda, R. Y.; Wang, J.-S.

2010-01-01

Summary Studies suggest that green tea polyphenols (GTP) or alphacalcidol is promising agent for preventing bone loss. Findings that GTP supplementation plus alphacalcidol administration increased bone mass via a decrease of oxidative stress and inflammation suggest a significant role of GTP plus alphacalcidol in bone health of patients with chronic inflammation. Introduction Studies have suggested that green tea polyphenols (GTP) or alphacalcidol are promising dietary supplements for preventing bone loss in women. However, the mechanism(s) related to the possible osteo-protective role of GTP plus D3 in chronic inflammation-induced bone loss is not well understood. Methods This study evaluated bioavailability, efficacy, and related mechanisms of GTP in combination with alphacalcidol in conserving bone loss in rats with chronic inflammation. A 12-week study of 2 (no GTP vs. 0.5% GTP in drinking water) × 2 (no alphacalcidol vs. 0.05 μg/kg alphacalcidol, 5×/week) factorial design in lipopolysaccharide-administered female rats was performed. In addition, a group receiving placebo administration was used to compare with a group receiving lipopolysaccharide administration only to evaluate the effect of lipopolysaccharide. Results Lipopolysaccharide administration resulted in lower values for bone mass, but higher values for serum tartrate-resistant acid phosphatase (TRAP), urinary 8-hydroxy-2′-deoxyguanosine, and mRNA expression of tumor necrosis factor-α and cyclooxygenase-2 in spleen. GTP supplementation increased urinary epigallocatechin and epicatechin concentrations. Both GTP supplementation and alphacalcidol administration resulted in a significant increase in bone mass, but a significant decrease in serum TRAP levels, urinary 8-hydroxydeoxyguanosine levels, and mRNA expression of tumor necrosis factor-α and cyclooxygenase-2 in spleen. A synergistic effect of GTP and alphacalcidol was observed in these parameters. Neither GTP nor alphacalcidol affected femoral bone area or serum osteocalcin. Conclusion We conclude that a bone-protective role of GTP plus alphacalcidol during chronic inflammation bone loss may be due to a reduction of oxidative stress damage and inflammation. PMID:20069278

Preclinical Activity of VX-787, a First-in-Class, Orally Bioavailable Inhibitor of the Influenza Virus Polymerase PB2 Subunit

PubMed Central

Byrn, Randal A.; Jones, Steven M.; Bennett, Hamilton B.; Bral, Chris; Clark, Michael P.; Jacobs, Marc D.; Kwong, Ann D.; Ledeboer, Mark W.; Leeman, Joshua R.; McNeil, Colleen F.; Murcko, Mark A.; Nezami, Azin; Perola, Emanuele; Rijnbrand, Rene; Saxena, Kumkum; Tsai, Alice W.; Zhou, Yi

2014-01-01

VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m7GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection. PMID:25547360

Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit.

PubMed

Byrn, Randal A; Jones, Steven M; Bennett, Hamilton B; Bral, Chris; Clark, Michael P; Jacobs, Marc D; Kwong, Ann D; Ledeboer, Mark W; Leeman, Joshua R; McNeil, Colleen F; Murcko, Mark A; Nezami, Azin; Perola, Emanuele; Rijnbrand, Rene; Saxena, Kumkum; Tsai, Alice W; Zhou, Yi; Charifson, Paul S

2015-03-01

VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m(7)GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mechanism of Selective Nickel Transfer from HypB to HypA, Escherichia coli [NiFe]-Hydrogenase Accessory Proteins.

PubMed

Lacasse, Michael J; Douglas, Colin D; Zamble, Deborah B

2016-12-13

[NiFe]-hydrogenase enzymes catalyze the reversible reduction of protons to molecular hydrogen and serve as a vital component of the metabolism of many pathogens. The synthesis of the bimetallic catalytic center requires a suite of accessory proteins, and the penultimate step, nickel insertion, is facilitated by the metallochaperones HypA and HypB. In Escherichia coli, nickel moves from a site in the GTPase domain of HypB to HypA in a process accelerated by GDP. To determine how the transfer of nickel is controlled, the impacts of HypA and nucleotides on the properties of HypB were examined. Integral to this work was His2Gln HypA, a mutant with attenuated nickel affinity that does not support hydrogenase production in E. coli. This mutation inhibits the translocation of nickel from HypB. H2Q-HypA does not modulate the apparent metal affinity of HypB, but the stoichiometry and stability of the HypB-nickel complex are modulated by the nucleotide. Furthermore, the HypA-HypB interaction was detected by gel filtration chromatography if HypB was loaded with GDP, but not a GTP analogue, and the protein complex dissociated upon binding of nickel to His2 of HypA. In contrast, a nucleotide does not modulate the binding of zinc to HypB, and loading zinc into the GTPase domain of HypB inhibits formation of the complex with HypA. These results demonstrate that GTP hydrolysis controls both metal binding and protein-protein interactions, conferring selective and directional nickel transfer during [NiFe]-hydrogenase biosynthesis.

Mutation analysis of inhibitory guanine nucleotide binding protein alpha (GNAI) loci in young and familial pituitary adenomas.

PubMed

Demir, Hande; Donner, Iikki; Kivipelto, Leena; Kuismin, Outi; Schalin-Jäntti, Camilla; De Menis, Ernesto; Karhu, Auli

2014-01-01

Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15-20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1, GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas.

The Era GTPase recognizes the GAUCACCUCC sequence and binds helix 45 near the 3; end of 16S rRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tu, Chao; Zhou, Xiaomei; Tarasov, Sergey G.

2012-03-26

Era, composed of a GTPase domain and a K homology domain, is essential for bacterial cell viability. It is required for the maturation of 16S rRNA and assembly of the 30S ribosomal subunit. We showed previously that the protein recognizes nine nucleotides (1531{sup AUCACCUCC}1539) near the 3{prime} end of 16S rRNA, and that this recognition stimulates GTP-hydrolyzing activity of Era. In all three kingdoms of life, the 1530{sup GAUCA}1534 sequence and helix 45 (h45) (nucleotides 1506-1529) are highly conserved. It has been shown that the 1530{sup GA}1531 to 1530{sup AG}1531 double mutation severely affects the viability of bacteria. However, whethermore » Era interacts with G1530 and/or h45 and whether such interactions (if any) contribute to the stimulation of Era's GTPase activity were not known. Here, we report two RNA structures that contain nucleotides 1506-1542 (RNA301), one in complex with Era and GDPNP (GNP), a nonhydrolysable GTP-analogue, and the other in complex with Era, GNP, and the KsgA methyltransferase. The structures show that Era recognizes 10 nucleotides, including G1530, and that Era also binds h45. Moreover, GTPase assay experiments show that G1530 does not stimulate Era's GTPase activity. Rather, A1531 and A1534 are most important for stimulation and h45 further contributes to the stimulation. Although G1530 does not contribute to the intrinsic GTPase activity of Era, its interaction with Era is important for binding and is essential for the protein to function, leading to the discovery of a new cold-sensitive phenotype of Era.« less

Mutation Analysis of Inhibitory Guanine Nucleotide Binding Protein Alpha (GNAI) Loci in Young and Familial Pituitary Adenomas

PubMed Central

Demir, Hande; Donner, Iikki; Kivipelto, Leena; Kuismin, Outi; Schalin-Jäntti, Camilla; De Menis, Ernesto; Karhu, Auli

2014-01-01

Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15–20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1 , GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas. PMID:25291362

Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saxena, Sunil K.; Horiuchi, Hisanori; Fukuda, Mitsunori

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays withmore » two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins.« less

Characterization of the PcCdc42 small G protein from Pneumocystis carinii, which interacts with the PcSte20 life cycle regulatory kinase

PubMed Central

Krajicek, Bryan J.; Kottom, Theodore J.; Villegas, Leah

2010-01-01

Pneumocystis carinii (Pc) causes severe pneumonia in immunocompromised hosts. The binding of Pc trophic forms to alveolar epithelial cells is a central feature of infection, inducing the expression and activation of PcSte20, a gene participating in mating, proliferation, and pseudohyphal growth. In related fungi, Ste20 proteins are generally activated by immediate upstream small G proteins of the Cdc42-like family. PcCdc42 has not been previously described in Pneumocystis. To address the potential role of such a G protein in Pneumocystis, PcCdc42 was cloned from a Pc cDNA library. Using the full-length 576-bp PcCdc42 cDNA sequence, a CHEF blot of genomic DNA yielded a single band, providing evidence that this gene is present as a single copy within the genome. The total length of PcCdc42 cDNA was 576 bp with an estimated molecular mass of ∼38 kDa. BLASTP analysis demonstrated greater than 80% homology with other fungal Cdc42p proteins. Northern analysis indicated equal mRNA expression in both cystic and trophic life forms. Heterologous expression of PcCdc42 in Saccharomyces cerevisiae (Sc) demonstrated that PcCdc42p was able to restore growth in an ScCdc42Δ yeast strain. Additional assays with purified PcCdc42 protein demonstrated GTP binding and intrinsic GTPase activity, which was partially but significantly suppressed by Clostridium difficile toxin B, characteristic of Cdc42 GTPases. Furthermore, PcCdc42 protein was also shown to bind to the downstream PCSte20 kinase partner in the presence (but not the absence) of GTP. These data indicate that Pc possesses a Cdc42 gene expressing an active G protein, which binds the downstream regulatory kinase PcSte20, important in Pc life cycle regulation. PMID:19915161

Issues and Prospects of microRNA-Based Biomarkers in Blood and Other Body Fluids

DTIC Science & Technology

2014-05-14

transported to the cytoplasm via a RanGTP-dependent double stranded RNA binding protein , exportin 5. The pre-miRNA is then further processed by another...usually one strand (the guide strand) is preferentially associated with the effector- protein Argonaute [15,16], as part of the RNA Induced Silencing...miRNAs were packaged in a way to protect them from RNase degradation, possibly in lipid vesicles or as part of a protein complex. 3.1. Exosomes and Other

Role of GGAP/PIKE-A in prostate cancer progression

DTIC Science & Technology

2009-05-01

linking alterations of GGAP2 activity to neo- plastic transformation. The GGAP2 locus at 12q13.3 is amplified in glioblastoma cell lines, primary glioma ...prostate cancer tissue lysates . Protein (1.5 mg of total) was incubated with anti-GGAP2 rabbit antibody and protein A agarose beads for 4 h before the...agarose pull-down assay. Purified proteins or cell lysates expressing wild-type and mutant GGAP2 were equilibrated in GTP binding buffer [20 mmol/L Tris

New alleles of mgm1: a gene encoding a protein with a GTP-binding domain related to dynamin.

PubMed

Backer, J S

1995-10-01

Three previously described genes that affect baker's yeast (Saccharomyces cerevisiae) mitochondrial DNA (mtDNA) or mitochondrial RNA, tpm2-1, mna1-1, and mgm-1-1, are shown to be alleles of the same gene. This report demonstrates that tpm2-1 does not affect recombination of mtDNA. Therefore, there is no evidence that this dynamin-like protein is involved in movement of mtDNA within a cell.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orellana, S.A.; Trilivas, I.; Brown, J.H.

Carbachol and guanine nucleotides stimulate formation of the (/sup 3/H)inositol phosphates IP, IP2, and IP3 in saponin-permeabilized monolayers labelled with (/sup 3/H) inositol. Carbachol alone has little effect on formation of the (/sup 3/H) inositol phosphates (IPs), but GTP..gamma..S causes synergistic accumulation of (/sup 3/H)IPs to levels similar to those seen in intact cells. GTP, GppNHp, and GTP..gamma..S all support formation of the (/sup 3/H)IPs, with or without hormone, but GTP..gamma..S is the most effective. In the presence of GTP..gamma..S, the effect of carbachol is dose-dependent. Half-maximal and maximal accumulation of the (/sup 3/H)IPs occur at approx. 5 ..mu..M andmore » approx. 100 ..mu..M carbachol, respectively; values close to those seen in intact cells. GTP..gamma..S alone stimulates formation of the (/sup 3/H)IPs after a brief lag time. The combination of GTP..gamma..S and carbachol both increases the rate of, and decreases the lag in, formation of the (/sup 3/H)IPs. LiCl increases (/sup 3/H)IP and IP2, but not IP3, accumulation; while 2,3-diphosphoglycerate substantially increases that of (/sup 3/H)IP3. GTP..gamma..S and carbachol cause formation of (/sup 3/H)IPs in the absence of Ca/sup + +/, but formation induced by GTP..gamma..S with or without carbachol is Ca/sup + +/-sensitive over a range of physiological concentrations. Although carbachol, Ca/sup + +/, and GTP..gamma..S all have effects on formation of (/sup 3/H)IPs, GTP..gamma..S appears to be a primary and obligatory regulator of phosphoinositide hydrolysis in the permeabilized 1321N1 astrocytoma cell.« less

Characterization of mouse and human GTP cyclohydrolase I genes. Mutations in patients with GTP cyclohydrolase I deficiency.

PubMed

Ichinose, H; Ohye, T; Matsuda, Y; Hori, T; Blau, N; Burlina, A; Rouse, B; Matalon, R; Fujita, K; Nagatsu, T

1995-04-28

GTP cyclohydrolase I is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in mammals. Previously, we reported three species of human GTP cyclohydrolase I cDNA in a human liver cDNA library (Togari, A., Ichinose, H., Matsumoto, S., Fujita, K., and Nagatsu, T. (1992) Biochem. Biophys. Res. Commun. 187, 359-365). Furthermore, very recently, we found that the GTP cyclohydrolase I gene is causative for hereditary progressive dystonia with marked diurnal fluctuation, also known as DOPA-responsive dystonia (Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M., Nomura, Y., Endo, K., Tanaka, H., Tsuji, S., Fujita, K., and Nagatsu, T. (1994) Nature Genetics 8, 236-242). To clarify the mechanisms that regulate transcription of the GTP cyclohydrolase I gene and to generate multiple species of mRNA, we isolated genomic DNA clones for the human and mouse GTP cyclohydrolase I genes. Structural analysis of the isolated clones revealed that the GTP cyclohydrolase I gene is encoded by a single copy gene and is composed of six exons spanning approximately 30 kilobases. We sequenced all exon/intron boundaries of the human and mouse genes. Structural analysis also demonstrated that the heterogeneity of GTP cyclohydrolase I mRNA is caused by an alternative usage of the splicing acceptor site at the sixth exon. The transcription start site of the mouse GTP cyclohydrolase I gene and the 5'-flanking sequences of the mouse and human genes were determined. We performed regional mapping of the mouse gene by fluorescence in situ hybridization, and the mouse GTP cyclohydrolase I gene was assigned to region C2-3 of mouse chromosome 14. We identified missense mutations in patients with GTP cyclohydrolase I deficiency and expressed mutated enzymes in Escherichia coli to confirm alterations in the enzyme activity.

Haemoglobin function in vertebrates: evolutionary changes in cellular regulation in hypoxia.

PubMed

Nikinmaa, M

2001-11-15

The evolution of erythrocytic hypoxia responses is reviewed by comparing the cellular control of haemoglobin-oxygen affinity in agnathans, teleost fish and terrestrial vertebrates. The most ancient response to hypoxic conditions appears to be an increase in cell volume, which increases the haemoglobin-oxygen affinity in lampreys. In teleost fish, an increase of cell volume in hypoxic conditions is also evident. The volume increase is coupled to an increase in erythrocyte pH. These changes are caused by an adrenergic activation of sodium/proton exchange across the erythrocyte membrane. The mechanism is important in acute hypoxia and is followed by a decrease in cellular adenosine triphosphate (ATP) and guanosine triphosphate (GTP) concentrations in continued hypoxia. In hypoxic bird embryos, the ATP levels are also reduced. The mechanisms by which hypoxia decreases cellular ATP and GTP concentrations remains unknown, although at least in bird embryos cAMP-dependent mechanisms have been implicated. In mammals, hypoxia responses appear to occur mainly via modulation of cellular organic phosphate concentrations. In moderate hypoxia, 2,3-diphosphoglycerate levels are increased as a result of alkalosis caused by increased ventilation.

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury.

PubMed

Pissarek, M; Reichelt, C; Krauss, G J; Illes, P

1998-11-23

ATP-dependent potassium (KATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate KATP channels. In addition, the effects of the KATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N2-5% CO2 instead of 95% O2-5% CO2 for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%),, probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (30 microM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 but not 30 microM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 microM) antagonized the effects of diazoxide (300 MicroM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 microM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO2. However, only the ATP/ADP ratio was decreased further by diazoxide (300 microM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of KATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival viaprotein and DNA synthesis.

The RhoGAP activity of CYK-4/MgcRacGAP functions non-canonically by promoting RhoA activation during cytokinesis

PubMed Central

Zhang, Donglei; Glotzer, Michael

2015-01-01

Cytokinesis requires activation of the GTPase RhoA. ECT-2, the exchange factor responsible for RhoA activation, is regulated to ensure spatiotemporal control of contractile ring assembly. Centralspindlin, composed of the Rho family GTPase-activating protein (RhoGAP) MgcRacGAP/CYK-4 and the kinesin MKLP1/ZEN-4, is known to activate ECT-2, but the underlying mechanism is not understood. We report that ECT-2-mediated RhoA activation depends on the ability of CYK-4 to localize to the plasma membrane, bind RhoA, and promote GTP hydrolysis by RhoA. Defects resulting from loss of CYK-4 RhoGAP activity can be rescued by activating mutations in ECT-2 or depletion of RGA-3/4, which functions as a conventional RhoGAP for RhoA. Consistent with CYK-4 RhoGAP activity contributing to GEF activation, the catalytic domains of CYK-4 and ECT-2 directly interact. Thus, counterintuitively, CYK-4 RhoGAP activity promotes RhoA activation. We propose that the most active form of the cytokinetic RhoGEF involves complex formation between ECT-2, centralspindlin and RhoA. DOI: http://dx.doi.org/10.7554/eLife.08898.001 PMID:26252513

Role of catechins on ET-1 induced stimulation of PLD and NADPH oxidase activities in pulmonary smooth muscle cells: Determination of the probable mechanism by molecular docking studies.

PubMed

Chakraborti, Sajal; Sarkar, Jaganmay; Bhuyan, Rajabrata; Chakraborti, Tapati

2017-12-05

Treatment of human pulmonary artery smooth muscle cells with ET-1 stimulated PLD and NADPH oxidase activities, which were inhibited upon pretreatment with bosentan (ET-1 receptor antagonist), FIPI (PLD inhibitor), apocynin (NADPH oxidase inhibitor) and EGCG & ECG (catechins having galloyl group), but not EGC & EC (catechins devoid of galloyl group). Herein, we determined the probable mechanism by which the galloyl group containing catechins inhibit ET-1 induced stimulation of PLD activity by molecular docking analyses based on our biochemical studies. ET-1 induced stimulation of PLD activity was inhibited by SecinH3 (inhibitor of cytohesin). Arf-6 and cytohesin-1 were associated in the cell membrane, which was not inhibited by the catechins during ET-1 treatment to the cells. However, EGCG and ECG inhibited binding of GTPγS with Arf-6 even in presence of cytohesin-1. The molecular docking analyses revealed that the galloyl group containing catechins (EGCG/ECG) with cytohesin1-Arf6GDP, but not the non-galloyl-containing catechins (EGC and EC), prevents GDP/GTP exchange in Arf-6 which seems to be an important mechanism for inhibition of ET-1 induced activation of PLD and subsequently increase in NADPH oxidase activities.

Structural rationale for the cross-resistance of tumor cells bearing the A399V variant of elongation factor eEF1A1 to the structurally unrelated didemnin B, ternatin, nannocystin A and ansatrienin B

NASA Astrophysics Data System (ADS)

Sánchez-Murcia, Pedro A.; Cortés-Cabrera, Álvaro; Gago, Federico

2017-10-01

At least four classes of structurally distinct natural products with potent antiproliferative activities target the translation elongation factor eEF1A1, which is best known as the G-protein that delivers amino acyl transfer RNAs (aa-tRNAs) to ribosomes during mRNA translation. We present molecular models in atomic detail that provide a common structural basis for the high-affinity binding of didemnin B, ternatin, ansatrienin B and nannocystin A to eEF1A1, as well as a rationale based on molecular dynamics results that accounts for the deleterious effect of replacing alanine 399 with valine. The proposed binding site, at the interface between domains I and III, is eminently hydrophobic and exists only in the GTP-bound conformation. Drug binding at this site is expected to disrupt neither loading of aa-tRNAs nor GTP hydrolysis but would give rise to stabilization of this particular conformational state, in consonance with reported experimental findings. The experimental solution of the three-dimensional structure of mammalian eEF1A1 has proved elusive so far and the highly homologous eEF1A2 from rabbit muscle has been crystallized and solved only as a homodimer in a GDP-bound conformation. Interestingly, in this dimeric structure the large interdomain cavity where the drugs studied are proposed to bind is occupied by a mostly hydrophobic α-helix from domain I of the same monomer. Since binding of this α-helix and any of these drugs to domain III of eEF1A(1/2) is, therefore, mutually exclusive and involves two distinct protein conformations, one intriguing possibility that emerges from our study is that the potent antiproliferative effect of these natural products may arise not only from inhibition of protein synthesis, which is the current dogma, but also from interference with some other non-canonical functions. From this standpoint, this type of drugs could be considered antagonists of eEF1A1/2 oligomerization, a hypothesis that opens up novel areas of research.

