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Sample records for gtp binding affects

  1. Partial characterization of GTP-binding proteins in Neurospora

    SciTech Connect

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-08-14

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. (/sup 35/S)GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of (/sup 35/S)GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin.

  2. Identification and isoprenylation of plant GTP-binding proteins.

    PubMed

    Biermann, B; Randall, S K; Crowell, D N

    1996-08-01

    To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies). PMID:8843944

  3. Subcellular distribution of small GTP binding proteins in pancreas: Identification of small GTP binding proteins in the rough endoplasmic reticulum

    SciTech Connect

    Nigam, S.K. )

    1990-02-01

    Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5{prime}-({gamma}-({sup 35}S)thio)triphosphate (GTP({gamma}-{sup 35}S)) was assayed in each fraction. Enrichment of GTP({gamma}-{sup 35}S) binding was greatest in the interfacial smooth microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a ({alpha}-{sup 32}P)GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP({gamma}-{sup 35}S) binding, as well as the small GTP binding proteins detected by the ({alpha}-{sup 32}P)GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMS were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP({gamma}-{sup 35}S) binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.

  4. Structural stabilization of GTP-binding domains in circularly permuted GTPases: Implications for RNA binding

    PubMed Central

    Anand, Baskaran; Verma, Sunil Kumar; Prakash, Balaji

    2006-01-01

    GTP hydrolysis by GTPases requires crucial residues embedded in a conserved G-domain as sequence motifs G1–G5. However, in some of the recently identified GTPases, the motif order is circularly permuted. All possible circular permutations were identified after artificially permuting the classical GTPases and subjecting them to profile Hidden Markov Model searches. This revealed G4–G5–G1–G2–G3 as the only possible circular permutation that can exist in nature. It was also possible to recognize a structural rationale for the absence of other permutations, which either destabilize the invariant GTPase fold or disrupt regions that provide critical residues for GTP binding and hydrolysis, such as Switch-I and Switch-II. The circular permutation relocates Switch-II to the C-terminus and leaves it unfastened, thus affecting GTP binding and hydrolysis. Stabilizing this region would require the presence of an additional domain following Switch-II. Circularly permuted GTPases (cpGTPases) conform to such a requirement and always possess an ‘anchoring’ C-terminal domain. There are four sub-families of cpGTPases, of which three possess an additional domain N-terminal to the G-domain. The biochemical function of these domains, based on available experimental reports and domain recognition analysis carried out here, are suggestive of RNA binding. The features that dictate RNA binding are unique to each subfamily. It is possible that RNA-binding modulates GTP binding or vice versa. In addition, phylogenetic analysis indicates a closer evolutionary relationship between cpGTPases and a set of universally conserved bacterial GTPases that bind the ribosome. It appears that cpGTPases are RNA-binding proteins possessing a means to relate GTP binding to RNA binding. PMID:16648363

  5. Uncoupling of gamma-aminobutyric acid B receptors from GTP-binding proteins by N-ethylmaleimide: effect of N-ethylmaleimide on purified GTP-binding proteins

    SciTech Connect

    Asano, T.; Ogasawara, N.

    1986-03-01

    Treatment of membranes from bovine cerebral cortex with N-ethylmaleimide (NEM) resulted in inhibition of gamma-aminobutyric acid (GABA) binding to GABAB receptors. The binding curve for increasing concentrations of agonist was shifted to the right by NEM treatment. Guanine nucleotide had little effect on the binding of GABA to NEM-treated membranes. The addition of purified GTP-binding proteins, which were the substrates of islet-activating protein (IAP), pertussis toxin, to the NEM-treated membranes caused a shift of the binding curve to the left, suggesting modification of GTP-binding proteins rather than receptors by NEM. The effect of NEM on two purified GTP-binding proteins, Gi (composed of three subunits with molecular weight of alpha, 41,000; beta, 35,000; gamma, 10,000) and Go (alpha, 39,000; beta, 35,000; gamma, 10,000) was studied. NEM did not significantly change guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding and GTPase activity of these two proteins. NEM-treated Gi and Go were not ADP-ribosylated by IAP and did not increase GABA binding to NEM-treated membranes. When alpha and beta gamma subunits were treated with NEM and then mixed with nontreated alpha and beta gamma to form Gi or Go, respectively, both oligomers with NEM-treated alpha-subunits lost their abilities to be IAP substrates and to couple to receptors. Results indicate that NEM uncoupled GTP-binding proteins from receptors by modifying alpha-subunits of GTP-binding proteins, and the site seemed to be on or near the site of ADP-ribosylation by IAP. When alpha and beta gamma subunits were treated with NEM and then mixed to form Gi or Go, GTP gamma S binding in the absence of Mg2+ and GTPase activity were changed, although they were not affected when oligomers were treated with NEM. Results suggest the existence of another sulfhydryl group which is protected from NEM by the association of subunits.

  6. Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes.

    PubMed

    Yoneyama, T; Hatakeyama, K

    2001-04-01

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed-forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively. PMID:11274478

  7. Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum.

    PubMed Central

    Lanoix, J; Roy, L; Paiement, J

    1989-01-01

    As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2508629

  8. Evidence for a vasopressin receptor-GTP binding protein complex

    SciTech Connect

    Fitzgerald, T.J.; Uhing, R.J.; Exton, J.H.

    1986-05-01

    Plasma membranes from the livers of rats were able to hydrolyze the ..gamma..-phosphate from guanosine-5'-triphosphate (GTP). The rate of GTP hydrolysis could be decreased to 10% of its initial rate by the addition of adenosine-5'-triphosphate with a concomitant decrease in the K/sub m/ for GTP from approx. 10/sup -3/ M to 10/sup -6/ M. The low K/sub m/ GTPase activity was inhibited by the addition of nonhydrolyzable analogs of GTP. In addition, the GTPase activity was stimulated from 10 to 30% over basal by the addition of vasopressin. A dose dependency curve showed that the maximum stimulation was obtained with 10/sup -8/ M vasopressin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized in the presence of (Phenylalanyl-3,4,5-/sup 3/H(N))vasopressin were subjected to sucrose gradient centrifugation, the majority of bound (/sup 3/H)vasopressin migrated with an approximate molecular weight of 300,000. Moreover, there was a GTPase activity that migrated with the bound (/sup 3/H)vasopressin. This peak of bound (/sup 3/H)vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10/sup -5/ M guanosine-5'-O-(3-thiotriphosphate). These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.

  9. GTP binding and hydrolysis kinetics of human septin 2.

    PubMed

    Huang, Yi-Wei; Surka, Mark C; Reynaud, Denis; Pace-Asciak, Cecil; Trimble, William S

    2006-07-01

    Septins are a family of conserved proteins that are essential for cytokinesis in a wide range of organisms including fungi, Drosophila and mammals. In budding yeast, where they were first discovered, they are thought to form a filamentous ring at the bridge between the mother and bud cells. What regulates the assembly and function of septins, however, has remained obscure. All septins share a highly conserved domain related to those found in small GTPases, and septins have been shown to bind and hydrolyze GTP, although the properties of this domain and the relationship between polymerization and GTP binding/hydrolysis is unclear. Here we show that human septin 2 is phosphorylated in vivo at Ser218 by casein kinase II. In addition, we show that recombinant septin 2 binds guanine nucleotides with a Kd of 0.28 microm for GTPgammaS and 1.75 microm for GDP. It has a slow exchange rate of 7 x 10(-5) s(-1) for GTPgammaS and 5 x 10(-4) s(-1) for GDP, and an apparent kcat value of 2.7 x 10(-4) s(-1), similar to those of the Ras superfamily of GTPases. Interestingly, the nucleotide binding affinity appears to be altered by phosphorylation at Ser218. Finally, we show that a single septin protein can form homotypic filaments in vitro, whether bound to GDP or GTP. PMID:16857012

  10. Immunochemical similarity of GTP-binding proteins from different systems

    SciTech Connect

    Kalinina, S.N.

    1986-06-20

    It was found that antibodies against the GTP-binding proteins of bovine retinal photoreceptor membranes blocked the inhibitory effect of estradiol on phosphodiesterase from rat and human uterine cytosol and prevented the cumulative effect of catecholamines and guanylyl-5'-imidodiphosphate on rat skeletal muscle adenylate cyclase. It was established by means of double radial immunodiffusion that these antibodies form a precipitating complex with purified bovine brain tubulin as well as with retinal preparations obtained from eyes of the bull, pig, rat, frog, some species of fish, and one reptile species. Bands of precipitation were not observed with these antibodies when retinal preparations from invertebrates (squid and octopus) were used as the antigens. The antibodies obtained interacted with the ..cap alpha..- and ..beta..-subunits of GTP-binding proteins from bovine retinal photoreceptor membranes.

  11. Light-dependent GTP-binding proteins in squid photoreceptors.

    PubMed Central

    Robinson, P R; Wood, S F; Szuts, E Z; Fein, A; Hamm, H E; Lisman, J E

    1990-01-01

    Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5'-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2124806

  12. 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.

  13. Timing of GTP binding and hydrolysis by translation termination factor RF3.

    PubMed

    Peske, Frank; Kuhlenkoetter, Stephan; Rodnina, Marina V; Wintermeyer, Wolfgang

    2014-02-01

    Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that--in contrast to a previous report--RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3-GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3-GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3-GTP-ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock. PMID:24214994

  14. Nonradioactive GTP binding assay to monitor activation of g protein-coupled receptors.

    PubMed

    Frang, Heini; Mukkala, Veli-Matti; Syystö, Rita; Ollikka, Pia; Hurskainen, Pertti; Scheinin, Mika; Hemmilä, Ilkka

    2003-04-01

    GPCRs represent important targets for drug discovery because GPCRs participate in a wide range of cellular signaling pathways that play a role in a variety of pathological conditions. A large number of screening assays have been developed in HTS laboratories for the identification of hits or lead compounds acting on GPCRs. One type of assay that has found relatively widespread application, due to its at least in part generic nature, relies on the use of a radioactive GTP analogue, [(35)S]GTPgammaS. The G-protein alpha subunit is an essential part of the interaction between receptor and G proteins in transmembrane signaling, where the activated receptor catalyzes the release of GDP from Galpha, thereby enabling the subsequent binding of GTP or a GTP analogue. [(35)S]GTPgammaS allows the extent of this interaction to be followed quantitatively by determining the amount of radioactivity associated with cell membranes. However, with the increased desire to move assays to nonradioactive formats, there is a considerable need to develop a nonradioactive GTP binding assay to monitor ligand-induced changes in GPCR activity. The Eu-GTP binding assay described here is based on TRF that exploits the unique fluorescence properties of lanthanide chelates, and provides a powerful alternative to assays using radioisotopes. In this article, we have used the human alpha(2A)-AR as a model GPCR system to evaluate the usefulness of this Eu-GTP binding assay. PMID:15090192

  15. Structural and functional similarities between the nucleotide-binding domains of CFTR and GTP-binding proteins.

    PubMed Central

    Carson, M R; Welsh, M J

    1995-01-01

    The opening and closing of the CFTR Cl- channel are regulated by ATP hydrolysis at its two nucleotide binding domains (NBDs). However, the mechanism and functional significance of ATP hydrolysis are unknown. Sequence similarity between the NBDs of CFTR and GTP-binding proteins suggested the NBDs might have a structure and perhaps a function like that of GTP-binding proteins. Based on this similarity, we predicted that the terminal residue of the LSGGQ motif in the NBDs of CFTR corresponds to a highly conserved glutamine residue in GTP-binding proteins that directly catalyzes the GTPase reaction. Mutations of this residue in NBD1 or NBD2, which were predicted to increase or decrease the rate of hydrolysis, altered the duration of channel closed and open times in a specific manner without altering ion conduction properties or ADP-dependent inhibition. These results suggest that the NBDs of CFTR, and consequently other ABC transporters, may have a structure and a function analogous to those of GTP-binding proteins. We conclude that the rates of ATP hydrolysis at NBD1 and at NBD2 determine the duration of the two states of the channel, closed and open, much as the rate of GTP hydrolysis by GTP-binding proteins determines the duration of their active state. Images FIGURE 3 FIGURE 4 PMID:8599650

  16. 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

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

    PubMed

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

    2015-10-01

    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 ttd14P75L mutant. The ttd14P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14P75L 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

  18. Timing of GTP binding and hydrolysis by translation termination factor RF3

    PubMed Central

    Peske, Frank; Kuhlenkoetter, Stephan; Rodnina, Marina V.; Wintermeyer, Wolfgang

    2014-01-01

    Protein synthesis in bacteria is terminated by release factors 1 or 2 (RF1/2), which, on recognition of a stop codon in the decoding site on the ribosome, promote the hydrolytic release of the polypeptide from the transfer RNA (tRNA). Subsequently, the dissociation of RF1/2 is accelerated by RF3, a guanosine triphosphatase (GTPase) that hydrolyzes GTP during the process. Here we show that—in contrast to a previous report—RF3 binds GTP and guanosine diphosphate (GDP) with comparable affinities. Furthermore, we find that RF3–GTP binds to the ribosome and hydrolyzes GTP independent of whether the P site contains peptidyl-tRNA (pre-termination state) or deacylated tRNA (post-termination state). RF3–GDP in either pre- or post-termination complexes readily exchanges GDP for GTP, and the exchange is accelerated when RF2 is present on the ribosome. Peptide release results in the stabilization of the RF3–GTP–ribosome complex, presumably due to the formation of the hybrid/rotated state of the ribosome, thereby promoting the dissociation of RF1/2. GTP hydrolysis by RF3 is virtually independent of the functional state of the ribosome and the presence of RF2, suggesting that RF3 acts as an unregulated ribosome-activated switch governed by its internal GTPase clock. PMID:24214994

  19. Direct Binding of GTP Cyclohydrolase and Tyrosine Hydroxylase

    PubMed Central

    Bowling, Kevin M.; Huang, Zhinong; Xu, Dong; Ferdousy, Faiza; Funderburk, Christopher D.; Karnik, Nirmala; Neckameyer, Wendi; O'Donnell, Janis M.

    2008-01-01

    The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation. PMID:18801743

  20. Importin {beta}-type nuclear transport receptors have distinct binding affinities for Ran-GTP

    SciTech Connect

    Hahn, Silvia; Schlenstedt, Gabriel

    2011-03-18

    Highlights: {yields} Determination of binding properties of nuclear transport receptor/Ran-GTP complexes. {yields} Biosensor measurements provide constants for dissociation, on-rates, and off-rates. {yields} The affinity of receptors for Ran-GTP is widely divergent. {yields} Dissociation constants differ for three orders of magnitude. {yields} The cellular concentration of yeast Ran is not limiting. -- Abstract: Cargos destined to enter or leave the cell nucleus are typically transported by receptors of the importin {beta} family to pass the nuclear pore complex. The yeast Saccharomyces cerevisiae comprises 14 members of this protein family, which can be divided in importins and exportins. The Ran GTPase regulates the association and dissociation of receptors and cargos as well as the transport direction through the nuclear pore. All receptors bind to Ran exclusively in its GTP-bound state and this event is restricted to the nuclear compartment. We determined the Ran-GTP binding properties of all yeast transport receptors by biosensor measurements and observed that the affinity of importins for Ran-GTP differs significantly. The dissociation constants range from 230 pM to 270 nM, which is mostly based on a variability of the off-rate constants. The divergent affinity of importins for Ran-GTP suggests the existence of a novel mode of nucleocytoplasmic transport regulation. Furthermore, the cellular concentration of {beta}-receptors and of other Ran-binding proteins was determined. We found that the number of {beta}-receptors altogether about equals the amounts of yeast Ran, but Ran-GTP is not limiting in the nucleus. The implications of our results for nucleocytoplasmic transport mechanisms are discussed.

  1. GTP binding to the. beta. -subunit of tubulin is greatly reduced in Alzheimers disease

    SciTech Connect

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

    1987-05-01

    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 photolabeled 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.

  2. 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.

  3. Characterization of membrane-bound small GTP-binding proteins from Nicotiana tabacum.

    PubMed Central

    Haizel, T; Merkle, T; Turck, F; Nagy, F

    1995-01-01

    We have cloned nine cDNAs encoding small GTP-binding proteins from leaf cDNA libraries of tobacco (Nicotiana tabacum). These cDNAs encode distinct proteins (22-25 kD) that display different levels of identity with members of the mammalian Rab family: Nt-Rab6 with Rab6 (83%), Nt-Rab7a-c with Rab7 (63-70%), and Nt-Rab11a-e with Rab11 (53-69%). Functionally important regions of these proteins, including the "effector binding" domain, the C-terminal Cys residues for membrane attachment, and the four regions involved in GTP-binding and hydrolysis, are highly conserved. Northern and western blot analyses show that these genes are expressed, although at slightly different levels, in all plant tissues examined. We demonstrate that the plant Rab5, Rab6, and Rab11 proteins, similar to their mammalian and yeast counterparts, are tightly bound to membranes and that they exhibit different solubilization characteristics. Furthermore, we show that the yeast GTPase-activating protein Gyp6, shown to be specifically required to control the GTP hydrolysis of the yeast Ypt6 protein, could interact with tobacco GTP-binding proteins. It increases in vitro the GTP hydrolysis rate of the wild-type Nt-Rab7 protein. In addition, it also increases, at different levels, the GTP hydrolysis rates of a Nt-Rab7m protein with a Rab6 effector domain and of two other chimaeric Nt-Rab6/Nt-Rab7 proteins. However, it does not interact with the wild-type Nt-Rab6 protein, which is most similar to the yeast Ypt6 protein. PMID:7784525

  4. Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1.

    PubMed Central

    Bischoff, F R; Krebber, H; Smirnova, E; Dong, W; Ponstingl, H

    1995-01-01

    RCC1 (the regulator of chromosome condensation) stimulates guanine nucleotide dissociation on the Ras-related nuclear protein Ran. Both polypeptides are components of a regulatory pathway that has been implicated in regulating DNA replication, onset of and exit from mitosis, mRNA processing and transport, and import of proteins into the nucleus. In a search for further members of the RCC1-Ran signal pathway, we have identified proteins of 23, 45 and 300 kDa which tightly bind to Ran-GTP but not Ran-GDP. The purified soluble 23 kDa Ran binding protein RanBP1 does not activate RanGTPase, but increases GTP hydrolysis induced by the RanGTPase-activating protein RanGAP1 by an order of magnitude. In the absence of RanGAP, it strongly inhibits RCC1-induced exchange of Ran-bound GTP. In addition, it forms a stable complex with nucleotide-free RCC1-Ran. With these properties, it differs markedly from guanine diphosphate dissociation inhibitors which preferentially prevent the exchange of protein-bound GDP and in some cases were shown to inhibit GAP-induced GTP hydrolysis. RanBP1 is the first member of a new class of proteins regulating the binding and hydrolysis of GTP by Ras-related proteins. Images PMID:7882974

  5. Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family.

    PubMed Central

    Chuang, T H; Xu, X; Kaartinen, V; Heisterkamp, N; Groffen, J; Bokoch, G M

    1995-01-01

    Philadelphia chromosome-positive leukemias result from the fusion of the BCR and ABL genes, which generates a functional chimeric molecule. The Abr protein is very similar to Bcr but lacks a structural domain which may influence its biological regulatory capabilities. Both Abr and Bcr have a GTPase-activating protein (GAP) domain similar to those found in other proteins that stimulate GTP hydrolysis by members of the Rho family of GTP-binding proteins, as well as a region of homology with the guanine nucleotide dissociation-stimulating domain of the DBL oncogene product. We purified as recombinant fusion proteins the GAP- and Dbl-homology domains of both Abr and Bcr. The Dbl-homology domains of Bcr and Abr were active in stimulating GTP binding to CDC42Hs, RhoA, Rac1, and Rac2 (rank order, CDC42Hs > RhoA > Rac1 = Rac2) but were inactive toward Rap1A and Ha-Ras. Both Bcr and Abr acted as GAPs for Rac1, Rac2, and CDC42Hs but were inactive toward RhoA, Rap1A, and Ha-Ras. Each individual domain bound in a noncompetitive manner to GTP-binding protein substrates. These data suggest the multifunctional Bcr and Abr proteins might interact simultaneously and/or sequentially with members of the Rho family to regulate and coordinate cellular signaling. Images Fig. 3 PMID:7479768

  6. Expression of the Xenopus GTP-binding protein gene Ran during embryogenesis.

    PubMed

    Onuma, Y; Nishihara, R; Takahashi, S; Tanegashima, K; Fukui, A; Asashima, M

    2000-06-01

    The Ran gene family encodes small GTP binding proteins that are associated with a variety of nuclear processes. We isolated a Xenopus Ran cDNA and analyzed the pattern of expression of this gene during embryogenesis. Ran is expressed maternally and later in the CNS, neural crest, mesenchyme, eyes, and otic vesicles. However, expression is not detected in the somites or the notochord. PMID:11180838

  7. Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome

    PubMed Central

    Wilden, Berthold; Savelsbergh, Andreas; Rodnina, Marina V.; Wintermeyer, Wolfgang

    2006-01-01

    The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP–GTP exchange would be required for translocation [Zavialov AV, Hauryliuk VV, Ehrenberg M (2005) J Biol 4:9]. We have reinvestigated GTP/GDP binding and show that EF-G binds GTP and GDP with affinities in the 20 to 40 μM range (37°C), in accordance with earlier reports. Furthermore, GDP exchange, which is extremely rapid on unbound EF-G, is retarded, rather than accelerated, on the ribosome, which, therefore, is not a nucleotide-exchange factor for EF-G. The EF-G·GDPNP complex, which is very labile, is stabilized 30,000-fold by binding to the ribosome. These findings, together with earlier kinetic results, reveal that EF-G enters the pretranslocation ribosome in the GTP-bound form and indicate that, upon ribosome-complex formation, the nucleotide-binding pocket of EF-G is closed, presumably in conjunction with GTPase activation. GTP hydrolysis is required for rapid tRNA–mRNA movement, and Pi release induces further rearrangements of both EF-G and the ribosome that are required for EF-G turnover. PMID:16940356

  8. Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

    PubMed Central

    Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Kim, Hyo Jeong; Ko, Myoung Seok; Seol, Wongi; Joe, Yeonsoo; Chung, Hun Taeg; Lee, Byung Ju; Moon, Chang Hoon; Cho, Wha Ja; Park, Jeong Woo

    2016-01-01

    The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate–containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase–dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and RabGAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling. PMID:26582392

  9. Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

    PubMed Central

    de Boor, Susanne; Knyphausen, Philipp; Kuhlmann, Nora; Wroblowski, Sarah; Brenig, Julian; Scislowski, Lukas; Baldus, Linda; Nolte, Hendrik; Krüger, Marcus; Lammers, Michael

    2015-01-01

    Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular Ran•GTP/Ran•GDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed. PMID:26124124

  10. Structure and Mutational Analysis of the Archaeal GTP:AdoCbi-P Guanylyltransferase (CobY) from Methanocaldococcus jannaschii: Insights into GTP Binding and Dimerization

    SciTech Connect

    Newmister, Sean A.; Otte, Michele M.; Escalante-Semerena, Jorge C.; Rayment, Ivan

    2012-02-08

    In archaea and bacteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucleotide loop assembly (NLA) pathway. In the archaeal and bacterial NLA pathways, two different guanylyltransferases catalyze the activation of the corrinoid. Structural and functional studies of the bifunctional bacterial guanylyltransferase that catalyze both ATP-dependent corrinoid phosphorylation and GTP-dependent guanylylation are available, but similar studies of the monofunctional archaeal enzyme that catalyzes only GTP-dependent guanylylation are not. Herein, the three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme from the archaeon Methanocaldococcus jannaschii (MjCobY) in complex with GTP is reported. The model identifies the location of the active site. An extensive mutational analysis was performed, and the functionality of the variant proteins was assessed in vivo and in vitro. Substitutions of residues Gly8, Gly153, or Asn177 resulted in {ge}94% loss of catalytic activity; thus, variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobY{sup G153D} had 10-fold higher affinity for GTP than MjCobY{sup WT} but failed to bind the corrinoid substrate. Results from Western blot analyses suggested that the above-mentioned substitutions render the protein unstable and prone to degradation; possible explanations for the observed instability of the variants are discussed within the framework of the three-dimensional crystal structure of MjCobY{sup G153D} in complex with GTP. The fold of MjCobY is strikingly similar to that of the N-terminal domain of Mycobacterium tuberculosis GlmU (MtbGlmU), a bifunctional acetyltransferase/uridyltransferase that catalyzes the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc).

  11. Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase

    SciTech Connect

    Kumar, K.P.; Chatterji, D. )

    1990-01-16

    Terbium(III) upon complexation with guanosine 5{prime}-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean K{sub d} between Tb(III) and GTP of 0.2 {mu}M, with three binding sites for TB(III) on GTP. {sup 31}P and {sup 1}H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a K{sub d} values of 4 {mu}M between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the {beta}-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 {angstrom} for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the {beta}-subunit of E. coli RNA polymerase was measured to be around 30 {angstrom}. This suggest that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate.

  12. Membrane-associated 41-kDa GTP-binding protein in collagen-induced platelet activation

    SciTech Connect

    Walker, G.; Bourguignon, L.Y. )

    1990-08-01

    Initially we established that the binding of collagen to human blood platelets stimulates both the rapid loss of PIP2 and the generation of inositol-4,5-bisphosphate (IP2) and inositol-1,4,5-triphosphate (IP3). These results indicate that the binding of collagen stimulates inositol phospholipid-specific phospholipase C during platelet activation. The fact that GTP or GTP-gamma-S augments, and pertussis toxin inhibits, collagen-induced IP3 formation suggests that a GTP-binding protein or (or proteins) may be directly involved in the regulation of phospholipase C-mediated phosphoinositide turnover in human platelets. We have used several complementary techniques to isolate and characterize a platelet 41-kDa polypeptide (or polypeptides) that has a number of structural and functional similarities to the regulatory alpha i subunit of the GTP-binding proteins isolated from bovine brain. This 41-kDa polypeptide (or polypeptides) is found to be closely associated with at least four membrane glycoproteins (e.g., gp180, gp110, gp95, and gp75) in a 330-kDa complex that can be dissociated by treatment with high salt plus urea. Most important, we have demonstrated that antilymphoma 41-kDa (alpha i subunit of GTP-binding proteins) antibody cross-reacts with the platelet 41-kDa protein (or proteins) and the alpha i subunit of bovine brain Gi alpha proteins, and blocks GTP/collagen-induced IP3 formation. These data provide strong evidence that the 41-kDa platelet GTP-binding protein (or proteins) is directly involved in collagen-induced signal transduction during platelet activation.

  13. A small GTP-binding protein from Arabidopsis thaliana functionally complements the yeast YPT6 null mutant.

    PubMed Central

    Bednarek, S Y; Reynolds, T L; Schroeder, M; Grabowski, R; Hengst, L; Gallwitz, D; Raikhel, N V

    1994-01-01

    A clone designated A.t.RAB6 encoding a small GTP-binding protein was isolated from a cDNA library of Arabidopsis thaliana leaf tissue. The predicted amino acid sequence was highly homologous to the mammalian and yeast counterparts, H.Rab6 and Ryh1/Ypt6, respectively. Lesser homology was found between the predicted Arabidopsis protein sequence and two small GTP-binding proteins isolated from plant species (44% homology to Zea mays Ypt1 and 43% homology to Nicotiana tabacum Rab5). Conserved stretches in the deduced amino acid sequence of A.t.Rab6 include four regions involved in GTP-binding, an effector region, and C-terminal cysteine residues required for prenylation and subsequent membrane attachment. Northern blot analysis demonstrated that A.t.Rab6 mRNA was expressed in root, leaf, stem, and flower tissues from A. thaliana with the highest levels present in roots. Escherichia coli produced histidine-tagged A.t.Rab6 protein-bound GTP, whereas a mutation in one of the guanine nucleotide-binding sites (asparagine122 to isoleucine) rendered it incapable of binding GTP. Functionally, the A.t.RAB6 gene was able to complement the temperature-sensitive phenotype of the YPT6 null mutant in yeast. The isolation of this gene will aid in the dissection of the machinery involved in soluble protein sorting at the trans-Golgi network of plants. PMID:8159788

  14. Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules

    SciTech Connect

    Darchen, F.; Hammel, F.; Monteils, M.P.; Scherman, D. ); Zahraoui, A.; Tavitian, A. )

    1990-08-01

    The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells, Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine {beta}-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTP-binding proteins, is involved in their biogenesis or in the regulated secretion process.

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

    SciTech Connect

    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 S 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.

  16. [Identification and isolation of GTP-binding regulator protein from plasma membranes of oocytes from the starfish Asterias amurensis].

    PubMed

    Lamash, N E

    2001-01-01

    A method for isolating a GTP-binding regulatory protein from starfish oocytes is described. The protein consists of three subunits with molecular weights of 40, 37, and about 8 kDa. It is shown that the 40-kDa subunit has a high GTPase activity and is susceptible to ADP-ribosylation by pertussis toxin. The latter property of this subunit proved to decrease upon its incubation with nonhydrolyzable GTP analogues. These data provide evidence that the plasma membrane of starfish oocytes contains a 40-kDa GTP-binding protein with properties characteristic of the alpha subunit of the inhibitory Gi protein. The role of this protein in the transmembrane signal transmission from the 1-methyladenine receptor to intracellular effectors is discussed. PMID:11236575

  17. Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+.