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking.

PubMed

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d(-/-) mouse. Copyright © 2012 Elsevier Ltd. All rights reserved.

The binding of Varp to VAMP7 traps VAMP7 in a closed, fusogenically inactive conformation

PubMed Central

Schäfer, Ingmar B.; Hesketh, Geoffrey G.; Bright, Nicholas A.; Gray, Sally R.; Pryor, Paul R.; Evans, Philip R; Luzio, J. Paul; Owen, David J.

2012-01-01

SNAREs provide energy and specificity to membrane fusion events. Fusogenic trans-SNARE complexes are assembled from Q-SNAREs embedded in one membrane and an R–SNARE embedded in the other. Regulation of membrane fusion events is crucial for intracellular trafficking. We identify the endosomal protein Varp as an R-SNARE-binding regulator of SNARE complex formation. Varp co-localises with and binds to VAMP7, an R-SNARE involved in both endocytic and secretory pathways. We present the structure of the second ankyrin repeat domain of mammalian Varp in complex with the cytosolic portion of VAMP7. The VAMP7 SNARE motif is trapped between Varp and the VAMP7 longin domain and hence Varp kinetically inhibits VAMP7’s ability to form SNARE complexes. This inhibition will be increased when Varp can also bind to other proteins present on the same membrane as the VAMP7 such as Rab32:GTP. PMID:23104059

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hepler, J.R.; Harden, T.K.

Formation of the inositol phosphates (InsP), InsP/sub 3/, InsP/sub 2/, and InsP/sub 1/ was increased in a concentration dependent manner (K/sub 0.5/ approx. 5 ..mu..M) by GTP..sigma..S in washed membranes prepared from /sup 3/H-inositol-prelabelled 1321N1 human astrocytoma cells. Both GTP..gamma..S and GppNHp stimulated InsP formation by 2-3 fold over control; GTP and GDP were much less efficacious and GMP had no effect. Although the muscarinic cholinergic receptor agonist carbachol had no effect in the absence of guanine nucleotide, in the presence of 10 ..mu..M GTP..gamma..S, carbachol stimulated (K/sub 0.5/ approx. 10 ..mu.. M) the formation of InsP above the levelmore » achieved with GTP..gamma..S alone. The effect of carbachol was completely blocked by atropine. The order of potency for a series of nucleotides for stimulation of InsP formation in the presence of 500 ..mu..M carbachol was GTP..gamma..S > GppNHp > GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate G/sub i/, had no effect on InsP formation in the presence of GTP..gamma..S or GTP..gamma..S plus carbachol. Histamine and bradykinin also stimulated InsP formation in the presence of GTP..gamma..S in washed membranes from 1321N1 cells. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not G/sub i/ is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells.« less

Evidence for the Involvement of Lfc and Tctex-1 in Axon Formation

PubMed Central

Conde, Cecilia; Arias, Cristina; Robin, Maria; Li, Aiqun; Saito, Masaki; Chuang, Jen-Zen; Nairn, Angus C.; Sung, Ching-Hwa; Cáceres, Alfredo

2013-01-01

RhoA and Rac play key and opposite roles during neuronal polarization. We now show that Lfc, a guanosine nucleotide exchange factor (GEF), localizes to the Golgi apparatus and growth cones of developing neurons and negatively regulates neurite sprouting and axon formation through a Rho signaling pathway. Tctex-1, a dynein light chain implicated in axon outgrowth by modulating actin dynamics and Rac activity, colocalizes and physically interacts with Lfc, thus inhibiting its GEF activity, decreasing Rho-GTP levels, and functionally antagonizing Lfc during neurite formation. PMID:20463241

Effects of acute amphetamine (AMPH) treatment on rat striatal dopamine (DA) receptor activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roseboom, P.H.; Iwaniec, L.M.; Ackerman, J.M.

1986-03-05

Upon administration of AMPH rats display a complex series of dose and time dependent behaviors and changes in dopaminergic activity. They found a decrease in D1 DA receptor-stimulated adenylate cyclase (DA-AC) activity in rat striatal membranes after acute in vivo AMPH at a dose and time of intense stereotyped behavior. The Ka for D1-AC activity increased and the Vmax decreased in striatal membranes from rats given 7.5 mg/kg AMPH i.p. and killed 1 hr later as compared to saline (SAL) controls. They examined whether the decrease of DA-AC was due to a change in receptor number or activation of GTP-bindingmore » protein, Ns. Female Holtzman rats were injected with SAL or 7.5 mg/kg AMPH and killed 1 hr later. A 27,000 x g striatal particulate fraction was prepared for AC assay or (/sup 3/H)DA binding with 10 nM spiroperidol. They found no difference in stimulation of AC by NaF, GTP or GppNHp at any dose tested in membranes from SAL- and AMPH-treated rats. Calmodulin-stimulated AC was also unchanged after AMPH. Specific binding at a saturating concentration of (/sup 3/H)DA was 191 +/- 31 and 117 +/- 14 fmol/mg prot in membranes from SAL- and AMPH-treated rats, respectively. This suggests an alteration is occurring at the level of the D1 receptor rather than at coupling of Ns with the AC catalytic subunit.« less

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

PubMed

Nie, Zhongzhen; Hirsch, Dianne S; Luo, Ruibai; Jian, Xiaoying; Stauffer, Stacey; Cremesti, Aida; Andrade, Josefa; Lebowitz, Jacob; Marino, Michael; Ahvazi, Bijan; Hinshaw, Jenny E; Randazzo, Paul A

2006-01-24

Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

PubMed Central

Burch, R M; Connor, J R; Axelrod, J

1988-01-01

Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

Ethylene Regulates Monomeric GTP-Binding Protein Gene Expression and Activity in Arabidopsis1

PubMed Central

Moshkov, Igor E.; Mur, Luis A.J.; Novikova, Galina V.; Smith, Aileen R.; Hall, Michael A.

2003-01-01

Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction. PMID:12692329

Sustained Release of Green Tea Polyphenols from Liposomal Nanoparticles; Release Kinetics and Mathematical Modelling.

PubMed

Prakash Upputuri, Ravi Theaj; Azad Mandal, Abul Kalam

2017-01-01

Background: Green tea polyphenols (GTP) are known to have several health benefits. In spite of these benefits, its application as a therapeutic agent is limited due to some of its limitations such as stability, bioavailability, and biotransformation. To overcome these limitations, liposomal nanoparticles have been used as a carrier of the GTP. Objective: Encapsulation of GTP to the liposomal nanoparticles in order to achieve a sustained release of the GTP and to determine the drug release kinetics and the mechanism of the release. Materials and Methods: GTP encapsulated liposomal nanoparticles were prepared using phosphatidyl choline and cholesterol. The synthesized particles were characterized for their particle size and morphology. In vitro release studies were carried out, followed by drug release kinetics, and determining the mechanism of release. In vitro , antioxidant assay was determined following 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Results: Atomic force microscope (AFM) and high resolution scanning electron microscope (HR SEM) images showed spherical particles of the size of 64.5 and 252 nm. An encapsulation efficiency as high as 77.7% was observed with GTP concentration of 5 mg.mL -1 . In vitro release studies showed that the loading concentrations of GTP were independent to the cumulative percentage of the drug release. GTP release by varying the pH and temperature showed a direct correlation between the release parameter and the percentage of drug release. The higher the pH and temperature, the higher was the percentage of the drug release. The release data showed a good correlation with Zero order kinetics and the mechanism of the release being anomalous mode. Radical scavenging activity of the released GTP showed a potent scavenging activity. Conclusion: GTP encapsulated liposomal nanoparticles could be used as a delivery vehicle for achieving a sustained release.

LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation.

PubMed

Milkereit, Ruth; Persaud, Avinash; Vanoaica, Liviu; Guetg, Adriano; Verrey, Francois; Rotin, Daniela

2015-05-22

Mammalian target of rapamycin 1 (mTORC1), a master regulator of cellular growth, is activated downstream of growth factors, energy signalling and intracellular essential amino acids (EAAs) such as Leu. mTORC1 activation occurs at the lysosomal membrane, and involves V-ATPase stimulation by intra-lysosomal EAA (inside-out activation), leading to activation of the Ragulator, RagA/B-GTP and mTORC1 via Rheb-GTP. How Leu enters the lysosomes is unknown. Here we identified the lysosomal protein LAPTM4b as a binding partner for the Leu transporter, LAT1-4F2hc (SLC7A5-SLAC3A2). We show that LAPTM4b recruits LAT1-4F2hc to lysosomes, leading to uptake of Leu into lysosomes, and is required for mTORC1 activation via V-ATPase following EAA or Leu stimulation. These results demonstrate a functional Leu transporter at the lysosome, and help explain the inside-out lysosomal activation of mTORC1 by Leu/EAA.

Eukaryotic Elongation Factor 1A Interacts with the Upstream Pseudoknot Domain in the 3′ Untranslated Region of Tobacco Mosaic Virus RNA

PubMed Central

Zeenko, Vladimir V.; Ryabova, Lyubov A.; Spirin, Alexander S.; Rothnie, Helen M.; Hess, Daniel; Browning, Karen S.; Hohn, Thomas

2002-01-01

The genomic RNA of tobacco mosaic virus (TMV), like that of other positive-strand RNA viruses, acts as a template for both translation and replication. The highly structured 3′ untranslated region (UTR) of TMV RNAs plays an important role in both processes; it is not polyadenylated but ends with a tRNA-like structure (TLS) preceded by a conserved upstream pseudoknot domain (UPD). The TLS of tobamoviral RNAs can be specifically aminoacylated and, in this state, can interact with eukaryotic elongation factor 1A (eEF1A)/GTP with high affinity. Using a UV cross-linking assay, we detected another specific binding site for eEF1A/GTP, within the UPDs of TMV and crucifer-infecting tobamovirus (crTMV), that does not require aminoacylation. A mutational analysis revealed that UPD pseudoknot conformation and some conserved primary sequence elements are required for this interaction. Its possible role in the regulation of tobamovirus gene expression and replication is discussed. PMID:11991996

Noribogaine is a G-protein biased κ-opioid receptor agonist.

PubMed

Maillet, Emeline L; Milon, Nicolas; Heghinian, Mari D; Fishback, James; Schürer, Stephan C; Garamszegi, Nandor; Mash, Deborah C

2015-12-01

Noribogaine is the long-lived human metabolite of the anti-addictive substance ibogaine. Noribogaine efficaciously reaches the brain with concentrations up to 20 μM after acute therapeutic dose of 40 mg/kg ibogaine in animals. Noribogaine displays atypical opioid-like components in vivo, anti-addictive effects and potent modulatory properties of the tolerance to opiates for which the mode of action remained uncharacterized thus far. Our binding experiments and computational simulations indicate that noribogaine may bind to the orthosteric morphinan binding site of the opioid receptors. Functional activities of noribogaine at G-protein and non G-protein pathways of the mu and kappa opioid receptors were characterized. Noribogaine was a weak mu antagonist with a functional inhibition constants (Ke) of 20 μM at the G-protein and β-arrestin signaling pathways. Conversely, noribogaine was a G-protein biased kappa agonist 75% as efficacious as dynorphin A at stimulating GDP-GTP exchange (EC50=9 μM) but only 12% as efficacious at recruiting β-arrestin, which could contribute to the lack of dysphoric effects of noribogaine. In turn, noribogaine functionally inhibited dynorphin-induced kappa β-arrestin recruitment and was more potent than its G-protein agonistic activity with an IC50 of 1 μM. This biased agonist/antagonist pharmacology is unique to noribogaine in comparison to various other ligands including ibogaine, 18-MC, nalmefene, and 6'-GNTI. We predict noribogaine to promote certain analgesic effects as well as anti-addictive effects at effective concentrations>1 μM in the brain. Because elevated levels of dynorphins are commonly observed and correlated with anxiety, dysphoric effects, and decreased dopaminergic tone, a therapeutically relevant functional inhibition bias to endogenously released dynorphins by noribogaine might be worthy of consideration for treating anxiety and substance related disorders. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling*

PubMed Central

Kanamarlapudi, Venkateswarlu; Thompson, Aiysha; Kelly, Eamonn; López Bernal, Andrés

2012-01-01

The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a Gs-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization. PMID:22523074

Regulation of Ras Exchange Factors and Cellular Localization of Ras Activation by Lipid Messengers in T Cells

PubMed Central

Jun, Jesse E.; Rubio, Ignacio; Roose, Jeroen P.

2013-01-01

The Ras-MAPK signaling pathway is highly conserved throughout evolution and is activated downstream of a wide range of receptor stimuli. Ras guanine nucleotide exchange factors (RasGEFs) catalyze GTP loading of Ras and play a pivotal role in regulating receptor-ligand induced Ras activity. In T cells, three families of functionally important RasGEFs are expressed: RasGRF, RasGRP, and Son of Sevenless (SOS)-family GEFs. Early on it was recognized that Ras activation is critical for T cell development and that the RasGEFs play an important role herein. More recent work has revealed that nuances in Ras activation appear to significantly impact T cell development and selection. These nuances include distinct biochemical patterns of analog versus digital Ras activation, differences in cellular localization of Ras activation, and intricate interplays between the RasGEFs during distinct T cell developmental stages as revealed by various new mouse models. In many instances, the exact nature of these nuances in Ras activation or how these may result from fine-tuning of the RasGEFs is not understood. One large group of biomolecules critically involved in the control of RasGEFs functions are lipid second messengers. Multiple, yet distinct lipid products are generated following T cell receptor (TCR) stimulation and bind to different domains in the RasGRP and SOS RasGEFs to facilitate the activation of the membrane-anchored Ras GTPases. In this review we highlight how different lipid-based elements are generated by various enzymes downstream of the TCR and other receptors and how these dynamic and interrelated lipid products may fine-tune Ras activation by RasGEFs in developing T cells. PMID:24027568

Arabidopsis ARF-GTP exchange factor, GNOM, mediates transport required for innate immunity and focal accumulation of syntaxin PEN1

PubMed Central

Nielsen, Mads Eggert; Feechan, Angela; Böhlenius, Henrik; Ueda, Takashi; Thordal-Christensen, Hans

2012-01-01

Penetration resistance to powdery mildew fungi, conferred by localized cell wall appositions (papillae), is one of the best-studied processes in plant innate immunity. The syntaxin PENETRATION (PEN)1 is required for timely appearance of papillae, which contain callose and extracellular membrane material, as well as PEN1 itself. Appearance of membrane material in papillae suggests secretion of exosomes. These are potentially derived from multivesicular bodies (MVBs), supported by our observation that ARA6-labeled organelles assemble at the fungal attack site. However, the trafficking components that mediate delivery of extracellular membrane material are unknown. Here, we show that the delivery is independent of PEN1 function. Instead, we find that application of brefeldin (BF)A blocks the papillary accumulation of GFP-PEN1–labeled extracellular membrane and callose, while impeding penetration resistance. We subsequently provide evidence indicating that the responsible BFA-sensitive ADP ribosylation factor–GTP exchange factor (ARF-GEF) is GNOM. Firstly, analysis of the transheterozygote gnomB4049/emb30-1 (gnomB/E) mutant revealed a delay in papilla formation and reduced penetration resistance. Furthermore, a BFA-resistant version of GNOM restored the BFA-sensitive papillary accumulation of GFP-PEN1 and callose. Our data, therefore, provide a link between GNOM and disease resistance. We suggest that papilla formation requires rapid reorganization of material from the plasma membrane mediated by GNOM. The papilla material is subsequently presumed to be sorted into MVBs and directed to the site of fungal attack, rendering the epidermal plant cell inaccessible for the invading powdery mildew fungus. PMID:22733775

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okajima, F.; Sho, K.; Kondo, Y.

1988-08-01

Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrationsmore » below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.« less

Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells

PubMed Central

Falkenburger, Björn H.; Jensen, Jill B.

2010-01-01

G protein–coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Gα subunit, are common for many different GPCRs. For Gq-coupled M1 muscarinic (acetylcholine) receptors (M1Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M1R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M1R signaling through Gq to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP2-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with ∼3,000 fluorescently labeled receptors, G proteins, or PLC molecules per µm2 of plasma membrane. Endogenous levels are much lower, 1–40 per µm2. Our kinetic model reproduces the time courses and concentration–response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M1R and Gβ FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Gαq greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Gαq subunits bound to PLC. PMID:20100890

Co-amplification of phosphoinositide 3-kinase enhancer A and cyclin-dependent kinase 4 triggers glioblastoma progression | Office of Cancer Genomics

Cancer.gov

Glioblastoma (GBM) is the most common primary brain tumor and has a dismal prognosis. Amplification of chromosome 12q13-q15 (Cyclin-dependent kinase 4 (CDK4) amplicon) is frequently observed in numerous human cancers including GBM. Phosphoinositide 3-kinase enhancer (PIKE) is a group of GTP-binding proteins that belong to the subgroup of centaurin GTPase family, encoded by CENTG1 located in CDK4 amplicon. However, the pathological significance of CDK4 amplicon in GBM formation remains incompletely understood.

Septin Organization and Functions in Budding Yeast

PubMed Central

Glomb, Oliver; Gronemeyer, Thomas

2016-01-01

The septins are a conserved family of GTP-binding proteins present in all eukaryotic cells except plants. They were originally discovered in the baker's yeast Saccharomyces cerevisiae that serves until today as an important model organism for septin research. In yeast, the septins assemble into a highly ordered array of filaments at the mother bud neck. The septins are regulators of spatial compartmentalization in yeast and act as key players in cytokinesis. This minireview summarizes the recent findings about structural features and cell biology of the yeast septins. PMID:27857941

The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

PubMed Central

Delgado Tascón, Julia; Nyffenegger-Jann, Naja J.; Hauck, Christof R.

2012-01-01

Background CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment. PMID:22448228

The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.

PubMed

Pils, Stefan; Kopp, Kathrin; Peterson, Lisa; Delgado Tascón, Julia; Nyffenegger-Jann, Naja J; Hauck, Christof R

2012-01-01

CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.

Angiotensin 2 directly increases rabbit renal brush-border membrane sodium transport: Presence of local signal transduction system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morduchowicz, G.A.; Sheikh-Hamad, D.; Dwyer, B.E.

1991-05-01

In the present study, the authors have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10(-11)-10(-7) M) was found to stimulate 22Na+ uptake by the isolated BBM vesicles directly. All did not affect the Na(+)-dependent BBM glucose uptake, and the effect of AII on BBM 22Na+ uptake was inhibited by amiloride, suggesting the involvement of Na+/H+ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na+/H+ antiport system. In searchmore » of the signal transduction mechanism, it was found that AII activated BBM phospholipase A2 (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-beta S or PTX abolished, the effects of AII on BBM PLA and 22Na+ uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBM 22Na+ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM 22Na+ uptake, suggesting the role of PLA activation in mediating AII's effect on BBM 22Na+ uptake. In summary, results of the present study show a direct stimulatory effect of AII on BBM Na+/H+ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.« less

Antioxidant and antiapoptotic effects of green tea polyphenols against azathioprine-induced liver injury in rats.

PubMed

El-Beshbishy, Hesham A; Tork, Ola M; El-Bab, Mohamed F; Autifi, Mohamed A

2011-04-01

Green tea polyphenols (GTP) is considered to have protective effects against several diseases. The hepatotoxicity of azathioprine (AZA) has been reported and was found to be associated with oxidative damage. This study was conducted to evaluate the role of GTP to protect against AZA-induced liver injury in rats. AZA was administered i.p. in a single dose (50mgkg(-1)) to adult male rats. AZA-intoxicated rats were orally administered GTP (either 100mgkg(-1)day(-1) or 300mgkg(-1)day(-1), for 21 consecutive days, started 7 days prior AZA injection). AZA administration to rats resulted in significant elevation of serum transaminases (sALT and sAST), alkaline phosphatase (sALP), depletion of hepatic reduced glutathione (GSH), catalase (CAT) and glutathione peroxidase (GPx), accumulation of oxidized glutathione (GSSG), elevation of lipid peroxides (LPO) expressed as malondialdehyde (MDA), reduction of the hepatic total antioxidant activity (TAA), decrease serum total proteins and elevation of liver protein carbonyl content. Significant rises in liver tumor necrosis factor-alpha (TNF-α) and caspase-3 levels were noticed in AZA-intoxicated rats. Treatment of the AZA-intoxicated rats with GTP significantly prevented the elevations of sALT, sAST and sALP, inhibited depletion of hepatic GSH, GPx, CAT and GSSG and inhibited MDA accumulation. Furthermore, GTP had normalized serum total proteins and hepatic TAA, CAT, TNF-α and caspase-3 levels of AZA-intoxicated rats. In addition, GTP prevented the AZA-induced apoptosis and liver injury as indicated by the liver histopathological analysis. The linear regression analysis showed significant correlation in either AZA-GTP100 or AZA-GTP300 groups between TNF-α and each of serum ALT, AST, ALP and total proteins and liver TAA, GPX, CAT, GSH, GSSG, MDA and caspase-3 levels. However, liver TNF-α produced non-significant correlation with the serum total proteins in both AZA-GTP100 and AZA-GTP300 groups. In conclusion, our data indicate that GTP protects against AZA-induced liver injury in rats through antioxidant, anti-inflammatory and antiapoptotic mechanisms. However, further merit investigations are needed to verify these results and to assess the efficacy of GTP therapy to counteract the liver injury and oxidative stress status. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

EHD proteins: Key conductors of endocytic transport

PubMed Central

Naslavsky, Naava; Caplan, Steve

2010-01-01

Regulation of endocytic transport is controlled by an elaborate network of proteins. Rab GTP-binding proteins and their effectors have well-defined roles in mediating specific endocytic transport steps, but until recently, less was known about the four mammalian dynamin-like C-terminal Eps15 Homology Domain (EHD) proteins that also regulate endocytic events. In recent years, however, great strides have been made in understanding the structure and function of these unique proteins. Indeed, a growing body of literature addresses EHD protein structure, interactions with binding partners, functions in mammalian cells, and the generation of various new model systems. Accordingly, this is now an opportune time to pause and review the function and mechanisms of action of EHD proteins, and to highlight some of the challenges and future directions for the field. PMID:21067929

Combination of selenium-enriched green tea polysaccharides and Huo-ji polysaccharides synergistically enhances antioxidant and immune activity in mice.

PubMed

Yuan, Chengfu; Li, Zhihong; Peng, Fan; Xiao, Fangxiang; Ren, Dongming; Xue, Hui; Chen, Tao; Mushtaq, Gohar; Kamal, Mohammad Amjad

2015-12-01

The aim of this study was to investigate the influence of a combination of selenium-enriched green tea polysaccharides (Se-GTP) and Huo-ji polysaccharides (HJP) on the immune function and antioxidant activity in mice. The results showed that the indices of spleen and thymus were markedly increased, and the activity of natural killer (NK) cell was promoted in mice treated with the combination of Se-GTP and HJP. The combined treatment of Se-GTP and HJP also reduced the content of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in splenocytes. In addition, the activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) were remarkably enhanced, and malondialdehyde (MDA) levels were significantly reduced in mice treated with combination of Se-GTP and HJP. Furthermore, the combined treatment of Se-GTP and HJP increased nuclear factor erythroid 2-related factor (Nrf2) expression at mRNA and protein levels in splenocytes. The effects of the combination treatment of Se-GTP and HJP in mice were stronger than with Se-GTP or HJP treatment alone. Our study suggests that the combined administration of Se-GTP and HJP can synergistically improve immune function and decrease the oxidative stress by enhancing the mechanisms involved in the clearance of free radicals. © 2015 Society of Chemical Industry.

Green tea polyphenols reduce body weight in rats by modulating obesity-related genes.

PubMed

Lu, Chuanwen; Zhu, Wenbin; Shen, Chwan-Li; Gao, Weimin

2012-01-01

Beneficial effects of green tea polyphenols (GTP) against obesity have been reported, however, the mechanism of this protection is not clear. Therefore, the objective of this study was to identify GTP-targeted genes in obesity using the high-fat-diet-induced obese rat model. A total of three groups (n = 12/group) of Sprague Dawley (SD) female rats were tested, including the control group (rats fed with low-fat diet), the HF group (rats fed with high-fat diet), and the HF+GTP group (rats fed with high-fat diet and GTP in drinking water). The HF group increased body weight as compared to the control group. Supplementation of GTP in the drinking water in the HF+GTP group reduced body weight as compared to the HF group. RNA from liver samples was extracted for gene expression analysis. A total of eighty-four genes related to obesity were analyzed using PCR array. Compared to the rats in the control group, the rats in the HF group had the expression levels of 12 genes with significant changes, including 3 orexigenic genes (Agrp, Ghrl, and Nr3c1); 7 anorectic genes (Apoa4, Cntf, Ghr, IL-1β, Ins1, Lepr, and Sort); and 2 genes that relate to energy expenditure (Adcyap1r1 and Adrb1). Intriguingly, the HF+GTP group restored the expression levels of these genes in the high-fat-induced obese rats. The protein expression levels of IL-1β and IL-6 in the serum samples from the control, HF, and HF+GTP groups confirmed the results of gene expression. Furthermore, the protein expression levels of superoxide dismutase-1 (SOD1) and catechol-O-methyltransferase (COMT) also showed GTP-regulated protective changes in this obese rat model. Collectively, this study revealed the beneficial effects of GTP on body weight via regulating obesity-related genes, anti-inflammation, anti-oxidant capacity, and estrogen-related actions in high-fat-induced obese rats.

Green Tea Polyphenols Reduce Body Weight in Rats by Modulating Obesity-Related Genes

PubMed Central

Lu, Chuanwen; Zhu, Wenbin; Shen, Chwan-Li; Gao, Weimin

2012-01-01

Beneficial effects of green tea polyphenols (GTP) against obesity have been reported, however, the mechanism of this protection is not clear. Therefore, the objective of this study was to identify GTP-targeted genes in obesity using the high-fat-diet-induced obese rat model. A total of three groups (n = 12/group) of Sprague Dawley (SD) female rats were tested, including the control group (rats fed with low-fat diet), the HF group (rats fed with high-fat diet), and the HF+GTP group (rats fed with high-fat diet and GTP in drinking water). The HF group increased body weight as compared to the control group. Supplementation of GTP in the drinking water in the HF+GTP group reduced body weight as compared to the HF group. RNA from liver samples was extracted for gene expression analysis. A total of eighty-four genes related to obesity were analyzed using PCR array. Compared to the rats in the control group, the rats in the HF group had the expression levels of 12 genes with significant changes, including 3 orexigenic genes (Agrp, Ghrl, and Nr3c1); 7 anorectic genes (Apoa4, Cntf, Ghr, IL-1β, Ins1, Lepr, and Sort); and 2 genes that relate to energy expenditure (Adcyap1r1 and Adrb1). Intriguingly, the HF+GTP group restored the expression levels of these genes in the high-fat-induced obese rats. The protein expression levels of IL-1β and IL-6 in the serum samples from the control, HF, and HF+GTP groups confirmed the results of gene expression. Furthermore, the protein expression levels of superoxide dismutase-1 (SOD1) and catechol-O-methyltransferase (COMT) also showed GTP-regulated protective changes in this obese rat model. Collectively, this study revealed the beneficial effects of GTP on body weight via regulating obesity-related genes, anti-inflammation, anti-oxidant capacity, and estrogen-related actions in high-fat-induced obese rats. PMID:22715380

GDP-tubulin incorporation into growing microtubules modulates polymer stability.

PubMed

Valiron, Odile; Arnal, Isabelle; Caudron, Nicolas; Job, Didier

2010-06-04

Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule "structural plasticity."

Increase in serotonin 5-HT sub 1A receptors in prefrontal and temporal cortices of brains from patients with chronic schizophrenia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Takeshi; Nishino, Naoki; Nakai, Hisao

1991-01-01

Binding studies with ({sup 3}H)8-hydroxy-2-(di-n-propylamino)tetralin (({sup 3}H)8-OH-DPAT), a specific serotonin{sub 1A} (5-HT{sub 1A}) receptor agonist, were done on the autopsied brains from control subjects and from patients with chronic schizophrenia. In the controls, representative Scatchard plots for the specific ({sup 3}H)8-OH-DPAT bindings in the prefrontal cortex and hippocampus revealed a single component of high affinity binding site. The ({sup 3}H)8-OH-DPAT bindings to the prefrontal cortex and hippocampus were potently inhibited by serotonin and 5-HT{sub 1A} agonists, while other neurotransmitters, 5-HT{sub 2} and 5-HT{sub 3} related compounds did not inhibit the binding. The bindings were decreased in the presence of 0.1mMmore » GTP and 0.1mM GppNHp but not in the presence of 0.1mM GMP. In the prefrontal and temporal cortices of schizophrenics, there was a significant increase in the specific ({sup 3}H)8-OH-DPAT binding, by 40% and 60%, respectively, with no change in the hippocampus, amygdala, cingulum, motor cortex, parietal or occipital cortex, as compared to findings in the controls.« less

A physiologically required G protein-coupled receptor (GPCR)-regulator of G protein signaling (RGS) interaction that compartmentalizes RGS activity.

PubMed

Croft, Wayne; Hill, Claire; McCann, Eilish; Bond, Michael; Esparza-Franco, Manuel; Bennett, Jeannette; Rand, David; Davey, John; Ladds, Graham

2013-09-20

G protein-coupled receptors (GPCRs) can interact with regulator of G protein signaling (RGS) proteins. However, the effects of such interactions on signal transduction and their physiological relevance have been largely undetermined. Ligand-bound GPCRs initiate by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins. Signaling is terminated by hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, a reaction catalyzed by RGS proteins. Using yeast as a tool to study GPCR signaling in isolation, we define an interaction between the cognate GPCR (Mam2) and RGS (Rgs1), mapping the interaction domains. This reaction tethers Rgs1 at the plasma membrane and is essential for physiological signaling response. In vivo quantitative data inform the development of a kinetic model of the GTPase cycle, which extends previous attempts by including GPCR-RGS interactions. In vivo and in silico data confirm that GPCR-RGS interactions can impose an additional layer of regulation through mediating RGS subcellular localization to compartmentalize RGS activity within a cell, thus highlighting their importance as potential targets to modulate GPCR signaling pathways.

Long-term treatment with green tea polyphenols modifies the gut microbiome of female sprague-dawley rats.

PubMed

Wang, Jincheng; Tang, Lili; Zhou, Hongyuan; Zhou, Jun; Glenn, Travis C; Shen, Chwan-Li; Wang, Jia-Sheng

2018-06-01

Green tea polyphenols (GTP) have been shown to exert a spectrum of health benefits to animals and humans. It is plausible that the beneficial effects of GTP are a result of its interaction with the gut microbiota. This study evaluated the effect of long-term treatment with GTP on the gut microbiota of experimental rats and the potential linkage between changes of the gut microbiota with the beneficial effects of GTP. Six-month-old Sprague-Dawley rats were randomly allocated into three dosing regimens (0, 0.5%, and 1.5% of GTP) and followed for 6 months. At the end of month 3 or month 6, half of the animals from each group were sacrificed and their colon contents were collected for microbiome analysis using 16S ribosomal RNA and shotgun metagenomic community sequencing. GTP treatment significantly decreased the biodiversity and modified the microbial community in a dose-dependent manner; similar patterns were observed at both sampling times. Multiple operational taxonomic units and phylotypes were modified: the phylotypes Bacteroidetes and Oscillospira, previously linked to the lean phenotype in human and animal studies, were enriched; and Peptostreptococcaceae previously linked to colorectal cancer phenotype was depleted in GTP treated groups in a dose-dependent manner. Several microbial gene orthologs were modified, among which genes related to energy production and conversion were consistently enriched in samples from month 6 in a dose-dependent manner. This study showed that long-term treatment with GTP induced a dose-dependent modification of the gut microbiome in experimental rats, which might be linked to beneficial effects of GTP. Copyright © 2018 Elsevier Inc. All rights reserved.

Cex1p facilitates Rna1p-mediated dissociation of the Los1p-tRNA-Gsp1p-GTP export complex.

PubMed

McGuire, Andrew T; Mangroo, Dev

2012-02-01

Nuclear tRNA export plays an essential role in key cellular processes such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage and development. Like other nuclear export processes, assembly of the nuclear tRNA export complex in the nucleus is dependent on Ran-GTP/Gsp1p-GTP, and dissociation of the export receptor-tRNA-Ran-GTP/Gsp1p-GTP complex in the cytoplasm requires RanBP1/Yrb1p and RanGAP/Rna1p to activate the GTPase activity of Ran-GTP/Gsp1p-GTP. The Saccharomyces cerevisiae Cex1p and Human Scyl1 have also been proposed to participate in unloading of the tRNA export receptors at the cytoplasmic face of the nuclear pore complex (NPC). Here, we provide evidence suggesting that Cex1p is required for activation of the GTPase activity of Gsp1p and dissociation of the receptor-tRNA-Gsp1p export complex in S. cerevisiae. The data suggest that Cex1p recruits Rna1p from the cytoplasm to the NPC and facilitates Rna1p activation of the GTPase activity of Gsp1p by enabling Rna1p to gain access to Gsp1p-GTP bound to the export receptor tRNA complex. It is possible that this tRNA unloading mechanism is conserved in evolutionarily diverse organisms and that other Gsp1p-GTP-dependent export processes use a pathway-specific component to recruit Rna1p to the NPC. © 2011 John Wiley & Sons A/S.

Synergistic effects and related bioactive mechanisms of Potentilla fruticosa Linn. leaves combined with green tea polyphenols studied with microbial test system (MTS).

PubMed

Liu, Ze-Hua; Luo, Zi-Wen; Li, Deng-Wu; Wang, Dong-Mei; Ji, Xia

2018-06-01

Previous research found Potentilla fruticosa leaf extracts (PFE) combined with green tea polyphenols (GTP) showed obvious synergistic effects based on chemical mechanisms. This study further confirmed the synergy of PFE + GTP viewed from bioactivities using the microbial test system (MTS). The MTS antioxidant activity results showed the combination of PFE + GTP exhibited synergistic effect and the ratio 3:1 showed the strongest synergy, which were in accordance with the results in H 2 O 2 production rate. The combination of PFE + GTP promoted CAT and SOD enzyme activity and their gene expression especially at the ratio 3:1. Therefore, the synergism of PFE + GTP may be due to the promotion of CAT and SOD genes expression which enhanced the CAT and SOD enzyme activities. These results confirmed the synergy of PFE + GTP and could provide theoretical basis to produce a compounded tea made of a mixture of leaves from Potentilla species.

A heteronuclear zero quantum coherence Nz-exchange experiment that resolves resonance overlap and its application to measure the rates of heme binding to the IsdC protein.

PubMed

Robson, Scott A; Peterson, Robert; Bouchard, Louis-S; Villareal, Valerie A; Clubb, Robert T

2010-07-21

Chemical exchange phenomena in NMR spectra can be quantitatively interpreted to measure the rates of ligand binding, as well as conformational and chemical rearrangements. In macromolecules, processes that occur slowly on the chemical shift time scale are frequently studied using 2D heteronuclear ZZ or N(z)-exchange spectroscopy. However, to successfully apply this method, peaks arising from each exchanging species must have unique chemical shifts in both dimensions, a condition that is often not satisfied in protein-ligand binding equilibria for (15)N nuclei. To overcome the problem of (15)N chemical shift degeneracy we developed a heteronuclear zero-quantum (and double-quantum) coherence N(z)-exchange experiment that resolves (15)N chemical shift degeneracy in the indirect dimension. We demonstrate the utility of this new experiment by measuring the heme binding kinetics of the IsdC protein from Staphylococcus aureus. Because of peak overlap, we could not reliably analyze binding kinetics using conventional methods. However, our new experiment resulted in six well-resolved systems that yielded interpretable data. We measured a relatively slow k(off) rate of heme from IsdC (<10 s(-1)), which we interpret as necessary so heme loaded IsdC has time to encounter downstream binding partners to which it passes the heme. The utility of using this new exchange experiment can be easily expanded to (13)C nuclei. We expect our heteronuclear zero-quantum coherence N(z)-exchange experiment will expand the usefulness of exchange spectroscopy to slow chemical exchange events that involve ligand binding.

Effects of transcriptional start site sequence and position on nucleotide-sensitive selection of alternative start sites at the pyrC promoter in Escherichia coli.

PubMed Central

Liu, J; Turnbough, C L

1994-01-01

In Escherichia coli, expression of the pyrC gene is regulated primarily by a translational control mechanism based on nucleotide-sensitive selection of transcriptional start sites at the pyrC promoter. When intracellular levels of CTP are high, pyrC transcripts are initiated predominantly with CTP at a site 7 bases downstream of the Pribnow box. These transcripts form a stable hairpin at their 5' ends that blocks ribosome binding. When the CTP level is low and the GTP level is high, conditions found in pyrimidine-limited cells, transcripts are initiated primarily with GTP at a site 9 bases downstream of the Pribnow box. These shorter transcripts are unable to form a hairpin at their 5' ends and are readily translated. In this study, we examined the effects of nucleotide sequence and position on the selection of transcriptional start sites at the pyrC promoter. We characterized promoter mutations that systematically alter the sequence at position 7 or 9 downstream of the Pribnow box or vary the spacing between the Pribnow box and wild-type transcriptional initiation region. The results reveal preferences for particular initiating nucleotides (ATP > or = GTP > UTP >> CTP) and for starting positions downstream of the Pribnow box (7 >> 6 and 8 > 9 > 10). The results indicate that optimal nucleotide-sensitive start site switching at the wild-type pyrC promoter is the result of competition between the preferred start site (position 7) that uses the poorest initiating nucleotide (CTP) and a weak start site (position 9) that uses a good initiating nucleotide (GTP). The sequence of the pyrC promoter also minimizes the synthesis of untranslatable transcripts and provides for maximum stability of the regulatory transcript hairpin. In addition, the results show that the effects of the mutations on pyrC expression and regulation are consistent with the current model for translational control. Possible effects of preferences for initiating nucleotides and start sites on the expression and regulation of other genes are discussed. Images PMID:7910603

ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells.

PubMed

Krey, Jocelyn F; Dumont, Rachel A; Wilmarth, Philip A; David, Larry L; Johnson, Kenneth R; Barr-Gillespie, Peter G

2018-01-24

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout ( rda )]. In rda/rda mice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered in rda/rda mice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated in rda/rda utricles compared with controls, and the level of ARF6-GTP was correlated with the severity of the rda/rda phenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle. SIGNIFICANCE STATEMENT Assembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in the Elmod1 gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia. Copyright © 2018 the authors 0270-6474/18/380843-15$15.00/0.

Unique regulatory properties of the UDP-glucose:. beta. -1,4-glucan synthetase of Acetobacter xylinum. [Acetobacter xylinum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benziman, M.; Aloni, Y.; Delmer, D.P.

1983-01-01

Conditions have been found for an extremely efficient transfer of glucose from UDP-glucose to a cellulosic ..beta..-1,4-glucan product, using enzyme preparations derived from cells of Acetobacter xylinum. Membrane fractions obtained by rupturing cells in the presence of 20% (w/v) polyethylene glycol-4000 (PEG-4000) exhibited UDP-glucose:..beta..-1,4-glucan synthetase activity 3- to 10-fold higher than those previously reported. Enzyme prepared in this fashion also shows a further marked activation by GTP. The activation (apparent K/sub alpha/ = 35 ..mu..M) is quite specific for GTP. A variety of other nucleotides and nucleotide derivatives had no effect on activity. Guanosine-5'-(lambda-thio)triphosphate, an analog of GTP, is evenmore » more efficient than GTP (K/sub alpha/ = 17 ..mu..M). Enzyme prepared in the absence of PEG-4000 does not respond to GTP because it lacks a protein factor essential for GTP activation. PEG-4000 promotes the interaction of the protein factor with the enzyme. The factor itself is devoid of synthetase activity and does not stimulate activity of the enzyme in the absence of GTP. Under optimal conditions, in the presence of GTP, factor, and PEG-4000, initial rates of enzyme activity that are 200 times higher than those previously reported can be achieved. Such rates exceed 40% of the in vivo rate of cellulose synthesis from glucose. 26 references, 3 figures, 3 tables.« less

Autophagy in Saccharomyces cerevisiae requires the monomeric GTP-binding proteins, Arl1 and Ypt6.