    PubMed Central

    Davidson, M M; Haslam, R J

    1994-01-01

    In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP-binding

  18. Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification.

    PubMed

    Uno, T; Nakasuji, A; Hara, W; Aizono, Y

    2000-04-01

    A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The fusion protein bound [(35)S] GTPgammaS and [(3)H] GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively. The binding of [(35)S] GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [(35)S] GTPgammaS and [(3)H] GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2). These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP. Arch. PMID:10737920

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

    SciTech Connect

    Mahy, N.; Woolkalis, M.; Thermos, K.; Carlson, K.; Manning, D.; Reisine, T.

    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 to 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.

  20. Identification of an essential Caulobacter crescentus gene encoding a member of the Obg family of GTP-binding proteins.

    PubMed Central

    Maddock, J; Bhatt, A; Koch, M; Skidmore, J

    1997-01-01

    We have identified an essential Caulobacter crescentus gene (cgtA) that encodes a member of a recently identified subfamily of GTPases (the Obg family) conserved from Bacteria to Archaea to humans. This evolutionary conservation between distantly related species suggests that this family of GTP-binding proteins possesses a fundamental, yet unknown, cellular role. In this report, we describe the isolation and sequence of the cgtA gene. The predicted CgtA protein displays striking similarity to the Obg family of small, monomeric GTP-binding proteins, both in the conserved guanine nucleotide-binding domains and throughout the N-terminal glycine-rich domain that is found in many members of the Obg family. Disruption of the cgtA gene was lethal, demonstrating that this gene is essential for cell growth. Immunoblot analysis revealed that CgtA protein levels remained constant throughout the C. crescentus cell cycle. PMID:9335292

  1. Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358

    PubMed Central

    Engelsma, Dieuwke; Bernad, Rafael; Calafat, Jero; Fornerod, Maarten

    2004-01-01

    Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. This interaction is stimulated by RanGTP but remains of a relatively low affinity. In order to identify strong signals, we screened a 15-mer random peptide library for CRM1 binding, both in the presence and absence of RanGTP. Under each condition, strikingly similar signals were enriched, conforming to the NES consensus sequence. A derivative of an NES selected in the absence of RanGTP exhibits very high affinity for CRM1 in vitro and stably binds without the requirement of RanGTP. Localisation studies and RNA interference demonstrate inefficient CRM1-mediated export and accumulation of CRM1 complexed with the high-affinity NES at nucleoporin Nup358. These results provide in vivo evidence for a nuclear export reaction intermediate. They suggest that NESs have evolved to maintain low affinity for CRM1 to allow efficient export complex disassembly and release from Nup358. PMID:15329671

  2. 6-Acetyldihydrohomopterin and sepiapterin affect some GTP cyclohydrolase I's and not others

    SciTech Connect

    Jacobson, K.B.; Manos, R.E.

    1988-01-01

    The first enzyme in pteridine biosynthesis, GTP cyclohydrolase I, is a likely site for regulation of pteridine biosynthesis to occur. GTP cyclohydrolase I responds to hormonal treatment and is found altered in a variety of mice with genetically based neurological and immunological disorders. Genetic loci can greatly modify the activity of GTP cyclohydrolase: Punch mutant in Drosophila hph-1 in mouse and atypical phenylketonuria in human. This report examines the ability of Ahp and sepiapterin to alter the activity of GTP cyclohydrolase I from mouse liver, rat liver and Drosophila head. 20 refs., 2 tabs.

  3. Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24.

    PubMed Central

    Zhao, Z S; Leung, T; Manser, E; Lim, L

    1995-01-01

    Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein beta gamma subunits. A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1. The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p. Cdc42p is required for alpha-factor-induced activation of FUS1.cdc24ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p12V mutants. Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway. The two-hybrid system revealed that Ste4p interacts with Cdc24p. We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling. PMID:7565673

  4. Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein, BCR, and Regulates Levels of Rac1-GTP*

    PubMed Central

    Gray, Elizabeth J.; Petsalaki, Evangelia; James, D. Andrew; Bagshaw, Richard D.; Stacey, Melissa M.; Rocks, Oliver; Gingras, Anne-Claude; Pawson, Tony

    2014-01-01

    SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein. PMID:25331951

  5. Inhibitory GTP binding protein G/sub i/ regulates US -adrenoceptor affinity towards US -agonists

    SciTech Connect

    Marbach, I.; Levitzki, A.

    1987-05-01

    Treatment of S-49 lymphoma cell membranes with pertussis toxin (PT) causes a three-fold reduction of US -adrenoceptor (US AR) affinity towards isoproterenol. A similar treatment with cholera toxin (CT) does not cause such a modulation. The effects were studied by the detailed analysis of SVI-cyanopindolol (CYP) binding curves in the absence and presence of increasing agonist concentrations. Thus, the authors were able to compare in detail the effects of G/sub s/ and G/sub i/ on the agonist-associated state of the US AR. In contrast to these findings, PT treatment does not have any effect on the displacement of SVI-CYP by (-)isoproterenol. These results demonstrate that the inhibitory GTP protein G/sub i/ modulates the US AR affinity towards US -agonists. This might be due to the association of G/sub i/ with the agonist-bound US AR x G/sub s/ x C complex within the membrane. This hypothesis, as well as others, is under investigation.

  6. Myristoylation of an inhibitory GTP-binding protein. alpha. subunit is essential for its membrane attachment

    SciTech Connect

    Jones, T.L.Z.; Simonds, W.F.; Merendino, J.J. Jr.; Brann, M.R.; Spiegel, A.M. )

    1990-01-01

    The authors transfected COS cells with cDNAs for the {alpha} subunits of stimulatory and inhibitory GTP-binding proteins, {alpha}{sub s} and {alpha}{sub i1}, respectively, and immunoprecipitated the metabolically labeled products with specific peptide antibodies. Cells were separated into particulate and soluble fractions before immunoprecipitation; ({sup 35}S)methionine-labeled {alpha}{sub s} and {alpha}{sub i} were both found primarily in the particulate fraction. ({sup 3}H)Myristate was incorporated into endogenous and transfected {alpha}{sub i} but could not be detected in {alpha}{sub s} even when it was overexpressed. They converted the second residue, glycine, of {alpha}{sub i1} into alanine by site-directed mutagenesis. Upon transfection of the mutant {alpha}{sub i1} into COS cells, the ({sup 35}S)methionine-labeled product was localized primarily to the soluble fraction, and, also unlike normal {alpha}{sub i1}, the mutant failed to incorporate ({sup 3}H)myristate. The unmyristoylated mutant {alpha}{sub i1} could still interact with the {beta}-{gamma} complex, since purified {beta}{gamma} subunits promoted pertussis toxin-catalyzed ADP-ribosylation of both the normal and mutant {alpha}{sub i1} subunits. These results indicate that myristoylation is critical for membrane attachment of {alpha}{sub i} but not {alpha}{sub s} subunits.

  7. GTP-binding of ARL-3 is activated by ARL-13 as a GEF and stabilized by UNC-119

    PubMed Central

    Zhang, Qing; Li, Yan; Zhang, Yuxia; Torres, Vicente E.; Harris, Peter C.; Ling, Kun; Hu, Jinghua

    2016-01-01

    Primary cilia are sensory organelles indispensable for organogenesis and tissue pattern formation. Ciliopathy small GTPase ARLs are proposed as prominent ciliary switches, which when disrupted result in dysfunctional cilia, yet how ARLs are activated remain elusive. Here, we discover a novel small GTPase functional module, which contains ARL-3, ARL-13, and UNC-119, localizes near the poorly understood inversin (InV)-like compartment in C. elegans. ARL-13 acts synergistically with UNC-119, but antagonistically with ARL-3, in regulating ciliogenesis. We demonstrate that ARL-3 is a unique small GTPase with unusual high intrinsic GDP release but low intrinsic GTP binding rate. Importantly, ARL-13 acts as a nucleotide exchange factor (GEF) of ARL-3, while UNC-119 can stabilize the GTP binding of ARL-3. We further show that excess inactivated ARL-3 compromises ciliogenesis. The findings reveal a novel mechanism that one ciliopathy GTPase ARL-13, as a GEF, coordinates with UNC-119, which may act as a GTP-binding stabilizing factor, to properly activate another GTPase ARL-3 in cilia, a regulatory process indispensable for ciliogenesis. PMID:27102355

  8. A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase

    PubMed Central

    Sabogal, Alex; Rio, Donald C

    2010-01-01

    Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site-specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP-binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP-binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP-binding residues in the central portion of the transposase coding region. This clone, amino acids 275–409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP-binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N-terminal THAP DNA-binding domain attached to an extended leucine zipper coiled-coil dimerization domain in the P element transposase, precisely delineating the DNA-binding and dimerization activities on the primary sequence. This study highlights the use of a GFP-based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions. PMID:20842711

  9. On the binding of BODIPY-GTP by the photosensory protein YtvA from the common soil bacterium Bacillus subtilis.

    PubMed

    Nakasone, Yusuke; Hellingwerf, Klaas J

    2011-01-01

    The YtvA protein, which is one of the proteins that comprises the network carrying out the signal transfer inducing the general stress response in Bacillus subtilis, is composed of an N-terminal LOV domain (that binds a flavin [FMN]) and a C-terminal STAS domain. This latter domain shows sequence features typical for a nucleotide (NTP) binding protein. It has been proposed (FEBS Lett., 580 [2006], 3818) that BODIPY-GTP can be used as a reporter for nucleotide binding to this site and that activation of the LOV domain by blue light is reflected in an alteration of the BODIPY-GTP fluorescence. Here we confirm that BODIPY-GTP indeed binds to YtvA, but rather nonspecifically, and not limited to the STAS domain. Blue-light modulation of fluorescence emission of YtvA-bound BODIPY-GTP is observed both in the full-length YtvA protein and in a truncated protein composed of the LOV-domain plus the LOV-STAS linker region (YtvA(1-147)) as a light-induced decrease in fluorescence emission. The isolated LOV domain (i.e. without the linker region) does not show such BODIPY-GTP fluorescence changes. Dialysis experiments have confirmed the blue-light-induced release of BODIPY-GTP from YtvA. PMID:21388385

  10. Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

    PubMed Central

    Burak, Matthew J.; Guja, Kip E.; Garcia-Diaz, Miguel

    2015-01-01

    8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala510 and Asn513, play differential roles in dNTP selectivity. Specifically, Ala510 and Asn513 facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases. PMID:26220180

  11. A Host Small GTP-binding Protein ARL8 Plays Crucial Roles in Tobamovirus RNA Replication

    PubMed Central

    Nishikiori, Masaki; Mori, Masashi; Dohi, Koji; Okamura, Hideyasu; Katoh, Etsuko; Naito, Satoshi; Meshi, Tetsuo; Ishikawa, Masayuki

    2011-01-01

    Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5′-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions. PMID:22174675

  12. Properties of SEPT9 isoforms and the requirement for GTP binding.

    PubMed

    Robertson, Claire; Church, Stewart W; Nagar, Hans A; Price, John; Hall, Peter A; Russell, S E Hilary

    2004-05-01

    Members of the evolutionarily conserved septin family of genes are emerging as key components of several cellular processes including membrane trafficking, cytokinesis, and cell-cycle control events. SEPT9 has been shown to have a complex genomic architecture, such that up to 15 different isoforms are possible by the shuffling of five alternate amino termini and three alternate carboxy termini. Genomic and transcriptional alterations of SEPT9 have been associated with neoplasia. The present study has used a Sept9-specific antibody to determine the pattern of isoform expression in a range of tumour cell lines. Western blot analysis indicated considerable variation in the relative amounts and isoform content of Sept9. Immunofluorescence studies showed a range of patterns of cytoplasmic localization ranging from mainly particulate to mainly filamentous. Expression constructs were also generated for each amino terminal isoform to investigate the patterns of localization of individual isoforms and the effects on cells of ectopic expression. The present study shows that the epsilon isoform appears filamentous in this overexpression system while the remaining isoforms are particulate and cytoplasmic. Transient transfection of individual constructs into tumour cell lines results in cell-cycle perturbation with a G2/M arrest and dramatic growth suppression, which was greatest in cell lines with the lowest amounts of endogenous Sept9. Similar phenotypic observations were made with GTP-binding mutants of all five N-terminal variants of Sept9. However, dramatic differences were observed in the kinetics of accumulation of wild-type versus mutant septin protein in transfected cells. In conclusion, the present study shows that the expression patterns of Sept9 protein are very varied in a panel of tumour cell lines and the functional studies are consistent with a model of septin function as a component of a molecular scaffold that contributes to diverse cellular functions

  13. A host small GTP-binding protein ARL8 plays crucial roles in tobamovirus RNA replication.

    PubMed

    Nishikiori, Masaki; Mori, Masashi; Dohi, Koji; Okamura, Hideyasu; Katoh, Etsuko; Naito, Satoshi; Meshi, Tetsuo; Ishikawa, Masayuki

    2011-12-01

    Tomato mosaic virus (ToMV), like other eukaryotic positive-strand RNA viruses, replicates its genomic RNA in replication complexes formed on intracellular membranes. Previous studies showed that a host seven-pass transmembrane protein TOM1 is necessary for efficient ToMV multiplication. Here, we show that a small GTP-binding protein ARL8, along with TOM1, is co-purified with a FLAG epitope-tagged ToMV 180K replication protein from solubilized membranes of ToMV-infected tobacco (Nicotiana tabacum) cells. When solubilized membranes of ToMV-infected tobacco cells that expressed FLAG-tagged ARL8 were subjected to immunopurification with anti-FLAG antibody, ToMV 130K and 180K replication proteins and TOM1 were co-purified and the purified fraction showed RNA-dependent RNA polymerase activity that transcribed ToMV RNA. From uninfected cells, TOM1 co-purified with FLAG-tagged ARL8 less efficiently, suggesting that a complex containing ToMV replication proteins, TOM1, and ARL8 are formed on membranes in infected cells. In Arabidopsis thaliana, ARL8 consists of four family members. Simultaneous mutations in two specific ARL8 genes completely inhibited tobamovirus multiplication. In an in vitro ToMV RNA translation-replication system, the lack of either TOM1 or ARL8 proteins inhibited the production of replicative-form RNA, indicating that TOM1 and ARL8 are required for efficient negative-strand RNA synthesis. When ToMV 130K protein was co-expressed with TOM1 and ARL8 in yeast, RNA 5'-capping activity was detected in the membrane fraction. This activity was undetectable or very weak when the 130K protein was expressed alone or with either TOM1 or ARL8. Taken together, these results suggest that TOM1 and ARL8 are components of ToMV RNA replication complexes and play crucial roles in a process toward activation of the replication proteins' RNA synthesizing and capping functions. PMID:22174675

  14. The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes

    PubMed Central

    Shibano, Takashi; Mamada, Hiroshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Taira, Masanori

    2015-01-01

    The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein. PMID:25946333

  15. Gene 33/Mig-6, a Transcriptionally Inducible Adapter Protein That Binds GTP-Cdc42 and Activates SAPK/JNK*

    PubMed Central

    Makkinje, Anthony; Quinn, Deborah A.; Chen, Ang; Cadilla, Carmen L.; Force, Thomas; Bonventre, Joseph V.; Kyriakis, John M.

    2013-01-01

    Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extra-cellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH2-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences. PMID:10749885

  16. A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley.

    PubMed

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

    2002-04-01

    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

  17. Purification, crystallization and preliminary crystallographic analysis of a GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus

    SciTech Connect

    Wu, Hao; Sun, Lei; Brouns, Stan J. J.; Fu, Sheng; Akerboom, Jasper; Li, Xuemei; Oost, John van der

    2007-03-01

    A GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus has been crystallized. Combined with biochemical analyses, it is expected that the structure of this protein will give insight in the function of a relatively unknown subfamily of the GTPase superfamily. A predicted GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus, termed SsGBP, has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion technique in the presence of 0.05 M cadmium sulfate and 0.8 M sodium acetate pH 7.5. A single-wavelength anomalous dispersion data set was collected to a maximum resolution of 2.0 Å using a single cadmium-incorporated crystal. The crystal form belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with approximate unit-cell parameters a = 65.0, b = 72.6, c = 95.9 Å and with a monomer in the asymmetric unit.

  18. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

    PubMed Central

    Ishizaki, T; Maekawa, M; Fujisawa, K; Okawa, K; Iwamatsu, A; Fujita, A; Watanabe, N; Saito, Y; Kakizuka, A; Morii, N; Narumiya, S

    1996-01-01

    The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo. Images PMID:8617235

  19. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

    PubMed Central

    Matsui, T; Amano, M; Yamamoto, T; Chihara, K; Nakafuku, M; Ito, M; Nakano, T; Okawa, K; Iwamatsu, A; Kaibuchi, K

    1996-01-01

    The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway. Images PMID:8641286

  20. Characteristics of intracellular Ca/sup 2 +/ release mediated by GTP

    SciTech Connect

    Rice, H.L.; Williamson, J.R.; Joseph, S.K.

    1987-05-01

    GTP (but not non-hydrolysable analogs) promotes microsomal Ca/sup 2 +/ release from several tissues provided polyethylene glycol (PEG) is present in the incubation medium. GTP-mediated Ca/sup 2 +/ release from insulinoma or rat liver microsomes is slow and proceeds only after a lag. Rapid Ca/sup 2 +/ release promoted by inositol trisphosphate occurs in microsomes from insulinoma but not liver unless GTP is present. Further experiments indicate that the effects of GTP are dependent on the ionic strength of the incubation medium, the intravesicular Ca/sup 2 +/ load, and are retained upon salt-washing or further purification of the microsomes. GTP-mediated Ca/sup 2 +/ release is halted by an excess of GTP..gamma..S added during the lag or at any stage of Ca/sup 2 +/ release indicating the continued requirement for GTP to sustain release. However, analogs do not promote Ca/sup 2 +/ re-accumulation when added after the release is complete. The relative potency with which analogs inhibit GTP-mediated Ca/sup 2 +/ release was similar to their ability to displace bound ..cap alpha../sup 32/P-GTP. 7-Methyl GTP was found to be relatively ineffective at releasing Ca/sup 2 +/ or displacing ..cap alpha../sup 32/P-GTP. PEG stimulated the rate of ..cap alpha../sup 32/P-GTP binding without affecting the equilibrium value. The lack of a similar effect on /sup 35/S-GTP-..gamma..S binding is consistent with previous studies suggesting that the step affected by PEG is GTP hydrolysis. Experiments on the purification of microsomal high affinity GTPase will be presented and the physiological relevance of this Ca/sup 2 +/ release mechanism will be assessed.

  1. Effects of temperature and ethanol on agonist and antagonist binding to rat heart muscarinic receptors in the absence and presence of GTP.

    PubMed Central

    Waelbroeck, M; Robberecht, P; Chatelain, P; De Neef, P; Christophe, J

    1985-01-01

    The effect of temperature on the binding of four agonists and three antagonists to rat heart muscarinic receptors was studied in the absence and presence of GTP. The binding of agonists to two states (or classes) of receptors, in the absence of GTP, led to enthalpy and entropy changes that decreased sharply above 25 degrees C, suggesting that agonists induced 'isomerization' reactions (large conformational changes and/or receptor-effector association). Both temperature increase and ethanol decreased hydrophobic interactions, thereby hindering binding and/or agonist-induced 'isomerization' reactions. Addition of GTP to the incubation medium also appeared to reverse (or prevent) 'isomerization' reactions. For agonist binding to the low-affinity state, in the presence of GTP, and for antagonist binding, the thermodynamic parameters observed could be readily explained by simple receptor-ligand associations; large entropy increases and small enthalpy increases, provoked by hydrophobic and ionic interactions, were partly neutralized by entropy and enthalpy decreases, due to hydrogen bonds and van der Waals interactions. The muscarinic antagonists used (atropine, n-methylscopolamine and dexetimide), being more hydrophobic molecules than the agonists tested (carbamylcholine, oxotremorine and pilocarpine), induced larger entropy changes or more negative enthalpy changes. PMID:4062907

  2. Effects of temperature and ethanol on agonist and antagonist binding to rat heart muscarinic receptors in the absence and presence of GTP.

    PubMed

    Waelbroeck, M; Robberecht, P; Chatelain, P; De Neef, P; Christophe, J

    1985-10-15

    The effect of temperature on the binding of four agonists and three antagonists to rat heart muscarinic receptors was studied in the absence and presence of GTP. The binding of agonists to two states (or classes) of receptors, in the absence of GTP, led to enthalpy and entropy changes that decreased sharply above 25 degrees C, suggesting that agonists induced 'isomerization' reactions (large conformational changes and/or receptor-effector association). Both temperature increase and ethanol decreased hydrophobic interactions, thereby hindering binding and/or agonist-induced 'isomerization' reactions. Addition of GTP to the incubation medium also appeared to reverse (or prevent) 'isomerization' reactions. For agonist binding to the low-affinity state, in the presence of GTP, and for antagonist binding, the thermodynamic parameters observed could be readily explained by simple receptor-ligand associations; large entropy increases and small enthalpy increases, provoked by hydrophobic and ionic interactions, were partly neutralized by entropy and enthalpy decreases, due to hydrogen bonds and van der Waals interactions. The muscarinic antagonists used (atropine, n-methylscopolamine and dexetimide), being more hydrophobic molecules than the agonists tested (carbamylcholine, oxotremorine and pilocarpine), induced larger entropy changes or more negative enthalpy changes. PMID:4062907

  3. X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTP-state microtubules upon binding

    PubMed Central

    Morikawa, Manatsu; Yajima, Hiroaki; Nitta, Ryo; Inoue, Shigeyuki; Ogura, Toshihiko; Sato, Chikara; Hirokawa, Nobutaka

    2015-01-01

    The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the ‘rigor conformation’, where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules. PMID:25777528

  4. How efficacious are 5-HT1B/D receptor ligands: an answer from GTP gamma S binding studies with stably transfected C6-glial cell lines.

    PubMed

    Pauwels, P J; Tardif, S; Palmier, C; Wurch, T; Colpaert, F C

    1997-01-01

    The intrinsic activity of a series of 5-hydroxytryptamine (serotonin, 5-HT) receptor ligands was analysed at recombinant h5-HT1B and h5-HT1D receptor sites using a [35S]GTP gamma S binding assay and membrane preparations of stably transfected C6-glial cell lines. Compounds either stimulated or inhibited [35S]GTP gamma S binding to a membrane preparation containing either h5-HT1B or h5-HT1D receptors. The potencies observed for most of the compounds at the h5-HT1B receptor subtype correlated with their potencies measured by inhibition of stimulated cAMP formation on intact cells. Apparent agonist potencies in the [35S]GTP gamma S binding assay to C6-glial/h5-HT1D membranes were, with the exception of 2-[5-[3-(4-methylsulphonylamino)benzyl-1 2,4-oxadiazol-5-yl]-1H-indol-3-yl] ethanamine (L694247), 5- to 13-times lower than in the cAMP assay on intact cells. This suggests that receptor coupling in the h5-HT1D membrane preparation is less efficient than that in the intact cell. It further appeared that 6-times more h5-HT1D than h5-HT1B binding sites were required to attain a similar, maximal (73%), 5-HT-stimulated [35S]GTP gamma S binding response: Hence, the h5-HT1B receptor in C6-glial cell membranes could be more efficiently coupled, even though some compounds more readily displayed intrinsic activity at h5-HT1D receptor sites [e.g. dihydroergotamine and (2'-methyl-4'-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127935)]. Efficacy differences were apparent for most of the compounds (sumatriptan, zolmitriptan, rizatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulfonamide (CP122638), dihydroergotamine, naratriptan and GR127935) that stimulated [35S]GTP gamma S binding compared to the native agonist 5-HT. The observed maximal responses were different for the h5-HT1B and h5-HT1D receptor subtypes. Few compounds behaved as full agonists: L694247, zolmitriptan and sumatriptan did so at

  5. Septin6 and Septin7 GTP binding proteins regulate AP-3- and ESCRT-dependent multivesicular body biogenesis.

    PubMed

    Traikov, Sofia; Stange, Christoph; Wassmer, Thomas; Paul-Gilloteaux, Perrine; Salamero, Jean; Raposo, Graça; Hoflack, Bernard

    2014-01-01

    Septins (SEPTs) form a family of GTP-binding proteins implicated in cytoskeleton and membrane organization, cell division and host/pathogen interactions. The precise function of many family members remains elusive. We show that SEPT6 and SEPT7 complexes bound to F-actin regulate protein sorting during multivesicular body (MVB) biogenesis. These complexes bind AP-3, an adapter complex sorting cargos destined to remain in outer membranes of maturing endosomes, modulate AP-3 membrane interactions and the motility of AP-3-positive endosomes. These SEPT-AP interactions also influence the membrane interaction of ESCRT (endosomal-sorting complex required for transport)-I, which selects ubiquitinated cargos for degradation inside MVBs. Whereas our findings demonstrate that SEPT6 and SEPT7 function in the spatial, temporal organization of AP-3- and ESCRT-coated membrane domains, they uncover an unsuspected coordination of these sorting machineries during MVB biogenesis. This requires the E3 ubiquitin ligase LRSAM1, an AP-3 interactor regulating ESCRT-I sorting activity and whose mutations are linked with Charcot-Marie-Tooth neuropathies. PMID:25380047

  6. A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent beta cells.

    PubMed Central

    Kowluru, A; Seavey, S E; Rhodes, C J; Metz, S A

    1996-01-01

    Recently we described roles for heterotrimeric and low-molecular-mass GTP-binding proteins in insulin release from normal rat islets. During these studies, we observed that a protein with an apparent molecular mass (37 kDa) similar to that of the beta subunit of trimeric GTP-binding proteins underwent phosphorylation in each of five classes of insulin-secreting cells. Incubation of the beta cell total membrane fraction or the isolated secretory granule fraction (but not the cytosolic fraction) with [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of this protein, which was selectively immunoprecipitated by an anti-serum directed against the common beta subunit of trimeric G-proteins. Disruption of the alpha beta gamma trimer (by pretreatment with either fluoroaluminate or guanosine 5'(-)[gamma-thio]triphosphate) prevented beta subunit phosphorylation. Based on differential sensitivities to pH, heat and the histidine-selective reagent diethyl pyrocarbonate (and reversal of the latter by hydroxylamine), the phosphorylated amino acid was presumptively identified as histidine. Incubation of pure beta subunit alone or in combination with the exogenous purified alpha subunit of transducin did not result in the phosphorylation of the beta subunit, but addition of the islet cell membrane fraction did support this event, suggesting that membrane localization (or a membrane-associated factor) is required for beta subunit phosphorylation. Incubation of phosphorylated beta subunit with G alpha.GDP accelerated the dephosphorylation of the beta subunit, accompanied by the formation of G alpha-GTP. Immunoblotting detected multiple alpha subunits (of Gi, G(o) and Gq) and at least one beta subunit in the secretory granule fraction of normal rat islets and insulinoma cells. These data describe a potential alternative mechanism for the activation of GTP-binding proteins in beta cells which contrasts with the classical receptor-agonist mechanism: G beta undergoes

  7. 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.

  8. Characterization of Human GTPBP3, a GTP-Binding Protein Involved in Mitochondrial tRNA Modification▿ †

    PubMed Central

    Villarroya, Magda; Prado, Silvia; Esteve, Juan M.; Soriano, Miguel A.; Aguado, Carmen; Pérez-Martínez, David; Martínez-Ferrandis, José I.; Yim, Lucía; Victor, Victor M.; Cebolla, Elvira; Montaner, Asunción; Knecht, Erwin; Armengod, M.-Eugenia

    2008-01-01

    Human GTPBP3 is an evolutionarily conserved, multidomain protein involved in mitochondrial tRNA modification. Characterization of its biochemical properties and the phenotype conferred by GTPBP3 inactivation is crucial to understanding the role of this protein in tRNA maturation and its effects on mitochondrial respiration. We show that the two most abundant GTPBP3 isoforms exhibit moderate affinity for guanine nucleotides like their bacterial homologue, MnmE, although they hydrolyze GTP at a 100-fold lower rate. This suggests that regulation of the GTPase activity, essential for the tRNA modification function of MnmE, is different in GTPBP3. In fact, potassium-induced dimerization of the G domain leads to stimulation of the GTPase activity in MnmE but not in GTPBP3. The GTPBP3 N-terminal domain mediates a potassium-independent dimerization, which appears as an evolutionarily conserved property of the protein family, probably related to the construction of the binding site for the one-carbon-unit donor in the modification reaction. Partial inactivation of GTPBP3 by small interfering RNA reduces oxygen consumption, ATP production, and mitochondrial protein synthesis, while the degradation of these proteins slightly increases. It also results in mitochondria with defective membrane potential and increased superoxide levels. These phenotypic traits suggest that GTPBP3 defects contribute to the pathogenesis of some oxidative phosphorylation diseases. PMID:18852288

  9. [Cloning and function analysis of the rice small GTP-binding protein gene OsPra2].