PubMed

Yang, Shu; Rosenwald, Anne G

2016-10-02

Macroautophagy/autophagy is a cellular degradation process that sequesters organelles or proteins into a double-membrane structure called the phagophore; this transient compartment matures into an autophagosome, which then fuses with the lysosome or vacuole to allow hydrolysis of the cargo. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that 2 monomeric GTP-binding proteins in Saccharomyces cerevisiae, Arl1 and Ypt6, which belong to the Arf/Arl/Sar protein family and the Rab family, respectively, and control endosome-trans-Golgi traffic, are also necessary for starvation-induced autophagy under high temperature stress. Using established autophagy-specific assays we found that cells lacking either ARL1 or YPT6, which exhibit synthetic lethality with one another, were unable to undergo autophagy at an elevated temperature, although autophagy proceeds normally at normal growth temperature; specifically, strains lacking one or the other of these genes are unable to construct the autophagosome because these 2 proteins are required for proper traffic of Atg9 to the phagophore assembly site (PAS) at the restrictive temperature. Using degron technology to construct an inducible arl1Δ ypt6Δ double mutant, we demonstrated that cells lacking both genes show defects in starvation-inducted autophagy at the permissive temperature. We also found Arl1 and Ypt6 participate in autophagy by targeting the Golgi-associated retrograde protein (GARP) complex to the PAS to regulate the anterograde trafficking of Atg9. Our data show that these 2 membrane traffic regulators have novel roles in autophagy.

Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hall, Justin; Brault, Amy; Vincent, Fabien

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a highmore » affinity (K D = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.« less

Alpha1B-adrenoceptor signaling and cell motility: GTPase function of Gh/transglutaminase 2 inhibits cell migration through interaction with cytoplasmic tail of integrin alpha subunits.

PubMed

Kang, Sung Koo; Yi, Kye Sook; Kwon, Nyoun Soo; Park, Kwang-Hyun; Kim, Uh-Hyun; Baek, Kwang Jin; Im, Mie-Jae

2004-08-27

A multifunctional enzyme, G(h), is a GTP-binding protein that couples to the alpha(1B)-adrenoreceptor and stimulates phospholipase C-delta1 but also displays transglutaminase 2 (TG2) activity. G(h)/TG2 has been implicated to play a role in cell motility. In this study we have examined which function of G(h)/TG2 is involved in this cellular response and the molecular basis. Treatment of human aortic smooth muscle cell with epinephrine inhibits migration to fibronectin and vitronectin, and the inhibition is blocked by the alpha(1)-adrenoreceptor antagonist prazosin or chloroethylclonidine. Up-regulation or overexpression of G(h)/TG2 in human aortic smooth muscle cells, DDT1-MF2, or human embryonic kidney cells, HEK 293 cells, results in inhibition of the migratory activity, and stimulation of the alpha(1B)-adrenoreceptor with the alpha(1) agonist further augments the inhibition of migration of human aortic smooth muscle cells and DDT1-MF2. G(h)/TG2 is coimmunoprecipitated by an integrin alpha(5) antibody and binds to the cytoplasmic tail peptide of integrins alpha(5), alpha(v), and alpha(IIb) subunits in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). Mutation of Lys-Arg residues in the GFFKR motif, present in the alpha(5)-tail, significantly reduces the binding of GTPgammaS-G(h)/TG2. Moreover, the motif-containing integrin alpha(5)-tail peptides block G(h)/TG2 coimmunoprecipitation and reverse the inhibition of the migratory activity of HEK 293 cells caused by overexpression G(h)/TG2. These results provide evidence that G(h) function initiates the modulation of cell motility via association of GTP-bound G(h)/TG2 with the GFFKR motif located in integrin alpha subunits.

The specific GTP requirement for inositol 1,4,5-trisphosphate-induced Ca2+ release from skinned vascular smooth muscle.

PubMed

Saida, K; Twort, C; van Breemen, C

1988-01-01

Exogenous GTP was required for the induction of Ca2+ release from smooth muscle SR by IP3 if endogenous GTP was depleted. NaN3 could function as a partial substitute for GTP as a cofactor for the IP3-induced Ca2+ release from the SR. In contrast to the IP3-induced Ca2+ release, caffeine-induced Ca2+ release from the SR did not require GTP. Pertussis toxin inhibited the IP3-induced Ca2+ release from the SR, whereas it had no effect on caffeine-induced Ca2+ release. These results indicate that in smooth muscle two different Ca2+ release-channels exist in the SR: (a) activated by IP3, and (b) activated by caffeine or Ca2+.

Neuroprotective Potential of Gentongping in Rat Model of Cervical Spondylotic Radiculopathy Targeting PPAR-γ Pathway.

PubMed

Sun, Wen; Zheng, Kang; Liu, Bin; Fan, Danping; Luo, Hui; Qu, Xiaoyuan; Li, Li; He, Xiaojuan; Yi, Jianfeng; Lu, Cheng

2017-01-01

Cervical spondylotic radiculopathy (CSR) is the most general form of spinal degenerative disease and is characterized by pain and numbness of the neck and arm. Gentongping (GTP) granule, as a classical Chinese patent medicine, has been widely used in curing CSR, whereas the underlying mechanism remains unclear. Therefore, the aim of this study is to explore the pharmacological mechanisms of GTP on CSR. The rat model of CSR was induced by spinal cord injury (SCI). Our results showed that GTP could significantly alleviate spontaneous pain as well as ameliorate gait. The HE staining and Western blot results showed that GTP could increase the quantity of motoneuron and enhance the activation of peroxisome proliferator-activated receptor gamma (PPAR- γ ) in the spinal cord tissues. Meanwhile, immunofluorescence staining analysis indicated that GTP could reduce the expression of TNF- α in the spinal cord tissues. Furthermore, the protein level of Bax was decreased whereas the protein levels of Bcl-2 and NF200 were increased after the GTP treatment. These findings demonstrated that GTP might modulate the PPAR- γ pathway by inhibiting the inflammatory response and apoptosis as well as by protecting the cytoskeletal integrity of the spinal cord, ultimately play a neuroprotective role in CSR.

17 CFR 260.7a-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.7a-3 Section 260.7a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.7a-3 Number of copies; filing; signatures; binding. (a) Three copies of the complete application...

17 CFR 260.4c-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.4c-3 Section 260.4c-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.4c-3 Number of copies; filing; signatures; binding. (a) Three copies of every application and of...

17 CFR 260.5a-3 - Number of copies; filing; signatures; binding.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Number of copies; filing; signatures; binding. 260.5a-3 Section 260.5a-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE... § 260.5a-3 Number of copies; filing; signatures; binding. (a) Three copies of each statement of...

Platelet-derived growth factor receptor mediates activation of ras through different signaling pathways in different cell types.

PubMed Central

Satoh, T; Fantl, W J; Escobedo, J A; Williams, L T; Kaziro, Y

1993-01-01

A series of pieces of evidence have shown that Ras protein acts as a transducer of the platelet-derived growth factor (PDGF) receptor-mediated signaling pathway: (i) formation of Ras.GTP is detected immediately on PDGF stimulation, and (ii) a dominant inhibitory mutant Ras, as well as a neutralizing anti-Ras antibody, can interfere with PDGF-induced responses. On the other hand, several signal transducing molecules including phosphatidylinositol 3-kinase (PI3-K), GTPase-activating protein (GAP), and phospholipase C gamma (PLC gamma) bind directly to the PDGF receptor and become tyrosine phosphorylated. Recently, it was shown that specific phosphorylated tyrosines of the PDGF receptor are responsible for interaction between the receptor and each signaling molecule. However, the roles of these signaling molecules have not been elucidated, and it remains unclear which molecules are implicated in the Ras pathway. In this study, we measured Ras activation in cell lines expressing mutant PDGF receptors that are deficient in coupling with specific molecules. In fibroblast CHO cells, a mutant receptor (Y708F/Y719F [PI3-K-binding sites]) was unable to stimulate Ras, whereas another mutant (Y739F [the GAP-binding site]) could do so, suggesting an indispensable role of PI3-K or a protein that binds to the same sites as PI3-K for PDGF-stimulated Ras activation. By contrast, both of the above mutants were capable of stimulating Ras protein in a pro-B-cell line, BaF3. Furthermore, a mutant receptor (Y977F/Y989F [PLC gamma-binding sites]) could fully activate Ras, and the direct activation of protein kinase C and calcium mobilization had almost no effect on the GDP/GTP state of Ras in this cell line. These results suggest that, in the pro-B-cell transfectants, each of the above pathways (PI3-K, GAP, and PLC gamma) can be eliminated without a loss of Ras activation. It remains unclear whether another unknown essential pathway which regulates Ras protein exists within BaF3 cells. Therefore, it is likely that several different PDGF receptor-mediated signaling pathways function upstream of Ras, and the extent of the contribution of each pathway for the regulation of Ras may differ among different cell types. Images PMID:8388543

Brain Region and Isoform-Specific Phosphorylation Alters Kalirin SH2 Domain Interaction Sites and Calpain Sensitivity

PubMed Central

Miller, Megan B.; Yan, Yan; Machida, Kazuya; Kiraly, Drew D.; Levy, Aaron D.; Wu, Yi I.; Lam, TuKiet T.; Abbott, Thomas; Koleske, Anthony J.; Eipper, Betty A.; Mains, Richard E.

2017-01-01

Kalirin7 (Kal7), a postsynaptic Rho GDP/GTP exchange factor (RhoGEF), plays a crucial role in long term potentiation and in the effects of cocaine on behavior and spine morphology. The KALRN gene has been linked to schizophrenia and other disorders of synaptic function. Mass spectrometry was used to quantify phosphorylation at 26 sites in Kal7 from individual adult rat nucleus accumbens and prefrontal cortex before and after exposure to acute or chronic cocaine. Region- and isoform-specific phosphorylation was observed along with region-specific effects of cocaine on Kal7 phosphorylation. Evaluation of the functional significance of multi-site phosphorylation in a complex protein like Kalirin is difficult. With the identification of five tyrosine phosphorylation (pY) sites, a panel of 71 SH2 domains was screened, identifying subsets that interacted with multiple pY sites in Kal7. In addition to this type of reversible interaction, endoproteolytic cleavage by calpain plays an essential role in long-term potentiation. Calpain cleaved Kal7 at two sites, separating the N-terminal domain, which affects spine length, and the PDZ binding motif from the GEF domain. Mutations preventing phosphorylation did not affect calpain sensitivity or GEF activity; phosphomimetic mutations at specific sites altered protein stability, increased calpain sensitivity and reduced GEF activity. PMID:28418645

Structures of the tRNA export factor in the nuclear and cytosolic states.

PubMed

Cook, Atlanta G; Fukuhara, Noemi; Jinek, Martin; Conti, Elena

2009-09-03

Transfer RNAs are among the most ubiquitous molecules in cells, central to decoding information from messenger RNAs on translating ribosomes. In eukaryotic cells, tRNAs are actively transported from their site of synthesis in the nucleus to their site of function in the cytosol. This is mediated by a dedicated nucleo-cytoplasmic transport factor of the karyopherin-beta family (Xpot, also known as Los1 in Saccharomyces cerevisiae). Here we report the 3.2 A resolution structure of Schizosaccharomyces pombe Xpot in complex with tRNA and RanGTP, and the 3.1 A structure of unbound Xpot, revealing both nuclear and cytosolic snapshots of this transport factor. Xpot undergoes a large conformational change on binding cargo, wrapping around the tRNA and, in particular, binding to the tRNA 5' and 3' ends. The binding mode explains how Xpot can recognize all mature tRNAs in the cell and yet distinguish them from those that have not been properly processed, thus coupling tRNA export to quality control.

NMR Chemical Exchange as a Probe for Ligand-Binding Kinetics in a Theophylline-Binding RNA Aptamer

PubMed Central

Latham, Michael P.; Zimmermann, Grant R.; Pardi, Arthur

2009-01-01

The apparent on- and off-rate constants for theophylline binding to its RNA aptamer in the absence of Mg2+ were determined here by 2D 1H-1H NMR ZZ-exchange spectroscopy. Analysis of the build-up rate of the exchange cross peaks for several base-paired imino protons in the RNA yielded an apparent kon of 600 M-1 s-1. This small apparent kon results from the free RNA existing as a dynamic equilibrium of inactive states rapidly interconverting with a low population of active species. The data here indicate that the RNA aptamer employs a conformational selection mechanism for binding theophylline in the absence of Mg2+. The kinetic data here also explain a very unusual property of this RNA-theophylline system, slow exchange on the NMR chemical shift timescale for a weak-binding complex. To our knowledge, it is unprecedented to have such a weak binding complex (Kd ≈ 3.0 mM at 15 °C) show slow exchange on the NMR chemical shift timescale, but the results clearly demonstrate that slow exchange and weak binding are readily rationalized by a small kon. Comparisons with other ligand-receptor interactions are presented. PMID:19317486

Novel Missense Mutation A789V in IQSEC2 Underlies X-Linked Intellectual Disability in the MRX78 Family

PubMed Central

Kalscheuer, Vera M.; James, Victoria M.; Himelright, Miranda L.; Long, Philip; Oegema, Renske; Jensen, Corinna; Bienek, Melanie; Hu, Hao; Haas, Stefan A.; Topf, Maya; Hoogeboom, A. Jeannette M.; Harvey, Kirsten; Walikonis, Randall; Harvey, Robert J.

2016-01-01

Disease gene discovery in neurodevelopmental disorders, including X-linked intellectual disability (XLID) has recently been accelerated by next-generation DNA sequencing approaches. To date, more than 100 human X chromosome genes involved in neuronal signaling pathways and networks implicated in cognitive function have been identified. Despite these advances, the mutations underlying disease in a large number of XLID families remained unresolved. We report the resolution of MRX78, a large family with six affected males and seven affected females, showing X-linked inheritance. Although a previous linkage study had mapped the locus to the short arm of chromosome X (Xp11.4-p11.23), this region contained too many candidate genes to be analyzed using conventional approaches. However, our X-chromosome exome resequencing, bioinformatics analysis and inheritance testing revealed a missense mutation (c.C2366T, p.A789V) in IQSEC2, encoding a neuronal GDP-GTP exchange factor for Arf family GTPases (ArfGEF) previously implicated in XLID. Molecular modeling of IQSEC2 revealed that the A789V substitution results in the insertion of a larger side-chain into a hydrophobic pocket in the catalytic Sec7 domain of IQSEC2. The A789V change is predicted to result in numerous clashes with adjacent amino acids and disruption of local folding of the Sec7 domain. Consistent with this finding, functional assays revealed that recombinant IQSEC2A789V was not able to catalyze GDP-GTP exchange on Arf6 as efficiently as wild-type IQSEC2. Taken together, these results strongly suggest that the A789V mutation in IQSEC2 is the underlying cause of XLID in the MRX78 family. PMID:26793055

Impact of point mutation P29S in RAC1 on tumorigenesis.

PubMed

Rajendran, Vidya; Gopalakrishnan, Chandrasekhar; Purohit, Rituraj

2016-11-01

A point mutation (P29S) in the RAS-related C3 botulinum toxin substrate 1 (RAC1) was considered to be a trigger for melanoma, a form of skin cancer with highest mortality rate. In this study, we have investigated the pathogenic role of P29S based on the conformational behavior of RAC1 protein toward guanosine triphosphate (GTP). Molecular interaction, molecular dynamics trajectory analysis (RMSD, RMSF, Rg, SASA, DSSP, and PCA), and shape analysis of binding pocket were performed to analyze the interaction energy and the dynamic behavior of native and mutant RAC1 at the atomic level. Due to this mutation, the RAC1 switch I region acquired more flexibility and, to compensate it, the switch II region becomes rigid in their conformational space, as a result of which the interaction energy of the protein for GTP increased. The overall results strongly implied that the changes in atomic conformation of the switch I and II regions in mutant RAC1 protein were a significant reason for its malignant transformation and tumorigenesis. We raised the opportunity for researchers to design possible therapeutic molecule by considering our findings.

Substrate-Induced Formation of Ribosomal Decoding Center for Accurate and Rapid Genetic Code Translation.

PubMed

Pavlov, Michael Y; Ehrenberg, Måns

2018-05-20

Accurate translation of genetic information is crucial for synthesis of functional proteins in all organisms. We use recent experimental data to discuss how induced fit affects accuracy of initial codon selection on the ribosome by aminoacyl transfer RNA in ternary complex ( T 3 ) with elongation factor Tu (EF-Tu) and guanosine-5'-triphosphate (GTP). We define actual accuracy ([Formula: see text]) of a particular protein synthesis system as its current accuracy and the effective selectivity ([Formula: see text]) as [Formula: see text] in the limit of zero ribosomal binding affinity for T 3 . Intrinsic selectivity ([Formula: see text]), defined as the upper thermodynamic limit of [Formula: see text], is determined by the free energy difference between near-cognate and cognate T 3 in the pre-GTP hydrolysis state on the ribosome. [Formula: see text] is much larger than [Formula: see text], suggesting the possibility of a considerable increase in [Formula: see text] and [Formula: see text] at negligible kinetic cost. Induced fit increases [Formula: see text] and [Formula: see text] without affecting [Formula: see text], and aminoglycoside antibiotics reduce [Formula: see text] and [Formula: see text] at unaltered [Formula: see text].

Straight and Curved Conformations of FtsZ Are Regulated by GTP Hydrolysis

PubMed Central

Lu, Chunlin; Reedy, Mary; Erickson, Harold P.

2000-01-01

FtsZ assembles in vitro into protofilaments that can adopt two conformations—the straight conformation, which can assemble further into two-dimensional protofilament sheets, and the curved conformation, which forms minirings about 23 nm in diameter. Here, we describe the structure of FtsZ tubes, which are a variation of the curved conformation. In the tube the curved protofilament forms a shallow helix with a diameter of 23 nm and a pitch of 18 or 24°. We suggest that this shallow helix is the relaxed structure of the curved protofilament in solution. We provide evidence that GTP favors the straight conformation while GDP favors the curved conformation. In particular, exclusively straight protofilaments and protofilament sheets are assembled in GMPCPP, a nonhydrolyzable GTP analog, or in GTP following chelation of Mg, which blocks GTP hydrolysis. Assembly in GDP produces exclusively tubes. The transition from straight protofilaments to the curved conformation may provide a mechanism whereby the energy of GTP hydrolysis is used to generate force for the constriction of the FtsZ ring in cell division. PMID:10613876

Aurora A regulates the activity of HURP by controlling the accessibility of its microtubule-binding domain.

PubMed

Wong, Jim; Lerrigo, Robert; Jang, Chang-Young; Fang, Guowei

2008-05-01

HURP is a spindle-associated protein that mediates Ran-GTP-dependent assembly of the bipolar spindle and promotes chromosome congression and interkinetochore tension during mitosis. We report here a biochemical mechanism of HURP regulation by Aurora A, a key mitotic kinase that controls the assembly and function of the spindle. We found that HURP binds to microtubules through its N-terminal domain that hyperstabilizes spindle microtubules. Ectopic expression of this domain generates defects in spindle morphology and function that reduce the level of tension across sister kinetochores and activate the spindle checkpoint. Interestingly, the microtubule binding activity of this N-terminal domain is regulated by the C-terminal region of HURP: in its hypophosphorylated state, C-terminal HURP associates with the microtubule-binding domain, abrogating its affinity for microtubules. However, when the C-terminal domain is phosphorylated by Aurora A, it no longer binds to N-terminal HURP, thereby releasing the inhibition on its microtubule binding and stabilizing activity. In fact, ectopic expression of this C-terminal domain depletes endogenous HURP from the mitotic spindle in HeLa cells in trans, suggesting the physiological importance for this mode of regulation. We concluded that phosphorylation of HURP by Aurora A provides a regulatory mechanism for the control of spindle assembly and function.

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs

PubMed Central

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-01-01

The 5’terminal oligopyrimidine (5’TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m7G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5’TOP motif, a cap analog (m7GTP), and a capped cytidine (m7GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5’TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5’TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis. DOI: http://dx.doi.org/10.7554/eLife.24146.001 PMID:28379136

[Alcohol intake and gamma -GTP observed from the viewpoint of an occupational physician].

PubMed

Oikawa, Takamitsu

2007-06-01

Alcohol is an important basic factor in health management at the workplace. The fact is, however, when alcohol is pervasive in a worker's daily life, effective measures are very difficult to carry out. We examined an intervention program based on serum y -GTP (IU/l) measurements at physical examination. Subjects were clients of the Keio Counseling Center in2005 (male, 5568: female, 1725). Among nondrinkers, gamma-GTP values were under 50 in 83% of men and 93% of women. Relative risk of lifestyle-related diseases (obesity, hypertension, hyperlipidemia, hyperuricemia, hyperglycemia and fatty liver) among male drinkers increased dramatically when gamma-GTP exceeded 50,with a further gradual increase for gamma-GTP over 100. Moreover, relative risk of over two concurrent diseases among obesity, hypertension, hyperlipidemia and hyperglycemia increased when gamma-GTP exceeded 25 and greatly increased beyond 50. While the findings suggest 25 or less as an ideal gamma-GTP values, a workplace program might more practically regard values over 50 as a threshold for management measures and values over 100 as indicating enforced management. At the workplace, management of other diseases including lifestyle-related diseases, alcoholism per se, and mental health issues needs to be carried out in a balanced, coordinated manner. Cooperation of related medical institutions and effective alcohol treatment program, and efforts to enlist the understanding and trust of all workers are needed.

Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling.

PubMed

Abbott, Jason R; Hodges, Timothy R; Daniels, R Nathan; Patel, Pratiq A; Kennedy, Jack Phillip; Howes, Jennifer E; Akan, Denis T; Burns, Michael C; Sai, Jiqing; Sobolik, Tammy; Beesetty, Yugandhar; Lee, Taekyu; Rossanese, Olivia W; Phan, Jason; Waterson, Alex G; Fesik, Stephen W

2018-06-01

Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report on structure-activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at sub-micromolar concentrations in vitro, increase levels of active RAS-GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway, resulting in a decrease in pERK1/2 T202/Y204 protein levels at higher compound concentrations.

Green tea polyphenols supplementation improves bone microstructure in orchidectomized middle-Aged rats

USDA-ARS?s Scientific Manuscript database

Our recent study shows that green tea polyphenols (GTP) attenuate trabecular bone loss in ovariectomized middle-aged female rats. To investigate whether GTP prevents bone loss in male rats, 40 rats with and without oriectomy (ORX) were assigned to 4 groups in a 2 (sham vs. ORX)× 2 (no GTP and 0.5% G...

Green tea polyphenols mitigate bone loss of female rats in a chronic inflammation-induced bone loss model

USDA-ARS?s Scientific Manuscript database

The purpose of this study was to explore bioavailability, efficacy, and molecular mechanisms of green tea polyphenols (GTP) related to preventing bone loss in rats with chronic inflammation. A 2 (placebo vs. lipopolysaccharide, LPS) × 2 (no GTP vs. 0.5% GTP in drinking water) factorial design using ...

Inhibitory effect of extracellular purine nucleotide and nucleoside concentrations on T cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weiler, Monica; Schmetzer, Helga; German Research Center for Environmental Health, Munich

The release of nucleic acids and derivatives after tissue-injury may affect cellular immune-response. We studied the impact of extracellular ribo-, desoxyribonucleotides and nucleosides on T-cell immunity. Peripheral-blood-mononuclear-cells (PBMCs) or isolated CD3{sup +}T-cells obtained from 6 healthy donors were stimulated via CD3/CD28 Dynabeads or dendritic cells (DCs) in the presence or absence of pyrimidine-, purine-nucleotides and -nucleosides (range 2–200 µM). Addition of deoxy-, guanosine-triphosphate (dGTP, GTP) and guanosine resulted concentration dependent in a complete, adenosine-triphosphate (ATP) in a partial inhibition of the induced T-cell-proliferation. Deoxyadenosine-triphosphate (dATP), adenosine and the pyrimidine-ribo- and -deoxyribonucleotides displayed no inhibitory capacity. Inhibitory effects of dGTP andmore » GTP, but not of guanosine and ATP were culture-media-dependent and could be almost abrogated by use of the serum-free lymphocyte-culture-media X-Vivo15 instead of RPMI1640 with standard-supplementation. In contrast to RPMI1640, X-Vivo15 resulted in a significant down-regulation of the cell-surface-located ectonucleotidases CD39 (Ecto-Apyrase) and CD73 (Ecto-5′-Nucleotidase), critical for the extracellular nucleotides-hydrolysis to nucleosides, explaining the loss of inhibition mediated by dGTP and GTP, but not Guanosine. In line with previous findings ATP was found to exert immunosuppressive effects on T-cell-proliferation. Purine-nucleotides, dGTP and GTP displayed a higher inhibitory capacity, but seem to be strictly dependent on the microenvironmental conditions modulating the responsiveness of the respective T-lymphocytes. Further evaluation of experimental and respective clinical settings should anticipate these findings.« less

Protective actions of green tea polyphenols and alfacalcidol on bone microstructure in female rats with chronic inflammation

USDA-ARS?s Scientific Manuscript database

This study investigated the effects of green tea polyphenols (GTP) and alfacalcidol on bone microstructure and strength along with possible mechanisms in rats with chronic inflammation. A 12-week study using a 2 (no GTP vs. 0.5%, w/v GTP in drinking water) × 2 (no alfacalcidol vs. 0.05 ug/kg alfacal...

Patterns of purine nucleotides in fish erythrocytes.

PubMed

Leray, C

1979-01-01

1. The purine nucleotides were determined in the whole blood of 9 fresh water teleosts and 2 marine selachians. 2. GTP and ATP accounted for 88-99% of the total erythrocytes purines. 3. The ATP/ADP ratio ranged from 11 to 60 in the erythrocytes of the fish examined. 4. GTP is widely distributed in fish erythrocytes but its level ranged from 1 to 33 nmol/mg Hb (0.4 to 9 mumol/ml erythrocyte). 5. Lepomis and Esox exhibited a GTP/ATP ratio as elevated as in Anguilla; moreover the concentration of GTP per mol of Hb (physiologically most indicative) is higher in Lepomis, Esox, Ictalurus and Silurus than in Anguilla.

Hormonal control of GTP cyclohydrolase I gene expression and enzyme activity during color pattern development in wings of Precis coenia.

PubMed

Sawada, H; Nakagoshi, M; Reinhardt, R K; Ziegler, I; Koch, P B

2002-06-01

Color patterns of butterfly wings are composed of single color points represented by each scale. In the case of Precis coenia, at the end of pupal development, different types of pigments are synthesized sequentially in the differently colored scales beginning with white (pterins) followed by red (ommatins) and then black (melanin). In order to explain how formation of these different colors is regulated, we examined the expression of an mRNA-encoding guanosine triphosphate-cyclohydrolase I (GTP-CH I; EC 3.5.4.16), the first key enzyme in the biosynthesis of pteridines, during pigment formation in the wings of P. coenia. The strongest positive signal was recognized around pigment formation one day before butterfly emergence. This GTP-CH I gene expression is paralleled by GTP-CH I enzyme activity measured in wing extracts. We also investigated the effect of 20-hydroxyecdysone on the expression of GTP-CH I mRNA and the enzyme activity during color formation. The results strongly suggest that the onset and duration of the expression of a GTP-CH I mRNA is triggered by a declining ecdysteroid hormone titer during late pupal development.

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

PubMed Central

Goffinet, Marine; Chinestra, Patrick; Lajoie-Mazenc, Isabelle; Medale-Giamarchi, Claire; Favre, Gilles; Faye, Jean-Charles

2008-01-01

Background The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. Results After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. Conclusion We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development. PMID:18377644

Structural basis for the binding of tryptophan-based motifs by δ-COP

PubMed Central

Suckling, Richard J.; Poon, Pak Phi; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

2015-01-01

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. PMID:26578768

N7-platinated ribonucleotides are not incorporated by RNA polymerases. New perspectives for a rational design of platinum antitumor drugs.

PubMed

Benedetti, Michele; Romano, Alessandro; De Castro, Federica; Girelli, Chiara R; Antonucci, Daniela; Migoni, Danilo; Verri, Tiziano; Fanizzi, Francesco P

2016-10-01

In this work, we assessed the capacity of RNA polymerases to use platinated ribonucleotides as substrates for RNA synthesis by testing the incorporation of the model compound [Pt(dien)(N7-5'-GTP)] (dien=diethylenetriamine; GTP=5'-guanosine triphosphate) into a natural RNA sequence. The yield of in vitro transcription operated by T7 RNA polymerase, on the LacZ (Escherichia coli gene encoding for β-galactosidase) sequence, decreases progressively with decreasing the concentration of natural GTP, in favor of the platinated nucleotide, [Pt(dien)(N7-5'-GTP)]. Comparison of the T7 RNA polymerase transcription activities for [Pt(dien)(N7-5'-GTP)] compound incorporation reaction test, with respect to the effect of a decreasing concentration of natural GTP, showed no major differences. A specific inhibitory effect of compound [Pt(dien)(N7-5'-GTP)] (which may pair the complementary base on the DNA strand, without being incorporated in the RNA by the T7 RNA polymerase) was evidenced. Our findings therefore suggest that RNA polymerases, unlike DNA polymerases, are unable to incorporate N7-platinated nucleotides into newly synthesized nucleic acids. In this respect, specifically designed N7-platinated nucleotides based compounds could be used in alternative to the classical platinum based drugs. This approach may offer a possible strategy to target specifically DNA, without affecting RNA, and is potentially able to better modulate pharmacological activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Safety Evaluation of Green Tea Polyphenols Consumption in Middle-aged Ovariectomized Rat Model.

PubMed

Shen, Chwan-Li; Brackee, Gordon; Song, Xiao; Tomison, Michael D; Finckbone, VelvetLee; Mitchell, Kelly T; Tang, Lili; Chyu, Ming-Chien; Dunn, Dale M; Wang, Jia-Sheng

2017-09-01

This work evaluates chronic safety in middle-aged ovariectomized rats supplemented with different dosages of green tea polyphenols (GTP) in drinking water. The experiment used 6-mo-old sham (n = 39) and ovariectomized (OVX, n = 143) female rats. All sham (n = 39) and 39 of the OVX animals received no GTP treatment and their samples were collected for outcome measures at baseline, 3 mo, and 6 mo (n = 13 per group for each). The remaining OVX animals were randomized into 4 groups receiving 0.15%, 0.5%, 1%, and 1.5% (n = 26 for each) of GTP (wt/vol), respectively, in drinking water for 3 and 6 mo. No mortality or abnormal treatment-related findings in clinical observations or ophthalmologic examinations were noted. No treatment-related macroscopic or microscopic findings were noted for animals administered 1.5% GTP supplementation. Throughout the study, there was no difference in the body weight among all OVX groups. In all OVX groups, feed intake and water consumption significantly decreased with GTP dose throughout the study period. At 6 mo, GTP intake did not affect hematology, clinical chemistry, and urinalysis, except for phosphorus and blood urea nitrogen (increased), total cholesterol, lactate dehydrogenase, and urine pH (decreased). This study reveals that the no-observed-adverse-effect level (NOAEL) of GTP is 1.5% (wt/vol) in drinking water, the highest dose used in this study. © 2017 Institute of Food Technologists®.

Stochastic flux analysis of chemical reaction networks

PubMed Central

2013-01-01

Background Chemical reaction networks provide an abstraction scheme for a broad range of models in biology and ecology. The two common means for simulating these networks are the deterministic and the stochastic approaches. The traditional deterministic approach, based on differential equations, enjoys a rich set of analysis techniques, including a treatment of reaction fluxes. However, the discrete stochastic simulations, which provide advantages in some cases, lack a quantitative treatment of network fluxes. Results We describe a method for flux analysis of chemical reaction networks, where flux is given by the flow of species between reactions in stochastic simulations of the network. Extending discrete event simulation algorithms, our method constructs several data structures, and thereby reveals a variety of statistics about resource creation and consumption during the simulation. We use these structures to quantify the causal interdependence and relative importance of the reactions at arbitrary time intervals with respect to the network fluxes. This allows us to construct reduced networks that have the same flux-behavior, and compare these networks, also with respect to their time series. We demonstrate our approach on an extended example based on a published ODE model of the same network, that is, Rho GTP-binding proteins, and on other models from biology and ecology. Conclusions We provide a fully stochastic treatment of flux analysis. As in deterministic analysis, our method delivers the network behavior in terms of species transformations. Moreover, our stochastic analysis can be applied, not only at steady state, but at arbitrary time intervals, and used to identify the flow of specific species between specific reactions. Our cases study of Rho GTP-binding proteins reveals the role played by the cyclic reverse fluxes in tuning the behavior of this network. PMID:24314153

A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions

PubMed Central

Swick, Lance; Kapatos, Gregory

2008-01-01

The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5′-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal α-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal α-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal α–helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1–96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1–42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1–42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1–GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4. PMID:16696853

The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy

PubMed Central

Kötting, Carsten; Kallenbach, Angela; Suveyzdis, Yan; Wittinghofer, Alfred; Gerwert, Klaus

2008-01-01

Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved “arginine finger” of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras·RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling “off” to the signaling “on” state with a rate of 3 s−1. The next step is the movement of the “arginine finger” into the active site of Ras with a rate of k2 = 0.8 s−1. Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound Pi intermediate forms. The switch-I reversal to the “off” state, the release of Pi, and the movement of arginine back into an aqueous environment is observed simultaneously with k3 = 0.1 s−1, the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the “arginine finger” movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a Pi-intermediate. PMID:18434546

Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA.

PubMed

Kinsella, B T; Erdman, R A; Maltese, W A

1991-05-25

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.

Stochastic flux analysis of chemical reaction networks.

PubMed

Kahramanoğulları, Ozan; Lynch, James F

2013-12-07

Chemical reaction networks provide an abstraction scheme for a broad range of models in biology and ecology. The two common means for simulating these networks are the deterministic and the stochastic approaches. The traditional deterministic approach, based on differential equations, enjoys a rich set of analysis techniques, including a treatment of reaction fluxes. However, the discrete stochastic simulations, which provide advantages in some cases, lack a quantitative treatment of network fluxes. We describe a method for flux analysis of chemical reaction networks, where flux is given by the flow of species between reactions in stochastic simulations of the network. Extending discrete event simulation algorithms, our method constructs several data structures, and thereby reveals a variety of statistics about resource creation and consumption during the simulation. We use these structures to quantify the causal interdependence and relative importance of the reactions at arbitrary time intervals with respect to the network fluxes. This allows us to construct reduced networks that have the same flux-behavior, and compare these networks, also with respect to their time series. We demonstrate our approach on an extended example based on a published ODE model of the same network, that is, Rho GTP-binding proteins, and on other models from biology and ecology. We provide a fully stochastic treatment of flux analysis. As in deterministic analysis, our method delivers the network behavior in terms of species transformations. Moreover, our stochastic analysis can be applied, not only at steady state, but at arbitrary time intervals, and used to identify the flow of specific species between specific reactions. Our cases study of Rho GTP-binding proteins reveals the role played by the cyclic reverse fluxes in tuning the behavior of this network.

SUMOylation of the mitochondrial fission protein Drp1 occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle

PubMed Central

Figueroa-Romero, Claudia; Iñiguez-Lluhí, Jorge A.; Stadler, Julia; Chang, Chuang-Rung; Arnoult, Damien; Keller, Peter J.; Hong, Yu; Blackstone, Craig; Feldman, Eva L.

2009-01-01

Dynamin-related protein (Drp) 1 is a key regulator of mitochondrial fission and is composed of GTP-binding, Middle, insert B, and C-terminal GTPase effector (GED) domains. Drp1 associates with mitochondrial fission sites and promotes membrane constriction through its intrinsic GTPase activity. The mechanisms that regulate Drp1 activity remain poorly understood but are likely to involve reversible post-translational modifications, such as conjugation of small ubiquitin-like modifier (SUMO) proteins. Through a detailed analysis, we find that Drp1 interacts with the SUMO-conjugating enzyme Ubc9 via multiple regions and demonstrate that Drp1 is a direct target of SUMO modification by all three SUMO isoforms. While Drp1 does not harbor consensus SUMOylation sequences, our analysis identified2 clusters of lysine residues within the B domain that serve as noncanonical conjugation sites. Although initial analysis indicates that mitochondrial recruitment of ectopically expressed Drp1 in response to staurosporine is unaffected by loss of SUMOylation, we find that Drp1 SUMOylation is enhanced in the context of the K38A mutation. This dominant-negative mutant, which is deficient in GTP binding and hydrolysis, does not associate with mitochondria and prevents normal mitochondrial fission. This finding suggests that SUMOylation of Drp1 is linked to its activity cycle and is influenced by Drp1 localization.—Figueroa-Romero, C., Iñiguez-Lluhí, J. A., Stadler, J., Chang, C.-R., Arnoult, D., Keller, P. J., Hong, Y., Blackstone, C., Feldman, E. L. SUMOylation of the mitochondrial fission protein Drp1 occurs at multiple nonconsensus sites within the B domain and is linked to its activity cycle. PMID:19638400

The GTP-bound and Sumoylated Form of the rab17 Small Molecular Weight GTPase Selectively Binds Syntaxin 2 in Polarized Hepatic WIF-B Cells*

PubMed Central

Striz, Anneliese C.; Tuma, Pamela L.

2016-01-01

A major focus for our laboratory is identifying the molecules and mechanisms that regulate polarized apical protein sorting in hepatocytes, the major epithelial cells of the liver. These trafficking pathways are regulated, in part, by small molecular weight rab GTPases. We chose to investigate rab17, whose expression is restricted to polarized epithelial cells, is enriched in liver, and has been implicated in regulating basolateral to apical transcytosis. To initiate our studies, we generated three recombinant adenoviruses expressing wild type, constitutively active (GTP bound), or dominant-negative (GDP bound) rab17. Immunoblotting revealed rab17 immunoreactive species at 25 kDa (the predicted rab17 molecular mass) and 40 kDa. We determined that mono-sumoylation of the 25-kDa rab17 is responsible for the shift in molecular mass, and that rab17 prenylation is required for sumoylation. We further determined that sumoylation selectively promotes interactions with syntaxin 2 (but not syntaxins 3 or 4) and that these interactions are nucleotide dependent. Furthermore, a K68R-mutated rab17 led to the redistribution of syntaxin 2 and 5′ nucleotidase from the apical membrane to subapical puncta, whereas multidrug resistance protein 2 distributions were not changed. Together these data are consistent with the proposed role of rab17 in vesicle fusion with the apical plasma membrane and further implicate sumoylation as an important mediator of protein-protein interactions. The selectivity in syntaxin binding and apical protein redistribution further suggests that rab17 and syntaxin 2 mediate fusion of transcytotic vesicles at the apical surface. PMID:26957544

Functional coupling of rat myometrial alpha 1-adrenergic receptors to Gh alpha/tissue transglutaminase 2 during pregnancy.

PubMed

Dupuis, Morgan; Lévy, Arlette; Mhaouty-Kodja, Sakina

2004-04-30

Gh alpha protein, which exhibits both transglutaminase and GTPase activities, represents a new class of GTP-binding proteins. In the present study, we characterized Gh alpha in rat uterine smooth muscle (myometrium) and followed its expression during pregnancy by reverse transcription-PCR and Western blot. We also measured transglutaminase and GTP binding functions and used a smooth muscle cell line to evaluate the role of Gh alpha in cell proliferation. The results show that pregnancy is associated with an up-regulation of Gh alpha expression at both the mRNA and protein level. Gh alpha induced during pregnancy is preferentially localized to the plasma membrane. This was found associated with an increased ability of plasma membrane preparations to catalyze Ca(2+)-dependent incorporation of [(3)H]putrescine into casein in vitro. In the cytosol, significant changes in the level of immunodetected Gh alpha and transglutaminase activity were seen only at term. Activation of alpha1-adrenergic receptors (alpha1-AR) enhanced photoaffinity labeling of plasma membrane Gh alpha. Moreover, the level of alpha1-AR-coupled Gh alpha increased progressively with pregnancy, which parallels the active period of myometrial cell proliferation. Overexpression of wild type Gh alpha in smooth muscle cell line DDT1-MF2 increased alpha1-AR-induced [(3)H]thymidine incorporation. A similar response was obtained in cells expressing the transglutaminase inactive mutant (C277S) of Gh alpha. Together, these findings underscore the role of Gh alpha as signal transducer of alpha1-AR-induced smooth muscle cell proliferation. In this context, pregnant rat myometrium provides an interesting physiological model to study the mechanisms underlying the regulation of the GTPase function of Gh alpha

Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons.

PubMed

Wang, Yan; Zhang, Heng; Shi, Meng; Liou, Yih-Cherng; Lu, Lei; Yu, Fengwei

2017-05-15

Pruning, whereby neurons eliminate their excess neurites, is central for the maturation of the nervous system. In Drosophila , sensory neurons, ddaCs, selectively prune their larval dendrites without affecting their axons during metamorphosis. However, it is unknown whether the secretory pathway plays a role in dendrite pruning. Here, we show that the small GTPase Arf1, an important regulator of the secretory pathway, is specifically required for dendrite pruning of ddaC/D/E sensory neurons but dispensable for apoptosis of ddaF neurons. Analyses of the GTP- and GDP-locked forms of Arf1 indicate that the cycling of Arf1 between GDP-bound and GTP-bound forms is essential for dendrite pruning. We further identified Sec71 as a guanine nucleotide exchange factor for Arf1 that preferentially interacts with its GDP-bound form. Like Arf1, Sec71 is also important for dendrite pruning, but not for apoptosis, of sensory neurons. Arf1 and Sec71 are interdependent for their localizations on Golgi. Finally, we show that the Sec71/Arf1-mediated trafficking process is a prerequisite for Rab5-dependent endocytosis to facilitate endocytosis and degradation of the cell-adhesion molecule Neuroglian (Nrg). © 2017. Published by The Company of Biologists Ltd.