    PubMed

    Zhao, Zhiqiang; Fu, Yaping; Yang, Kun; Zhang, Yuman; Yan, Yongsheng; Fang, Rongxiang; Sun, Zongxiu; Chen, Xiaoying

    2008-12-01

    Gene expression in rice roots under nutritional stress was studied using micro array techniques. The results showed that when re-supplied with sufficient amounts of nutrition after nutrition stress, the expression of OsPra2 (a small G protein which is homologous with Pea Pra2 protein) decreased in the plants root tissue. The cDNA sequence of the OsPra2 gene and its promoter, which is about 1 kb upstream of the translation origin point, was obtained using RT-PCR and PCR approaches. The OsPra2 protein contains four conserved GTP/GDP binding domains and specific domain of Rab small G protein family. The expression of OsPra2 and GST fusion protein in onion epidermal cells showed that OsPra2 protein was localized in the membrane and nucleus of the cell. The fusion expression of OsPra2 promoter and GUS reporter gene in transgenic rice suggested that the OsPra2 promoter allowed GUS expression in coleoptiles and roots. Compared with wild type rice, OsPra2 over expressed transgenic rice showed an obvious dwarf phenotype which resembles the BR deficient rice. PMID:19306571

  10. rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

    PubMed

    Kinsella, B T; Maltese, W A

    1992-02-25

    p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic. PMID:1740442

  11. Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

    PubMed Central

    Sahyoun, N; McDonald, O B; Farrell, F; Lapetina, E G

    1991-01-01

    A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release. Images PMID:1901412

  12. Rab1 Small GTP-Binding Protein Regulates Cell Surface Trafficking of the Human Calcium-Sensing Receptor

    PubMed Central

    Zhuang, Xiaolei; Adipietro, Kaylin A.; Datta, Shomik; Northup, John K.; Ray, Kausik

    2010-01-01

    The human calcium-sensing receptor (hCaR) is a family-3/C G-protein-coupled receptor that regulates Ca2+ homeostasis by controlling parathyroid hormone secretion. Here we investigated the role of Rab1, a small GTP-binding protein that specifically regulates protein transport from the endoplasmic reticulum to the Golgi, in cell surface transport of the hCaR. Cell surface expression of hCaR transiently expressed in human embryonic kidney 293 cells was strongly augmented by coexpression of Rab1 and attenuated by disruption of endogenous Rab1 function by expression of the dominant-negative Rab1N124I mutant or depletion of Rab1 with small interfering RNA. Rab1N124I expression also partially attenuated cell surface expression and signaling response to gain-of-function mutants of hCaR with truncated carboxyl-terminal sequences at positions 895 and 903. These carboxyl-tail truncations are similar to a deletion between residues S895 and V1075 found in a patient family causing autosomal dominant hypocalcemia. In addition, coexpression with wild-type Rab1 increased cell surface expression of the loss-of-function missense mutation R185Q, located on the hCaR amino-terminal extracellular ligand-binding domain (ECD), which causes familial hypocalciuric hypercalcemia. Truncated hCaR variants containing either the ECD with the first transmembrane helix or only the ECD also display Rab1-dependent cell surface expression or secretion into the culture medium, respectively. These data reveal a role for Rab1 in hCaR trafficking from the endoplasmic reticulum to the Golgi that regulates receptor cell surface expression and thereby cell signaling responsiveness to extracellular calcium. PMID:20861236

  13. Role of the Rab GTP-Binding Protein Ypt3 in the Fission Yeast Exocytic Pathway and Its Connection to Calcineurin Function

    PubMed Central

    Cheng, Hong; Sugiura, Reiko; Wu, Wenlian; Fujita, Masaaki; Lu, Yabin; Sio, Susie O.; Kawai, Rena; Takegawa, Kaoru; Shuntoh, Hisato; Kuno, Takayoshi

    2002-01-01

    A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1+ leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes. PMID:12181359

  14. Rim1 and rabphilin-3 bind Rab3-GTP by composite determinants partially related through N-terminal alpha -helix motifs.

    PubMed

    Wang, X; Hu, B; Zimmermann, B; Kilimann, M W

    2001-08-31

    Rim1 is a protein of the presynaptic active zone, the area of the plasma membrane specialized for neurotransmitter exocytosis, and interacts with Rab3, a small GTPase implicated in neurotransmitter vesicle dynamics. Here, we have studied the molecular determinants of Rim1 that are responsible for Rab3 binding, employing surface plasmon resonance and recombinant, bacterially expressed Rab3 and Rim1 proteins. A site that binds GTP- but not GDP-saturated Rab3 was localized to a short alpha-helical sequence near the Rim1 N terminus (amino acids 19-55). Rab3 isoforms A, C, and D were bound with similar affinities (K(d) = 1-2 microm). Low affinity binding of Rab6A-GTP was also observed (K(d) = 16 microm), whereas Rab1B, -5, -7, -8, or -11A did not bind. Adjacent sequences up to amino acid 387, encompassing differentially spliced sequences, the zinc finger module, and the SGAWFF motif of Rim1, did not significantly contribute to the strength or the specificity of Rab3 binding, whereas a point mutation within the helix (R33G) abolished binding. This Rab3 binding site of Rim1 is reminiscent of the N-terminal alpha-helix that is part of the Rab3-binding region of rabphilin-3, and indeed we observed low affinity, specific binding of Rab3A (K(d) on the order of magnitude of 10-100 microm) to this region of rabphilin-3 alone (amino acids 40-88), whereas additional sequences up to amino acid 178 are needed for high affinity Rab3A binding to rabphilin-3 (K(d) = 10-20 nm). In contrast, an N-terminal alpha-helix motif in aczonin, with sequence similarity to the Rab3-binding site of Rim1, did not bind Rab3A, -C, or -D or several other Rab proteins. These results were qualitatively confirmed in pull-down experiments with native, prenylated Rab3 from brain lysate in Triton X-100. Munc13 bound to the zinc finger domain of Rim1 but not to the rabphilin-3 or aczonin zinc fingers. Pull-down experiments from brain lysate in the presence of cholate as detergent detected binding to

  15. The N-terminal peptide of mammalian GTP cyclohydrolase I is an autoinhibitory control element and contributes to binding the allosteric regulatory protein GFRP.

    PubMed

    Higgins, Christina E; Gross, Steven S

    2011-04-01

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity. PMID:21163945

  16. The N-terminal Peptide of Mammalian GTP Cyclohydrolase I Is an Autoinhibitory Control Element and Contributes to Binding the Allosteric Regulatory Protein GFRP*

    PubMed Central

    Higgins, Christina E.; Gross, Steven S.

    2011-01-01

    GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity. PMID:21163945

  17. Tandem duplications of a degenerated GTP-binding domain at the origin of GTPase receptors Toc159 and thylakoidal SRP

    SciTech Connect

    Hernandez Torres, Jorge Maldonado, Monica Alexandra Arias; Chomilier, Jacques

    2007-12-14

    The evolutionary origin of some nuclear encoded proteins that translocate proteins across the chloroplast envelope remains unknown. Therefore, sequences of GTPase proteins constituting the Arabidopsis thaliana translocon at the outer membrane of chloroplast (atToc) complexes were analyzed by means of HCA. In particular, atToc159 and related proteins (atToc132, atToc120, and atToc90) do not have proven homologues of prokaryotic or eukaryotic ancestry. We established that the three domains commonly referred to as A, G, and M originate from the GTPase G domain, tandemly repeated, and probably evolving toward an unstructured conformation in the case of the A domain. It resulted from this study a putative common ancestor for these proteins and a new domain definition, in particular the splitting of A into three domains (A1, A2, and A3), has been proposed. The family of Toc159, previously containing A. thaliana and Pisum sativum, has been extended to Medicago truncatula and Populus trichocarpa and it has been revised for Oryza sativa. They have also been compared to GTPase subunits involved in the cpSRP system. A distant homology has been revealed among Toc and cpSRP GTP-hydrolyzing proteins of A. thaliana, and repetitions of a GTPase domain were also found in cpSRP protein receptors, by means of HCA analysis.

  18. A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network.

    PubMed

    Guo, Yusong; Zanetti, Giulia; Schekman, Randy

    2013-01-01

    Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.DOI:http://dx.doi.org/10.7554/eLife.00160.001. PMID:23326640

  19. [35S]GTP gamma S binding studies of amphiphilic drugs-activated Gi proteins: a caveat.

    PubMed

    Manetti, Dina; Di Cesare Mannelli, Lorenzo; Dei, Silvia; Guandalini, Luca; Martini, Elisabetta; Banchelli, Martina; Ghelardini, Carla

    2009-04-15

    This paper documents a serious problem met during the testing of Gi protein-activating properties of a new series of synthetic compounds by measuring the induced binding of [(35)S]GTPgammaS to different subtypes of Gi protein. The problem arose from the strong affinity between [(35)S]GTPgammaS and the tested compounds, that are characterized by several (2-4) positive charges and high lipophilicity. Apparently, such affinity yields insoluble, labelled complexes that, also in the absence of Gi protein, are retained on the filters and give rise to false positive results. PMID:19289280

  20. Leukotriene BLT2 Receptor Monomers Activate the Gi2 GTP-binding Protein More Efficiently than Dimers*

    PubMed Central

    Arcemisbéhère, Laure; Sen, Tuhinadri; Boudier, Laure; Balestre, Marie-Noëlle; Gaibelet, Gérald; Detouillon, Emilie; Orcel, Hélène; Mendre, Christiane; Rahmeh, Rita; Granier, Sébastien; Vivès, Corinne; Fieschi, Franck; Damian, Marjorie; Durroux, Thierry; Banères, Jean-Louis; Mouillac, Bernard

    2010-01-01

    Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B4 receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory Gi2 protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human α5 integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB4 binding in the presence of the purified G protein Gαi2. The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5′-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified Gαi2β1γ2 protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal. PMID:20026606

  1. Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP–ASAP1 complex

    PubMed Central

    Luo, Ruibai; Ahvazi, Bijan; Amariei, Diana; Shroder, Deborah; Burrola, Beatriz; Losert, Wolfgang; Randazzo, Paul A.

    2006-01-01

    Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a kcat of 57±5 s−1 and a Km of 2.2±0.5 μM determined by steady-state kinetics and a kcat of 56±7 s−1 determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4−), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than Km. Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the kcat. Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP–ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed. PMID:17112341

  2. Structure of Escherichia coli dGTP Triphosphohydrolase

    PubMed Central

    Singh, Deepa; Gawel, Damian; Itsko, Mark; Hochkoeppler, Alejandro; Krahn, Juno M.; London, Robert E.; Schaaper, Roel M.

    2015-01-01

    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 study, 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 DNA with high affinity (Kd ∼ 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 Km 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. Our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding. PMID:25694425

  3. The GTP-bound and Sumoylated Form of the rab17 Small Molecular Weight GTPase Selectively Binds Syntaxin 2 in Polarized Hepatic WIF-B Cells.

    PubMed

    Striz, Anneliese C; Tuma, Pamela L

    2016-04-29

    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

  4. Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

    PubMed Central

    Coorssen, J R; Davidson, M M; Haslam, R J

    1990-01-01

    Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from

  5. Gene 33/Mig-6, a transcriptionally inducible adapter protein that binds GTP-Cdc42 and activates SAPK/JNK. A potential marker transcript for chronic pathologic conditions, such as diabetic nephropathy. Possible role in the response to persistent stress.

    PubMed

    Makkinje, A; Quinn, D A; Chen, A; Cadilla, C L; Force, T; Bonventre, J V; Kyriakis, J M

    2000-06-01

    Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extracellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH(2)-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences. PMID:10749885

  6. Studies on tissue transglutaminases: interaction of erythrocyte type-2 transglutaminase with GTP.

    PubMed Central

    Bergamini, C M; Signorini, M

    1993-01-01

    Ca2+ and GTP are the main modulators of type-2 transglutaminases. To study the interaction of the enzyme with GTP, we have employed periodate-oxidized GTP as an affinity-label probe. Dialdehyde GTP bound irreversibly to type-2 transglutaminase in a time-dependent way with 1:1 stoichiometry at complete modification. The reaction took place in the absence, but was more rapid in the presence, of cyanoborohydride. Native GTP prevented incorporation of dialdehyde GTP, and Ca2+ significantly slowed down the reaction rate. The modified enzyme displayed decreased sensitivity to Ca2+, with a sigmoid saturation curve. We conclude that type-2 transglutaminase has a single GTP-binding site, the modification of which by dialdehyde GTP mimics nucleotide binding to the enzyme. Images Figure 1 PMID:8097088

  7. Decameric GTP cyclohydrolase I forms complexes with two pentameric GTP cyclohydrolase I feedback regulatory proteins in the presence of phenylalanine or of a combination of tetrahydrobiopterin and GTP.

    PubMed

    Yoneyama, T; Hatakeyama, K

    1998-08-01

    The activity of GTP cyclohydrolase I is inhibited by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and stimulated by phenylalanine through complex formation with GTP cyclohydrolase I feedback regulatory protein (GFRP). Gel filtration experiments as well as enzyme activity measurements showed that the number of subunits of GFRP in both the inhibitory and stimulatory complexes is equal to that of GTP cyclohydrolase I. Because GFRP is a pentamer and GTP cyclohydrolase I was shown here by cross-linking experiments to be a decamer, the results indicate that two molecules of a pentameric GFRP associate with one molecule of GTP cyclohydrolase I. Gel filtration analysis suggested that the complex has a radius of gyration similar to that of the enzyme itself. These observations support our model that one molecule of GFRP binds to each of the two outer faces of the torus-shaped GTP cyclohydrolase I. For formation of the inhibitory protein complex, both BH4 and GTP were required; the median effective concentrations of BH4 and GTP were 2 and 26 microM, respectively. BH4 was the most potent of biopterins with different oxidative states. Among GTP analogues, dGTP as well as guanosine 5'-O-(3'-thiotriphosphate) exhibited similar inducibility compared with GTP, whereas other nucleotide triphosphates had no effect. On the other hand, phenylalanine alone was enough for formation of the stimulatory protein complex, and positive cooperativity was found for the phenylalanine-induced protein complex formation. Phenylalanine was the most potent of the aromatic amino acids. PMID:9685352

  8. Impaired chromosome partitioning and synchronization of DNA replication initiation in an insertional mutant in the Vibrio harveyi cgtA gene coding for a common GTP-binding protein.

    PubMed Central

    Słomińska, Monika; Konopa, Grazyna; Wegrzyn, Grzegorz; Czyz, Agata

    2002-01-01

    The Vibrio harveyi cgtA gene product belongs to a subfamily of small GTP-binding proteins, called Obg-like proteins. Members of this subfamily are present in diverse organisms ranging from bacteria to humans. On the other hand, the functions of these proteins in the regulation of cellular processes are largely unknown. Genes coding for these proteins are essential in almost all bacteria investigated thus far. However, a viable V. harveyi insertional mutant in the cgtA gene was described recently. Therefore, this mutant gives a unique opportunity to study functions of a member of the subfamily of Obg-like proteins. Here we demonstrate that the mutant cells often form long filaments with expanded, non-partitioned or rarely partitioned chromosomes. Such a phenotype suggests impairment of the mechanism of chromosome partition. Flow cytometric studies revealed that synchronization of chromosome replication initiation is also significantly disturbed in the cgtA mutant. Moreover, in contrast to wild-type V. harveyi, inhibition of chromosome replication and/or of cell division in the mutant bacteria caused significant increase in the number of large cells, suggesting that the cgtA gene product may be involved in the coupling of cell growth to chromosome replication and cell division. These results indicate that CgtA, an Obg-like GTP-binding protein, plays an important role in the regulation of chromosomal functions. PMID:11879184

  9. GTP cyclohydrolase I feedback regulatory protein-dependent and -independent inhibitors of GTP cyclohydrolase I.

    PubMed

    Yoneyama, T; Wilson, L M; Hatakeyama, K

    2001-04-01

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates the feedback inhibition of GTP cyclohydrolase I activity by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) through protein complex formation. Since guanine and BH4 have a common pyrimidine ring structure, we examined the inhibitory effect of guanine and its analogs on the enzyme activity. Guanine, 8-hydroxyguanine, 8-methylguanine, and 8-bromoguanine inhibited the enzyme activity in a GFRP-dependent and pH-dependent manner and induced complex formation between GTP cyclohydrolase I and GFRP. The type of inhibition by this group is a mixed type. All these properties were shared with BH4. In striking contrast, inhibition by 8-azaguanine and 8-mercaptoguanine was GFRP-independent and pH-independent. The type of inhibition by 8-azaguanine and 8-mercaptoguanine was a competitive type. The two compounds did not induce complex formation between the enzyme and GFRP. These results demonstrate that guanine compounds of the first group bind to the BH4-binding site of the GTP cyclohydrolase I/GFRP complex, whereas 8-azaguanine and 8-mercaptoguanine bind to the active site of the enzyme. Finally, the possible implications in Lesch-Nyhan syndrome and Parkinson diseases of the inhibition of GTP cyclohydrolase I by guanine and 8-hydroxyguanine are discussed. PMID:11361142

  10. Proteins that interact with GTP during sporulation of Bacillus subtilis

    SciTech Connect

    Mitchell, C.; Vary, J.C. )

    1989-06-01

    During sporulation of Bacillus subtilis, several proteins were shown to interact with GTP in specific ways. UV light was used to cross-link ({alpha}-{sup 32}P)GTP to proteins in cell extracts at different stages of growth. After electrophoresis, 11 bands of radioactivity were found in vegetative cells, 4 more appeared during sporulation, and only 9 remained in mature spores. Based on the labeling pattern with or without UV light to cross-link either ({alpha}-{sup 32}P)GTP or ({gamma}-{sup 32}P)GTP, 11 bands of radioactivity were apparent guanine nucleotide-binding proteins, and 5 bands appeared to be phosphorylated and/or guanylated. Similar results were found with Bacillus megaterium. Assuming the GTP might be a type of signal for sporulation, it could interact with and regulate proteins by at least three mechanisms.

  11. Pentoxifylline affects idarubicin binding to DNA.

    PubMed

    Gołuński, Grzegorz; Borowik, Agnieszka; Lipińska, Andrea; Romanik, Monika; Derewońko, Natalia; Woziwodzka, Anna; Piosik, Jacek

    2016-04-01

    Anticancer drug idarubicin - derivative of doxorubicin - is commonly used in treatment of numerous cancer types. However, in contrast to doxorubicin, its biophysical properties are not well established yet. Additionally, potential direct interactions of idarubicin with other biologically active aromatic compounds, such as pentoxifylline - representative of methylxanthines - were not studied at all. Potential formation of such hetero-aggregates may result in sequestration of the anticancer drug and, in consequence, reduction of its biological activity. This work provide description of the idarubicin biophysical properties as well as assess influence of pentoxifylline on idarubicin interactions with DNA. To achieve these goals we employed spectrophotometric methods coupled with analysis with the appropriate mathematical models as well as flow cytometry and Ames test. Obtained results show influence of pentoxifylline on idarubicin binding to DNA and are well in agreement with the data previously published for other aromatic ligands. Additionally it may be hypothesized that direct interactions between idarubicin and pentoxifylline may influence the anticancer drug biological activity. PMID:26921593

  12. 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

  13. Selective Impairment of a Subset of Ran-GTP-binding Domains of Ran-binding Protein 2 (Ranbp2) Suffices to Recapitulate the Degeneration of the Retinal Pigment Epithelium (RPE) Triggered by Ranbp2 Ablation*

    PubMed Central

    Patil, Hemangi; Saha, Arjun; Senda, Eugene; Cho, Kyoung-in; Haque, MdEmdadul; Yu, Minzhong; Qiu, Sunny; Yoon, Dosuk; Hao, Ying; Peachey, Neal S.; Ferreira, Paulo A.

    2014-01-01

    Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2−/−, with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2−/− background. Among the transgenic lines produced, only TgRBD2/3*-HA::RPE-cre::Ranbp2−/−-expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2−/−. By contrast, TgRBD2/3*-HA expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2−/− and TgRBD2/3*-HA::RPE-cre::Ranbp2−/− share proteostatic deregulation of Ran GTPase, serotransferrin, and γ-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases

  14. A potential role for guanine nucleotide-binding protein in the regulation of endosomal proton transport.

    PubMed Central

    Gurich, R W; Codina, J; DuBose, T D

    1991-01-01

    The effects of guanosine 5'-triphosphate (GTP) and GTP-gamma-S, known activators of GTP binding proteins, on proton transport were investigated in endosome-enriched vesicles (endosomes). Endosomes were prepared from rabbit renal cortex following the intravenous injection of FITC-dextran. The rate of intravesicular acidification was determined by measuring changes in fluorescence of FITC-dextran. Both GTP and GTP-gamma-S stimulated significantly the initial rate of proton transport. In contrast, GDP-beta-S, which does not activate GTP binding proteins, inhibited proton transport. The rank order of stimulation was GTP-gamma-S greater than GTP greater than control greater than GDP-beta-S. GTP-gamma-S stimulation of proton transport was also observed under conditions in which chloride entry was eliminated, i.e., 0 mM external chloride concentration in the presence of potassium/valinomycin voltage clamping. GTP-gamma-S did not affect proton leak in endosomes as determined by collapse of H+ ATPase-generated pH gradients. ADP ribosylation by treatment of endosomal membranes with pertussis toxin revealed two substrates corresponding to the 39-41 kD region and comigrating with alpha i subunits. Pretreatment of the membranes with pertussis toxin had no effect on proton transport in the absence of GTP or GTP-gamma-S. However, pretreatment with pertussis toxin blocked the stimulation of proton transport by GTP. In contrast, as reported in other membranes by others previously, pertussis toxin did not prevent the stimulation of proton transport by GTP-gamma-S. These findings, taken together, indicate that GTP binding proteins are present in endosomal membranes derived from renal cortex and that activation of G protein by GTP and GTP-gamma-S stimulates proton transport in a rank order identical to that reported for other transport pathways modulated by Gi proteins. Therefore, these studies suggest that G proteins are capable of stimulating the vacuolar H ATPase of endosomes

  15. A polymorphism of the GTP-cyclohydrolase I feedback regulator gene alters transcriptional activity and may affect response to SSRI antidepressants.

    PubMed

    McHugh, P C; Joyce, P R; Deng, X; Kennedy, M A

    2011-06-01

    Tetrahydrobiopterin (BH(4)) is an essential cofactor for synthesis of many neurotransmitters including serotonin. In serotonergic neurons, BH(4) is tightly regulated by GTP-cyclohydrolase I feedback regulator (GFRP). Given the pivotal role of the serotonergic system in mood disorders and selective serotonin reuptake inhibitors (SSRIs) antidepressant function, we tested the hypothesis that GFRP gene (GCHFR) variants would modify response to antidepressants in subjects with major depression. Two single nucleotide polymorphisms (rs7164342 and rs7163862) in the GCHFR promoter were identified and occurred as two haplotypes (GA or TT). A multiple regression analysis revealed that homozygous individuals for the TT haplotype were less likely to respond to the SSRI fluoxetine than to the tricyclic antidepressant nortriptyline (P = 0.037). Moreover, the TT haplotype showed a reduced transcription rate in luciferase reporter gene assays, which may impact on BH(4)-mediated neurotransmitter production, thus suggesting a biological process through which GCHFR promoter variants might influence antidepressant response. PMID:20351752

  16. Inhibitory heterotrimeric GTP-binding proteins inhibit hydrogen peroxide-induced apoptosis by up-regulation of Bcl-2 via NF-{kappa}B in H1299 human lung cancer cells

    SciTech Connect

    Seo, Mi Ran; Nam, Hyo-Jung; Kim, So-Young; Juhnn, Yong-Sung

    2009-04-03

    Inhibitory heterotrimeric GTP-binding proteins (Gi proteins) mediate a variety of signaling pathways by coupling receptors and effectors to regulate cellular proliferation, differentiation, and apoptosis. However, the role of Gi proteins in the modulation of hydrogen peroxide-induced apoptosis is not clearly understood. Thus, we investigated the effect of Gi proteins on hydrogen peroxide-induced apoptosis and the underlying mechanisms in H1299 human lung cancer cells. The stable expression of constitutively active alpha subunits of Gi1 (G{alpha}i1QL), Gi2, or Gi3 inhibited hydrogen peroxide-induced apoptosis. The expression of G{alpha}i1QL up-regulated Bcl-2 expression, and the knockdown of Bcl-2 with siRNA abolished the anti-apoptotic effect of G{alpha}i1QL. G{alpha}i1 induced the transcription of Bcl-2 by activation of NF-{kappa}B, which resulted from an increase in NF-{kappa}B p50 protein. We conclude that G{alpha}i1 inhibits hydrogen peroxide-induced apoptosis of H1299 lung cancer cells by up-regulating the transcription of Bcl-2 through a p50-mediated NF-{kappa}B activation.

  17. Mechanisms of calcium release induced by GTP and inositol 1,4,5-trisphosphate

    SciTech Connect

    Gill, D.L.; Chueh, S.H.; Mullaney, J.M.; Mallet, M.K.

    1987-05-01

    Recent studies show that Ca/sup 2 +/ efflux from ER is controlled by a sensitive and specific guanine nucleotide regulatory mechanism. Using microsomes of permeabilized cells derived from N1E-115 neuroblastoma or DDT/sub 1/MF-2 smooth muscle cell lines, both GTP and IP/sub 3/ effect Ca/sup 2 +/ release from a common intracellular pool; however, the mechanisms of activation of Ca/sup 2 +/ release by the two agents appear distinct with regard to several parameters. Studies using liver microsomes are currently investigating whether similar distinctions between the actions of IP/sub 3/ and GTP exist in other cell types. At present it is unknown if GTP-activated Ca/sup 2 +/ release is mediated by a G-protein-like activity. Studies indicate that such release is not altered by pertussis toxin. Since GTP..gamma..S is inactive and blocks the action of GTP, a modified G-protein activation process must be invoked. Current investigations are attempting to identify the protein(s) involved in GTP-mediated Ca/sup 2 +/ release by direct photo-crosslinking experiments using (..cap alpha..-/sup 32/P)GTP. Successful labeling of many nucleotide-binding proteins has been accomplished; most but not all labeling is displaced by ATP. GTP-specifically labeled proteins are being assessed as candidates for the GTP-mediated release process.

  18. Identification of a GTP-binding protein. cap alpha. subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

    SciTech Connect

    Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.; Simon, M.I.

    1988-05-01

    Recent molecular cloning of cDNA for the ..cap alpha.. subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein ..cap alpha.. subunit, which they refer to as G/sub z/..cap alpha.., by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ ..cap alpha..-subunit cDNA. The deduced amino acid sequence of G/sub z/..cap alpha.. is 41-67% identical with those of other known G-protein ..cap alpha.. subunits. However, the 355-residue G/sub z/..cap alpha.. lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein ..cap alpha.. subunits. They suggest that G/sub z/..cap alpha.., which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.

  19. Invited review: Activation of G proteins by GTP and the mechanism of Gα-catalyzed GTP hydrolysis.

    PubMed

    Sprang, Stephen R

    2016-08-01

    This review addresses the regulatory consequences of the binding of GTP to the alpha subunits (Gα) of heterotrimeric G proteins, the reaction mechanism of GTP hydrolysis catalyzed by Gα and the means by which GTPase activating proteins (GAPs) stimulate the GTPase activity of Gα. The high energy of GTP binding is used to restrain and stabilize the conformation of the Gα switch segments, particularly switch II, to afford stable complementary to the surfaces of Gα effectors, while excluding interaction with Gβγ, the regulatory binding partner of GDP-bound Gα. Upon GTP hydrolysis, the energy of these conformational restraints is dissipated and the two switch segments, particularly switch II, become flexible and are able to adopt a conformation suitable for tight binding to Gβγ. Catalytic site pre-organization presents a significant activation energy barrier to Gα GTPase activity. The glutamine residue near the N-terminus of switch II (Glncat ) must adopt a conformation in which it orients and stabilizes the γ phosphate and the water nucleophile for an in-line attack. The transition state is probably loose with dissociative character; phosphoryl transfer may be concerted. The catalytic arginine in switch I (Argcat ), together with amide hydrogen bonds from the phosphate binding loop, stabilize charge at the β-γ bridge oxygen of the leaving group. GAPs that harbor "regulator of protein signaling" (RGS) domains, or structurally unrelated domains within G protein effectors that function as GAPs, accelerate catalysis by stabilizing the pre-transition state for Gα-catalyzed GTP hydrolysis, primarily by restraining Argcat and Glncat to their catalytic conformations. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 449-462, 2016. PMID:26996924

  20. Dual use of GTP hydrolysis by elongation factor G on the ribosome

    PubMed Central

    Cunha, Carlos E.; Belardinelli, Riccardo; Peske, Frank; Holtkamp, Wolf; Wintermeyer, Wolfgang; Rodnina, Marina V.

    2013-01-01

    Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor. PMID:26824016

  1. Dual use of GTP hydrolysis by elongation factor G on the ribosome.

    PubMed

    Cunha, Carlos E; Belardinelli, Riccardo; Peske, Frank; Holtkamp, Wolf; Wintermeyer, Wolfgang; Rodnina, Marina V

    2013-01-01

    Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor. PMID:26824016

  2. Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis.