Analysis of RNA binding by the dengue virus NS5 RNA capping enzyme.

PubMed

Henderson, Brittney R; Saeedi, Bejan J; Campagnola, Grace; Geiss, Brian J

2011-01-01

Flaviviruses are small, capped positive sense RNA viruses that replicate in the cytoplasm of infected cells. Dengue virus and other related flaviviruses have evolved RNA capping enzymes to form the viral RNA cap structure that protects the viral genome and directs efficient viral polyprotein translation. The N-terminal domain of NS5 possesses the methyltransferase and guanylyltransferase activities necessary for forming mature RNA cap structures. The mechanism for flavivirus guanylyltransferase activity is currently unknown, and how the capping enzyme binds its diphosphorylated RNA substrate is important for deciphering how the flavivirus guanylyltransferase functions. In this report we examine how flavivirus NS5 N-terminal capping enzymes bind to the 5' end of the viral RNA using a fluorescence polarization-based RNA binding assay. We observed that the K(D) for RNA binding is approximately 200 nM Dengue, Yellow Fever, and West Nile virus capping enzymes. Removal of one or both of the 5' phosphates reduces binding affinity, indicating that the terminal phosphates contribute significantly to binding. RNA binding affinity is negatively affected by the presence of GTP or ATP and positively affected by S-adensyl methoninine (SAM). Structural superpositioning of the dengue virus capping enzyme with the Vaccinia virus VP39 protein bound to RNA suggests how the flavivirus capping enzyme may bind RNA, and mutagenesis analysis of residues in the putative RNA binding site demonstrate that several basic residues are critical for RNA binding. Several mutants show differential binding to 5' di-, mono-, and un-phosphorylated RNAs. The mode of RNA binding appears similar to that found with other methyltransferase enzymes, and a discussion of diphosphorylated RNA binding is presented.

Green tea polyphenols supplementation and Tai Chi exercise for postmenopausal osteopenic women: safety and quality of life report

PubMed Central

2010-01-01

Background Evidence suggests that both green tea polyphenols (GTP) and Tai Chi (TC) exercise may benefit bone health in osteopenic women. However, their safety in this population has never been systematically investigated. In particular, there have been hepatotoxicity concerns related to green tea extract. This study was to evaluate the safety of 24 weeks of GTP supplementation combined with TC exercise in postmenopausal osteopenic women, along with effects on quality of life in this population. Methods 171 postmenopausal women with osteopenia were randomly assigned to 4 treatment arms for 24 weeks: (1) Placebo (500 mg starch/day), (2) GTP (500 mg GTP/day), (3) Placebo + TC (placebo plus TC training at 60 min/session, 3 sessions/week), and (4) GTP + TC (GTP plus TC training). Safety was examined by assessing liver enzymes (aspartate aminotransferase, alanine aminotransferase), alkaline phosphatase, and total bilirubin at baseline and every 4 weeks. Kidney function (urea nitrogen and creatinine), calcium, and inorganic phosphorus were also assessed at the same times. Qualify of life using SF-36 questionnaire was evaluated at baseline, 12, and 24 weeks. A mixed model of repeated measures ANOVA was applied for analysis. Results 150 subjects completed the study (12% attrition rate). The compliance rates for study agents and TC exercise were 89% and 83%, respectively. Neither GTP supplementation nor TC exercise affected liver or kidney function parameters throughout the study. No adverse event due to study treatment was reported by the participants. TC exercise significantly improved the scores for role-emotional and mental health of subjects, while no effect on quality of life was observed due to GTP supplementation. Conclusions GTP at a dose of 500 mg/day and/or TC exercise at 3 hr/week for 24 weeks appear to be safe in postmenopausal osteopenic women, particularly in terms of liver and kidney functions. TC exercise for 24 weeks (3 hr/wk) significantly improved quality of life in terms of role-emotional and mental health in these subjects. ClinicalTrials.gov identifier: NCT00625391. PMID:21143878

Localizing Carbohydrate Binding Sites in Proteins Using Hydrogen/Deuterium Exchange Mass Spectrometry

NASA Astrophysics Data System (ADS)

Zhang, Jingjing; Kitova, Elena N.; Li, Jun; Eugenio, Luiz; Ng, Kenneth; Klassen, John S.

2016-01-01

The application of hydrogen/deuterium exchange mass spectrometry (HDX-MS) to localize ligand binding sites in carbohydrate-binding proteins is described. Proteins from three bacterial toxins, the B subunit homopentamers of Cholera toxin and Shiga toxin type 1 and a fragment of Clostridium difficile toxin A, and their interactions with native carbohydrate receptors, GM1 pentasaccharides (β-Gal-(1→3)-β-GalNAc-(1→4)[α-Neu5Ac-(2→3)]-β-Gal-(1→4)-Glc), Pk trisaccharide (α-Gal-(1→4)-β-Gal-(1→4)-Glc) and CD-grease (α-Gal-(1→3)-β-Gal-(1→4)-β-GlcNAcO(CH2)8CO2CH3), respectively, served as model systems for this study. Comparison of the differences in deuterium uptake for peptic peptides produced in the absence and presence of ligand revealed regions of the proteins that are protected against deuterium exchange upon ligand binding. Notably, protected regions generally coincide with the carbohydrate binding sites identified by X-ray crystallography. However, ligand binding can also result in increased deuterium exchange in other parts of the protein, presumably through allosteric effects. Overall, the results of this study suggest that HDX-MS can serve as a useful tool for localizing the ligand binding sites in carbohydrate-binding proteins. However, a detailed interpretation of the changes in deuterium exchange upon ligand binding can be challenging because of the presence of ligand-induced changes in protein structure and dynamics.

Arsenite Stress Down-regulates Phosphorylation and 14-3-3 Binding of Leucine-rich Repeat Kinase 2 (LRRK2), Promoting Self-association and Cellular Redistribution*

PubMed Central

Mamais, Adamantios; Chia, Ruth; Beilina, Alexandra; Hauser, David N.; Hall, Christine; Lewis, Patrick A.; Cookson, Mark R.; Bandopadhyay, Rina

2014-01-01

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function. PMID:24942733

CHMP6 and VPS4A mediate recycling of Ras to the plasma membrane to promote growth factor signaling

PubMed Central

Zheng, Ze-Yi; Cheng, Chiang-Min; Fu, Xin-Rong; Chen, Liuh-Yow; Xu, Lizhong; Terrillon, Sonia; Wong, Stephen T.; Bar-Sagi, Dafna; Songyang, Zhou; Chang, Eric C.

2011-01-01

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III, were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding-loop and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound N-Ras, but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked EGFR recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling. PMID:22231449

Effects of metal on the biochemical properties of Helicobacter pylori HypB, a maturation factor of [NiFe]-hydrogenase and urease.

PubMed

Sydor, Andrew M; Liu, Jenny; Zamble, Deborah B

2011-03-01

The biosyntheses of the [NiFe]-hydrogenase and urease enzymes in Helicobacter pylori require several accessory proteins for proper construction of the nickel-containing metallocenters. The hydrogenase accessory proteins HypA and HypB, a GTPase, have been implicated in the nickel delivery steps of both enzymes. In this study, the metal-binding properties of H. pylori HypB were characterized, and the effects of metal binding on the biochemical behavior of the protein were examined. The protein can bind stoichiometric amounts of Zn(II) or Ni(II), each with nanomolar affinity. Mutation of Cys106 and His107, which are located between two major GTPase motifs, results in undetectable Ni(II) binding, and the Zn(II) affinity is weakened by 2 orders of magnitude. These two residues are also required for the metal-dependent dimerization observed in the presence of Ni(II) but not Zn(II). The addition of metals to the protein has distinct impacts on GTPase activity, with zinc significantly reducing GTP hydrolysis to below detectable levels and nickel only slightly altering the k(cat) and K(m) of the reaction. The regulation of HypB activities by metal binding may contribute to the maturation of the nickel-containing enzymes.

Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

PubMed Central

2015-01-01

ABSTRACT Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms. PMID:26055114

Suppression of Chemotaxis by SSeCKS via Scaffolding of Phosphoinositol Phosphates and the Recruitment of the Cdc42 GEF, Frabin, to the Leading Edge

PubMed Central

Ko, Hyun-Kyung; Guo, Li-wu; Su, Bing; Gao, Lingqiu; Gelman, Irwin H.

2014-01-01

Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS-null MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS-null leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS-null MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS-null MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement. PMID:25356636

Transforming growth factor‐β enhances Rho‐kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

PubMed Central

Shaifta, Yasin; MacKay, Charles E.; Irechukwu, Nneka; O'Brien, Katie A.; Wright, David B.; Ward, Jeremy P. T.

2017-01-01

Key points Transforming growth‐factor‐β (TGF‐β) and RhoA/Rho‐kinase are independently implicated in the airway hyper‐responsiveness associated with asthma, but how these proteins interact is not fully understood.We examined the effects of pre‐treatment with TGF‐β on expression and activity of RhoA, Rho‐kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin‐induced contraction, in airway smooth muscle.TGF‐β enhanced bradykinin‐induced RhoA translocation, Rho‐kinase‐dependent phosphorylation and contraction, but partially suppressed bradykinin‐induced RhoA activity (RhoA‐GTP content).TGF‐β enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF‐β on RhoA and Rho‐kinase activity and contraction, respectively.ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin‐sensitized mice.ARHGEF1 is a key TGF‐β target gene, an important regulator of Rho‐kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper‐responsiveness. Abstract Transforming growth factor‐β (TGF‐β), RhoA/Rho‐kinase and Src‐family kinases (SrcFK) have independently been implicated in airway hyper‐responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF‐β pre‐treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho‐kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF‐β pre‐treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20, MYPT‐1 and the actin‐severing protein cofilin, but not of RhoA, ROCK2 or c‐Src. In hASMCs, acute treatment with BK triggered subcellular translocation of ARHGEF1 and RhoA and enhanced auto‐phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20, but induced de‐phosphorylation of cofilin. TGF‐β pre‐treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA‐GTP content, SrcFK auto‐phosphorylation and cofilin de‐phosphorylation. In hASMCs, an ARHGEF1 small interfering RNA suppressed the effects of BK and TGF‐β on RhoA‐GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF‐β on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMCs of asthmatic patients and in lungs of ovalbumin‐sensitized mice. Our data indicate that TGF‐β enhances BK‐induced contraction, RhoA translocation and Rho‐kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA‐GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper‐responsiveness is worthy of further investigation. PMID:29071730

Implications of low great toe pressures in clinical practice.

PubMed

Varatharajan, Nandanan; Pillay, Sugendran; Hitos, Kerry; Fletcher, John P

2006-04-01

The aim of this study was to observe the outcome of patients with a great toe pressure (GTP) reading of less than 40 mmHg. Between 2002 and 2004, of 4,714 patients assessed in the Westmead Vascular Laboratory, 365 (7.7%) had GTP measurements for assessment of possible critical limb ischaemia. There were 56 limbs in 40 patients with a GTP measurement of less than 40 mmHg, and this group was studied to assess outcome factors of death, requirement for major or minor amputation, arterial reconstructive surgery, clinical stability, or documented improvement from the initial assessment. Patients requiring a major amputation had an average GTP of 13 mmHg and a toe brachial index (TBI) of 0.08, whereas those not undergoing a major amputation had an average GTP of 23.6 mmHg and TBI of 0.15. This association was weaker when an initial single evaluation was used as opposed to two or more serial assessments. Patients with and without a major amputation had an average ankle brachial index of 0.16 and 0.53, respectively. Diabetes mellitus requiring insulin and cerebrovascular disease were risk factors for major amputations. Low GTP was associated with a greater risk of major amputations. Two or more serial assessments were found to be of greater value than an initial single assessment.

Climate change impact of livestock CH4 emission in India: Global temperature change potential (GTP) and surface temperature response.

PubMed

Kumari, Shilpi; Hiloidhari, Moonmoon; Kumari, Nisha; Naik, S N; Dahiya, R P

2018-01-01

Two climate metrics, Global surface Temperature Change Potential (GTP) and the Absolute GTP (AGTP) are used for studying the global surface temperature impact of CH 4 emission from livestock in India. The impact on global surface temperature is estimated for 20 and 100 year time frames due to CH 4 emission. The results show that the CH 4 emission from livestock, worked out to 15.3 Tg in 2012. In terms of climate metrics GTP of livestock-related CH 4 emission in India in 2012 were 1030 Tg CO 2 e (GTP 20 ) and 62 Tg CO 2 e (GTP 100 ) at the 20 and 100 year time horizon, respectively. The study also illustrates that livestock-related CH 4 emissions in India can cause a surface temperature increase of up to 0.7mK and 0.036mK over the 20 and 100 year time periods, respectively. The surface temperature response to a year of Indian livestock emission peaks at 0.9mK in the year 2021 (9 years after the time of emission). The AGTP gives important information in terms of temperature change due to annual CH 4 emissions, which is useful when comparing policies that address multiple gases. Copyright © 2017 Elsevier Inc. All rights reserved.

Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, C.; Matozaki, T.; Nagao, M.

1987-09-01

Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced /sup 125/I-(Tyr/sup 1/)somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate (Gpp(NH)p)>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg/sup 2 +/. When pancreatic acini were treated withmore » 1 ..mu..g/ml pertussis toxin for 4 h, subsequent /sup 125/I-(Tyr/sup 1/)somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor.« less

Volatile anesthetics interfere with muscarinic receptor-g protein interactions in rat heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anthony, B.L.

The influence of halothane and enflurane (0.5-8%) on muscarinic receptor binding in rat atrium was studied using (/sup 3/H) methylscopolamine ((/sup 3/H)MS). Anesthetic-gas mixtures were blown over membrane suspensions for 20 min before and during the binding assays. Halothane and enflurane increased the affinity of cardiac muscarinic receptors for (/sup 3/H)MS by slowing the rate of dissociation. These anesthetics did not affect the affinity of the receptor for carbamylcholine, but significantly reduced the sensitivity of agonist binding to regulation by guanine nucleotides. For example, the fraction of receptors displaying high affinity agonist binding was decreased by a GTP analog frommore » 0.64 to 0.43 in the absence, but only to 0.52 in the presence of 2% halothane. The binding of a radiolabeled agonist, (/sup 3/H)oxotremorine-M, was reduced by 50% by halothane, while its sensitivity to guanine nucleotides was reduced by at least 100 fold. The diminution of the guanine nucleotide effect may reflect a stabilization of the receptor-G proteincomplex due to either a direct action on the receptor complex or to an alteration of the physical state of the membrane. It is also possible that the ability of the G protein to bind guanine nucleotides is adversely affected by anesthetic agents.« less

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation--A Novel Role for NDK.

PubMed

Mishra, Saurabh; Jakkala, Kishor; Srinivasan, Ramanujam; Arumugam, Muthu; Ranjeri, Raghavendra; Gupta, Prabuddha; Rajeswari, Haryadi; Ajitkumar, Parthasarathi

2015-01-01

Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.

NDK Interacts with FtsZ and Converts GDP to GTP to Trigger FtsZ Polymerisation - A Novel Role for NDK

PubMed Central

Mishra, Saurabh; Jakkala, Kishor; Srinivasan, Ramanujam; Arumugam, Muthu; Ranjeri, Raghavendra; Gupta, Prabuddha; Rajeswari, Haryadi; Ajitkumar, Parthasarathi

2015-01-01

Introduction Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation. Methods Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively. Results NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK’s NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK. Conclusion Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented. PMID:26630542

Cloning and analysis of 16 Rab genes from macronuclear DNA of Euplotes octocarinatus.

PubMed

Zhi, Hui; Wang, Wei; Li, Lingyan; Chai, Baofeng; Sun, Yonghua; Liang, Aihua

2005-08-01

Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus. Sixteen Rab genes were obtained. They share 87.6-99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well.

Rapid increase of inositol 1,4,5-trisphosphate in the HeLa cells after hypergravity exposure

NASA Technical Reports Server (NTRS)

Kumei, Yasuhiro; Whitson, Peggy A.; Cintron, Nitza M.; Sato, Atsushige

1990-01-01

The IP3 level in HeLa cells has been elevated through the application in hypergravity in a time-dependent manner. The data obtained for the hydrolytic products of PIP2, IP3, and DG are noted to modulate c-myc gene expression. It is also established that the cAMP accumulation by the IBMX in hypergravity-exposed cells was suppressed relative to the control. In light of IP3 increase and cAMP decrease results, a single GTP-binding protein may play a role in the hypergravity signal transduction of HeLa cells by stimulating PLC while inhibiting adenylate cyclase.

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ti, Shih-Chieh; Pamula, Melissa C.; Howes, Stuart C.

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. In this research, we have purified and characterized tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments.more » Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.« less

A Collapsin Response Mediator Protein 2 Isoform Controls Myosin II-Mediated Cell Migration and Matrix Assembly by Trapping ROCK II

PubMed Central

Morgan-Fisher, Marie; Wait, Robin; Couchman, John R.; Wewer, Ulla M.

2012-01-01

Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells. PMID:22431514

Dynamin: possible mechanism of "Pinchase" action.

PubMed

Kozlov, M M

1999-07-01

Dynamin is a GTPase playing an essential role in ubiquitous intra cellular processes involving separation of vesicles from plasma membranes and membranes of cellular compartments. Recent experimental progress (. Cell. 93:1021-1029;. Cell. 94:131-141) has made it possible to attempt to understand the action of dynamin in physical terms. Dynamin molecules are shown to bind to a lipid membrane, to self-assemble into a helicoidal structure constricting the membrane into a tubule, and, as a result of GTP hydrolysis, to mediate fission of this tubule (). In a similar way, dynamin is supposed to mediate fission of a neck connecting an endocytic bud and the plasma membrane, i.e., to complete endocytosis. We suggest a mechanism of this "pinchase" action of dynamin. We propose that, as a result of GTP hydrolysis, dynamin undergoes a conformational change manifested in growth of the pitch of the dynamin helix. We show that this gives rise to a dramatic change of shape of the tubular membrane constricted inside the helix, resulting in a local tightening of the tubule, which is supposed to promote its fission. We treat this model in terms of competing elasticities of the dynamin helix and the tubular membrane and discuss the predictions of the model in relation to the previous views on the mechanism of dynamin action.

A role for POR1, a Rac1-interacting protein, in ARF6-mediated cytoskeletal rearrangements.

PubMed Central

D'Souza-Schorey, C; Boshans, R L; McDonough, M; Stahl, P D; Van Aelst, L

1997-01-01

The ARF6 GTPase, the least conserved member of the ADP ribosylation factor (ARF) family, associates with the plasma membrane and intracellular endosome vesicles. Mutants of ARF6 defective in GTP binding and hydrolysis have a marked effect on endocytic trafficking and the gross morphology of the peripheral membrane system. Here we report that expression of the GTPase-defective mutant of ARF6, ARF6(Q67L), remodels the actin cytoskeleton by inducing actin polymerization at the cell periphery. This cytoskeletal rearrangement was inhibited by co-expression of ARF6(Q67L) with deletion mutants of POR1, a Rac1-interacting protein involved in membrane ruffling, but not with the dominant-negative mutant of Rac1, Rac1(S17N). A synergistic effect between POR1 and ARF6 for the induction of actin polymerization was detected. Furthermore, we observed that ARF6 interacts directly with POR1 and that this interaction was GTP dependent. These findings indicate that ARF6 and Rac1 function on distinct signaling pathways to mediate cytoskeletal reorganization, and suggest a role for POR1 as an important regulatory element in orchestrating cytoskeletal rearrangements at the cell periphery induced by ARF6 and Rac1. PMID:9312003

Mechanisms of kinetic stabilization by the drugs paclitaxel and vinblastine.

PubMed

Castle, Brian T; McCubbin, Seth; Prahl, Louis S; Bernens, Jordan N; Sept, David; Odde, David J

2017-05-01

Microtubule-targeting agents (MTAs), widely used as biological probes and chemotherapeutic drugs, bind directly to tubulin subunits and "kinetically stabilize" microtubules, suppressing the characteristic self-assembly process of dynamic instability. However, the molecular-level mechanisms of kinetic stabilization are unclear, and the fundamental thermodynamic and kinetic requirements for dynamic instability and its elimination by MTAs have yet to be defined. Here we integrate a computational model for microtubule assembly with nanometer-scale fluorescence microscopy measurements to identify the kinetic and thermodynamic basis of kinetic stabilization by the MTAs paclitaxel, an assembly promoter, and vinblastine, a disassembly promoter. We identify two distinct modes of kinetic stabilization in live cells, one that truly suppresses on-off kinetics, characteristic of vinblastine, and the other a "pseudo" kinetic stabilization, characteristic of paclitaxel, that nearly eliminates the energy difference between the GTP- and GDP-tubulin thermodynamic states. By either mechanism, the main effect of both MTAs is to effectively stabilize the microtubule against disassembly in the absence of a robust GTP cap. © 2017 Castle et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Recombinant viral RdRps can initiate RNA synthesis from circular templates

PubMed Central

RANJITH-KUMAR, C.T.; KAO, C.C.