    PubMed Central

    Vulevic, B; Correia, J J

    1997-01-01

    that assembly with GTP/2 M glycerol and with taxol is consistent with conformational rearrangements in 3-6% of the total amino acids in the heterodimer. In addition, taxol binding contributes to the thermodynamics of the overall process by reducing the delta H degree and delta S degree for microtubule assembly. In the presence of GMPCPP or GMPCP, tubulin subunits associate with extensive conformational rearrangement, corresponding to 10% and 26% of the total amino acids in the heterodimer, respectively, which gives rise to a large loss of configurational entropy. An alternative, and probably preferable, interpretation of these data is that, especially with GMPCP-tubulin, additional isomerization or protonation events are induced by the presence of the methylene moiety and linked to microtubule assembly. Structural analysis shows that GTP hydrolysis is not required for sheet closure into a microtubule cylinder, but only increases the probability of this event occurring. Sheet extensions and sheet polymers appear to have a similar average length under various conditions, suggesting that the minimum cooperative unit for closure of sheets into a microtubule cylinder is approximately 400 nm long. Because of their low level of occurrence, sheets are not expected to significantly affect the thermodynamics of assembly. PMID:9138581

  3. Mutation of the Zinc-Binding Metalloprotease Motif Affects Bacteroides fragilis Toxin Activity but Does Not Affect Propeptide Processing

    PubMed Central

    Franco, Augusto A.; Buckwold, Simy L.; Shin, Jai W.; Ascon, Miguel; Sears, Cynthia L.

    2005-01-01

    To evaluate the role of the zinc-binding metalloprotease in Bacteroides fragilis toxin (BFT) processing and activity, the zinc-binding consensus sequences (H348, E349, H352, G355, H358, and M366) were mutated by site-directed-mutagenesis. Our results indicated that single point mutations in the zinc-binding metalloprotease motif do not affect BFT processing but do reduce or eliminate BFT biologic activity in vitro. PMID:16041055

  4. Modification of amino groups in EF-Tu.GTP and the ternary complex EF-Tu.GTP.valyl-tRNAVal.

    PubMed

    Antonsson, B; Leberman, R

    1984-06-15

    In an attempt to describe the binding region of EF-Tu . GTP for aminoacyl-tRNA, the epsilon-amino groups of the lysine residues of the protein molecule in the GTP and ternary complexes were modified with ethyl acetimidate. Using [14C]ethyl acetimidate, tryptic digestion, fractionation of peptides by high-performance liquid chromatography, and amino acid analysis, all reactive lysine residues could be unambiguously identified. 19 of the 23 lysine residues of EF-Tu were labelled under conditions for ternary complex stability. Of these only 8 showed differences in reactivity between free and complexed EF-Tu . GTP. In the ternary complex lysine residues 208 and 390 [Jones, M. D., Petersen, T. E., Nielsen, K. M., Magnusson, S., Sotterup-Jensen, L., Gausing, K. and Clark, B. F. C. (1980) Eur. J. Biochem. 108, 507-526] showed an increase in reactivity (60% and 30% respectively) and residues 2, 4, 237, 248, 263, and 282 showed a decrease in reactivity (between 85% and 37%) compared to the values observed with EF-Tu . GTP. The greatest changes in reactivity were observed for lysine residues 2, 4 and 263. These data can be combined with the available structural information to identify possible areas of contact between the protein and nucleic acid moieties in the ternary complex. PMID:6430701

  5. Noncanonical Myo9b-RhoGAP Accelerates RhoA GTP Hydrolysis by a Dual-Arginine-Finger Mechanism.

    PubMed

    Yi, Fengshuang; Kong, Ruirui; Ren, Jinqi; Zhu, Li; Lou, Jizhong; Wu, Jane Y; Feng, Wei

    2016-07-31

    The GTP hydrolysis activities of Rho GTPases are stimulated by GTPase-activating proteins (GAPs), which contain a RhoGAP domain equipped with a characteristic arginine finger and an auxiliary asparagine for catalysis. However, the auxiliary asparagine is missing in the RhoGAP domain of Myo9b (Myo9b-RhoGAP), a unique motorized RhoGAP that specifically targets RhoA for controlling cell motility. Here, we determined the structure of Myo9b-RhoGAP in complex with GDP-bound RhoA and magnesium fluoride. Unexpectedly, Myo9b-RhoGAP contains two arginine fingers at its catalytic site. The first arginine finger resembles the one within the canonical RhoGAP domains and inserts into the nucleotide-binding pocket of RhoA, whereas the second arginine finger anchors the Switch I loop of RhoA and interacts with the nucleotide, stabilizing the transition state of GTP hydrolysis and compensating for the lack of the asparagine. Mutating either of the two arginine fingers impaired the catalytic activity of Myo9b-RhoGAP and affected the Myo9b-mediated cell migration. Our data indicate that Myo9b-RhoGAP accelerates RhoA GTP hydrolysis by a previously unknown dual-arginine-finger mechanism, which may be shared by other noncanonical RhoGAP domains lacking the auxiliary asparagine. PMID:27363609

  6. Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

    SciTech Connect

    Kikuchi, M.; Ishii, S. )

    1989-12-01

    By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

  7. Structures and reaction mechanisms of GTP cyclohydrolases.

    PubMed

    Gräwert, Tobias; Fischer, Markus; Bacher, Adelbert

    2013-04-01

    GTP cyclohydrolases generate the first committed intermediates for the biosynthesis of certain vitamins/cofactors (folic acid, riboflavin, deazaflavin, and tetrahydrobiopterin), deazapurine antibiotics, some t-RNA bases (queuosine, archaeosine), and the phytotoxin, toxoflavin. They depend on divalent cations for hydrolytic opening of the imidazole ring of the substrate, guanosine triphosphate (GTP). Surprisingly, the ring opening reaction is not the rate-limiting step for GTP cyclohydrolases I and II whose mechanism have been studied in some detail. GTP cyclohydrolase I, Ib, and II are potential targets for novel anti-infectives. Genetic factors modulating the activity of human GTP cyclohydrolase are highly pleiotropic, since the signal transponders whose biosyntheses require their participation (nitric oxide, catecholamines) impact a very wide range of physiological phenomena. Recent studies suggest that human GTP cyclohydrolase may become an oncology target. PMID:23457054

  8. Light- and GTP-activated hydrolysis of phosphatidylinositol bisphosphate in squid photoreceptor membranes

    SciTech Connect

    Baer, K.M.; Saibil, H.R.

    1988-01-05

    Light stimulates the hydrolysis of exogenous, (/sup 3/H)inositol-labeled phosphatidylinositol bisphosphate (PtdInsP2) added to squid photoreceptor membranes, releasing inositol trisphosphate (InsP3). At free calcium levels of 0.05 microM or greater, hydrolysis of the labeled lipid is stimulated up to 4-fold by GTP and light together, but not separately. This activity is the biochemical counterpart of observations on intact retina showing that a rhodopsin-activated GTP-binding protein is involved in visual transduction in invertebrates, and that InsP3 release is correlated with visual excitation and adaptation. Using an in vitro assay, we investigated the calcium and GTP dependence of the phospholipase activity. At calcium concentrations between 0.1 and 0.5 microM, some hydrolysis occurs independently of GTP and light, with a light- and GTP-activated component superimposed. At 1 microM calcium there is no background activity, and hydrolysis absolutely requires both GTP and light. Ion exchange chromatography on Dowex 1 (formate form) of the water-soluble products released at 1 microM calcium reveals that the product is almost entirely InsP3. Invertebrate rhodopsin is homologous in sequence and function to vertebrate visual pigment, which modulates the concentration of cyclic GMP through the mediation of the GTP-binding protein transducin. While there is some evidence that light also modulates PtdInsP2 content in vertebrate photoreceptors, the case for its involvement in phototransduction is stronger for the invertebrate systems. The results reported here support the scheme of rhodopsin----GTP-binding protein----phospholipase C activation in invertebrate photoreceptors.

  9. RanGTP aids anaphase entry through Ubr5-mediated protein turnover

    PubMed Central

    Jiang, Hao; He, Xiaonan; Feng, Di

    2015-01-01

    RanGTP is known to regulate the spindle assembly checkpoint (SAC), but the underlying molecular mechanism is unclear. BuGZ stabilizes SAC protein Bub3 through direct interaction and facilitates its mitotic function. Here we show that RanGTP promotes the turnover of BuGZ and Bub3 in metaphase, which in turn facilitates metaphase-to-anaphase transition. BuGZ and Bub3 interact with either importin-β or an E3 ubiquitin ligase, Ubr5. RanGTP promotes the dissociation of importin-β from BuGZ and Bub3 in metaphase. This results in increased binding of BuGZ and Bub3 to Ubr5, leading to ubiquitination and subsequent turnover of both proteins. We propose that elevated metaphase RanGTP levels use Ubr5 to couple overall chromosome congression to SAC silencing. PMID:26438829

  10. Steady-state theory of the interference of GTP hydrolysis in the mechanism of microtubule assembly.

    PubMed Central

    Hill, T L; Carlier, M F

    1983-01-01

    A model is presented for the interference of GTP hydrolysis in the mechanism of microtubule assembly. This model is suggested by previous results showing that both GTP and GDP are present at microtubule ends because of GTP hydrolysis and that tubulin does not bind to a GDP-bound end. The analytical theory developed here is aimed at calculation of the steady-state subunit flux at one end of the polymer. The GTP/GDP features just mentioned result in a nonlinear plot of the flux versus tubulin concentration. Microtubules are predicted to exhibit a different kinetic behavior below and above the critical concentration, which can be considered as a transition between two regimes. PMID:6580643

  11. Carboxymethyl modification of konjac glucomannan affects water binding properties.

    PubMed

    Xiao, Man; Dai, Shuhong; Wang, Le; Ni, Xuewen; Yan, Wenli; Fang, Yapeng; Corke, Harold; Jiang, Fatang

    2015-10-01

    The water binding properties of konjac glucomannan (KGM) and carboxymethyl konjac glucomannan (CMKGM) are important for their application in food, pharmaceutical, and chemical engineering fields. The equilibrium moisture content of CMKGM was lower than that of KGM at the relative humidity in the range 30-95% at 25°C. The water absorption and solubility of CMKGM in water solution were lower than that of KGM at 25°C. Carboxymethyl modification of KGM reduces the water adsorption, absorption, and solubility. Both carboxymethylation and deacetylation could confer hydrophobicity for CMKGM. These data provide the basis for expanding CMKGM application. PMID:26076594

  12. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  13. Invited review: Mechanisms of GTP hydrolysis and conformational transitions in the dynamin superfamily.

    PubMed

    Daumke, Oliver; Praefcke, Gerrit J K

    2016-08-01

    Dynamin superfamily proteins are multidomain mechano-chemical GTPases which are implicated in nucleotide-dependent membrane remodeling events. A prominent feature of these proteins is their assembly- stimulated mechanism of GTP hydrolysis. The molecular basis for this reaction has been initially clarified for the dynamin-related guanylate binding protein 1 (GBP1) and involves the transient dimerization of the GTPase domains in a parallel head-to-head fashion. A catalytic arginine finger from the phosphate binding (P-) loop is repositioned toward the nucleotide of the same molecule to stabilize the transition state of GTP hydrolysis. Dynamin uses a related dimerization-dependent mechanism, but instead of the catalytic arginine, a monovalent cation is involved in catalysis. Still another variation of the GTP hydrolysis mechanism has been revealed for the dynamin-like Irga6 which bears a glycine at the corresponding position in the P-loop. Here, we highlight conserved and divergent features of GTP hydrolysis in dynamin superfamily proteins and show how nucleotide binding and hydrolysis are converted into mechano-chemical movements. We also describe models how the energy of GTP hydrolysis can be harnessed for diverse membrane remodeling events, such as membrane fission or fusion. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 580-593, 2016. PMID:27062152

  14. Spastin Binds to Lipid Droplets and Affects Lipid Metabolism

    PubMed Central

    Papadopoulos, Chrisovalantis; Orso, Genny; Mancuso, Giuseppe; Herholz, Marija; Gumeni, Sentiljana; Tadepalle, Nimesha; Jüngst, Christian; Tzschichholz, Anne; Schauss, Astrid; Höning, Stefan; Trifunovic, Aleksandra; Daga, Andrea; Rugarli, Elena I.

    2015-01-01

    Mutations in SPAST, encoding spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). HSP is characterized by weakness and spasticity of the lower limbs, owing to progressive retrograde degeneration of the long corticospinal axons. Spastin is a conserved microtubule (MT)-severing protein, involved in processes requiring rearrangement of the cytoskeleton in concert to membrane remodeling, such as neurite branching, axonal growth, midbody abscission, and endosome tubulation. Two isoforms of spastin are synthesized from alternative initiation codons (M1 and M87). We now show that spastin-M1 can sort from the endoplasmic reticulum (ER) to pre- and mature lipid droplets (LDs). A hydrophobic motif comprised of amino acids 57 through 86 of spastin was sufficient to direct a reporter protein to LDs, while mutation of arginine 65 to glycine abolished LD targeting. Increased levels of spastin-M1 expression reduced the number but increased the size of LDs. Expression of a mutant unable to bind and sever MTs caused clustering of LDs. Consistent with these findings, ubiquitous overexpression of Dspastin in Drosophila led to bigger and less numerous LDs in the fat bodies and increased triacylglycerol levels. In contrast, Dspastin overexpression increased LD number when expressed specifically in skeletal muscles or nerves. Downregulation of Dspastin and expression of a dominant-negative variant decreased LD number in Drosophila nerves, skeletal muscle and fat bodies, and reduced triacylglycerol levels in the larvae. Moreover, we found reduced amount of fat stores in intestinal cells of worms in which the spas-1 homologue was either depleted by RNA interference or deleted. Taken together, our data uncovers an evolutionarily conserved role of spastin as a positive regulator of LD metabolism and open up the possibility that dysfunction of LDs in axons may contribute to the pathogenesis of HSP. PMID:25875445

  15. Arabidopsis AtADF1 is functionally affected by mutations on actin binding sites.

    PubMed

    Dong, Chun-Hai; Tang, Wei-Ping; Liu, Jia-Yao

    2013-03-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A, R137/A) form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization. PMID:23190411

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

    SciTech Connect

    Simonetti, Angelita; Fabbretti, Attilio; Hazemann, Isabelle; Jenner, Lasse; Gualerzi, Claudio O.; Klaholz, Bruno P.

    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 IF2 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.

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

    PubMed

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

    2015-12-25

    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

  18. Nuclear relaxation rates study of GTP(gamma F)-tubulin interaction using 19F-nuclear magnetic resonance.

    PubMed

    Monasterio, O

    1989-07-01

    To study the relationship between the exchangeable GTP binding site (E-site) and the high affinity metal binding site we synthesized P3-fluoro P1-5'-guanosine tripaosphate (GTP(gamma F), an analog of GTP. Our results show that this analog binds to the exchangeable GTP binding site of calf brain tubulin. The values of the dissociation constant and the stoichiometry of the GTP(gamma F)-Mn(II) complex as determined by EPR spectroscopy were 1.64 x 10(-4) M and one mole of manganese per mole of nucleotide, respectively. The distance separating the high-affinity binding site for the divalent metal ion and the exchangeable nucleotide binding site was evaluated by using high-resolution 19F-NMR. The 31P- and 19F-NMR spectra of GTP(gamma F) were studied, both the fluorine and the gamma-phosphate were split in a doublet with a coupling constant of 936 Hz. Tubulin purified by the method of Weisenberg (Weisenberg, R.C., and Timashef, S.N. (1970) Biochemistry 9, 4110-4116) was treated with colchicine to stabilize it, GTP(gamma F) was added and the 254.1 MHz 19fluorine relaxation rates measured within the first four hours. Longitudinal and transversal relaxation rates were determined in the presence of colchicine-tubulin-Mn(II), (paramagnetic complex), or the ternary complex with magnesium (diamagnetic complex). The analysis of the temperature-dependent relaxation data indicates that the metal and the exchangeable nucleotide binding sites are separated by a maximal distance of 6 at 35 degrees C, to 8.1 A at 12 degrees C. PMID:2619317

  19. Membrane curvature induced by Arf1-GTP is essential for vesicle formation

    PubMed Central

    Beck, Rainer; Sun, Zhe; Adolf, Frank; Rutz, Chistoph; Bassler, Jochen; Wild, Klemens; Sinning, Irmgard; Hurt, Ed; Brügger, Britta; Béthune, Julien; Wieland, Felix

    2008-01-01

    The GTPase Arf1 is considered as a molecular switch that regulates binding and release of coat proteins that polymerize on membranes to form transport vesicles. Here, we show that Arf1-GTP induces positive membrane curvature and find that the small GTPase can dimerize dependent on GTP. Investigating a possible link between Arf dimerization and curvature formation, we isolated an Arf1 mutant that cannot dimerize. Although it was capable of exerting the classical role of Arf1 as a coat receptor, it could not mediate the formation of COPI vesicles from Golgi-membranes and was lethal when expressed in yeast. Strikingly, this mutant was not able to deform membranes, suggesting that GTP-induced dimerization of Arf1 is a critical step inducing membrane curvature during the formation of coated vesicles. PMID:18689681

  20. Structure of a GTP-dependent Bacterial PEP-carboxykinase from Corynebacterium glutamicum

    SciTech Connect

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

    2008-06-23

    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 guanine 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.

  1. Activation of adenylate cyclase by dopamine, GTP, NaF and forskolin in striatal membranes of neonatal, adult and senescent rats.

    PubMed

    Nomura, Y; Makihata, J; Segawa, T

    1984-11-13

    Dopamine (DA) caused a significant activation of striatal adenylate cyclase in neonatal and adult but not in senescent rats. GTP activated cyclase at the adult stage but not at both neonatal and senescent stages. NaF and forskolin activated cyclase at every stage. The coupling mechanism between DA1 receptors and catalytic units of cyclase seems to become functional at the neonatal stage but GTP recognition and/or binding sites lack in stimulatory GTP binding protein in neonatal and senescent membranes. PMID:6543337

  2. GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits

    PubMed Central

    Holtkamp, Wolf; Cunha, Carlos E; Peske, Frank; Konevega, Andrey L; Wintermeyer, Wolfgang; Rodnina, Marina V

    2014-01-01

    Elongation factor G (EF-G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF-G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF-G mutants and translocation-specific antibiotics to investigate timing and energetics of translocation. We show that EF-G–GTP facilitates synchronous movements of peptidyl-tRNA on the two subunits into an early post-translocation state, which resembles a chimeric state identified by structural studies. EF-G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF-G. Our results reveal two distinct modes for utilizing the energy of EF-G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit. PMID:24614227

  3. GTP hydrolysis by EF-G synchronizes tRNA movement on small and large ribosomal subunits.

    PubMed

    Holtkamp, Wolf; Cunha, Carlos E; Peske, Frank; Konevega, Andrey L; Wintermeyer, Wolfgang; Rodnina, Marina V

    2014-05-01

    Elongation factor G (EF-G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF-G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF-G mutants and translocation-specific antibiotics to investigate timing and energetics of translocation. We show that EF-G-GTP facilitates synchronous movements of peptidyl-tRNA on the two subunits into an early post-translocation state, which resembles a chimeric state identified by structural studies. EF-G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF-G. Our results reveal two distinct modes for utilizing the energy of EF-G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit. PMID:24614227

  4. Seasonal difference in brain serotonin transporter binding predicts symptom severity in patients with seasonal affective disorder.

    PubMed

    Mc Mahon, Brenda; Andersen, Sofie B; Madsen, Martin K; Hjordt, Liv V; Hageman, Ida; Dam, Henrik; Svarer, Claus; da Cunha-Bang, Sofi; Baaré, William; Madsen, Jacob; Hasholt, Lis; Holst, Klaus; Frokjaer, Vibe G; Knudsen, Gitte M

    2016-05-01

    Cross-sectional neuroimaging studies in non-depressed individuals have demonstrated an inverse relationship between daylight minutes and cerebral serotonin transporter; this relationship is modified by serotonin-transporter-linked polymorphic region short allele carrier status. We here present data from the first longitudinal investigation of seasonal serotonin transporter fluctuations in both patients with seasonal affective disorder and in healthy individuals. Eighty (11)C-DASB positron emission tomography scans were conducted to quantify cerebral serotonin transporter binding; 23 healthy controls with low seasonality scores and 17 patients diagnosed with seasonal affective disorder were scanned in both summer and winter to investigate differences in cerebral serotonin transporter binding across groups and across seasons. The two groups had similar cerebral serotonin transporter binding in the summer but in their symptomatic phase during winter, patients with seasonal affective disorder had higher serotonin transporter than the healthy control subjects (P = 0.01). Compared to the healthy controls, patients with seasonal affective disorder changed their serotonin transporter significantly less between summer and winter (P < 0.001). Further, the change in serotonin transporter was sex- (P = 0.02) and genotype- (P = 0.04) dependent. In the patients with seasonal affective disorder, the seasonal change in serotonin transporter binding was positively associated with change in depressive symptom severity, as indexed by Hamilton Rating Scale for Depression - Seasonal Affective Disorder version scores (P = 0.01). Our findings suggest that the development of depressive symptoms in winter is associated with a failure to downregulate serotonin transporter levels appropriately during exposure to the environmental stress of winter, especially in individuals with high predisposition to affective disorders.media-1vid110.1093/brain/aww043_video_abstractaww043_video

  5. 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

  6. Gene size differentially affects the binding of yeast transcription factor tau to two intragenic regions.

    PubMed Central

    Baker, R E; Camier, S; Sentenac, A; Hall, B D

    1987-01-01

    Yeast transcription factor tau (transcription factor IIIC) specifically interacts with tRNA genes, binding to both the A block and the B block elements of the internal promoter. To study the influence of A block-B block spacing, we analyzed the binding of purified tau protein to a series of internally deleted yeast tRNA(3Leu) genes with A and B blocks separated by 0 to 74 base pairs. Optimal binding occurred with genes having A block-B block distances of 30-60 base pairs; the relative helical orientation of the A and B blocks was unimportant. Results from DNase I "footprinting" and lambda exonuclease protection experiments were consistent with these findings and further revealed that changes in A block-B block distance primarily affect the ability of tau to interact with A block sequences; B block interactions are unaltered. When the A block-B block distance is 17 base pairs or less, tau interacts with a sequence located 15 base pairs upstream of the normal A block, and a new RNA initiation site is observed by in vitro transcription. We propose that the initial binding of tau to the B block activates transcription by enhancing its ability to bind at the A block, and that the A block interaction ultimately directs initiation by RNA polymerase III. Images PMID:2827154

  7. Characterization of an Fe(2+)-dependent archaeal-specific GTP cyclohydrolase, MptA, from Methanocaldococcus jannaschii.

    PubMed

    Grochowski, Laura L; Xu, Huimin; Leung, Kapo; White, Robert H

    2007-06-01

    The first step in the biosynthesis of pterins in bacteria and plants is the conversion of GTP to 7,8-dihydro-d-neopterin triphosphate catalyzed by GTP cyclohydrolase I (GTPCHI). Although GTP has been shown to be a precursor of pterins in archaea, homologues of GTPCHI have not been identified in most archaeal genomes. Here we report the identification of a new GTP cyclohydrolase that converts GTP to 7,8-dihydro-d-neopterin 2',3'-cyclic phosphate, the first intermediate in methanopterin biosynthesis in methanogenic archaea. The enzyme from Methanocaldococcus jannaschii is designated MptA to indicate that it catalyzes the first step in the biosynthesis of methanopterin. MptA is the archetype of a new class of GTP cyclohydrolases that catalyzes a series of reactions most similar to that seen with GTPCHI but unique in that the cyclic phosphate is the product. MptA was found to require Fe2+ for activity. Mutation of conserved histidine residues H200N, H293N, and H295N, expected to be involved in Fe2+ binding, resulted in reduced enzymatic activity but no reduction in the amount of bound iron. PMID:17497938

  8. The Cytoplasmic Domain of Anthrax Toxin Receptor 1 Affects Binding of the Protective Antigen▿

    PubMed Central

    Go, Mandy Y.; Chow, Edith M. C.; Mogridge, Jeremy

    2009-01-01

    The protective antigen (PA) component of anthrax toxin binds the I domain of the receptor ANTXR1. Integrin I domains convert between open and closed conformations that bind ligand with high and low affinities, respectively; this process is regulated by signaling from the cytoplasmic domains. To assess whether intracellular signals might influence the interaction between ANTXR1 and PA, we compared two splice variants of ANTXR1 that differ only in their cytoplasmic domains. We found that cells expressing ANTXR1 splice variant 1 (ANTXR1-sv1) bound markedly less PA than did cells expressing a similar level of the shorter splice variant ANTXR1-sv2. ANTXR1-sv1 but not ANTXR1-sv2 associated with the actin cytoskeleton, although disruption of the cytoskeleton did not affect binding of ANTXR-sv1 to PA. Introduction of a cytoplasmic domain missense mutation found in the related receptor ANTXR2 in a patient with juvenile hyaline fibromatosis impaired actin association and increased binding of PA to ANTXR1-sv1. These results suggest that ANTXR1 has two affinity states that may be modulated by cytoplasmic signals. PMID:18936178

  9. Binding of ATP by pertussis toxin and isolated toxin subunits

    SciTech Connect

    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; 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.

  10. Over-expression of GTP-cyclohydrolase 1 feedback regulatory protein attenuates LPS and cytokine-stimulated nitric oxide production.

    PubMed

    Nandi, Manasi; Kelly, Peter; Vallance, Patrick; Leiper, James

    2008-02-01

    GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate-limiting step for the de novo production of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS). The GTP-CH1-BH(4) pathway is emerging as an important regulator in a number of pathologies associated with over-production of nitric oxide (NO) and hence a more detailed understanding of this pathway may lead to novel therapeutic targets for the treatment of certain vascular diseases. GTP-CH1 activity can be inhibited by BH(4) through its protein-protein interactions with GTP-CH1 regulatory protein (GFRP), and transcriptional and post-translational modification of both GTP-CH1 and GFRP have been reported in response to proinflammatory stimuli. However, the functional significance of GFRP/GTP-CH1 interactions on NO pathways has not yet been demonstrated. We aimed to investigate whether over-expression of GFRP could affect NO production in living cells. Over-expression of N-terminally Myc-tagged recombinant human GFRP in the murine endothelial cell line sEnd 1 resulted in no significant effect on basal BH(4) nor NO levels but significantly attenuated the rise in BH(4) and NO observed following lipopolysaccharide and cytokine stimulation of cells. This study demonstrates that GFRP can play a direct regulatory role in iNOS-mediated NO synthesis and suggests that the allosteric regulation of GTP-CH1 activity by GFRP may be an important mechanism regulating BH(4) and NO levels in vivo. PMID:18372436

  11. Identification of cDNA encoding an additional. alpha. subunit of a human GTP-binding protein: Expression of three. alpha. sub i subtypes in human tissues and cell lines

    SciTech Connect

    Kim, S.; Ang, S.L.; Bloch, D.B.; Bloch, K.D.; Kawahara, Y.; Tolman, C.; Lee, R.; Seidman, J.G.; Neer, E.J. )

    1988-06-01

    The guanine nucleotide-binding proteins (G proteins), which mediate hormonal regulation of many membrane functions, are composed of {alpha}, {beta}, and {gamma} subunits. The authors have cloned and characterized cDNA from a human T-cell library encoding a form of {alpha}{sub i} that is different from the human {alpha}{sub i} subtypes previously reported. {alpha}{sub i} is the {alpha} subunit of a class of G proteins that inhibits adenylate cyclase and regulates other enzymes and ion channels. This cDNA encodes a polypeptide of 354 amino acids and is assigned to encode the {alpha}{sub i-3} subtype of G proteins on the basis of its similarity to other {alpha}{sub i}-like cDNAs and the presence of a predicted site for ADP ribosylation by pertussis toxin. They have determined the expression of mRNA for this and two other subtypes of human {alpha}{sub i} ({alpha}{sub i-1} and {alpha}{sub i-2}) in a variety of human fetal tissues and in human cell lines. All three {alpha}{sub i} subtypes were present in the tissues tested. However, analysis of individual cell types reveals specificity of {alpha}{sub i-1} expression. mRNA for {alpha}{i-1} is absent in T cells, B cells, and monocytes but is present in other cell lines. The finding of differential expression of {alpha}{sub i-1} genes may permit characterization of distinct physiological roles for this {alpha}{sub i} subunit. mRNA for {alpha}{sub i-2} and {alpha}{sub i-3} was found in all the primary and transformed cell lines tested. Thus, some cells contain all three {alpha}{sub i} subtypes. This observation raises the question of how cells prevent cross talk among receptors that are coupled to effectors through such similar {alpha} proteins.