2006-01-01

The crystal structure of the recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. We made circularized RNA templates and found that they were efficiently used by the HCV RdRp to synthesize product RNAs that are significantly longer than the template, suggesting that RdRp could exist in an open conformation prior to template binding. RNA synthesis using circular RNA templates had properties similar to those previously documented for linear RNA, including a need for higher GTP concentration for initiation, usage of GTP analogs, sensitivity to salt, and involvement of active-site residues for product formation. Some products were resistant to challenge with the template competitor heparin, indicating that the elongation complexes remain bound to template and are competent for RNA synthesis. Other products were not elongated in the presence of heparin, indicating that the elongation complex was terminated. Lastly, recombinant RdRps from two other flaviviruses and from the Pseudomonas phage φ6 also could use circular RNA templates for RNA-dependent RNA synthesis, although the φ6 RdRp could only use circular RNAs made from the 3′-terminal sequence of the φ6 genome. PMID:16373481

Odorants selectively activate distinct G protein subtypes in olfactory cilia.

PubMed

Schandar, M; Laugwitz, K L; Boekhoff, I; Kroner, C; Gudermann, T; Schultz, G; Breer, H

1998-07-03

Chemoelectrical signal transduction in olfactory neurons appears to involve intracellular reaction cascades mediated by heterotrimeric GTP-binding proteins. In this study attempts were made to identify the G protein subtype(s) in olfactory cilia that are activated by the primary (odorant) signal. Antibodies directed against the alpha subunits of distinct G protein subtypes interfered specifically with second messenger reponses elicited by defined subsets of odorants; odor-induced cAMP-formation was attenuated by Galphas antibodies, whereas Galphao antibodies blocked odor-induced inositol 1,4, 5-trisphosphate (IP3) formation. Activation-dependent photolabeling of Galpha subunits with [alpha-32P]GTP azidoanilide followed by immunoprecipitation using subtype-specific antibodies enabled identification of particular individual G protein subtypes that were activated upon stimulation of isolated olfactory cilia by chemically distinct odorants. For example odorants that elicited a cAMP response resulted in labeling of a Galphas-like protein, whereas odorants that elicited an IP3 response led to the labeling of a Galphao-like protein. Since odorant-induced IP3 formation was also blocked by Gbeta antibodies, activation of olfactory phospholipase C might be mediated by betagamma subunits of a Go-like G protein. These results indicate that different subsets of odorants selectively trigger distinct reaction cascades and provide evidence for dual transduction pathways in olfactory signaling.

DAMGO binding to mouse brain membranes: influence of salts, guanine nucleotides, substance P, and substance P fragments.

PubMed

Krumins, S A; Kim, D C; Igwe, O J; Larson, A A

1993-01-01

Substance P (SP) appears to mediate many processes of the central nervous system, including pain. This report deals with modulation of opioid binding in the mouse brain by SP and SP fragments, as well as by salts and guanine nucleotides. Binding studies of the selective mu opioid receptor agonist [D-Ala2, MePhe4,Gly(ol)5]enkephalin (DAMGO) to mouse brain membrane preparations demonstrated that guanine nucleotide modulation of DAMGO binding affinity was modified by SP. However, SP had little or no influence on inhibition of DAMGO binding induced by salts, such as MgCl2, CaCl2, or NaCl. By replacing GTP with GppNHp, SP (0.1 nM) produced multiple affinity forms of the DAMGO receptor, while at a higher concentration (10 nM), SP lost its influence on DAMGO binding. Furthermore, 0.1 nM SP changed DAMGO binding parameters in a medium containing NaCl, CaCl2, and GppNHp such that the high- and low-affinity conformations of the receptor converted to a single site following the addition of SP to the incubation medium. While the C-terminal SP fragment SP(5-11) was without effect, the N-terminal SP fragments SP(1-9) and SP(1-7) appeared to imitate SP in modifying GppNHp-modulated DAMGO binding. These results suggest that SP functions as a modulator of opioid binding at the mu receptor and it appears that the N-terminus of SP plays a role in the modulatory process.

NADPH oxidase activation in neutrophils: Role of the Phosphorylation of its subunits.

PubMed

Belambri, Sahra A; Rolas, Loïc; Raad, Houssam; Hurtado-Nedelec, Margarita; Dang, Pham My-Chan; El-Benna, Jamel

2018-05-14

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O 2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O 2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47 phox , p67 phox , p40 phox and Rac2) with the transmembrane proteins (p22 phox and gp91 phox , which form the cytochrome b 558 ). gp91 phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space in order to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47 phox and p40 phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, i.e., gp91 phox , p22 phox , p47 phox , p67 phox and p40 phox , in the activation of this enzyme. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Evidence for a G protein-coupled diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) receptor binding site in lung membranes from rat.

PubMed

Laubinger, W; Reiser, G

1999-01-29

Nucleotide receptors are of considerable importance in the treatment of lung diseases, such as cystic fibrosis. Because diadenosine polyphosphates may also be of significance as signalling molecules in lung, as they are in a variety of tissues, in the present work we investigated the binding sites for [3H]diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) in plasma membranes from rat lung and studied their possible coupling to G proteins. We present evidence for a single high-affinity binding site for [3H]Ap4A with similar affinity for other diadenosine polyphosphates ApnA (n = 2 to 6). Displacement studies with different nucleotides revealed that the [3H]Ap4A binding site was different from P2X and P2Y2 receptor binding sites. Pretreatment of lung membranes with GTPgammaS or GTP in the presence of Mg2+ increased the Ki for Ap4A from 91 nM to 5.1 microM, which is indicative of G protein coupling. The putative coupling to G proteins was further confirmed by the enhancement of [35S]GTPgammaS binding (to Galpha proteins) to lung membranes by Ap4A (63% increase over basal) in a concentration-dependent manner. Therefore, our data for the first time provide evidence of a G protein-coupled Ap4A binding site in lung membranes.

Suppressors of dGTP Starvation in Escherichia coli

PubMed Central

Itsko, Mark

2017-01-01

ABSTRACT dGTP starvation, a newly discovered phenomenon in which Escherichia coli cells are starved specifically for the DNA precursor dGTP, leads to impaired growth and, ultimately, cell death. Phenomenologically, it represents an example of nutritionally induced unbalanced growth: cell mass amplifies normally as dictated by the nutritional status of the medium, but DNA content growth is specifically impaired. The other known example of such a condition, thymineless death (TLD), involves starvation for the DNA precursor dTTP, which has been found to have important chemotherapeutic applications. Experimentally, dGTP starvation is induced by depriving an E. coli gpt optA1 strain of its required purine source, hypoxanthine. In our studies of this phenomenon, we noted the emergence of a relatively high frequency of suppressor mutants that proved resistant to the treatment. To study such suppressors, we used next-generation sequencing on a collection of independently obtained mutants. A significant fraction was found to carry a defect in the PurR transcriptional repressor, controlling de novo purine biosynthesis, or in its downstream purEK operon. Thus, upregulation of de novo purine biosynthesis appears to be a major mode of overcoming the lethal effects of dGTP starvation. In addition, another large fraction of the suppressors contained a large tandem duplication of a 250- to 300-kb genomic region that included the purEK operon as well as the acrAB-encoded multidrug efflux system. Thus, the suppressive effects of the duplications could potentially involve beneficial effects of a number of genes/operons within the amplified regions. IMPORTANCE Concentrations of the four precursors for DNA synthesis (2′-deoxynucleoside-5′-triphosphates [dNTPs]) are critical for both the speed of DNA replication and its accuracy. Previously, we investigated consequences of dGTP starvation, where the DNA precursor dGTP was specifically reduced to a low level. Under this condition, E. coli cells continued cell growth but eventually developed a DNA replication defect, leading to cell death due to formation of unresolvable DNA structures. Nevertheless, dGTP-starved cultures eventually resumed growth due to the appearance of resistant mutants. Here, we used whole-genome DNA sequencing to identify the responsible suppressor mutations. We show that the majority of suppressors can circumvent death by upregulating purine de novo biosynthesis, leading to restoration of dGTP to acceptable levels. PMID:28373271

Reducing the heterogeneity of xylan through processing.

PubMed

Zhang, Wei; Johnson, Amanda M; Barone, Justin R; Renneckar, Scott

2016-10-05

Glycerol thermal processing (GTP) of hardwood biomass at temperatures between 200 and 240°C facilitated stepwise biopolymer fractionation, while limiting significant degradation of the major hemicellulose, glucuronoxylan, into water-extractable oligosaccharides. After GTP pretreatment and sequential water and organic solvent extraction, up to 80% of the initial xylan remained in the pretreated biomass. The majority of the xylan from GTP pretreated and water/solvent extracted biomass was removed using a mild alkali extraction and the composition was compared to xylan directly isolated from untreated hardwood. The precipitated xylan from the neutralized alkaline filtrate was isolated as a water insoluble xylan portion (WIX). The residual xylan dissolved in the neutralized filtrate was precipitated in cold methanol and recovered as the water soluble xylan portion (WSX). Results showed that xylan in WIX was in a polymeric form with a number average degree of polymerization (DP) over 100, whereas the WSX had a much lower average DP of 27 (ca) and contained more substitution. As the processing severity increased during GTP pretreatment, the proportion of WIX increased and the purity of the xylan within the WIX sample reached 84% based on compositional analysis. FT-IR analysis of WIX revealed that xylan isolated after GTP contained peaks related to a reduced carbonyl signal compared to the control. Furthermore, crude WSX contained less xylan with more lignin contamination at severe GTP conditions. The recovery of the xylan in two portions facilitated a preferential purification strategy resulting in WIX with an extremely narrow polydispersity index between 1.1 and 1.25, dependent upon the GTP severity. This study provided insight into fractionating higher molecular weight xylan that may serve value-added applications such as healthcare materials and advanced packaging. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

PubMed

Long, Aaron; Klimova, Nina; Kristian, Tibor

2017-10-01

NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

DOE PAGES

Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; ...

2014-11-17

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2’-deoxyguanosine found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate E. coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerase discriminates between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities,more » nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine (Cy) and 8-oxodGTP(syn) utilizes its Hoogsteen edge to base pair with adenine (Ad). Here in this paper we utilized time-lapse crystallography to follow 8-oxo-dGTP insertion opposite Ad or Cy with human DNA pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxodGTP utilizes charge modulation during insertion that can lead to a blocked DNA repair intermediate.« less

Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

PubMed

Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

2014-01-01

In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

Different Effects of Guanine Nucleotides (GDP and GTP) on Protein-Mediated Mitochondrial Proton Leak

PubMed Central

Woyda-Ploszczyca, Andrzej M.; Jarmuszkiewicz, Wieslawa

2014-01-01

In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP. PMID:24904988

Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

NASA Astrophysics Data System (ADS)

Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; Shock, David D.; Kim, Taejin; Schlick, Tamar; Wilson, Samuel H.

2015-01-01

Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

GTPase Sar1 regulates the trafficking and secretion of the virulence factor gp63 in Leishmania.

PubMed

Parashar, Smriti; Mukhopadhyay, Amitabha

2017-07-21

Metalloprotease gp63 ( Leishmania donovani gp63 (Ldgp63)) is a critical virulence factor secreted by Leishmania However, how newly synthesized Ldgp63 exits the endoplasmic reticulum (ER) and is secreted by this parasite is unknown. Here, we cloned, expressed, and characterized the GTPase LdSar1 and other COPII components like LdSec23, LdSec24, LdSec13, and LdSec31 from Leishmania to understand their role in ER exit of Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds to LdSec23, which binds, in turn, with LdSec24(1-702) to form a prebudding complex. Moreover, LdSec13 specifically interacted with His 6 -LdSec31(1-603), and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine 594/595 and valine 597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24(703-966), and GFP-Ldgp63 L594A/L595A or GFP-Ldgp63 V597S mutants failed to exit from the ER. Moreover, Ldgp63-containing COPII vesicle budding from the ER was inhibited by LdSar1:T34N in an in vitro budding assay, indicating that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we coexpressed RFP-Ldgp63 along with LdSar1:WT-GFP or LdSar1:T34N-GFP and found that LdSar1:T34N overexpression blocks Ldgp63 trafficking and secretion in Leishmania Finally, we noted significantly compromised survival of LdSar1:T34N-GFP-overexpressing transgenic parasites in macrophages. Taken together, these results indicated that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63 ER exit and subsequent secretion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers.

PubMed

Pang, T; Su, X; Wakabayashi, S; Shigekawa, M

2001-05-18

The Na+/H+ exchangers (NHEs) comprise a family of transporters that catalyze cell functions such as regulation of the pH and volume of a cell and epithelial absorption of Na+ and bicarbonate. Ubiquitous calcineurin B homologous protein (CHP or p22) is co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or NHE3 independently of its myristoylation and Ca2+ binding, and its binding site was identified as the juxtamembrane region within the carboxyl-terminal cytoplasmic domain of exchangers. CHP binding-defective mutations of NHE1-3 or CHP depletion by injection of the competitive CHP-binding region of NHE1 into Xenopus oocytes resulted in a dramatic reduction (>90%) in the Na+/H+ exchange activity. The data suggest that CHP serves as an essential cofactor, which supports the physiological activity of NHE family members.

The Role of Magnesium for Geometry and Charge in GTP Hydrolysis, Revealed by Quantum Mechanics/Molecular Mechanics Simulations

PubMed Central

Rudack, Till; Xia, Fei; Schlitter, Jürgen; Kötting, Carsten; Gerwert, Klaus

2012-01-01

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and Pi in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and Pi release back to the diphosphate. Furthermore, the Ras·Mg2+ complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger. PMID:22853907

Physical and functional connection between auxilin and dynamin during endocytosis

PubMed Central

Sever, Sanja; Skoch, Jesse; Newmyer, Sherri; Ramachandran, Rajesh; Ko, David; McKee, Mary; Bouley, Richard; Ausiello, Dennis; Hyman, Bradley T; Bacskai, Brian J

2006-01-01

During clathrin-mediated endocytosis, the GTPase dynamin promotes formation of clathrin-coated vesicles, but its mode of action is unresolved. We provide evidence that a switch in three functional states of dynamin (dimers, tetramers, rings/spirals) coordinates its GTPase cycle. Dimers exhibit negative cooperativity whereas tetramers exhibit positive cooperativity with respect to GTP. Our study identifies tetramers as the kinetically most stable GTP-bound conformation of dynamin, which is required to promote further assembly into higher order structures such as rings or spirals. In addition, using fluorescence lifetime imaging microscopy, we show that interactions between dynamin and auxilin in cells are GTP-, endocytosis- and tetramer-dependent. Furthermore, we show that the cochaperone activity of auxilin is required for constriction of clathrin-coated pits, the same early step in endocytosis known to be regulated by the lifetime of dynamin:GTP. Together, our findings support the model that the GTP-bound conformation of dynamin tetramers stimulates formation of constricted coated pits at the plasma membrane by regulating the chaperone activity of hsc70/auxilin. PMID:16946707

Modulatory efficacy of green tea polyphenols on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide-induced oral carcinogenesis-A therapeutic approach.

PubMed

Srinivasan, Periasamy; Sabitha, Kuruvimalai Ekambaram; Shyamaladevi, Chennam Srinivasulu

2006-08-25

Green tea polyphenols (GTP) has been used as a chemopreventive agent world wide against chemically induced cancer. The present study is aimed to understand the therapeutic action of GTP on glycoconjugates and immunological markers in 4-Nitroquinoline 1-oxide (4-NQO)-induced oral cancer over a period of 30 days at 200mg/kg, p.o., Oral cancer was induced by painting 4-NQO for 8 weeks followed by administration of GTP after 22 weeks, for 30 days. Glycoconjugates such as hexose, hexosamine, sialicacid, fucose and mucoprotein were analysed. Expression of glycoconjugates was examined through histology and SDS-PAGE. Immunological markers such as circulating immune complex and mast cell density were studied. Oral cancer-induced animals showed a significant increase in levels of glycoconjugates and its expression, similar to that observed for immunological markers. Treatment with GTP altered the expression of glycoconjugates as well as immunological markers. The results suggest that GTP modulates both the expression of glycoconjugates and immunological markers resulting in regression of oral cancer.

Off-target effect of the Epac agonist 8-pCPT-2'-O-Me-cAMP on P2Y12 receptors in blood platelets.

PubMed

Herfindal, Lars; Nygaard, Gyrid; Kopperud, Reidun; Krakstad, Camilla; Døskeland, Stein Ove; Selheim, Frode

2013-08-09

The primary target of the cAMP analogue 8-pCPT-2'-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2'-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2'-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2'O-Me-cAMP binding into the purinergic platelet receptor P2Y12 revealed that the analogue docks similar to the P2Y12 antagonist 2MeSAMP. The 8-pCPT-2'-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2'-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2'-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y12 receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2'-O-Me-cAMP did not affect platelet activation at similar concentrations. Copyright © 2013 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bollinger, Jonathan A.; Stevens, Mark J.

We report that microtubules exhibit a dynamic instability between growth and catastrophic depolymerization. GTP-tubulin (αβ-dimer bound to GTP) self-assembles, but dephosphorylation of GTP- to GDP-tubulin within the tubule results in destabilization. While the mechanical basis for destabilization is not fully understood, one hypothesis is that dephosphorylation causes tubulin to change shape, frustrating bonds and generating stress. To test this idea, we perform molecular dynamics simulations of microtubules built from coarse-grained models of tubulin, incorporating a small compression of α-subunits associated with dephosphorylation in experiments. We find that this shape change induces depolymerization of otherwise stable systems via unpeeling “ram's horns”more » characteristic of microtubules. Depolymerization can be averted by caps with uncompressed α-subunits, i.e., GTP-rich end regions. Thus, the shape change is sufficient to yield microtubule behavior.« less

GTP regeneration influences interactions of microtubules, neurofilaments, and microtubule-associated proteins in vitro.

PubMed

Flynn, G; Purich, D L

1987-11-15

Interactions of microtubules, neurofilaments, and microtubule-associated proteins were investigated by turbidity and falling-ball viscometry measurements. We found evidence of endogenous GTPase activity in neurofilaments and microtubule-associated proteins (MAPs) in preparations that do not include urea or heat treatment, respectively. The absence or presence of either adenyl-5'-yl imidodiphosphonic acid or a GTP-regenerating system markedly influenced observed polymerization and gelation characteristics. Most significantly, the apparent viscosity of neurofilament and microtubule samples did not display a biphasic optimal MAP concentration profile when a GTP-regenerating system was operant. Likewise, GTP regeneration promoted the recovery of gelation following mechanical disruption of neurofilament/MAP/microtubule mixtures. These and other observations require some reassessment of proposed roles for microtubule-associated proteins in modulating neurofilament-microtubule interactions in vitro.

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

PubMed Central

Thévenod, F; Gasser, K W; Hopfer, U

1990-01-01

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation. PMID:2264815

Two classes of binding sites for [3H]substance P in rat cerebral cortex.

PubMed

Geraghty, D P; Burcher, E

1993-01-22

The binding characteristics of [3H]substance P ([3H]SP) were investigated in membranes prepared from rat cerebral cortex. Binding of [3H]SP reached equilibrium after 50 min at 25 degrees C and was saturable at 8 nM. Saturation data could be resolved into high affinity (equilibrium dissociation constant, Kd, 0.22 nM) and low affinity sites (Kd, 2.65 nM). The low affinity sites were more numerous than the high affinity sites, with a ratio of 4:1. The non-hydrolyzable GTP analogue GppNHp had no effect on binding, indicating that the high and low affinity sites are not guanine nucleotide-regulated states of the same (NK-1) receptor. The low affinity sites are unlikely to represent NK-3 receptors since coincubation with the selective NK-3 receptor agonist senktide did not alter the biphasic nature of [3H]SP binding. The rank order of potency for inhibition of [3H]SP (2 nM) binding was SP > or = [Sar9, Met(O2)11]-SP > or = physalaemin > SP(3-11) > NP gamma = [Ala3]-SP > or = SP(4-11) > or = NPK > or = SP(5-11) > or = NKB approximately NKA > SP(1-9), compatible with binding to an NK-1 site. N-terminal fragments and non-amidated analogues were ineffective competitors for [3H]SP binding. However, competition data for several peptides including substance P (SP) and the NK-1 selective agonist [Sar9, Met(O2)11]-SP could be resolved into two components.(ABSTRACT TRUNCATED AT 250 WORDS)

A web server for analysis, comparison and prediction of protein ligand binding sites.