  12. GABA(B) receptor subunit 1 binds to proteins affected in 22q11 deletion syndrome.

    PubMed

    Zunner, Dagmar; Deschermeier, Christina; Kornau, Hans-Christian

    2010-03-01

    GABA(B) receptors mediate slow inhibitory effects of the neurotransmitter gamma-aminobutyric acid (GABA) on synaptic transmission in the central nervous system. They function as heterodimeric G-protein-coupled receptors composed of the seven-transmembrane domain proteins GABA(B1) and GABA(B2), which are linked through a coiled-coil interaction. The ligand-binding subunit GABA(B1) is at first retained in the endoplasmic reticulum and is transported to the cell surface only upon assembly with GABA(B2). Here, we report that GABA(B1), via the coiled-coil domain, can also bind to soluble proteins of unknown function, that are affected in 22q11 deletion/DiGeorge syndrome and are therefore referred to as DiGeorge critical region 6 (DGCR6). In transfected neurons the GABA(B1)-DGCR6 association resulted in a redistribution of both proteins into intracellular clusters. Furthermore, the C-terminus of GABA(B2) interfered with the novel interaction, consistent with heterodimer formation overriding transient DGCR6-binding to GABA(B1). Thus, sequential coiled-coil interactions may direct GABA(B1) into functional receptors. PMID:20036641

  13. Hand proximity differentially affects visual working memory for color and orientation in a binding task.

    PubMed

    Kelly, Shane P; Brockmole, James R

    2014-01-01

    Observers determined whether two sequentially presented arrays of six lines were the same or different. Differences, when present, involved either a swap in the color of two lines or a swap in the orientation of two lines. Thus, accurate change detection required the binding of color and orientation information for each line within visual working memory. Holding viewing distance constant, the proximity of the arrays to the hands was manipulated. Placing the hands near the to-be-remembered array decreased participants' ability to remember color information, but increased their ability to remember orientation information. This pair of results indicates that hand proximity differentially affects the processing of various types of visual information, a conclusion broadly consistent with functional and anatomical differences in the magnocellular and parvocellular pathways. It further indicates that hand proximity affects the likelihood that various object features will be encoded into integrated object files. PMID:24795671

  14. Haemoglobin polymorphisms affect the oxygen-binding properties in Atlantic cod populations

    PubMed Central

    Andersen, Øivind; Wetten, Ola Frang; De Rosa, Maria Cristina; Andre, Carl; Carelli Alinovi, Cristiana; Colafranceschi, Mauro; Brix, Ole; Colosimo, Alfredo

    2008-01-01

    A major challenge in evolutionary biology is to identify the genes underlying adaptation. The oxygen-transporting haemoglobins directly link external conditions with metabolic needs and therefore represent a unique system for studying environmental effects on molecular evolution. We have discovered two haemoglobin polymorphisms in Atlantic cod populations inhabiting varying temperature and oxygen regimes in the North Atlantic. Three-dimensional modelling of the tetrameric haemoglobin structure demonstrated that the two amino acid replacements Met55β1Val and Lys62β1Ala are located at crucial positions of the α1β1 subunit interface and haem pocket, respectively. The replacements are proposed to affect the oxygen-binding properties by modifying the haemoglobin quaternary structure and electrostatic feature. Intriguingly, the same molecular mechanism for facilitating oxygen binding is found in avian species adapted to high altitudes, illustrating convergent evolution in water- and air-breathing vertebrates to reduction in environmental oxygen availability. Cod populations inhabiting the cold Arctic waters and the low-oxygen Baltic Sea seem well adapted to these conditions by possessing the high oxygen affinity Val55–Ala62 haplotype, while the temperature-insensitive Met55–Lys62 haplotype predominates in the southern populations. The distinct distributions of the functionally different haemoglobin variants indicate that the present biogeography of this ecologically and economically important species might be seriously affected by global warming. PMID:19033139

  15. GTP cyclohydrolase I inhibition by the prototypic inhibitor 2, 4-diamino-6-hydroxypyrimidine. Mechanisms and unanticipated role of GTP cyclohydrolase I feedback regulatory protein.

    PubMed

    Xie, L; Smith, J A; Gross, S S

    1998-08-14

    2,4-Diamino-6-hydroxypyrimidine (DAHP) is considered to be a selective and direct-acting inhibitor of GTP cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in the pathway for synthesis of tetrahydrobiopterin (BH4). Accordingly, DAHP has been widely employed to distinguish whether de novo BH4 synthesis is required in a given biological system. Although it has been assumed that DAHP inhibits GTPCH by direct competition with substrate GTP, this has never been formally demonstrated. In view of apparent structural homology between DAHP and BH4, we questioned whether DAHP may mimic BH4 in its inhibition of GTPCH by an indirect mechanism, involving interaction with a recently cloned 9.5-kDa protein termed GTPCH Feedback Regulatory Protein (GFRP). We show by reverse transcription-polymerase chain reaction that GFRP mRNA is constitutively expressed in rat aortic smooth muscle cells and further induced by treatment with immunostimulants. Moreover, functional GFRP is expressed and immunostimulant-induced BH4 accumulates in sufficient quantity to trigger feedback inhibition of GTPCH. Studies with DAHP reveal that GFRP is also essential to achieve potent inhibition of GTPCH. Indeed, DAHP inhibits GTPCH by dual mechanisms. At a relatively low concentration, DAHP emulates BH4 and engages the GFRP-dependent feedback inhibitory system; at higher concentrations, DAHP competes directly for binding with GTP substrate. This knowledge predicts that DAHP would preferably target GTPCH in tissues with abundant GFRP. PMID:9694862

  16. 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

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

    DOE PAGESBeta

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

    2015-06-16

    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 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.« less

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

    SciTech Connect

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

    2015-06-16

    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 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.

  19. Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Chang, D.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

  20. Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1.

    PubMed

    Hauk, Glenn; Bowman, Gregory D

    2015-01-01

    The DEAD-box RNA helicase Ded1, which is essential in yeast and known as DDX3 in humans, shuttles between the nucleus and cytoplasm and takes part in several basic processes including RNA processing and translation. A key interacting partner of Ded1 is the exportin Xpo1, which together with the GTP-bound state of the small GTPase Ran, facilitates unidirectional transport of Ded1 out of the nucleus. Here we demonstrate that Xpo1 and Ran[GTP] together reduce the RNA-stimulated ATPase and helicase activities of Ded1. Binding and inhibition of Ded1 by Xpo1 depend on the affinity of the Ded1 nuclear export sequence (NES) for Xpo1 and the presence of Ran[GTP]. Association with Xpo1/Ran[GTP] reduces RNA-stimulated ATPase activity of Ded1 by increasing the apparent KM for the RNA substrate. Despite the increased KM, the Ded1:Xpo1:Ran[GTP] ternary complex retains the ability to bind single stranded RNA, suggesting that Xpo1/Ran[GTP] may modulate the substrate specificity of Ded1. These results demonstrate that, in addition to transport, exportins such as Xpo1 also have the capability to alter enzymatic activities of their cargo. PMID:26120835

  1. High-Density Lipoprotein Binds to Mycobacterium avium and Affects the Infection of THP-1 Macrophages.

    PubMed

    Ichimura, Naoya; Sato, Megumi; Yoshimoto, Akira; Yano, Kouji; Ohkawa, Ryunosuke; Kasama, Takeshi; Tozuka, Minoru

    2016-01-01

    High-density lipoprotein (HDL) is involved in innate immunity toward various infectious diseases. Concerning bacteria, HDL is known to bind to lipopolysaccharide (LPS) and to neutralize its physiological activity. On the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. However, the pathogenicity of Mycobacterium avium (M. avium) infection, which tends to increase worldwide, remains poorly studied. Here we report that HDL indicated a stronger interaction with M. avium than that with other Gram-negative bacteria containing abundant LPS. A binding of apolipoprotein (apo) A-I, the main protein component of HDL, with a specific lipid of M. avium might participate in this interaction. HDL did not have a direct bactericidal activity toward M. avium but attenuated the engulfment of M. avium by THP-1 macrophages. HDL also did not affect bacterial killing after ingestion of live M. avium by THP-1 macrophage. Furthermore, HDL strongly promoted the formation of lipid droplets in M. avium-infected THP-1 macrophages. These observations provide new insights into the relationship between M. avium infection and host lipoproteins, especially HDL. Thus, HDL may help M. avium to escape from host innate immunity. PMID:27516907

  2. High-Density Lipoprotein Binds to Mycobacterium avium and Affects the Infection of THP-1 Macrophages

    PubMed Central

    Ichimura, Naoya; Sato, Megumi; Yoshimoto, Akira; Yano, Kouji; Ohkawa, Ryunosuke; Kasama, Takeshi

    2016-01-01

    High-density lipoprotein (HDL) is involved in innate immunity toward various infectious diseases. Concerning bacteria, HDL is known to bind to lipopolysaccharide (LPS) and to neutralize its physiological activity. On the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. However, the pathogenicity of Mycobacterium avium (M. avium) infection, which tends to increase worldwide, remains poorly studied. Here we report that HDL indicated a stronger interaction with M. avium than that with other Gram-negative bacteria containing abundant LPS. A binding of apolipoprotein (apo) A-I, the main protein component of HDL, with a specific lipid of M. avium might participate in this interaction. HDL did not have a direct bactericidal activity toward M. avium but attenuated the engulfment of M. avium by THP-1 macrophages. HDL also did not affect bacterial killing after ingestion of live M. avium by THP-1 macrophage. Furthermore, HDL strongly promoted the formation of lipid droplets in M. avium-infected THP-1 macrophages. These observations provide new insights into the relationship between M. avium infection and host lipoproteins, especially HDL. Thus, HDL may help M. avium to escape from host innate immunity. PMID:27516907

  3. Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding*

    PubMed Central

    Houde, Damian; Peng, Yucai; Berkowitz, Steven A.; Engen, John R.

    2010-01-01

    Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin γ1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcγRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcγRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications. PMID:20103567

  4. Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP.

    PubMed

    Maita, Nobuo; Okada, Kengo; Hatakeyama, Kazuyuki; Hakoshima, Toshio

    2002-02-01

    In the presence of phenylalanine, GTP cyclohydrolase I feedback regulatory protein (GFRP) forms a stimulatory 360-kDa complex with GTP cyclohydrolase I (GTPCHI), which is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. The crystal structure of the stimulatory complex reveals that the GTPCHI decamer is sandwiched by two GFRP homopentamers. Each GFRP pentamer forms a symmetrical five-membered ring similar to beta-propeller. Five phenylalanine molecules are buried inside each interface between GFRP and GTPCHI, thus enhancing the binding of these proteins. The complex structure suggests that phenylalanine-induced GTPCHI x GFRP complex formation enhances GTPCHI activity by locking the enzyme in the active state. PMID:11818540

  5. Arf1-GTP-induced Tubule Formation Suggests a Function of Arf Family Proteins in Curvature Acquisition at Sites of Vesicle Budding*

    PubMed Central

    Krauss, Michael; Jia, Jun-Yong; Roux, Aurélien; Beck, Rainer; Wieland, Felix T.; De Camilli, Pietro; Haucke, Volker

    2008-01-01

    ADP-ribosylation factor (Arf) and related small GTPases play crucial roles in membrane traffic within the exo- and endocytic pathways. Arf proteins in their GTP-bound state are associated with curved membrane buds and tubules, frequently together with effector coat proteins to which they bind. Here we report that Arf1 is found on membrane tubules originating from the Golgi complex where it colocalizes with COPI and GGA1 vesicle coat proteins. Arf1 also induces tubulation of liposomes in vitro. Mutations within the amino-terminal amphipathic helix (NTH) of Arf1 affect the number of Arf1-positive tubules in vivo and its property to tubulate liposomes. Moreover, hydrophilic substitutions within the hydrophobic part of its NTH impair Arf1-catalyzed budding of COPI vesicles in vitro. Our data indicate that GTP-controlled local induction of high curvature membranes is an important property of Arf1 that might be shared by a subgroup of Arf/Arl family GTPases. PMID:18693248

  6. The dynamin-binding domains of Dap160/intersectin affect bulk membrane retrieval in synapses

    PubMed Central

    Winther, Åsa M. E.; Jiao, Wei; Vorontsova, Olga; Rees, Kathryn A.; Koh, Tong-Wey; Sopova, Elena; Schulze, Karen L.; Bellen, Hugo J.; Shupliakov, Oleg

    2013-01-01

    Summary Dynamin-associated protein 160 kDa (Dap160)/intersectin interacts with several synaptic proteins and affects endocytosis and synapse development. The functional role of the different protein interaction domains is not well understood. Here we show that Drosophila Dap160 lacking the dynamin-binding SH3 domains does not affect the development of the neuromuscular junction but plays a key role in synaptic vesicle recycling. dap160 mutants lacking dynamin-interacting domains no longer accumulate dynamin properly at the periactive zone, and it becomes dispersed in the bouton during stimulation. This is accompanied by a reduction in uptake of the dye FM1-43 and an accumulation of large vesicles and membrane invaginations. However, we do not observe an increase in the number of clathrin-coated intermediates. We also note a depression in evoked excitatory junction potentials (EJPs) during high-rate stimulation, accompanied by aberrantly large miniature EJPs. The data reveal the important role of Dap160 in the targeting of dynamin to the periactive zone, where it is required to suppress bulk synaptic vesicle membrane retrieval during high-frequency activity. PMID:23321638

  7. Proteome-wide Discovery and Characterizations of Nucleotide-binding Proteins with Affinity-labeled Chemical Probes

    PubMed Central

    Xiao, Yongsheng; Guo, Lei; Jiang, Xinning; Wang, Yinsheng

    2013-01-01

    Nucleotide-binding proteins play pivotal roles in many cellular processes including cell signaling. However, targeted study of sub-proteome of nucleotide-binding proteins, especially protein kinases and GTP-binding proteins, remained challenging. Here, we reported a general strategy in using affinity-labeled chemical probes to enrich, identify, and quantify ATP- and GTP-binding proteins in the entire human proteome. Our results revealed that the ATP/GTP affinity probes facilitated the identification of 100 GTP-binding proteins and 206 kinases with the use of low mg quantities of lysate of HL-60 cells. In combination with the use of SILAC-based quantitative proteomics method, we assessed the ATP/GTP binding selectivities of nucleotide-binding proteins at the global proteome scale. Our results confirmed known and, more importantly, unveiled new ATP/GTP-binding preferences of hundreds of nucleotide-binding proteins. Additionally, our strategy led to the identification of three and one unique nucleotide-binding motifs for kinases and GTP-binding proteins, respectively, and the characterizations of the nucleotide binding selectivities of individual motifs. Our strategy for capturing and characterizing ATP/GTP-binding proteins should be generally applicable for those proteins that can interact with other nucleotides. PMID:23413923

  8. Ribosome-induced tuning of GTP hydrolysis by a translational GTPase.

    PubMed

    Maracci, Cristina; Peske, Frank; Dannies, Ev; Pohl, Corinna; Rodnina, Marina V

    2014-10-01

    GTP hydrolysis by elongation factor Tu (EF-Tu), a translational GTPase that delivers aminoacyl-tRNAs to the ribosome, plays a crucial role in decoding and translational fidelity. The basic reaction mechanism and the way the ribosome contributes to catalysis are a matter of debate. Here we use mutational analysis in combination with measurements of rate/pH profiles, kinetic solvent isotope effects, and ion dependence of GTP hydrolysis by EF-Tu off and on the ribosome to dissect the reaction mechanism. Our data suggest that--contrary to current models--the reaction in free EF-Tu follows a pathway that does not involve the critical residue H84 in the switch II region. Binding to the ribosome without a cognate codon in the A site has little effect on the GTPase mechanism. In contrast, upon cognate codon recognition, the ribosome induces a rearrangement of EF-Tu that renders GTP hydrolysis sensitive to mutations of Asp21 and His84 and insensitive to K(+) ions. We suggest that Asp21 and His84 provide a network of interactions that stabilize the positions of the γ-phosphate and the nucleophilic water, respectively, and thus play an indirect catalytic role in the GTPase mechanism on the ribosome. PMID:25246550

  9. Ribosome-induced tuning of GTP hydrolysis by a translational GTPase

    PubMed Central

    Maracci, Cristina; Peske, Frank; Dannies, Ev; Pohl, Corinna; Rodnina, Marina V.

    2014-01-01

    GTP hydrolysis by elongation factor Tu (EF-Tu), a translational GTPase that delivers aminoacyl-tRNAs to the ribosome, plays a crucial role in decoding and translational fidelity. The basic reaction mechanism and the way the ribosome contributes to catalysis are a matter of debate. Here we use mutational analysis in combination with measurements of rate/pH profiles, kinetic solvent isotope effects, and ion dependence of GTP hydrolysis by EF-Tu off and on the ribosome to dissect the reaction mechanism. Our data suggest that—contrary to current models—the reaction in free EF-Tu follows a pathway that does not involve the critical residue H84 in the switch II region. Binding to the ribosome without a cognate codon in the A site has little effect on the GTPase mechanism. In contrast, upon cognate codon recognition, the ribosome induces a rearrangement of EF-Tu that renders GTP hydrolysis sensitive to mutations of Asp21 and His84 and insensitive to K+ ions. We suggest that Asp21 and His84 provide a network of interactions that stabilize the positions of the γ-phosphate and the nucleophilic water, respectively, and thus play an indirect catalytic role in the GTPase mechanism on the ribosome. PMID:25246550

  10. Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells.

    PubMed

    Guo, Chuanliang; Xue, Yan; Yang, Guanheng; Yin, Shang; Shi, Wansheng; Cheng, Yan; Yan, Xiaoshuang; Fan, Shuyue; Zhang, Huijun; Zeng, Fanyi

    2016-08-01

    Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs. PMID:26289635

  11. Water molecules inside protein structure affect binding of monosaccharides with HIV-1 antibody 2G12.

    PubMed

    Ueno-Noto, Kaori; Takano, Keiko

    2016-10-01

    Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X-ray crystallographic data are insufficient for analyzing a series of ligand-protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand-antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand-antibody interaction. Our results indicate the fact that d-fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc. PMID:27388036

  12. The Chromatin-binding Protein HMGN1 Regulates the Expression of Methyl CpG-binding Protein 2 (MECP2) and Affects the Behavior of Mice*

    PubMed Central

    Abuhatzira, Liron; Shamir, Alon; Schones, Dustin E.; Schäffer, Alejandro A.; Bustin, Michael

    2011-01-01

    High mobility group N1 protein (HMGN1), a nucleosomal-binding protein that affects the structure and function of chromatin, is encoded by a gene located on chromosome 21 and is overexpressed in Down syndrome, one of the most prevalent genomic disorders. Misexpression of HMGN1 affects the cellular transcription profile; however, the biological function of this protein is still not fully understood. We report that HMGN1 modulates the expression of methyl CpG-binding protein 2 (MeCP2), a DNA-binding protein known to affect neurological functions including autism spectrum disorders, and whose alterations in HMGN1 levels affect the behavior of mice. Quantitative PCR and Western analyses of cell lines and brain tissues from mice that either overexpress or lack HMGN1 indicate that HMGN1 is a negative regulator of MeCP2 expression. Alterations in HMGN1 levels lead to changes in chromatin structure and histone modifications in the MeCP2 promoter. Behavior analyses by open field test, elevated plus maze, Reciprocal Social Interaction, and automated sociability test link changes in HMGN1 levels to abnormalities in activity and anxiety and to social deficits in mice. Targeted analysis of the Autism Genetic Resource Exchange genotype collection reveals a non-random distribution of genotypes within 500 kbp of HMGN1 in a region affecting its expression in families predisposed to autism spectrum disorders. Our results reveal that HMGN1 affects the behavior of mice and suggest that epigenetic changes resulting from altered HMGN1 levels could play a role in the etiology of neurodevelopmental disorders. PMID:22009741

  13. A single-nucleotide variation in a p53-binding site affects nutrient-sensitive human SIRT1 expression

    PubMed Central

    Naqvi, Asma; Hoffman, Timothy A.; DeRicco, Jeremy; Kumar, Ajay; Kim, Cuk-Seong; Jung, Saet-Byel; Yamamori, Tohru; Kim, Young-Rae; Mehdi, Fardeen; Kumar, Santosh; Rankinen, Tuomo; Ravussin, Eric; Irani, Kaikobad

    2010-01-01

    The SIRTUIN1 (SIRT1) deacetylase responds to changes in nutrient availability and regulates mammalian physiology and metabolism. Human and mouse SIRT1 are transcriptionally repressed by p53 via p53 response elements in their proximal promoters. Here, we identify a novel p53-binding sequence in the distal human SIRT1 promoter that is required for nutrient-sensitive SIRT1 transcription. In addition, we show that a common single-nucleotide (C/T) variation in this sequence affects nutrient deprivation-induced SIRT1 transcription, and calorie restriction-induced SIRT1 expression. The p53-binding sequence lies in a region of the SIRT1 promoter that also binds the transcriptional repressor Hypermethylated-In-Cancer-1 (HIC1). Nutrient deprivation increases occupancy by p53, while decreasing occupancy by HIC1, of this region of the promoter. HIC1 and p53 compete with each other for promoter occupancy. In comparison with the T variation, the C variation disrupts the mirror image symmetry of the p53-binding sequence, resulting in decreased binding to p53, decreased nutrient sensitivity of the promoter and impaired calorie restriction-stimulated tissue expression of SIRT1 and SIRT1 target genes AMPKα2 and PGC-1β. Thus, a common SNP in a novel p53-binding sequence in the human SIRT1 promoter affects nutrient-sensitive SIRT1 expression, and could have a significant impact on calorie restriction-induced, SIRT1-mediated, changes in human metabolism and physiology. PMID:20693263

  14. Hydrolysis of GTP associated with the formation of tubulin oligomers is involved in microtubule nucleation.

    PubMed Central

    Carlier, M F; Didry, D; Pantaloni, D

    1997-01-01

    Hydrolysis of GTP is known to accompany microtubule assembly. Here we show that hydrolysis of GTP is also associated with the formation of linear oligomers of tubulin, which are precursors (prenuclei) in microtubule assembly. The hydrolysis of GTP on these linear oligomers inhibits the lateral association of GTP-tubulin that leads to the formation of a bidimensional lattice. Therefore GTP hydrolysis interferes with the nucleation of microtubules. Linear oligomers are also formed in mixtures of GTP-tubulin and GDP-tubulin. The hydrolysis of GTP associated with heterologous interactions between GTP-tubulin and GDP-tubulin in the cooligomer takes place at a threefold faster rate than upon homologous interactions between GTP-tubulins. The implication of these results in a model of vectorial GTP hydrolysis in microtubule assembly is discussed. Images FIGURE 7 PMID:9199805

  15. Bile acid salt binding with colesevelam HCl is not affected by suspension in common beverages.

    PubMed

    Hanus, Martin; Zhorov, Eugene

    2006-12-01

    It has been previously reported that anions in common beverages may bind to bile acid sequestrants (BAS), reducing their capacity for binding bile acid salts. This study examined the ability of the novel BAS colesevelam hydrochloride (HCl), in vitro, to bind bile acid sodium salts following suspension in common beverages. Equilibrium binding was evaluated under conditions of constant time and varying concentrations of bile acid salts in simulated intestinal fluid (SIF). A stock solution of sodium salts of glycochenodeoxycholic acid (GCDC), taurodeoxycholic acid (TDC), and glycocholic acid (GC), was added to each prepared sample of colesevelam HCl. Bile acid salt binding was calculated by high-performance liquid chromatography (HPLC) analysis. Kinetics experiments were conducted using constant initial bile acid salt concentrations and varying binding times. The affinity, capacity, and kinetics of colesevelam HCl binding for GCDC, TDC, and GC were not significantly altered after suspension in water, carbonated water, Coca-Cola, Sprite, grape juice, orange juice, tomato juice, or Gatorade. The amount of bile acid sodium salt bound as a function of time was unchanged by pretreatment with any beverage tested. The in vitro binding characteristics of colesevelam HCl are unchanged by suspension in common beverages. PMID:16937334

  16. Intracellular GTP level determines cell's fate toward differentiation and apoptosis

    SciTech Connect

    Meshkini, Azadeh; Yazdanparast, Razieh Nouri, Kazem

    2011-06-15

    Since the adequate supply of guanine nucleotides is vital for cellular activities, limitation of their syntheses would certainly result in modulation of cellular fate toward differentiation and apoptosis. The aim of this study was to set a correlation between the intracellular level of GTP and the induction of relevant signaling pathways involved in the cell's fate toward life or death. In that regard, we measured the GTP level among human leukemia K562 cells exposed to mycophenolic acid (MPA) or 3-hydrogenkwadaphnin (3-HK) as two potent inosine monophosphate dehydrogenase inhibitors. Our results supported the maturation of the cells when the intracellular GTP level was reduced by almost 30-40%. Under these conditions, 3-HK and/or MPA caused up-regulation of PKC{alpha} and PI3K/AKT pathways. Furthermore, co-treatment of cells with hypoxanthine plus 3-HK or MPA, which caused a reduction of about 60% in the intracellular GTP levels, led to apoptosis and activation of mitochondrial pathways through inverse regulation of Bcl-2/Bax expression and activation of caspase-3. Moreover, our results demonstrated that attenuation of GTP by almost 60% augmented the intracellular ROS and nuclear localization of p21 and subsequently led to cell death. These results suggest that two different threshold levels of GTP are needed for induction of differentiation and/or ROS-associated apoptosis. - Graphical abstract: Display Omitted

  17. Coordination of two sequential ester-transfer reactions: exogenous guanosine binding promotes the subsequent omegaG binding to a group I intron.

    PubMed

    Bao, Penghui; Wu, Qi-Jia; Yin, Ping; Jiang, Yanfei; Wang, Xu; Xie, Mao-Hua; Sun, Tao; Huang, Lin; Mo, Ding-Ding; Zhang, Yi

    2008-12-01

    Self-splicing of group I introns is accomplished by two sequential ester-transfer reactions mediated by sequential binding of two different guanosine ligands, but it is yet unclear how the binding is coordinated at a single G-binding site. Using a three-piece trans-splicing system derived from the Candida intron, we studied the effect of the prior GTP binding on the later omegaG binding by assaying the ribozyme activity in the second reaction. We showed that adding GTP simultaneously with and prior to the esterified omegaG in a substrate strongly accelerated the second reaction, suggesting that the early binding of GTP facilitates the subsequent binding of omegaG. GTP-mediated facilitation requires C2 amino and C6 carbonyl groups on the Watson-Crick edge of the base but not the phosphate or sugar groups, suggesting that the base triple interactions between GTP and the binding site are important for the subsequent omegaG binding. Strikingly, GTP binding loosens a few local structures of the ribozyme including that adjacent to the base triple, providing structural basis for a rapid exchange of omegaG for bound GTP. PMID:18978026

  18. 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

  19. Stability of the Octameric Structure Affects Plasminogen-Binding Capacity of Streptococcal Enolase

    PubMed Central

    Law, Ruby H. P.; Casey, Lachlan W.; Valkov, Eugene; Bertozzi, Carlo; Stamp, Anna; Jovcevski, Blagojce; Aquilina, J. Andrew; Whisstock, James C.; Walker, Mark J.; Kobe, Bostjan

    2015-01-01

    Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation. PMID:25807546

  20. Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

    PubMed Central

    Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Joseph J.

    2012-01-01

    To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and 19F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan (5FW). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that 5FW incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when 5FW was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. 19F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each 5FW in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody–antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody–antigen complexes with altered function that may not be discernible by other biophysical techniques. PMID:22769726

  1. Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.

    PubMed

    Rocheleau, Gail; Petrillo, Jessica; Guogas, Laura; Gehrke, Lee

    2004-08-01

    The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity. PMID:15254175

  2. Disruption of NAD+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

    PubMed Central

    Phadke, Manali; Krynetskaia, Natalia; Mishra, Anurag; Barrero, Carlos; Merali, Salim; Gothe, Scott A; Krynetskiy, Evgeny

    2015-01-01

    AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+ (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners. PMID:26629320

  3. Higher-Order Septin Assembly Is Driven by GTP-Promoted Conformational Changes: Evidence From Unbiased Mutational Analysis in Saccharomyces cerevisiae

    PubMed Central

    Weems, Andrew D.; Johnson, Courtney R.; Argueso, Juan Lucas; McMurray, Michael A.