PubMed

Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

2016-03-25

One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

A Salmonella typhimurium-translocated Glycerophospholipid:Cholesterol Acyltransferase Promotes Virulence by Binding to the RhoA Protein Switch Regions

DOE Office of Scientific and Technical Information (OSTI.GOV)

LaRock, Doris L.; Brzovic, Peter S.; Levin, Itay

Salmonella enterica serovar typhimurium translocates a glycerophospholipid: cholesterol acyltransferase (SseJ) into the host cytosol after its entry into mammalian cells. SseJ is recruited to the cytoplasmic face of the host cell phagosome membrane where it is activated upon binding the small GTPase, RhoA. SseJ is regulated similarly to cognate eukaryotic effectors, as only the GTP-bound form of RhoA family members stimulates enzymatic activity. Using NMR and biochemistry, this work demonstrates that SseJ competes effectively with Rhotekin, ROCK, and PKN1 in binding to a similar RhoA surface. The RhoA surface that binds SseJ includes the regulatory switch regions that control activationmore » of mammalian effectors. These data were used to create RhoA mutants with altered SseJ binding and activation. This structure-function analysis supports a model in which SseJ activation occurs predominantly through binding to residues within switch region II. We further defined the nature of the interaction between SseJ and RhoA by constructing SseJ mutants in the RhoA binding surface. These data indicate that SseJ binding to RhoA is required for recruitment of SseJ to the endosomal network and for full Salmonella virulence for inbred susceptible mice, indicating that regulation of SseJ by small GTPases is an important virulence strategy of this bacterial pathogen. The dependence of a bacterial effector on regulation by a mammalian GTPase defines further how intimately host pathogen interactions have coevolved through similar and divergent evolutionary strategies.« less

Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5 prime -O-(3-thiotriphosphate)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baldassare, J.J.; Henderson, P.A.; Fisher, G.J.

1989-01-10

The effects of thrombin and GTP{gamma}S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous ({sup 3}H)inositol-labeled membranes or with lipid vesicles containing either ({sup 3}H)phosphatidylinositol or ({sup 3}H)phosphatidylinositol 4,5-bisphosphate. GTP{gamma}S (1 {mu}M) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP{sub 3}), inositol bisphosphate (IP{sub 2}), or inositol phosphate (IP) from ({sup 3}H)inositol-labeled membranes. IP{sub 2} and IP{sub 3}, but not IP, from ({sup 3}H)inositol-labeled membranes were, however, stimulated 3-fold by GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). A higher concentration of GTP{gamma}S (100 {mu}M) alone also stimulatedmore » IP{sub 2} and IP{sub 3}, but not IP, release. In the presence of 1 mM calcium, release of IP{sub 2} and IP{sub 3} was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}) by platelet membrane associated PLC was also markedly enhanced by GTP{gamma}S (100 {mu}M) or GTP{gamma}S (1 {mu}M) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP{sub 2} was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP{gamma}S (100 {mu}M) or calcium (1 mM) dependent PIP{sub 2} breakdown, while TPA inhibited GTP{gamma}S-dependent but not calcium-dependent phospholipase C activity.« less

Quantitative analysis of the interactions between prenyl Rab9, GDP dissociation inhibitor-alpha, and guanine nucleotides.

PubMed

Shapiro, A D; Pfeffer, S R

1995-05-12

Rab9 is a Ras-like GTPase required for the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network. Rab9 occurs in the cytosol as a complex with GDP dissociation inhibitor (GDI), which we have shown delivers prenyl Rab9 to late endosomes in a functional form. We report here basal rate constants for guanine nucleotide dissociation and GTP hydrolysis for prenyl Rab9. Both rate constants were influenced in part by the hydrophobic environment of the prenyl group. Guanine nucleotide dissociation and GTP hydrolysis rates were lower in the presence of lipid; detergent stimulated intrinsic nucleotide exchange. GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 2.4-fold. GDI-alpha associated with prenyl Rab9 with a KD of 60 nM in 0.1% Lubrol and 23 nM in 0.02% Lubrol. In 0.1% Lubrol, GDI-alpha inhibited GDP dissociation half maximally at 72 +/- 18 nM, consistent with the KD determinations. These data suggest that GDI-alpha associates with prenyl Rab9 with a KD of < or = 23 nM under physiological conditions. Finally, a previously uncharacterized minor form of GDI-alpha inhibited GDP dissociation from prenyl Rab9 by 1.9-fold and bound prenyl Rab9 with a KD of 67 nM in 0.1% Lubrol.

Minimal determinants for binding activated G-alpha from the structure of a G-alpha-i1/peptide dimer†

PubMed Central

Johnston, Christopher A.; Lobanova, Ekaterina S.; Shavkunov, Alexander S.; Low, Justin; Ramer, J. Kevin; Blaesius, Rainer; Fredericks, Zoey; Willard, Francis S.; Kuhlman, Brian; Arshavsky, Vadim Y.; Siderovski, David P.

2008-01-01

G-proteins cycle between an inactive GDP-bound state and active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage-display to identify a series of peptides that bind Gα subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069–1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP·AlF4−- and GTPγS-bound states of Gαi subunits. KB-1753 blocks interaction of Gαtransducin with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated Gα in vitro. The crystal structure of KB-1753 bound to Gαi1·GDP·AlF4− reveals binding to a conserved hydrophobic groove between switch II and α3 helices, and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for Gαi subunits. PMID:16981699

Identification and Characterization of a G Protein-binding Cluster in α7 Nicotinic Acetylcholine Receptors.

PubMed

King, Justin R; Nordman, Jacob C; Bridges, Samuel P; Lin, Ming-Kuan; Kabbani, Nadine

2015-08-14

α7 nicotinic acetylcholine receptors (nAChRs) play an important role in synaptic transmission and inflammation. In response to ligands, this receptor channel opens to conduct cations into the cell but desensitizes rapidly. In recent studies we show that α7 nAChRs bind signaling proteins such as heterotrimeric GTP-binding proteins (G proteins). Here, we demonstrate that direct coupling of α7 nAChRs to G proteins enables a downstream calcium signaling response that can persist beyond the expected time course of channel activation. This process depends on a G protein-binding cluster (GPBC) in the M3-M4 loop of the receptor. A mutation of the GPBC in the α7 nAChR (α7345-348A) abolishes interaction with Gαq as well as Gβγ while having no effect on receptor synthesis, cell-surface trafficking, or α-bungarotoxin binding. Expression of α7345-348A, however, did significantly attenuate the α7 nAChR-induced Gαq calcium signaling response as evidenced by a decrease in PLC-β activation and IP3R-mediated calcium store release in the presence of the α7 selective agonist choline. Taken together, the data provides new evidence for the existence of a GPBC in nAChRs serving to promote intracellular signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mixed-mode sorption of hydroxylated atrazine degradation products to sell: A mechanism for bound residue

USGS Publications Warehouse

Lerch, R.N.; Thurman, E.M.; Kruger, E.L.

1997-01-01

This study tested the hypothesis that sorption of hydroxylated atrazine degradation products (HADPs: hydroxyatrazine, HA; deethylhydroxyatrazine, DEHA; and deisopropylhydroxyatrazine, DIHA) to soils occurs by mixed-mode binding resulting from two simultaneous mechanisms: (1) cation exchange and (2) hydrophobic interaction. The objective was to use liquid chromatography and soil extraction experiments to show that mixed-mode binding is the mechanism controlling HADP sorption to soils and is also a mechanism for bound residue. Overall, HADP binding to solid-phase extraction (SPE) sorbents occurred in the order: cation exchange >> octadecyl (C18) >> cyanopropyl. Binding to cation exchange SPE and to a high-performance liquid chromatograph octyl (C8) column showed evidence for mixed-mode binding. Comparison of soil extracted by 0.5 M KH2P04, pH 7.5, or 25% aqueous CH3CN showed that, for HA and DIHA, cation exchange was a more important binding mechanism to soils than hydrophobic interaction. Based on differences between several extractants, the extent of HADP mixed-mode binding to soil occurred in the following order: HA > DIHA > DEHA. Mixed-mode extraction recovered 42.8% of bound atrazine residues from aged soil, and 88% of this fraction was identified as HADPs. Thus, a significant portion of bound atrazine residues in soils is sorbed by the mixed-mode binding mechanisms.

Calexcitin interaction with neuronal ryanodine receptors.

PubMed Central

Nelson, T J; Zhao, W Q; Yuan, S; Favit, A; Pozzo-Miller, L; Alkon, D L

1999-01-01

Calexcitin (CE), a Ca2+- and GTP-binding protein, which is phosphorylated during memory consolidation, is shown here to co-purify with ryanodine receptors (RyRs) and bind to RyRs in a calcium-dependent manner. Nanomolar concentrations of CE released up to 46% of the 45Ca label from microsomes preloaded with 45CaCl2. This release was Ca2+-dependent and was blocked by antibodies against the RyR or CE, by the RyR inhibitor dantrolene, and by a seven-amino-acid peptide fragment corresponding to positions 4689-4697 of the RyR, but not by heparin, an Ins(1,4,5)P3-receptor antagonist. Anti-CE antibodies, in the absence of added CE, also blocked Ca2+ release elicited by ryanodine, suggesting that the CE and ryanodine binding sites were in relative proximity. Calcium imaging with bis-fura-2 after loading CE into hippocampal CA1 pyramidal cells in hippocampal slices revealed slow, local calcium transients independent of membrane depolarization. Calexcitin also released Ca2+ from liposomes into which purified RyR had been incorporated, indicating that CE binding can be a proximate cause of Ca2+ release. These results indicated that CE bound to RyRs and suggest that CE may be an endogenous modulator of the neuronal RyR. PMID:10393102

Inhibition of GTPase Rac1 in endothelium by 6-mercaptopurine results in immunosuppression in nonimmune cells: new target for an old drug.

PubMed

Marinković, Goran; Kroon, Jeffrey; Hoogenboezem, Mark; Hoeben, Kees A; Ruiter, Matthijs S; Kurakula, Kondababu; Otermin Rubio, Iker; Vos, Mariska; de Vries, Carlie J M; van Buul, Jaap D; de Waard, Vivian

2014-05-01

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are well established immunosuppressive drugs. Common understanding of their immunosuppressive properties is largely limited to immune cells. However, in this study, the mechanism underlying the protective role of 6-MP in endothelial cell activation is investigated. Because 6-MP and its derivative 6-thioguanosine-5'-triphosphate (6-T-GTP) were shown to block activation of GTPase Rac1 in T lymphocytes, we focused on Rac1-mediated processes in endothelial cells. Indeed, 6-MP and 6-T-GTP decreased Rac1 activation in endothelial cells. As a result, the compounds inhibited TNF-α-induced downstream signaling via JNK and reduced activation of transcription factors c-Jun, activating transcription factor-2 and, in addition, NF κ-light-chain-enhancer of activated B cells (NF-κB), which led to decreased transcription of proinflammatory cytokines. Moreover, 6-MP and 6-T-GTP selectively decreased TNF-α-induced VCAM-1 but not ICAM-1 protein levels. Rac1-mediated generation of cell membrane protrusions, which form docking structures to capture leukocytes, also was reduced by 6-MP/6-T-GTP. Consequently, leukocyte transmigration was inhibited after 6-MP/6-T-GTP treatment. These data underscore the anti-inflammatory effect of 6-MP and 6-T-GTP on endothelial cells by blocking Rac1 activation. Our data provide mechanistic insight that supports development of novel Rac1-specific therapeutic approaches against chronic inflammatory diseases.

Delta Alert: Expanding Gerotrauma Criteria to Improve Patient Outcomes: A 2-Year Study.

PubMed

Wiles, Lynn L; Day, Mark D

Because of their decreased physical reserve and increased risk of complications, the geriatric trauma patient (GTP) population warrants heightened awareness by clinical staff. The purpose of this study is to determine whether the institution of a third-tier trauma protocol results in a change in GTP outcomes, complications, and mortality rates. Researchers conducted a retrospective review of 2 years of data from the trauma registry, hospital quality improvement audits, and patient charts to examine what, if any, patient outcomes were impacted by the institution of the expanded GTP protocol. Sample homogeneity was determined. Emergency department (ED) length of stay and time to the operating room decreased in the protocol cohort. The rate of complications decreased from 16.4% preprotocol to 1.6% postprotocol. Discharge to home rates in the GTP population improved from 31% preprotocol to nearly 77% postimplementation of the protocol. The expanded GTP protocol front loads evaluation and resuscitation to be consistent with ED trauma protocols already in place. By fast-tracking radiology and laboratory testing, patients injuries are identified and the appropriate consultations are initiated. Appropriate inpatient nursing unit placement is identified or treatment and discharge from the ED are expedited. The expanded GTP protocol provided early and comprehensive evaluation and interventions for GTPs who fall outside of traditional trauma alert criteria. Patients spend less time in the ED and the hospital. Patients had decreased length of stay in the ED, less complications, and return to home rates showed significant improvement after the protocol was implemented.

Identification of RhoGAP22 as an Akt-Dependent Regulator of Cell Motility in Response to Insulin▿‡

PubMed Central

Rowland, Alexander F.; Larance, Mark; Hughes, William E.; James, David E.

2011-01-01

Insulin exerts many of its metabolic actions via the canonical phosphatidylinositide 3 kinase (PI3K)/Akt pathway, leading to phosphorylation and 14-3-3 binding of key metabolic targets. We previously identified a GTPase-activating protein (GAP) for Rac1 called RhoGAP22 as an insulin-responsive 14-3-3 binding protein. Insulin increased 14-3-3 binding to RhoGAP22 fourfold, and this effect was PI3K dependent. We identified two insulin-responsive 14-3-3 binding sites (pSer16 and pSer395) within RhoGAP22, and mutagenesis studies revealed a complex interplay between the phosphorylation at these two sites. Mutating Ser16 to alanine blocked 14-3-3 binding to RhoGAP22 in vivo, and phosphorylation at Ser16 was mediated by the kinase Akt. Overexpression of a mutant RhoGAP22 that was unable to bind 14-3-3 reduced cell motility in NIH-3T3 fibroblasts, and this effect was dependent on a functional GAP domain. Mutation of the catalytic arginine of the GAP domain of RhoGAP22 potentiated growth factor-stimulated Rac1 GTP loading. We propose that insulin and possibly growth factors such as platelet-derived growth factor may play a novel role in regulating cell migration and motility via the Akt-dependent phosphorylation of RhoGAP22, leading to modulation of Rac1 activity. PMID:21969604

Mechanism of the Exchange Reaction in HRAS from Multiscale Modeling

PubMed Central

Kapoor, Abhijeet; Travesset, Alex

2014-01-01

HRAS regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Understanding the transition mechanism is central for the design of small molecules to inhibit the formation of RAS-driven tumors. Using a multiscale approach involving coarse-grained (CG) simulations, all-atom classical molecular dynamics (CMD; total of 3.02 µs), and steered molecular dynamics (SMD) in combination with Principal Component Analysis (PCA), we identified the structural features that determine the nucleotide (GDP) exchange reaction. We show that weakening the coupling between the SwitchI (residues 25–40) and SwitchII (residues 59–75) accelerates the opening of SwitchI; however, an open conformation of SwitchI is unstable in the absence of guanine nucleotide exchange factors (GEFs) and rises up towards the bound nucleotide to close the nucleotide pocket. Both I21 and Y32, play a crucial role in SwitchI transition. We show that an open SwitchI conformation is not necessary for GDP destabilization but is required for GDP/Mg escape from the HRAS. Further, we present the first simulation study showing displacement of GDP/Mg away from the nucleotide pocket. Both SwitchI and SwitchII, delays the escape of displaced GDP/Mg in the absence of GEF. Based on these results, a model for the mechanism of GEF in accelerating the exchange process is hypothesized. PMID:25272152

Electrostatic Interactions Positively Regulate K-Ras Nanocluster Formation and Function▿

PubMed Central

Plowman, Sarah J.; Ariotti, Nicholas; Goodall, Andrew; Parton, Robert G.; Hancock, John F.

2008-01-01

The organization of Ras proteins into plasma membrane nanoclusters is essential for high-fidelity signal transmission, but whether the nanoscale enviroments of different Ras nanoclusters regulate effector interactions is unknown. We show using high-resolution spatial mapping that Raf-1 is recruited to and retained in K-Ras-GTP nanoclusters. In contrast, Raf-1 recruited to the plasma membrane by H-Ras is not retained in H-Ras-GTP nanoclusters. Similarly, upon epidermal growth factor receptor activation, Raf-1 is preferentially recruited to K-Ras-GTP and not H-Ras-GTP nanoclusters. The formation of K-Ras-GTP nanoclusters is inhibited by phosphorylation of S181 in the C-terminal polybasic domain or enhanced by blocking S181 phosphorylation, with a concomitant reduction or increase in Raf-1 plasma membrane recruitment, respectively. Phosphorylation of S181 does not, however, regulate in vivo interactions with the nanocluster scaffold galectin-3 (Gal3), indicating separate roles for the polybasic domain and Gal3 in driving K-Ras nanocluster formation. Together, these data illustrate that Ras nanocluster composition regulates effector recruitment and highlight the importance of lipid/protein nanoscale environments to the activation of signaling cascades. PMID:18458061

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

PubMed

Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke; Kita, Yoshiko; Yonezawa, Yasushi; Tokunaga, Masao

2007-04-10

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

Differentiation of neuroblastoma cell line N1E-115 involves several signaling cascades.

PubMed

Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Joo-ho; Pollak, Arnold; Freilinger, Angelika; Hengstschläger, Markus; Lubec, Gert

2005-03-01

No systematic searches for differential expression of signaling proteins (SP) in undifferentiated vs. differentiated cell lineages were published and herein we used protein profiling for this purpose. The NIE-115 cell line was cultivated and an aliquot was differentiated with dimethylsulfoxide (DMSO), that is known to lead to a neuronal phenotype. Cell lysates were prepared, run on two-dimensional gel electrophoresis followed by MALDI-TOF-TOF identification of proteins and maps of identified SPs were generated. Seven SPs were comparable, 27 SPs: GTP-binding/Ras-related proteins, kinases, growth factors, calcium binding proteins, phosphatase-related proteins were observed in differentiated NIE-115 cells and eight SPs of the groups mentioned above were observed in undifferentiated cells only. Switching-on/off of several individual SPs from different signaling cascades during the differentiation process is a key to understand mechanisms involved. The findings reported herein are challenging in vitro and in vivo studies to confirm a functional role for deranged SPs.

Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions.

PubMed

Gillette, William K; Esposito, Dominic; Abreu Blanco, Maria; Alexander, Patrick; Bindu, Lakshman; Bittner, Cammi; Chertov, Oleg; Frank, Peter H; Grose, Carissa; Jones, Jane E; Meng, Zhaojing; Perkins, Shelley; Van, Que; Ghirlando, Rodolfo; Fivash, Matthew; Nissley, Dwight V; McCormick, Frank; Holderfield, Matthew; Stephen, Andrew G

2015-11-02

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA.

PubMed

Booth, David S; Cheng, Yifan; Frankel, Alan D

2014-12-08

The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-Ran(GTP) nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy. Unexpectedly, Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer. The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication. The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression.

Plant Defense Response to Fungal Pathogens (Activation of Host-Plasma Membrane H+-ATPase by Elicitor-Induced Enzyme Dephosphorylation).

PubMed Central

Vera-Estrella, R.; Barkla, B. J.; Higgins, V. J.; Blumwald, E.

1994-01-01

Elicitor preparations containing the avr5 gene products from race 4 of Cladosporium fulvum and tomato (Lycopersicon esculentum L.) cells near isogenic for the resistance gene Cf5 were used to investigate events following the treatment of host plasma membranes with elicitor. A 4-fold increase in H+-ATPase activity, coincident with the acidification of the extracellular medium, was detected immediately after elicitor treatment. The elicitor-induced stimulation of the plasma membrane H+-ATPase was inhibited by okadaic acid but not by staurosporine, suggesting that protein dephosphorylation was required for increased H+-ATPase activity. This observation was confirmed by [gamma]-32P labeling and immunodetection of the plasma membrane H+-ATPase. Effects of guanidine nucleotide analogs and mastoparan on the ATPase activity suggested the role of GTP-binding proteins in mediating the putative elicitor-receptor binding, resulting in activation of a phosphatase(s), which in turn stimulates the plasma membrane H+-ATPase by dephosphorylation. PMID:12232073