    2014-01-01

    Septin proteins bind GTP and heterooligomerize into filaments with conserved functions across a wide range of eukaryotes. Most septins hydrolyze GTP, altering the oligomerization interfaces; yet mutations designed to abolish nucleotide binding or hydrolysis by yeast septins perturb function only at high temperatures. Here, we apply an unbiased mutational approach to this problem. Mutations causing defects at high temperature mapped exclusively to the oligomerization interface encompassing the GTP-binding pocket, or to the pocket itself. Strikingly, cold-sensitive defects arise when certain of these same mutations are coexpressed with a wild-type allele, suggestive of a novel mode of dominance involving incompatibility between mutant and wild-type molecules at the septin–septin interfaces that mediate filament polymerization. A different cold-sensitive mutant harbors a substitution in an unstudied but highly conserved region of the septin Cdc12. A homologous domain in the small GTPase Ran allosterically regulates GTP-binding domain conformations, pointing to a possible new functional domain in some septins. Finally, we identify a mutation in septin Cdc3 that restores the high-temperature assembly competence of a mutant allele of septin Cdc10, likely by adopting a conformation more compatible with nucleotide-free Cdc10. Taken together, our findings demonstrate that GTP binding and hydrolysis promote, but are not required for, one-time events—presumably oligomerization-associated conformational changes—during assembly of the building blocks of septin filaments. Restrictive temperatures impose conformational constraints on mutant septin proteins, preventing new assembly and in certain cases destabilizing existing assemblies. These insights from yeast relate directly to disease-causing mutations in human septins. PMID:24398420

  4. UMP kinase from Streptococcus pneumoniae: evidence for co-operative ATP binding and allosteric regulation

    PubMed Central

    2004-01-01

    UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP and ADP. In prokaryotes, the reaction is carried out by a hexameric enzyme, activated by GTP and inhibited by UTP. In the present study, Streptococcus pneumoniae UMP kinase was studied as a target for antibacterial research and its interest was confirmed by the demonstration of the essentiality of the gene for cell growth. In the presence of MnCl2 or MgCl2, the saturation kinetics of recombinant purified UMP kinase was hyperbolic for UMP (Km=0.1 mM) and sigmoidal for ATP (the substrate concentration at half-saturation S0.5=9.4±0.7 mM and n=1.9±0.1 in the presence of MgCl2). GTP increased the affinity for ATP and decreased the Hill coefficient (n). UTP decreased the affinity for ATP and only slightly increased the Hill coefficient. The kcat (175±13 s−1 in the presence of MgCl2) was not affected by the addition of GTP or UTP, whose binding site was shown to be different from the active site. The hydrodynamic radius of the protein similarly decreased in the presence of ATP or GTP. There was a shift in the pH dependence of the activity when the ATP concentration was switched from low to high. These results support the hypothesis of an allosteric transition from a conformation with low affinity for ATP to a form with high affinity, which would be induced by the presence of ATP or GTP. PMID:15324307

  5. Exploring potassium-dependent GTP hydrolysis in TEES family GTPases.

    PubMed

    Rafay, Abu; Majumdar, Soneya; Prakash, Balaji

    2012-01-01

    GTPases are important regulatory proteins that hydrolyze GTP to GDP. A novel GTP-hydrolysis mechanism is employed by MnmE, YqeH and FeoB, where a potassium ion plays a role analogous to the Arginine finger of the Ras-RasGAP system, to accelerate otherwise slow GTP hydrolysis rates. In these proteins, two conserved asparagines and a 'K-loop' present in switch-I, were suggested as attributes of GTPases employing a K(+)-mediated mechanism. Based on their conservation, a similar mechanism was suggested for TEES family GTPases. Recently, in Dynamin, Fzo1 and RbgA, which also conserve these attributes, a similar mechanism was shown to be operative. Here, we probe K(+)-activated GTP hydrolysis in TEES (TrmE-Era-EngA-YihA-Septin) GTPases - Era, EngB and the two contiguous G-domains, GD1 and GD2 of YphC (EngA homologue) - and also in HflX, another GTPase that also conserves the same attributes. While GD1-YphC and Era exhibit a K(+)-mediated activation of GTP hydrolysis, surprisingly GD2-YphC, EngB and HflX do not. Therefore, the attributes identified thus far, do not necessarily predict a K(+)-mechanism in GTPases and hence warrant extensive structural investigations. PMID:23650596

  6. The Binding Ring Illusion: assimilation affects the perceived size of a circular array

    PubMed Central

    Caplovitz, Gideon P

    2013-01-01

    Our perception of an object’s size arises from the integration of multiple sources of visual information including retinal size, perceived distance and its size relative to other objects in the visual field. This constructive process is revealed through a number of classic size illusions such as the Delboeuf Illusion, the Ebbinghaus Illusion and others illustrating size constancy. Here we present a novel variant of the Delbouef and Ebbinghaus size illusions that we have named the Binding Ring Illusion. The illusion is such that the perceived size of a circular array of elements is underestimated when superimposed by a circular contour – a binding ring – and overestimated when the binding ring slightly exceeds the overall size of the array. Here we characterize the stimulus conditions that lead to the illusion, and the perceptual principles that underlie it. Our findings indicate that the perceived size of an array is susceptible to the assimilation of an explicitly defined superimposed contour. Our results also indicate that the assimilation process takes place at a relatively high level in the visual processing stream, after different spatial frequencies have been integrated and global shape has been constructed. We hypothesize that the Binding Ring Illusion arises due to the fact that the size of an array of elements is not explicitly defined and therefore can be influenced (through a process of assimilation) by the presence of a superimposed object that does have an explicit size. PMID:24555042

  7. Size and molecular flexibility affect the binding of ellagitannins to bovine serum albumin.

    PubMed

    Dobreva, Marina A; Green, Rebecca J; Mueller-Harvey, Irene; Salminen, Juha-Pekka; Howlin, Brendan J; Frazier, Richard A

    2014-09-17

    Binding to bovine serum albumin of monomeric (vescalagin and pedunculagin) and dimeric ellagitannins (roburin A, oenothein B, and gemin A) was investigated by isothermal titration calorimetry and fluorescence spectroscopy, which indicated two types of binding sites. Stronger and more specific sites exhibited affinity constants, K1, of 10(4)-10(6) M(-1) and stoichiometries, n1, of 2-13 and dominated at low tannin concentrations. Weaker and less-specific binding sites had K2 constants of 10(3)-10(5) M(-1) and stoichiometries, n2, of 16-30 and dominated at higher tannin concentrations. Binding to stronger sites appeared to be dependent on tannin flexibility and the presence of free galloyl groups. Positive entropies for all but gemin A indicated that hydrophobic interactions dominated during complexation. This was supported by an exponential relationship between the affinity, K1, and the modeled hydrophobic accessible surface area and by a linear relationship between K1 and the Stern-Volmer quenching constant, K(SV). PMID:25162485

  8. Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation

    PubMed Central

    2004-01-01

    Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes. We demonstrate that such complexes are also present in secretions of cultured cells. To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs. The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids. In gel filtration, proSAP behaved as an oligomer and proCD as a monomer. ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions. ProSAP did not change the elution of mature cathepsin D. Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions. At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer. The dissociation of the first site was characterized by a KD1 of 5.8±2.9×10−8 M−1 (calculated for the monomer). ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D. Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments. Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D. In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate. PMID:15255780

  9. Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

    SciTech Connect

    Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Jr., Joseph J.

    2012-10-16

    To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and {sup 19}F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ({sup 5F}W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that {sup 5F}W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when {sup 5F}W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. {sup 19}F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each {sup 5F}W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

  10. GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

    PubMed

    Walker, M W; Bobak, D A; Tsai, S C; Moss, J; Vaughan, M

    1992-02-15

    ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo. PMID:1737779

  11. Mg2+ binds to the surface of thymidylate synthase and affects hydride transfer at the interior active site

    PubMed Central

    Wang, Zhen; Sapienza, Paul J.; Abeysinghe, Thelma; Luzum, Calvin; Lee, Andrew L.; Finer-Moore, Janet S.; Stroud, Robert M.; Kohen, Amnon

    2013-01-01

    Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e. the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg2+ has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg2+ binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg2+ near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg2+ binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg2+ to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg2+ accelerates the hydride transfer by ca. 7-fold but does not affect the magnitude or temperature-dependence of the intrinsic kinetic isotope effect. These results suggest that Mg2+ facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg2+ concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg2+ with the glutamyl-tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with poly-glutamyl substitutes as species-specific antibiotic drugs. PMID:23611499

  12. Cardiac Myosin Binding Protein C Phosphorylation Affects Cross-Bridge Cycle's Elementary Steps in a Site-Specific Manner

    PubMed Central

    Wang, Li; Sadayappan, Sakthivel; Kawai, Masakata

    2014-01-01

    Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases. PMID:25420047

  13. The neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation.

    PubMed

    Dunzendorfer-Matt, Theresia; Mercado, Ellen L; Maly, Karl; McCormick, Frank; Scheffzek, Klaus

    2016-07-01

    Neurofibromatosis type 1 (NF1) and Legius syndrome are related diseases with partially overlapping symptoms caused by alterations of the tumor suppressor genes NF1 (encoding the protein neurofibromin) and SPRED1 (encoding sprouty-related, EVH1 domain-containing protein 1, Spred1), respectively. Both proteins are negative regulators of Ras/MAPK signaling with neurofibromin functioning as a Ras-specific GTPase activating protein (GAP) and Spred1 acting on hitherto undefined components of the pathway. Importantly, neurofibromin has been identified as a key protein in the development of cancer, as it is genetically altered in a large number of sporadic human malignancies unrelated to NF1. Spred1 has previously been demonstrated to interact with neurofibromin via its N-terminal Ena/VASP Homology 1 (EVH1) domain and to mediate membrane translocation of its target dependent on its C-terminal Sprouty domain. However, the region of neurofibromin required for the interaction with Spred1 has remained unclear. Here we show that the EVH1 domain of Spred1 binds to the noncatalytic (GAPex) portion of the GAP-related domain (GRD) of neurofibromin. Binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity. Our study points to a potential targeting function of the GAPex subdomain of neurofibromin that is present in all known canonical RasGAPs. PMID:27313208

  14. A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding.

    PubMed

    Amblar, Mónica; Arraiano, Cecília M

    2005-01-01

    Exoribonuclease II (RNase II), encoded by the rnb gene, is a ubiquitous enzyme that is responsible for 90% of the hydrolytic activity in Escherichia coli crude extracts. The E. coli strain SK4803, carrying the mutant allele rnb296, has been widely used in the study of the role of RNase II. We determined the DNA sequence of rnb296 and cloned this mutant gene in an expression vector. Only a point mutation in the coding sequence of the gene was detected, which results in the single substitution of aspartate 209 for asparagine. The mutant and the wild-type RNase II enzymes were purified, and their 3' to 5' exoribonucleolytic activity, as well as their RNA binding capability, were characterized. We also studied the metal dependency of the exoribonuclease activity of RNase II. The results obtained demonstrated that aspartate 209 is absolutely essential for RNA hydrolysis, but is not required for substrate binding. This is the first evidence of an acidic residue that is essential for the activity of RNase II-like enzymes. The possible involvement of this residue in metal binding at the active site of the enzyme is discussed. These results are particularly relevant at this time given that no structural or mutational analysis has been performed for any protein of the RNR family of exoribonucleases. PMID:15654875

  15. The neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation

    PubMed Central

    Dunzendorfer-Matt, Theresia; Mercado, Ellen L.; Maly, Karl; McCormick, Frank; Scheffzek, Klaus

    2016-01-01

    Neurofibromatosis type 1 (NF1) and Legius syndrome are related diseases with partially overlapping symptoms caused by alterations of the tumor suppressor genes NF1 (encoding the protein neurofibromin) and SPRED1 (encoding sprouty-related, EVH1 domain-containing protein 1, Spred1), respectively. Both proteins are negative regulators of Ras/MAPK signaling with neurofibromin functioning as a Ras-specific GTPase activating protein (GAP) and Spred1 acting on hitherto undefined components of the pathway. Importantly, neurofibromin has been identified as a key protein in the development of cancer, as it is genetically altered in a large number of sporadic human malignancies unrelated to NF1. Spred1 has previously been demonstrated to interact with neurofibromin via its N-terminal Ena/VASP Homology 1 (EVH1) domain and to mediate membrane translocation of its target dependent on its C-terminal Sprouty domain. However, the region of neurofibromin required for the interaction with Spred1 has remained unclear. Here we show that the EVH1 domain of Spred1 binds to the noncatalytic (GAPex) portion of the GAP-related domain (GRD) of neurofibromin. Binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity. Our study points to a potential targeting function of the GAPex subdomain of neurofibromin that is present in all known canonical RasGAPs. PMID:27313208

  16. 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

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

    PubMed

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

    2016-04-01

    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

  18. Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters.

    PubMed

    Thangasamy, Saminathan; Chen, Pei-Wei; Lai, Ming-Hsing; Chen, Jychian; Jauh, Guang-Yuh

    2012-07-01

    Tiller initiation and panicle development are important agronomical traits for grain production in Oryza sativa L. (rice), but their regulatory mechanisms are not yet fully understood. In this study, T-DNA mutant and RNAi transgenic approaches were used to functionally characterize a unique rice gene, LAGGING GROWTH AND DEVELOPMENT 1 (LGD1). The lgd1 mutant showed slow growth, reduced tiller number and plant height, altered panicle architecture and reduced grain yield. The fewer unelongated internodes and cells in lgd1 led to respective reductions in tiller number and to semi-dwarfism. Several independent LGD1-RNAi lines exhibited defective phenotypes similar to those observed in lgd1. Interestingly, LGD1 encodes multiple transcripts with different transcription start sites (TSSs), which were validated by RNA ligase-mediated rapid amplification of 5' and 3' cDNA ends (RLM-RACE). Additionally, GUS assays and a luciferase promoter assay confirmed the promoter activities of LGD1.1 and LGD1.5. LGD1 encoding a von Willebrand factor type A (vWA) domain containing protein is a single gene in rice that is seemingly specific to grasses. GFP-tagged LGD1 isoforms were predominantly detected in the nucleus, and weakly in the cytoplasm. In vitro northwestern analysis showed the RNA-binding activity of the recombinant C-terminal LGD1 protein. Our results demonstrated that LGD1 pleiotropically regulated rice vegetative growth and development through both the distinct spatiotemporal expression patterns of its multiple transcripts and RNA binding activity. Hence, the study of LGD1 will strengthen our understanding of the molecular basis of the multiple transcripts, and their corresponding polypeptides with RNA binding activity, that regulate pleiotropic effects in rice. PMID:22409537

  19. Calcium affects OX1 orexin (hypocretin) receptor responses by modifying both orexin binding and the signal transduction machinery

    PubMed Central

    Putula, Jaana; Pihlajamaa, Tero; Kukkonen, Jyrki P

    2014-01-01

    Background and Purpose One of the major responses upon orexin receptor activation is Ca2+ influx, and this influx seems to amplify the other responses mediated by orexin receptors. However, the reduction in Ca2+, often used to assess the importance of Ca2+ influx, might affect other properties, like ligand−receptor interactions, as suggested for some GPCR systems. Hence, we investigated the role of the ligand−receptor interaction and Ca2+ signal cascades in the apparent Ca2+ requirement of orexin-A signalling. Experimental Approach Receptor binding was assessed in CHO cells expressing human OX1 receptors with [125I]-orexin-A by conventional ligand binding as well as scintillation proximity assays. PLC activity was determined by chromatography. Key Results Both orexin receptor binding and PLC activation were strongly dependent on the extracellular Ca2+ concentration. The relationship between Ca2+ concentration and receptor binding was the same as that for PLC activation. However, when Ca2+ entry was reduced by depolarizing the cells or by inhibiting the receptor-operated Ca2+ channels, orexin-A-stimulated PLC activity was much more strongly inhibited than orexin-A binding. Conclusions and Implications Ca2+ plays a dual role in orexin signalling by being a prerequisite for both ligand−receptor interaction and amplifying orexin signals via Ca2+ influx. Some previous results obtained utilizing Ca2+ chelators have to be re-evaluated based on the results of the current study. From a drug discovery perspective, further experiments need to identify the target for Ca2+ in orexin-A−OX1 receptor interaction and its mechanism of action. PMID:25132134

  20. Glycation of Ribonuclease A affects its enzymatic activity and DNA binding ability.

    PubMed

    Dinda, Amit Kumar; Tripathy, Debi Ranjan; Dasgupta, Swagata

    2015-11-01

    Prolonged non-enzymatic glycation of proteins results in the formation of advanced glycation end products (AGEs) that cause several diseases. The glycation of Ribonuclease A (RNase A) at pH 7.4 and 37 °C with ribose, glucose and fructose has been monitored by UV-vis, fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization spectroscopy-time of flight (MALDI-TOF) methods. The enzymatic activity and DNA binding ability of glycated RNase A was also investigated by an agarose gel-based assay. A precipitation assay examined the ribonucleolytic activity of the glycated enzyme. An increase in incubation time resulted in the formation of high molecular weight AGEs with a decrease in ribonucleolytic activity. Ribose exhibits the highest potency as a glycating agent and showed the greatest reduction in the ribonucleolytic activity of the enzyme. Interestingly, glycated RNase A was unable to bind with the ribonuclease inhibitor (RI) and DNA. The glycated form of the protein was also found to be ineffective in DNA melting unlike native RNase A. PMID:26365067

  1. Dimethyl sulfoxide: an antagonist in scintillation proximity assay [(35)S]-GTPgammaS binding to rat 5-HT(6) receptor cloned in HEK-293 cells?

    PubMed

    Mereghetti, Ilario; Cagnotto, Alfredo; Mennini, Tiziana

    2007-03-15

    We have tested by [(35)S]-GTPgammaS binding the intrinsic activity of three full agonists (serotonin, 5-methoxytryptamine and 5-methoxy-2-methyl-N,N-dimethyltryptamine) on rat 5-HT(6) receptors cloned in HEK-293 cells, using the scintillation proximity assay. Serotonin and 5-methoxytryptamine are soluble in water, while the agonist 5-methoxy-2-methyl-N,N-dimethyltryptamine is soluble in dimethyl sulfoxide (DMSO). In [(35)S]-GTPgammaS binding 5-HT and 5-methoxytryptamine were able to increase basal binding, while 5-methoxy-2-methyl-N,N-dimethyltryptamine surprisingly showed an inverse agonist activity. So we have tested 5-HT and 5-methoxytryptamine in the presence of DMSO: in this condition the two agonists behaved as antagonists. This interfering effect of DMSO was not observed when GTP-europium filtration binding was used in place of scintillation proximity assay using [(35)S]-GTPgammaS. In addition, DMSO did not affect [(3)H]-5HT binding or cAMP accumulation in cloned HEK-293 cells expressing rat 5-HT(6) receptors. In conclusion, we demonstrated that DMSO, the most common solvent used to dissolve compounds insoluble in water, interferes with the method of scintillation proximity assay using [(35)S]-GTPgammaS. DMSO does not affect basal signal, nor the GTPgammaS binding itself, as indicated by the experiments with GTP-europium. Therefore its interfering effect is likely to occur at the binding of antibodies in the scintillation proximity assay. PMID:17049618

  2. Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

    PubMed Central

    Patel, A.; Hashimoto, H.; Zhang, X.; Cheng, X.

    2016-01-01

    Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides. PMID:27372763

  3. TANK-binding kinase-1 broadly affects oyster immune response to bacteria and viruses.

    PubMed

    Tang, Xueying; Huang, Baoyu; Zhang, Linlin; Li, Li; Zhang, Guofan

    2016-09-01

    As a benthic filter feeder of estuaries, the immune system of oysters provides one of the best models for studying the genetic and molecular basis of the innate immune pathway in marine invertebrates and examining the influence of environmental factors on the immune system. Here, the molecular function of molluscan TANK-binding kinase-1 (TBK1) (which we named CgTBK1) was studied in the Pacific oyster, Crassostrea gigas. Compared with known TBK1 proteins in other model organisms, CgTBK1 contains a conserved S-TKc domain and a coiled coil domain at the N- and C-terminals but lacks an important ubiquitin domain. Quantitative real-time PCR analysis revealed that the expression level of CgTBK1 was ubiquitous in all selected tissues, with highest expression in the gills. CgTBK1 expression was significantly upregulated in response to infections with Vibrio alginolyticus, ostreid herpesvirus 1 (OsHV-1 reference strain and μvar), and polyinosinic:polycytidylic acid sodium salt, suggesting its broad function in immune response. Subcellular localization showed the presence of CgTBK1 in the cytoplasm of HeLa cells, suggesting its potential function as the signal transducer between the receptor and transcription factor. We further demonstrated that CgTBK1 interacted with CgSTING in HEK293T cells, providing evidence that CgTBK1 could be activated by direct binding to CgSTING. In summary, we characterized the TBK1 gene in C. gigas and demonstrated its role in the innate immune response to pathogen infections. PMID:27422757

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

    SciTech Connect

    Sano, Hiroyuki; Peck, Grantley R.; Blachon, Stephanie; Lienhard, Gustav E.

    2015-09-25

    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 two 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.

  5. Ca2+-dependent Calmodulin Binding to FcRn Affects Immunoglobulin G Transport in the Transcytotic Pathway

    PubMed Central

    Dickinson, Bonny L.; Claypool, Steven M.; D'Angelo, June A.; Aiken, Martha L.; Venu, Nanda; Yen, Elizabeth H.; Wagner, Jessica S.; Borawski, Jason A.; Pierce, Amy T.; Hershberg, Robert; Blumberg, Richard S.

    2008-01-01

    The Fcγ receptor FcRn transports immunoglobulin G (IgG) so as to avoid lysosomal degradation and to carry it bidirectionally across epithelial barriers to affect mucosal immunity. Here, we identify a calmodulin-binding site within the FcRn cytoplasmic tail that affects FcRn trafficking. Calmodulin binding to the FcRn tail is direct, calcium-dependent, reversible, and specific to residues comprising a putative short amphipathic α-helix immediately adjacent to the membrane. FcRn mutants with single residue substitutions in this motif, or FcRn mutants lacking the cytoplasmic tail completely, exhibit a shorter half-life and attenuated transcytosis. Chemical inhibitors of calmodulin phenocopy the mutant FcRn defect in transcytosis. These results suggest a novel mechanism for regulation of IgG transport by calmodulin-dependent sorting of FcRn and its cargo away from a degradative pathway and into a bidirectional transcytotic route. PMID:18003977

  6. A functional MiR-124 binding-site polymorphism in IQGAP1 affects human cognitive performance.

    PubMed

    Yang, Lixin; Zhang, Rui; Li, Ming; Wu, Xujun; Wang, Jianhong; Huang, Lin; Shi, Xiaodong; Li, Qingwei; Su, Bing

    2014-01-01

    As a product of the unique evolution of the human brain, human cognitive performance is largely a collection of heritable traits. Rather surprisingly, to date there have been no reported cases to highlight genes that underwent adaptive evolution in humans and which carry polymorphisms that have a marked effect on cognitive performance. IQ motif containing GTPase activating protein 1 (IQGAP1), a scaffold protein, affects learning and memory in a dose-dependent manner. Its expression is regulated by miR-124 through the binding sites in the 3'UTR, where a SNP (rs1042538) exists in the core-binding motif. Here we showed that this SNP can influence the miR-target interaction both in vitro and in vivo. Individuals carrying the derived T alleles have higher IQGAP1 expression in the brain as compared to the ancestral A allele carriers. We observed a significant and male-specific association between rs1042538 and tactile performances in two independent cohorts. Males with the derived allele displayed higher tactual performances as compared to those with the ancestral allele. Furthermore, we found a highly diverged allele-frequency distribution of rs1042538 among world human populations, likely caused by natural selection and/or recent population expansion. These results suggest that current human populations still carry sequence variations that affect cognitive performances and that these genetic variants may likely have been subject to comparatively recent natural selection. PMID:25222038

  7. GTP Cyclohydrolase I Phosphorylation and Interaction with GTP Cyclohydrolase Feedback Regulatory Protein Provide Novel Regulation of Endothelial Tetrahydrobiopterin and Nitric Oxide

    PubMed Central

    Li, Li; Rezvan, Amir; Salerno, John C.; Husain, Ahsan; Kwon, Kihwan; Jo, Hanjoong; Harrison, David G.; Chen, Wei

    2009-01-01

    Rationale GTP cyclohydrolase I (GTPCH-1) is the rate-limiting enzyme involved in de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases and aromatic amino acid hydroxylases. GTPCH-1 undergoes negative feedback regulation by its end-product BH4 via interaction with the GTP cyclohydrolase feedback regulatory protein (GFRP). Such a negative feedback mechanism should maintain cellular BH4 levels within a very narrow range; however, we recently identified a phosphorylation site (S81) on human GTPCH-1 that markedly increases BH4 production in response to laminar shear. Objective To define how S81 phosphorylation alters GTPCH-1 enzyme activity and how this is modulated by GFRP. Methods and Results Using prokaryotically expressed proteins, we found that the GTPCH-1 phospho-mimetic mutant (S81D) has increased enzyme activity, reduced binding to GFRP and resistance to inhibition by GFRP compared to wild-type GTPCH-1. Using siRNA or overexpressing plasmids, GFRP was shown to modulate phosphorylation of GTPCH-1, BH4 levels and nitric oxide (NO) production in human endothelial cells. Laminar, but not oscillatory shear stress caused dissociation of GTPCH-1 and GFRP, promoting GTPCH-1 phosphorylation. We also found that both GTPCH-1 phosphorylation and GFRP down-regulation prevents eNOS uncoupling in response to oscillatory shear. Finally oscillatory shear was associated with impaired GTPCH-1 phosphorylation and reduced BH4 levels in vivo. Conclusion These studies provide a new mechanism for regulation of endothelial GTPCH-1 by its phosphorylation and interplay with GFRP. This mechanism allows for escape from GFRP negative feedback and permits large amounts of BH4 to be produced in response to laminar shear stress. PMID:19926872

  8. Starch-binding domain affects catalysis in two Lactobacillus alpha-amylases.

    PubMed

    Rodríguez-Sanoja, R; Ruiz, B; Guyot, J P; Sanchez, S

    2005-01-01

    A new starch-binding domain (SBD) was recently described in alpha-amylases from three lactobacilli (Lactobacillus amylovorus, Lactobacillus plantarum, and Lactobacillus manihotivorans). Usually, the SBD is formed by 100 amino acids, but the SBD sequences of the mentioned lactobacillus alpha-amylases consist of almost 500 amino acids that are organized in tandem repeats. The three lactobacillus amylase genes share more than 98% sequence identity. In spite of this identity, the SBD structures seem to be quite different. To investigate whether the observed differences in the SBDs have an effect on the hydrolytic capability of the enzymes, a kinetic study of L. amylovorus and L. plantarum amylases was developed, with both enzymes acting on several starch sources in granular and gelatinized forms. Results showed that the amylolytic capacities of these enzymes are quite different; the L. amylovorus alpha-amylase is, on average, 10 times more efficient than the L. plantarum enzyme in hydrolyzing all the tested polymeric starches, with only a minor difference in the adsorption capacities. PMID:15640201

  9. Starch-Binding Domain Affects Catalysis in Two Lactobacillus α-Amylases

    PubMed Central

    Rodríguez-Sanoja, R.; Ruiz, B.; Guyot, J. P.; Sanchez, S.

    2005-01-01

    A new starch-binding domain (SBD) was recently described in α-amylases from three lactobacilli (Lactobacillus amylovorus, Lactobacillus plantarum, and Lactobacillus manihotivorans). Usually, the SBD is formed by 100 amino acids, but the SBD sequences of the mentioned lactobacillus α-amylases consist of almost 500 amino acids that are organized in tandem repeats. The three lactobacillus amylase genes share more than 98% sequence identity. In spite of this identity, the SBD structures seem to be quite different. To investigate whether the observed differences in the SBDs have an effect on the hydrolytic capability of the enzymes, a kinetic study of L. amylovorus and L. plantarum amylases was developed, with both enzymes acting on several starch sources in granular and gelatinized forms. Results showed that the amylolytic capacities of these enzymes are quite different; the L. amylovorus α-amylase is, on average, 10 times more efficient than the L. plantarum enzyme in hydrolyzing all the tested polymeric starches, with only a minor difference in the adsorption capacities. PMID:15640201

  10. Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.

    PubMed

    Martin, Katherine R; Kantari-Mimoun, Chahrazade; Yin, Min; Pederzoli-Ribeil, Magali; Angelot-Delettre, Fanny; Ceroi, Adam; Grauffel, Cédric; Benhamou, Marc; Reuter, Nathalie; Saas, Philippe; Frachet, Philippe; Boulanger, Chantal M; Witko-Sarsat, Véronique

    2016-05-13

    Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease. PMID:26961880

  11. Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity.

    PubMed Central

    Schwedock, J S; Liu, C; Leyh, T S; Long, S R

    1994-01-01

    The nodulation genes nodP and nodQ are required for production of Rhizobium meliloti nodulation (Nod) factors. These sulfated oligosaccharides act as morphogenic signals to alfalfa, the symbiotic host of R. meliloti. In previous work, we have shown that nodP and nodQ encode ATP sulfurylase, which catalyzes the formation of APS (adenosine 5'-phosphosulfate) and PPi. In the subsequent metabolic reaction, APS is converted to PAPS (3'-phosphoadenosine 5'-phosphosulfate) by APS kinase. In Escherichia coli, cysD and cysN encode ATP sulfurylase; cysC encodes APS kinase. Here, we present genetic, enzymatic, and sequence similarity data demonstrating that nodP and nodQ encode both ATP sulfurylase and APS kinase activities and that these enzymes associate into a multifunctional protein complex which we designate the sulfate activation complex. We have previously described the presence of a putative GTP-binding site in the nodQ sequence. The present report also demonstrates that GTP enhances the rate of PAPS synthesis from ATP and sulfate (SO4(2-)) by NodP and NodQ expressed in E. coli. Thus, GTP is implicated as a metabolic requirement for synthesis of the R. meliloti Nod factors. Images PMID:7961471

  12. A Nucleotide Binding Motif in Hepatitis C Virus (HCV) NS4B Mediates HCV RNA Replication

    PubMed Central

    Einav, Shirit; Elazar, Menashe; Danieli, Tsafi; Glenn, Jeffrey S.

    2004-01-01

    Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the virus's nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral strategies. PMID:15452248

  13. A cyst nematode effector binds to diverse plant proteins, increases nematode susceptibility and affects root morphology.

    PubMed

    Pogorelko, Gennady; Juvale, Parijat S; Rutter, William B; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Mitchum, Melissa G; Baum, Thomas J

    2016-08-01

    Cyst nematodes are plant-parasitic roundworms that are of significance in many cropping systems around the world. Cyst nematode infection is facilitated by effector proteins secreted from the nematode into the plant host. The cDNAs of the 25A01-like effector family are novel sequences that were isolated from the oesophageal gland cells of the soybean cyst nematode (Heterodera glycines). To aid functional characterization, we identified an orthologous member of this protein family (Hs25A01) from the closely related sugar beet cyst nematode H. schachtii, which infects Arabidopsis. Constitutive expression of the Hs25A01 CDS in Arabidopsis plants caused a small increase in root length, accompanied by up to a 22% increase in susceptibility to H. schachtii. A plant-expressed RNA interference (RNAi) construct targeting Hs25A01 transcripts in invading nematodes significantly reduced host susceptibility to H. schachtii. These data document that Hs25A01 has physiological functions in planta and a role in cyst nematode parasitism. In vivo and in vitro binding assays confirmed the specific interactions of Hs25A01 with an Arabidopsis F-box-containing protein, a chalcone synthase and the translation initiation factor eIF-2 β subunit (eIF-2bs), making these proteins probable candidates for involvement in the observed changes in plant growth and parasitism. A role of eIF-2bs in the mediation of Hs25A01 virulence function is further supported by the observation that two independent eIF-2bs Arabidopsis knock-out lines were significantly more susceptible to H. schachtii. PMID:26575318

  14. Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

    PubMed Central

    Pratt-Hyatt, Matthew; Pai, Dave A.; Haeusler, Rebecca A.; Wozniak, Glenn G.; Good, Paul D.; Miller, Erin L.; McLeod, Ian X.; Yates, John R.; Hopper, Anita K.; Engelke, David R.

    2013-01-01

    The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification. PMID:23898186

  15. Four novel cystic fibrosis mutations in splice junction sequences affecting the CFTR nucleotide binding folds

    SciTech Connect

    Doerk, T.; Wulbrand, U.; Tuemmler, B. )

    1993-03-01

    Single cases of the four novel splice site mutations 1525[minus]1 G [r arrow] A (intron 9), 3601[minus]2 A [r arrow] G (intron 18), 3850[minus]3 T [r arrow] G (intron 19), and 4374+1 G [r arrow] T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and Germany descent. The nucleotide substitutions at the +1, [minus]1, and [minus]2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850[minus]3 T-to-G change was discovered in a very mildly affected 33-year-old [Delta]F508 compound heterozygote, suggesting that the T-to-G transversion at the less conserved [minus]3 position of the acceptor splice site may retain some wildtype function. 13 refs., 1 fig., 2 tabs.

  16. “DNA Binding Region” of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint

    PubMed Central

    Masuda, Takaaki; Xu, Xiaoling; Dimitriadis, Emilios K.; Lahusen, Tyler; Deng, Chu-Xia

    2016-01-01

    The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress. PMID:26884712

  17. A JUMONJI Protein with E3 Ligase and Histone H3 Binding Activities Affects Transposon Silencing in Arabidopsis.

    PubMed

    Kabelitz, Tina; Brzezinka, Krzysztof; Friedrich, Thomas; Górka, Michał; Graf, Alexander; Kappel, Christian; Bäurle, Isabel

    2016-05-01

    Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity. PMID:26979329

  18. Synergistic activation by serotonin and GTP analogue and inhibition by phorbol ester of cyclic Ca2+ rises in hamster eggs.

    PubMed Central

    Miyazaki, S; Katayama, Y; Swann, K

    1990-01-01

    1. Synergistic activation of a GTP-binding protein (G protein) by external serotonin (5-hydroxytryptamine, 5-HT) and internally applied guanosine-5'-O-(3-thiotriphosphate (GTP gamma S) in hamster eggs was demonstrated by the facilitation of repetitive increases in cytoplasmic Ca2+ as measured by their associated hyperpolarizing responses (HRs) and by aequorin luminescence. 2. Rapid application of 70 nM-5-HT caused a single HR of 10-12 s duration and with a delay of 80 s. The critical concentration of 5-HT to cause an HR was 50 nM. 3. With 10 microM-5-HT four to six HRs were often elicited with a delay to the first HR of 8-30 s. HRs disappeared after prolonged or repeated application of 5-HT, indicating an apparent desensitization. 4. 5-HT-induced HRs were completely inhibited by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (TPA) (100 nM). Conversely, the PKC inhibitor sphingosine (2 microM) enhanced the series of HRs by shortening the delay to the first HR (3-9 s) and by causing more HRs. 5. Ionophoretic injection of GTP gamma S into the egg usually produced a large HR with a delay of 120-240 s followed by a series of much smaller HRs. When 5-HT was applied within 1 min of injection of GTP gamma S. 70 nM-5-HT induced a number of large HRs and even 1 nM-5-HT could induce HR(s). In contrast, when 5-HT was applied after the size of GTP gamma S-induced HRs had declined, as much as 10 microM-5-HT could only elicit a single large HR. Thus, GTP gamma S apparently caused a sensitization and then a desensitization of the action of 5-HT. 6. GTP gamma S-induced Ca2+ transients were facilitated when injected in the presence of 5-HT concentrations as low as 0.1 nM. The time delay to the first HR was 65 s in 0.1 nM-5-HT or 4 s in 100 nM-5-HT whereas it was 170 s without 5-HT (mean values). The magnitude as well as frequency of HRs succeeding the first HR was enhanced by 5-HT at concentrations above 0.01 nM. 7. TPA (100 nM) blocked the GTP gamma S-plus-5

  19. Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis, and Concurrently Affect the Pathogen's Growth

    PubMed Central

    Kumar, Krishan; Tharad, Megha; Ganapathy, Swetha; Ram, Geeta; Narayan, Azeet; Khan, Jameel Ahmad; Pratap, Rana; Ghosh, Anamika; Samuchiwal, Sachin Kumar; Kumar, Sushil; Bhalla, Kuhulika; Gupta, Deepti; Natarajan, Krishnamurthy; Singh, Yogendra; Ranganathan, Anand

    2009-01-01

    Background The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung. Methodology/Principal Findings During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage. Conclusions/Significance While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide's binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine

  20. 19F nuclear magnetic resonance measurement of the distance between the E-site GTP and the high-affinity Mg2+ in tubulin.

    PubMed

    Monasterio, O

    1987-09-22

    The distance separating the divalent metal ion high-affinity binding site and the exchangeable nucleotide binding site on tubulin was evaluated by using high-resolution 19F NMR. The 31P and 19F NMR spectra of guanosine 5'-(gamma-fluorotriphosphate) [GTP (gamma F)] were studied. Both the fluorine and the gamma-phosphate were split into a doublet with a coupling constant of 936 Hz. Tubulin purified according to the method of Weisenberg [Weisenberg, R.C., & Timasheff, S.N. (1970) Biochemistry 9, 4110-4116] was incubated with 1 mM Mn2+. After one cycle of assembly, Mn2+ replaced Mg2+ only partially, i.e., 60% at the high-affinity binding site. After colchicine treatment of tubulin to stabilize it, GTP(gamma F) was added, and the 254-MHz fluorine-19 relaxation rates were measured within the first 4 h. Longitudinal and transversal relaxation rates were determined at two concentrations of GTP(gamma F) and variable concentrations of colchicine-tubulin-Mn(II) (paramagnetic complex) or the ternary complex with magnesium (diamagnetic complex). The analysis of the relaxation data indicates that the rate of exchange of GTP(gamma F) from the exchangeable nucleotide site has a lower limit of 8.7 X 10(4) s-1 and the metal and exchangeable nucleotide binding sites are separated by an upper distance between 6 and 8 A. These data confirm that the high-affinity divalent cation site is situated in the same locus as that of the exchangeable nucleotide, forming a metal-nucleotide complex. PMID:3689763

  1. /sup 19/F nuclear magnetic resonance measurement of the distance between the E-site GTP and the high-affinity Mg/sup 2 +/ in tubulin

    SciTech Connect

    Monasterio, O.

    1987-09-22

    The distance separating the divalent metal ion high-affinity binding site and the exchangeable nucleotide binding site on tubulin was evaluated by using high-resolution /sup 19/F NMR. The /sup 31/P and /sup 19/F NMR spectra of guanosine 5'-(..gamma..-fluorotriphosphate) (GTP(..gamma..F)) were studied. Both the fluorine and the ..gamma..-phosphate were split into a doublet with a coupling constant of 936 Hz. Tubulin purified according to the method of Weisenberg was incubated with 1 mM Mn/sup 2 +/. After one cycle of assembly, Mn/sup 2 +/ only partially, i.e., 60% at the high-affinity binding site. After colchicine treatment of tubulin to stabilize it, GTP(..gamma..F) was added, and the 254-MHz fluorine-19 relaxation rates were measured within the first 4 h. Longitudinal and transversal relaxation rates were determined at two concentrations of GTP(..gamma..F) and variable concentrations of colchicine-tubulin-Mn(II) (paramagnetic complex) or the ternary complex with magnesium diamagnetic complex). The analysis of the relaxation data indicates that the rate of exchange of GTP(..gamma..F) from the exchangeable nucleotide site has a lower limit of 8.7 x 10/sup 4/ s/sup -1/ and the metal and exchangeable nucleotide binding sites are separated by an upper distance between 6 and 8 A. These data confirm that the high-affinity divalent cation site is situated in the same locus as that of the exchangeable nucleotide, forming a metal-nucleotide complex.

  2. GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long-lived activated state

    PubMed Central

    Hansen, Erik C.; Seamon, Kyle J.; Cravens, Shannen L.; Stivers, James T.

    2014-01-01

    The HIV-1 restriction factor sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is a tetrameric protein that catalyzes the hydrolysis of all dNTPs to the deoxynucleoside and tripolyphosphate, which effectively depletes the dNTP substrates of HIV reverse transcriptase. Here, we establish that SAMHD1 is activated by GTP binding to guanine-specific activator sites (A1) as well as coactivation by substrate dNTP binding to a distinct set of nonspecific activator sites (A2). Combined activation by GTP and dNTPs results in a long-lived tetrameric form of SAMHD1 that persists for hours, even after activating nucleotides are withdrawn from the solution. These results reveal an ordered model for assembly of SAMHD1 tetramer from its inactive monomer and dimer forms, where GTP binding to the A1 sites generates dimer and dNTP binding to the A2 and catalytic sites generates active tetramer. Thus, cellular regulation of active SAMHD1 is not determined by GTP alone but instead, the levels of all dNTPs and the generation of a persistent tetramer that is not in equilibrium with free activators. The significance of the long-lived activated state is that SAMHD1 can remain active long after dNTP pools have been reduced to a level that would lead to inactivation. This property would be important in resting CD4+ T cells, where dNTP pools are reduced to nanomolar levels to restrict infection by HIV-1. PMID:24753578

  3. Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I

    PubMed Central

    2014-01-01

    Background Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. Methods Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ∆folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. Results Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. Conclusions

  4. Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP.

    PubMed

    Bader, G; Schiffmann, S; Herrmann, A; Fischer, M; Gütlich, M; Auerbach, G; Ploom, T; Bacher, A; Huber, R; Lemm, T

    2001-10-01

    Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory protein (GFRP). The 2.6 A resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the interaction of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins. PMID:11580249

  5. Real-time detection reveals that effectors couple dynamin's GTP-dependent conformational changes to the membrane

    PubMed Central

    Ramachandran, Rajesh; Schmid, Sandra L

    2008-01-01

    The GTPase dynamin is a mechanochemical enzyme involved in membrane fission, but the molecular nature of its membrane interactions and their regulation by guanine nucleotides and protein effectors remain poorly characterized. Using site-directed fluorescence labeling and several independent fluorescence spectroscopic techniques, we have developed robust assays for the detection and real-time monitoring of dynamin–membrane and dynamin–dynamin interactions. We show that dynamin interacts preferentially with highly curved, PIP2-dense membranes and inserts partially into the lipid bilayer. Our kinetic measurements further reveal that cycles of GTP binding and hydrolysis elicit major conformational rearrangements in self-assembled dynamin that favor dynamin–membrane association and dissociation, respectively. Sorting nexin 9, an abundant dynamin partner, transiently stabilizes dynamin on the membrane at the onset of stimulated GTP hydrolysis and may function to couple dynamin's mechanochemical conformational changes to membrane destabilization. Amphiphysin I has the opposite effect. Thus, dynamin's mechanochemical properties on a membrane surface are dynamically regulated by its GTPase cycle and major binding partners. PMID:18079695

  6. Fluctuating capacity and advance decision-making in Bipolar Affective Disorder - Self-binding directives and self-determination.

    PubMed

    Gergel, Tania; Owen, Gareth S

    2015-01-01

    For people with Bipolar Affective Disorder, a self-binding (advance) directive (SBD), by which they commit themselves to treatment during future episodes of mania, even if unwilling, can seem the most rational way to deal with an imperfect predicament. Knowing that mania will almost certainly cause enormous damage to themselves, their preferred solution may well be to allow trusted others to enforce treatment and constraint, traumatic though this may be. No adequate provision exists for drafting a truly effective SBD and efforts to establish such provision are hampered by very valid, but also paralysing ethical, clinical and legal concerns. Effectively, the autonomy and rights of people with bipolar are being 'protected' through being denied an opportunity to protect themselves. From a standpoint firmly rooted in the clinical context and experience of mania, this article argues that an SBD, based on a patient-centred evaluation of capacity to make treatment decisions (DMC-T) and grounded within the clinician-patient relationship, could represent a legitimate and ethically coherent form of self-determination. After setting out background information on fluctuating capacity, mania and advance directives, this article proposes a framework for constructing such an SBD, and considers common objections, possible solutions and suggestions for future research. PMID:25939286

  7. Fluctuating capacity and advance decision-making in Bipolar Affective Disorder — Self-binding directives and self-determination

    PubMed Central

    Gergel, Tania; Owen, Gareth S.

    2015-01-01

    For people with Bipolar Affective Disorder, a self-binding (advance) directive (SBD), by which they commit themselves to treatment during future episodes of mania, even if unwilling, can seem the most rational way to deal with an imperfect predicament. Knowing that mania will almost certainly cause enormous damage to themselves, their preferred solution may well be to allow trusted others to enforce treatment and constraint, traumatic though this may be. No adequate provision exists for drafting a truly effective SBD and efforts to establish such provision are hampered by very valid, but also paralysing ethical, clinical and legal concerns. Effectively, the autonomy and rights of people with bipolar are being ‘protected’ through being denied an opportunity to protect themselves. From a standpoint firmly rooted in the clinical context and experience of mania, this article argues that an SBD, based on a patient-centred evaluation of capacity to make treatment decisions (DMC-T) and grounded within the clinician–patient relationship, could represent a legitimate and ethically coherent form of self-determination. After setting out background information on fluctuating capacity, mania and advance directives, this article proposes a framework for constructing such an SBD, and considers common objections, possible solutions and suggestions for future research. PMID:25939286

  8. Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission*♦

    PubMed Central

    Meinecke, Michael; Boucrot, Emmanuel; Camdere, Gamze; Hon, Wai-Ching; Mittal, Rohit; McMahon, Harvey T.

    2013-01-01

    Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission. PMID:23297414

  9. The Protein Partners of GTP Cyclohydrolase I in Rat Organs

    PubMed Central

    Du, Jianhai; Teng, Ru-Jeng; Lawrence, Matt; Guan, Tongju; Xu, Hao; Ge, Ying; Shi, Yang

    2012-01-01

    Objective GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis and has been shown to be a promising therapeutic target in ischemic heart disease, hypertension, atherosclerosis and diabetes. The endogenous GCH1-interacting partners have not been identified. Here, we determined endogenous GCH1-interacting proteins in rat. Methods and Results A pulldown and proteomics approach were used to identify GCH1 interacting proteins in rat liver, brain, heart and kidney. We demonstrated that GCH1 interacts with at least 17 proteins including GTP cyclohydrolase I feedback regulatory protein (GFRP) in rat liver by affinity purification followed by proteomics and validated six protein partners in liver, brain, heart and kidney by immunoblotting. GCH1 interacts with GFRP and very long-chain specific acyl-CoA dehydrogenase in the liver, tubulin beta-2A chain in the liver and brain, DnaJ homolog subfamily A member 1 and fatty aldehyde dehydrogenase in the liver, heart and kidney and eukaryotic translation initiation factor 3 subunit I (EIF3I) in all organs tested. Furthermore, GCH1 associates with mitochondrial proteins and GCH1 itself locates in mitochondria. Conclusion GCH1 interacts with proteins in an organ dependant manner and EIF3I might be a general regulator of GCH1. Our finding indicates GCH1 might have broader functions beyond tetrahydrobiopterin biosynthesis. PMID:22479495

  10. Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits

    PubMed Central

    Gao, Ning; Zavialov, Andrey V.; Ehrenberg, Måns; Frank, Joachim

    2008-01-01

    Summary After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a GTP-hydrolysis dependent manner. Based on a previous cryo-electron microscopy (cryo-EM) study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF containing post-termination ribosome triggers an inter-domain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9 Å (FSC at 0.5 cutoff) cryo-EM map of a 50S EFG GDPNP RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the non-cleavable GTP analogue GDPNP. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in tRNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G’s domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits. PMID:17996252

  11. A Novel Domain in Translational GTPase BipA Mediates Interaction with the 70S Ribosome and Influences GTP Hydrolysis

    SciTech Connect

    deLivron, M.; Makanji, H; Lane, M; Robinson, V

    2009-01-01

    BipA is a universally conserved prokaryotic GTPase that exhibits differential ribosome association in response to stress-related events. It is a member of the translation factor family of GTPases along with EF-G and LepA. BipA has five domains. The N-terminal region of the protein, consisting of GTPase and {beta}-barrel domains, is common to all translational GTPases. BipA domains III and V have structural counterparts in EF-G and LepA. However, the C-terminal domain (CTD) of the protein is unique to the BipA family. To investigate how the individual domains of BipA contribute to the biological properties of the protein, deletion constructs were designed and their GTP hydrolysis and ribosome binding properties assessed. Data presented show that removal of the CTD abolishes the ability of BipA to bind to the ribosome and that ribosome complex formation requires the surface provided by domains III and V and the CTD. Additional mutational analysis was used to outline the BipA-70S interaction surface extending across these domains. Steady state kinetic analyses revealed that successive truncation of domains from the C-terminus resulted in a significant increase in the intrinsic GTP hydrolysis rate and a loss of ribosome-stimulated GTPase activity. These results indicate that, similar to other translational GTPases, the ribosome binding and GTPase activities of BipA are tightly coupled. Such intermolecular regulation likely plays a role in the differential ribosome binding by the protein.

  12. Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii

    PubMed Central

    Singarapu, Kiran K.; Otte, Michele M.; Tonelli, Marco; Westler, William M.; Escalante-Semerena, Jorge C.; Markley, John L.

    2015-01-01

    GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time. PMID:26513744

  13. The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2-ezrin binding and dexamethasone stimulated NHE3 activity.

    PubMed

    Yang, Jianbo; Sarker, Rafiquel; Singh, Varsha; Sarker, Prateeti; Yin, Jianyi; Chen, Tian-E; Chaerkady, Raghothama; Li, Xuhang; Tse, C Ming; Donowitz, Mark

    2015-08-15

    In the brush border of intestinal and kidney epithelial cells, scaffolding proteins ezrin, Na(+)-H(+) exchanger regulatory factor (NHERF)1 and NHERF2 play important roles in linking transmembrane proteins to the cytoskeleton and assembling signalling regulatory complexes. The last 30 carboxyl residues of NHERF1 and NHERF2 form the EBDs [ezrin, radixin and moesin (ERM)-binding domain]. The current study found that NHERF1/2 contain an ERM-binding regulatory sequence (EBRS), which facilitates the interaction between the EBD and ezrin. The EBRSs are located within 24 and 19 residues immediately upstream of EBDs for NHERF1 and NHERF2 respectively. In OK (opossum kidney) epithelial cells, EBRSs are necessary along with the EBD to distribute NHERF1 and NHERF2 exclusively to the apical domain. Furthermore, phosphorylation of Ser(303) located in the EBRS of NHERF2, decreases the binding affinity for ezrin, dislocates apical NHERF2 into the cytosol and increases the NHERF2 microvillar mobility rate. Moreover, increased phosphorylation of Ser(303) was functionally significant preventing acute stimulation of NHE3 (Na(+)-H(+) exchanger 3) activity by dexamethasone. PMID:26251448

  14. The NHERF2 sequence adjacent and upstream of the ERM-binding domain affects NHERF2–ezrin binding and dexamethasone stimulated NHE3 activity

    PubMed Central

    Yang, Jianbo; Sarker, Rafiquel; Singh, Varsha; Sarker, Prateeti; Yin, Jianyi; Chen, Tian-E; Chaerkady, Raghothama; Li, Xuhang; Tse, C. Ming; Donowitz, Mark

    2015-01-01

    In the brush border of intestinal and kidney epithelial cells, scaffolding proteins ezrin, Na+-H+ exchanger regulatory factor (NHERF)1 and NHERF2 play important roles in linking transmembrane proteins to the cytoskeleton and assembling signalling regulatory complexes. The last 30 carboxyl residues of NHERF1 and NHERF2 form the EBDs [ezrin, radixin and moesin (ERM)-binding domain]. The current study found that NHERF1/2 contain an ERM-binding regulatory sequence (EBRS), which facilitates the interaction between the EBD and ezrin. The EBRSs are located within 24 and 19 residues immediately upstream of EBDs for NHERF1 and NHERF2 respectively. In OK (opossum kidney) epithelial cells, EBRSs are necessary along with the EBD to distribute NHERF1 and NHERF2 exclusively to the apical domain. Furthermore, phosphorylation of Ser303 located in the EBRS of NHERF2, decreases the binding affinity for ezrin, dislocates apical NHERF2 into the cytosol and increases the NHERF2 microvillar mobility rate. Moreover, increased phosphorylation of Ser303 was functionally significant preventing acute stimulation of NHE3 (Na+-H+ exchanger 3) activity by dexamethasone. PMID:26251448

  15. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    SciTech Connect

    Lindeberg, M.; Jacobs, M. )

    1989-04-01

    A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5{prime}-({gamma}-thio) triphosphate (GTP-{gamma}-S). Both the binding affinity and the pattern of binding specificity for GTP and GTP analogs are similar to animal G-proteins, and two zucchini membrane proteins are recognized in western blots by antiserum specific for the {sigma} subunit of platelet G{sub s} protein. GTP-{gamma}-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al{sup +3} and F{sup {minus}} agents known to activate G-proteins - decreased NPA specific binding by ca. 15%. In tests of in vitro auxin transport employing zucchini plasma membrane vesicles, AlF{sup {minus}}{sub 4} strongly inhibited {sup 3}H-indoleacetic acid nor accumulation; GTP-{gamma}-S effects on this system will be discussed.

  16. Imipramine treatment differentially affects platelet /sup 3/H-imipramine binding and serotonin uptake in depressed patients

    SciTech Connect

    Suranyi-Cadotte, B.E.; Quirion, R.; Nair, N.P.V.; Lafaille, F.; Schwartz, G.

    1985-02-25

    Uptake of serotonin and /sup 3/H-imipramine binding in platelets of depressed patients were investigated simultaneously with changes in clinical state. Both V/sub max/ for serotonin uptake and B/sub max/ for /sup 3/H-imipramine binding were significantly lower in unmedicated depressed patients with respect to normal subjects. Successful treatment with imipramine led to a significant increase in B/sub max/ for /sup 3/H-imipramine binding, without significant change in V/sub max/ for serotonin uptake. B/sub max/ values increased to the normal range following complete, rather than partial clinical improvement. These data indicate that successful antidepressant treatment may increase the density of /sup 3/H-imipramine binding sites on platelets by a process which is independent of the uptake of serotonin. 29 references, 1 table.

  17. Cooperative substrate binding by a diguanylate cyclase.

    PubMed

    Oliveira, Maycon C; Teixeira, Raphael D; Andrade, Maxuel O; Pinheiro, Glaucia M S; Ramos, Carlos H I; Farah, Chuck S

    2015-01-30

    XAC0610, from Xanthomonas citri subsp. citri, is a large multi-domain protein containing one GAF (cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA) domain, four PAS (Per-Arnt-Sim) domains and one GGDEF domain. This protein has a demonstrable in vivo and in vitro diguanylate cyclase (DGC) activity that leads to the production of cyclic di-GMP (c-di-GMP), a ubiquitous bacterial signaling molecule. Analysis of a XacΔ0610 knockout strain revealed that XAC0610 plays a role in the regulation of Xac motility and resistance to H2O2. Site-directed mutagenesis of a conserved DGC lysine residue (Lys759 in XAC0610) resulted in a severe reduction in XAC0610 DGC activity. Furthermore, experimental and in silico analyses suggest that XAC0610 is not subject to allosteric product inhibition, a common regulatory mechanism for DGC activity control. Instead, steady-state kinetics of XAC0610 DGC activity revealed a positive cooperative effect of the GTP substrate with a dissociation constant for the binding of the first GTP molecule (K1) approximately 5× greater than the dissociation constant for the binding of the second GTP molecule (K2). We present a general kinetics scheme that should be used when analyzing DGC kinetics data and propose that cooperative GTP binding could be a common, though up to now overlooked, feature of these enzymes that may in some cases offer a physiologically relevant mechanism for regulation of DGC activity in vivo. PMID:25463434

  18. Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP.

    PubMed

    Milstien, S; Jaffe, H; Kowlessur, D; Bonner, T I

    1996-08-16

    The activity of GTP cyclohydrolase I, the initial enzyme of the de novo pathway for biosynthesis of tetrahydrobiopterin, the cofactor required for aromatic amino acid hydroxylations and nitric oxide synthesis, is sensitive to end-product feedback inhibition by tetrahydrobiopterin. This inhibition by tetrahydrobiopterin is mediated by the GTP cyclohydrolase I feedback regulatory protein GFRP, previously named p35 (Harada, T., Kagamiyama, H., and Hatakeyama, K. (1993) Science 260, 1507-1510), and -phenylalanine specifically reverses the tetrahydrobiopterin-dependent inhibition. As a first step in the investigation of the physiological role of this unique mechanism of regulation, a convenient procedure has been developed to co-purify to homogeneity both GTP cyclohydrolase I and GFRP from rat liver. GTP cyclohydrolase I and GFRP exist in a complex which can be bound to a GTP-affinity column from which GTP cyclohydrolase I and GFRP are separately and selectively eluted. GFRP is dissociated from the GTP agarose-bound complex with 0.2 NaCl, a concentration of salt which also effectively blocks the tetrahydrobiopterin-dependent inhibitory activity of GFRP. GTP cyclohydrolase I is then eluted from the GTP-agarose column with GTP. Both GFRP and GTP cyclohydrolase I were then purified separately to near homogeneity by sequential high performance anion exchange and gel filtration chromatography. GFRP was found to have a native molecular mass of 20 kDa and consist of a homodimer of 9.5-kDa subunits. Based on peptide sequences obtained from purified GFRP, oligonucleotides were synthesized and used to clone a cDNA from a rat liver cDNA library by polymerase chain reaction-based methods. The cDNA contained an open reading frame that encoded a novel protein of 84 amino acids (calculated molecular mass 9665 daltons). This protein when expressed in Escherichia coli as a thioredoxin fusion protein had tetrahydrobiopterin-dependent GTP cyclohydrolase I inhibitory activity. Northern

  19. The fatty acid amide hydrolase C385A variant affects brain binding of the positron emission tomography tracer [11C]CURB

    PubMed Central

    Boileau, Isabelle; Tyndale, Rachel F; Williams, Belinda; Mansouri, Esmaeil; Westwood, Duncan J; Foll, Bernard Le; Rusjan, Pablo M; Mizrahi, Romina; De Luca, Vincenzo; Zhou, Qian; Wilson, Alan A; Houle, Sylvain; Kish, Stephen J; Tong, Junchao

    2015-01-01

    The common functional single-nucleotide polymorphism (rs324420, C385A) of the endocannabinoid inactivating enzyme fatty acid amide hydrolase (FAAH) has been associated with anxiety disorder relevant phenotype and risk for addictions. Here, we tested whether the FAAH polymorphism affects in vivo binding of the FAAH positron emission tomography (PET) probe [11C]CURB ([11C-carbonyl]-6-hydroxy-[1,10-biphenyl]-3-yl cyclohexylcarbamate (URB694)). Participants (n=24) completed one [11C]CURB/PET scan and were genotyped for rs324420. Relative to C/C (58%), A-allele carriers (42%) had 23% lower [11C]CURB binding (λk3) in brain. We report evidence that the genetic variant rs324420 in FAAH is associated with measurable differences in brain FAAH binding as per PET [11C]CURB measurement. PMID:26036940

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

    DOE PAGESBeta

    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

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

    SciTech Connect

    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 RNA 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.

  2. Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP.

    PubMed

    Bao, Jie; Ryu, Dewey D Y

    2005-02-20

    The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use. PMID:15643625

  3. Specific interactions between lactose repressor protein and DNA affected by ligand binding: ab initio molecular orbital calculations.

    PubMed

    Ohyama, Tatsuya; Hayakawa, Masato; Nishikawa, Shin; Kurita, Noriyuki

    2011-06-01

    Transcription mechanisms of gene information from DNA to mRNA are essentially controlled by regulatory proteins such as a lactose repressor (LacR) protein and ligand molecules. Biochemical experiments elucidated that a ligand binding to LacR drastically changes the mechanism controlled by LacR, although the effect of ligand binding has not been clarified at atomic and electronic levels. We here investigated the effect of ligand binding on the specific interactions between LacR and operator DNA by the molecular simulations combined with classical molecular mechanics and ab initio fragment molecular orbital methods. The results indicate that the binding of anti-inducer ligand strengthens the interaction between LacR and DNA, which is consistent with the fact that the binding of anti-inducer enhances the repression of gene transcription by LacR. It was also elucidated that hydrating water molecules existing between LacR and DNA contribute to the specific interactions between LacR and DNA. PMID:21328406

  4. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly.

    PubMed

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

    2014-01-01

    Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers-termed here escortins-to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. PMID:25144938

  5. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    PubMed Central

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

    2014-01-01

    Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers—termed here escortins—to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. DOI: http://dx.doi.org/10.7554/eLife.03473.001 PMID:25144938

  6. GTP Cyclohydrolase I Expression, Protein, and Activity Determine Intracellular Tetrahydrobiopterin Levels, Independent of GTP Cyclohydrolase Feedback Regulatory Protein Expression

    PubMed Central

    Tatham, Amy L.; Crabtree, Mark J.; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J.; Channon, Keith M.

    2009-01-01

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r2 = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r2 = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  7. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression. PMID:19286659

  8. Phosphorylation of serine residues affects the conformation of the calmodulin binding domain of human protein 4.1.

    PubMed

    Vetter, S W; Leclerc, E

    2001-08-01

    We have previously characterized the calcium-dependent calmodulin (CaM)-binding domain (Ser76-Ser92) of the 135-kDa human protein 4.1 isoform using fluorescence spectroscopy and chemically synthesized nonphosphorylated or serine phosphorylated peptides [Leclerc, E. & Vetter, S. (1998) Eur. J. Biochem. 258, 567-671]. Here we demonstrate that phosphorylation of two serine residues within the 17-residue peptide alters their ability to adopt alpha helical conformation in a position-dependent manner. The helical content of the peptides was determined by CD-spectroscopy and found to increase from 36 to 45% for the Ser80 phosphorylated peptide and reduce to 28% for the Ser84 phosphorylated peptide; the di-phosphorylated peptide showed 32% helical content. Based on secondary structure prediction methods we propose that initial helix formation involves the central residues Leu82-Phe86. The ability of the peptides to adopt alpha helical conformations did not correlate with the observed binding affinities to CaM. We suggest that the reduced CaM-binding affinities observed for the phosphorylated peptides are more likely to be the result of unfavorable sterical and electrostatic interactions introduced into the CaM peptide-binding interface by the phosphate groups, rather than being due to the effect of phosphorylation on the secondary structure of the peptides. PMID:11488924

  9. A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions.

    PubMed

    Swick, Lance; Kapatos, Gregory

    2006-06-01

    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 alpha-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal alpha-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal alpha-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

  10. Alteration of tropomyosin-binding properties of tropomodulin-1 affects its capping ability and localization in skeletal myocytes.

    PubMed

    Moroz, Natalia A; Novak, Stefanie M; Azevedo, Ricardo; Colpan, Mert; Uversky, Vladimir N; Gregorio, Carol C; Kostyukova, Alla S

    2013-02-15

    Tropomodulin (Tmod) is an actin-capping protein that binds to the two tropomyosins (TM) at the pointed end of the actin filament to prevent further actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying actin filament dependent processes such as muscle contraction and intracellular transport. The capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1-4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of actin pointed-end polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the actin-capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the actin filament depends on its TM-binding affinity. PMID:23271735

  11. Alteration of Tropomyosin-binding Properties of Tropomodulin-1 Affects Its Capping Ability and Localization in Skeletal Myocytes*

    PubMed Central

    Moroz, Natalia A.; Novak, Stefanie M.; Azevedo, Ricardo; Colpan, Mert; Uversky, Vladimir N.; Gregorio, Carol C.; Kostyukova, Alla S.

    2013-01-01

    Tropomodulin (Tmod) is an actin-capping protein that binds to the two tropomyosins (TM) at the pointed end of the actin filament to prevent further actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying actin filament dependent processes such as muscle contraction and intracellular transport. The capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1–4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of actin pointed-end polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the actin-capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the actin filament depends on its TM-binding affinity. PMID:23271735

  12. Phosphorylation Affects DNA-Binding of the Senescence-Regulating bZIP Transcription Factor GBF1

    PubMed Central

    Smykowski, Anja; Fischer, Stefan M.; Zentgraf, Ulrike

    2015-01-01

    Massive changes in the transcriptome of Arabidopsis thaliana during onset and progression of leaf senescence imply a central role for transcription factors. While many transcription factors are themselves up- or down-regulated during senescence, the bZIP transcription factor G-box-binding factor 1 (GBF1/bZIP41) is constitutively expressed in Arabidopsis leaf tissue but at the same time triggers the onset of leaf senescence, suggesting posttranscriptional mechanisms for senescence-specific GBF1 activation. Here we show that GBF1 is phosphorylated by the threonine/serine CASEIN KINASE II (CKII) in vitro and that CKII phosphorylation had a negative effect on GBF1 DNA-binding to G-boxes of two direct target genes, CATALASE2 and RBSCS1a. Phosphorylation mimicry at three serine positions in the basic region of GBF1 also had a negative effect on DNA-binding. Kinase assays revealed that CKII phosphorylates at least one serine in the basic domain but has additional phosphorylation sites outside this domain. Two different ckII α subunit1 and one α subunit2 T-DNA insertion lines showed no visible senescence phenotype, but in all lines the expression of the senescence marker gene SAG12 was remarkably diminished. A model is presented suggesting that senescence-specific GBF1 activation might be achieved by lowering the phosphorylation of GBF1 by CKII. PMID:27135347

  13. The kinesin-13 KLP10A motor regulates oocyte spindle length and affects EB1 binding without altering microtubule growth rates.

    PubMed

    Do, Kevin K; Hoàng, Kim Liên; Endow, Sharyn A

    2014-01-01

    Kinesin-13 motors are unusual in that they do not walk along microtubules, but instead diffuse to the ends, where they remove tubulin dimers, regulating microtubule dynamics. Here we show that Drosophila kinesin-13 klp10A regulates oocyte meiosis I spindle length and is haplo-insufficient - KLP10A, reduced by RNAi or a loss-of-function P element insertion mutant, results in elongated and mispositioned oocyte spindles, and abnormal cortical microtubule asters and aggregates. KLP10A knockdown by RNAi does not significantly affect microtubule growth rates in oocyte spindles, but, unexpectedly, EB1 binding and unbinding are slowed, suggesting a previously unobserved role for kinesin-13 in mediating EB1 binding interactions with microtubules. Kinesin-13 may regulate spindle length both by disassembling subunits from microtubule ends and facilitating EB1 binding to plus ends. We also observe an increased number of paused microtubules in klp10A RNAi knockdown spindles, consistent with a reduced frequency of microtubule catastrophes. Overall, our findings indicate that reduced kinesin-13 decreases microtubule disassembly rates and affects EB1 interactions with microtubules, rather than altering microtubule growth rates, causing spindles to elongate and abnormal cortical microtubule asters and aggregates to form. PMID:24907370

  14. The structure of YqeH: An AtNOS1/AtNOA1 ortholog that couples GTP hydrolysis to molecular recognition

    SciTech Connect

    Sudhamsu, J.; Lee, G.I.; Klessig, D.F.; Crane, B.R.

    2009-03-27

    AtNOS1/AtNOA1 was identified as a nitric oxide-generating enzyme in plants, but that function has recently been questioned. To resolve issues surrounding AtNOA1 activity, we report the biochemical properties and a 2.36 {angstrom} resolution crystal structure of a bacterial AtNOA1 ortholog (YqeH). Geobacillus YqeH fused to a putative AtNOA1 leader peptide complements growth and morphological defects of Atnoa1 mutant plants. YqeH does not synthesize nitric oxide from L-arginine but rather hydrolyzes GTP. The YqeH structure reveals a circularly permuted GTPase domain and an unusual C-terminal {beta}-domain. A small N-terminal domain, disordered in the structure, binds zinc. Structural homology among the C-terminal domain, the RNA-binding regulator TRAP, and the hypoxia factor pVHL define a recognition module for peptides and nucleic acids. TRAP residues important for RNA binding are conserved by the YqeH C-terminal domain, whose positioning is coupled to GTP hydrolysis. YqeH and AtNOA1 probably act as G-proteins that regulate nucleic acid recognition and not as nitric-oxide synthases.

  15. Spindle Assembly in the Absence of a RanGTP Gradient Requires Localized CPC Activity

    PubMed Central

    Maresca, Thomas J.; Groen, Aaron C.; Gatlin, Jesse C.; Ohi, Ryoma; Mitchison, Timothy J.; Salmon, Edward D.

    2009-01-01

    Summary During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin [1, 2]. It is generally accepted that this RanGTP gradient is essential for organizing the spindle since it locally activates critical spindle assembly factors [3–5]. Here, we show in Xenopus egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin, and a CPC-dependent signal predominantly generated from centromeric chromatin. PMID:19540121

  16. Identification of a second GTP-bound magnesium ion in archaeal initiation factor 2

    PubMed Central

    Dubiez, Etienne; Aleksandrov, Alexey; Lazennec-Schurdevin, Christine; Mechulam, Yves; Schmitt, Emmanuelle

    2015-01-01

    Eukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eukaryotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In archaea, orthologs of eIF5 are not found and aIF2 GTPase activity is thought to be non-assisted. However, no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2γ, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2γ. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2. PMID:25690901

  17. Cardiomyocyte GTP Cyclohydrolase 1 Protects the Heart Against Diabetic Cardiomyopathy.

    PubMed

    Wu, Hsiang-En; Baumgardt, Shelley L; Fang, Juan; Paterson, Mark; Liu, Yanan; Du, Jianhai; Shi, Yang; Qiao, Shigang; Bosnjak, Zeljko J; Warltier, David C; Kersten, Judy R; Ge, Zhi-Dong

    2016-01-01

    Diabetic cardiomyopathy increases the risk of heart failure and death. At present, there are no effective approaches to preventing its development in the clinic. Here we report that reduction of cardiac GTP cyclohydrolase 1 (GCH1) degradation by genetic and pharmacological approaches protects the heart against diabetic cardiomyopathy. Diabetic cardiomyopathy was induced in C57BL/6 wild-type mice and transgenic mice with cardiomyocyte-specific overexpression of GCH1 with streptozotocin, and control animals were given citrate buffer. We found that diabetes-induced degradation of cardiac GCH1 proteins contributed to adverse cardiac remodeling and dysfunction in C57BL/6 mice, concomitant with decreases in tetrahydrobiopterin, dimeric and phosphorylated neuronal nitric oxide synthase, sarcoplasmic reticulum Ca(2+) handling proteins, intracellular [Ca(2+)]i, and sarcoplasmic reticulum Ca(2+) content and increases in phosphorylated p-38 mitogen-activated protein kinase and superoxide production. Interestingly, GCH-1 overexpression abrogated these detrimental effects of diabetes. Furthermore, we found that MG 132, an inhibitor for 26S proteasome, preserved cardiac GCH1 proteins and ameliorated cardiac remodeling and dysfunction during diabetes. This study deepens our understanding of impaired cardiac function in diabetes, identifies GCH1 as a modulator of cardiac remodeling and function, and reveals a new therapeutic target for diabetic cardiomyopathy. PMID:27295516

  18. Identification of Proteins Interacting with GTP Cyclohydrolase I

    PubMed Central

    Du, Jianhai; Xu, Hao; Wei, Na; Wakim, Bassam; Halligan, Brian; Pritchard, Kirkwood A.; Shi, Yang

    2009-01-01

    GTP cyclohydrolase I (GCH-1) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, an essential cofactor for nitric oxide synthase and aromatic amino acid hydroxylase. To explore the interactome of GCH-1, we established a HEK293 cell line stably expressing tetracycline-inducible FLAG-GCH-1. FLAG-GCH-1 and associated proteins were immunoprecipitated and analyzed by liquid chromatography/tandem mass spectrometry. Twenty-nine proteins, derived from different subcellular components such as cytosol, membranes, nucleus and mitochondria were identified to interact with GCH-1. Cell fractionation studies also showed that GCH-1 was present in the cytosol, membranes and nucleus. Gene ontology analysis revealed that GCH-1 interactome was involved in a variety of biological processes such as signal transduction, apoptosis, metabolism, transport and cell organization. To our knowledge, this study is the first to provide a comprehensive analysis of the GCH-1 interactome. Findings expand the number and diversity of proteins that are known to associate with GCH-1. PMID:19442649

  19. Interaction of human GTP cyclohydrolase I with its splice variants

    PubMed Central

    Pandya, Maya J.; Golderer, Georg; Werner, Ernst R.; Werner-Felmayer, Gabriele

    2006-01-01

    Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity. PMID:16848765

  20. Cardiomyocyte GTP Cyclohydrolase 1 Protects the Heart Against Diabetic Cardiomyopathy

    PubMed Central

    Wu, Hsiang-En; Baumgardt, Shelley L.; Fang, Juan; Paterson, Mark; Liu, Yanan; Du, Jianhai; Shi, Yang; Qiao, Shigang; Bosnjak, Zeljko J.; Warltier, David C.; Kersten, Judy R.; Ge, Zhi-Dong

    2016-01-01

    Diabetic cardiomyopathy increases the risk of heart failure and death. At present, there are no effective approaches to preventing its development in the clinic. Here we report that reduction of cardiac GTP cyclohydrolase 1 (GCH1) degradation by genetic and pharmacological approaches protects the heart against diabetic cardiomyopathy. Diabetic cardiomyopathy was induced in C57BL/6 wild-type mice and transgenic mice with cardiomyocyte-specific overexpression of GCH1 with streptozotocin, and control animals were given citrate buffer. We found that diabetes-induced degradation of cardiac GCH1 proteins contributed to adverse cardiac remodeling and dysfunction in C57BL/6 mice, concomitant with decreases in tetrahydrobiopterin, dimeric and phosphorylated neuronal nitric oxide synthase, sarcoplasmic reticulum Ca2+ handling proteins, intracellular [Ca2+]i, and sarcoplasmic reticulum Ca2+ content and increases in phosphorylated p-38 mitogen-activated protein kinase and superoxide production. Interestingly, GCH-1 overexpression abrogated these detrimental effects of diabetes. Furthermore, we found that MG 132, an inhibitor for 26S proteasome, preserved cardiac GCH1 proteins and ameliorated cardiac remodeling and dysfunction during diabetes. This study deepens our understanding of impaired cardiac function in diabetes, identifies GCH1 as a modulator of cardiac remodeling and function, and reveals a new therapeutic target for diabetic cardiomyopathy. PMID:27295516

  1. NLRP7 affects trophoblast lineage differentiation, binds to overexpressed YY1 and alters CpG methylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maternal-effect mutations in NLRP7 cause rare biparentally inherited hydatidiform moles (BiHMs), abnormal pregnancies containing hypertrophic vesicular trophoblast but no embryo. BiHM trophoblasts display abnormal DNA methylation patterns affecting maternally methylated germline differentially methy...

  2. Hypertrophic cardiomyopathy mutations in the calponin-homology domain of ACTN2 affect actin binding and cardiomyocyte Z-disc incorporation

    PubMed Central

    Haywood, Natalie J.; Wolny, Marcin; Rogers, Brendan; Trinh, Chi H.; Shuping, Yu; Edwards, Thomas A.; Peckham, Michelle

    2016-01-01

    α-Actinin-2 (ACTN2) is the only muscle isoform of α-actinin expressed in cardiac muscle. Mutations in this protein have been implicated in mild to moderate forms of hypertrophic cardiomyopathy (HCM). We have investigated the effects of two mutations identified from HCM patients, A119T and G111V, on the secondary and tertiary structure of a purified actin binding domain (ABD) of ACTN2 by circular dichroism and X-ray crystallography, and show small but distinct changes for both mutations. We also find that both mutants have reduced F-actin binding affinity, although the differences are not significant. The full length mEos2 tagged protein expressed in adult cardiomyocytes shows that both mutations additionally affect Z-disc localization and dynamic behaviour. Overall, these two mutations have small effects on structure, function and behaviour, which may contribute to a mild phenotype for this disease. PMID:27287556

  3. Inactivation of Cyclic Di-GMP Binding Protein TDE0214 Affects the Motility, Biofilm Formation, and Virulence of Treponema denticola

    PubMed Central

    Bian, Jiang; Liu, Xiangyang; Cheng, Yi-Qiang

    2013-01-01

    As a ubiquitous second messenger, cyclic dimeric GMP (c-di-GMP) has been studied in numerous bacteria. The oral spirochete Treponema denticola, a periodontal pathogen associated with human periodontitis, has a complex c-di-GMP signaling network. However, its function remains unexplored. In this report, a PilZ-like c-di-GMP binding protein (TDE0214) was studied to investigate the role of c-di-GMP in the spirochete. TDE0214 harbors a PilZ domain with two signature motifs: RXXXR and DXSXXG. Biochemical studies showed that TDE0214 binds c-di-GMP in a specific manner, with a dissociation constant (Kd) value of 1.73 μM, which is in the low range compared to those of other reported c-di-GMP binding proteins. To reveal the role of c-di-GMP in T. denticola, a TDE0214 deletion mutant (TdΔ214) was constructed and analyzed in detail. First, swim plate and single-cell tracking analyses showed that TdΔ214 had abnormal swimming behaviors: the mutant was less motile and reversed more frequently than the wild type. Second, we found that biofilm formation of TdΔ214 was substantially repressed (∼6.0-fold reduction). Finally, in vivo studies using a mouse skin abscess model revealed that the invasiveness and ability to induce skin abscesses and host humoral immune responses were significantly attenuated in TdΔ214, indicative of the impact that TDE0214 has on the virulence of T. denticola. Collectively, the results reported here indicate that TDE0214 plays important roles in motility, biofilm formation, and virulence of the spirochete. This report also paves a way to further unveil the roles of the c-di-GMP signaling network in the biology and pathogenicity of T. denticola. PMID:23794624

  4. Glycosylation of Skp1 affects its conformation and promotes binding to a model f-box protein.

    PubMed

    Sheikh, M Osman; Schafer, Christopher M; Powell, John T; Rodgers, Karla K; Mooers, Blaine H M; West, Christopher M

    2014-03-18

    In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline 143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2 regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies. Interaction studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1 revealed preferential binding to the glycosylated isoforms of Skp1. This difference correlated with the increased α-helical and decreased β-sheet content of glycosylated Skp1s based on circular dichroism and increased folding order based on small-angle X-ray scattering. A comparison of the molecular envelopes of fully glycosylated Skp1 and the apoprotein indicated that both isoforms exist as an antiparallel dimer that is more compact and extended in the glycosylated state. Analytical gel filtration and chemical cross-linking studies showed a growing tendency of less modified isoforms to dimerize. Considering that regions of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds when bound to F-box proteins, we propose that glycosylation, which occurs adjacent to the F-box binding site, influences the spectrum of energetically similar conformations that vary inversely in their propensity to dock with Fbs1 or another Skp1. Glycosylation may thus influence Skp1 function by modulating F-box protein binding in cells. PMID:24506136

  5. ACBD3 Interaction with TBC1 Domain 22 Protein Is Differentially Affected by Enteroviral and Kobuviral 3A Protein Binding

    PubMed Central

    Greninger, Alexander L.; Knudsen, Giselle M.; Betegon, Miguel; Burlingame, Alma L.; DeRisi, Joseph L.

    2013-01-01

    ABSTRACT Despite wide sequence divergence, multiple picornaviruses use the Golgi adaptor acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GCP60) to recruit phosphatidylinositol 4-kinase class III beta (PI4KIIIβ/PI4KB), a factor required for viral replication. The molecular basis of this convergent interaction and the cellular function of ACBD3 are not fully understood. Using affinity purification-mass spectrometry, we identified the putative Rab33 GTPase-activating proteins TBC1D22A and TBC1D22B as ACBD3-interacting factors. Fine-scale mapping of binding determinants within ACBD3 revealed that the interaction domains for TBC1D22A/B and PI4KB are identical. Affinity purification confirmed that PI4KB and TBC1D22A/B interactions with ACBD3 are mutually exclusive, suggesting a possible regulatory mechanism for recruitment of PI4KB. The C-terminal Golgi dynamics (GOLD) domain of ACBD3 has been previously shown to bind the 3A replication protein from Aichi virus. We find that the 3A proteins from several additional picornaviruses, including hepatitis A virus, human parechovirus 1, and human klassevirus, demonstrate an interaction with ACBD3 by mammalian two-hybrid assay; however, we also find that the enterovirus and kobuvirus 3A interactions with ACBD3 are functionally distinct with respect to TBC1D22A/B and PI4KB recruitment. These data reinforce the notion that ACBD3 organizes numerous cellular functionalities and that RNA virus replication proteins likely modulate these interactions by more than one mechanism. PMID:23572552

  6. Glycosylation of Skp1 Affects Its Conformation and Promotes Binding to a Model F-Box Protein

    PubMed Central

    2015-01-01

    In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline 143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2 regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies. Interaction studies with the soluble mammalian F-box protein Fbs1/Fbg1/OCP1 revealed preferential binding to the glycosylated isoforms of Skp1. This difference correlated with the increased α-helical and decreased β-sheet content of glycosylated Skp1s based on circular dichroism and increased folding order based on small-angle X-ray scattering. A comparison of the molecular envelopes of fully glycosylated Skp1 and the apoprotein indicated that both isoforms exist as an antiparallel dimer that is more compact and extended in the glycosylated state. Analytical gel filtration and chemical cross-linking studies showed a growing tendency of less modified isoforms to dimerize. Considering that regions of free Skp1 are intrinsically disordered and Skp1 can adopt distinct folds when bound to F-box proteins, we propose that glycosylation, which occurs adjacent to the F-box binding site, influences the spectrum of energetically similar conformations that vary inversely in their propensity to dock with Fbs1 or another Skp1. Glycosylation may thus influence Skp1 function by modulating F-box protein binding in cells. PMID:24506136

  7. von Hippel–Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

    PubMed Central

    Mousnier, Aurélie; Kubat, Nicole; Massias-Simon, Aurélie; Ségéral, Emmanuel; Rain, Jean-Christophe; Benarous, Richard; Emiliani, Stéphane; Dargemont, Catherine

    2007-01-01

    HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin–proteasome pathway. Here, we identify von Hippel–Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel–Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin–VHL–proteasome pathway in the integration–transcription transition of the viral replication cycle. PMID:17698809

  8. Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus.

    PubMed

    Schrauwen, Eefje J A; Richard, Mathilde; Burke, David F; Rimmelzwaan, Guus F; Herfst, Sander; Fouchier, Ron A M

    2016-01-01

    Receptor-binding preference and stability of hemagglutinin have been implicated as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for α2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission. PMID:26792744

  9. Inhibition of GDP/GTP exchange on G alpha subunits by proteins containing G-protein regulatory motifs.

    PubMed

    Natochin, M; Gasimov, K G; Artemyev, N O

    2001-05-01

    A novel Galpha binding consensus sequence, termed G-protein regulatory (GPR) or GoLoco motif, has been identified in a growing number of proteins, which are thought to modulate G-protein signaling. Alternative roles of GPR proteins as nucleotide exchange factors or as GDP dissociation inhibitors for Galpha have been proposed. We investigated the modulation of the GDP/GTP exchange of Gialpha(1), Goalpha, and Gsalpha by three proteins containing GPR motifs (GPR proteins), LGN-585-642, Pcp2, and RapIGAPII-23-131, to elucidate the mechanisms of GPR protein function. The GPR proteins displayed similar patterns of interaction with Gialpha(1) with the following order of affinities: Gialpha(1)GDP > Gialpha(1)GDPAlF(4)(-) > or = Gialpha(1)GTPgammaS. No detectable binding of the GPR proteins to Gsalpha was observed. LGN-585-642, Pcp2, and RapIGAPII-23-131 inhibited the rates of spontaneous GTPgammaS binding and blocked GDP release from Gialpha(1) and Goalpha. The inhibitory effects of the GPR proteins on Gialpha(1) were significantly more potent, indicating that Gi might be a preferred target for these modulators. Our results suggest that GPR proteins are potent GDP dissociation inhibitors for Gialpha-like Galpha subunits in vitro, and in this capacity they may inhibit GPCR/Gi protein signaling in vivo. PMID:11318657

  10. Mutants affecting nucleotide recognition by T7 DNA polymerase.

    PubMed

    Donlin, M J; Johnson, K A

    1994-12-13

    Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in

  11. DNA Binding by Sgf11 Protein Affects Histone H2B Deubiquitination by Spt-Ada-Gcn5-Acetyltransferase (SAGA)*

    PubMed Central

    Koehler, Christian; Bonnet, Jacques; Stierle, Matthieu; Romier, Christophe; Devys, Didier; Kieffer, Bruno

    2014-01-01

    The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is a transcription coactivator that contains a histone H2B deubiquitination activity mediated by its Ubp8 subunit. Full enzymatic activity requires the formation of a quaternary complex, the deubiquitination module (DUBm) of SAGA, which is composed of Ubp8, Sus1, Sgf11, and Sgf73. The crystal structures of the DUBm have shed light on the structure/function relationship of this complex. Specifically, both Sgf11 and Sgf73 contain zinc finger domains (ZnF) that appear essential for the DUBm activity. Whereas Sgf73 N-terminal ZnF is important for DUBm stability, Sgf11 C-terminal ZnF appears to be involved in DUBm function. To further characterize the role of these two zinc fingers, we have solved their structure by NMR. We show that, contrary to the previously reported structures, Sgf73 ZnF adopts a C2H2 coordination with unusual tautomeric forms for the coordinating histidines. We further report that the Sgf11 ZnF, but not the Sgf73 ZnF, binds to nucleosomal DNA with a binding interface composed of arginine residues located within the ZnF α-helix. Mutational analyses both in vitro and in vivo provide evidence for the functional relevance of our structural observations. The combined interpretation of our results leads to an uncommon ZnF-DNA interaction between the SAGA DUBm and nucleosomes, thus providing further functional insights into SAGA's epigenetic modulation of the chromatin structure. PMID:24509845

  12. TRICHOME BIREFRINGENCE-LIKE27 affects aluminum sensitivity by modulating the O-acetylation of xyloglucan and aluminum-binding capacity in Arabidopsis.

    PubMed

    Zhu, Xiao Fang; Sun, Ying; Zhang, Bao Cai; Mansoori, Nasim; Wan, Jiang Xue; Liu, Yu; Wang, Zhi Wei; Shi, Yuan Zhi; Zhou, Yi Hua; Zheng, Shao Jian

    2014-09-01

    Xyloglucan (XyG) has been reported to contribute to the aluminum (Al)-binding capacity of the cell wall in Arabidopsis (Arabidopsis thaliana). However, the influence of O-acetylation of XyG, accomplished by the putative O-acetyltransferase TRICHOME BIREFRINGENCE-LIKE27 (TBL27 [AXY4]), on its Al-binding capacity is not known. In this study, we found that the two corresponding TBL27 mutants, axy4-1 and axy4-3, were more Al sensitive than wild-type Columbia-0 plants. TBL27 was expressed in roots as well as in leaves, stems, flowers, and siliques. Upon Al treatment, even within 30 min, TBL27 transcript accumulation was strongly down-regulated. The mutants axy4-1 and axy4-3 accumulated significantly more Al in the root and wall, which could not be correlated with pectin content or pectin methylesterase activity, as no difference in the mutants was observed compared with the wild type when exposed to Al stress. The increased Al accumulation in the wall of the mutants was found to be in the hemicellulose fraction. While the total sugar content of the hemicellulose fraction did not change, the O-acetylation level of XyG was reduced by Al treatment. Taken together, we conclude that modulation of the O-acetylation level of XyG influences the Al sensitivity in Arabidopsis by affecting the Al-binding capacity in the hemicellulose. PMID:25006026

  13. COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation.

    PubMed

    Pérez-Pérez, Rafael; Lobo-Jarne, Teresa; Milenkovic, Dusanka; Mourier, Arnaud; Bratic, Ana; García-Bartolomé, Alberto; Fernández-Vizarra, Erika; Cadenas, Susana; Delmiro, Aitor; García-Consuegra, Inés; Arenas, Joaquín; Martín, Miguel A; Larsson, Nils-Göran; Ugalde, Cristina

    2016-08-30

    Mitochondrial respiratory chain (MRC) complexes I, III, and IV associate into a variety of supramolecular structures known as supercomplexes and respirasomes. While COX7A2L was originally described as a supercomplex-specific factor responsible for the dynamic association of complex IV into these structures to adapt MRC function to metabolic variations, this role has been disputed. Here, we further examine the functional significance of COX7A2L in the structural organization of the mammalian respiratory chain. As in the mouse, human COX7A2L binds primarily to free mitochondrial complex III and, to a minor extent, to complex IV to specifically promote the stabilization of the III2+IV supercomplex without affecting respirasome formation. Furthermore, COX7A2L does not affect the biogenesis, stabilization, and function of the individual oxidative phosphorylation complexes. These data show that independent regulatory mechanisms for the biogenesis and turnover of different MRC supercomplex structures co-exist. PMID:27545886

  14. Binding of the wheat germ lectin to Cryptococcus neoformans chitooligomers affects multiple mechanisms required for fungal pathogenesis

    PubMed Central

    Fonseca, Fernanda L.; Guimarães, Allan J.; Kmetzsch, Lívia; Dutra, Fabianno F.; Silva, Fernanda D.; Taborda, Carlos P.; Araujo, Glauber de S.; Frases, Susana; Staats, Charley C.; Bozza, Marcelo T.; Schrank, Augusto; Vainstein, Marilene H.; Nimrichter, Leonardo; Casadevall, Arturo; Rodrigues, Marcio L.

    2015-01-01

    The principal capsular component of Cryptococcus neoformans, glucuronoxylomannan (GXM), interacts with surface glycans, including chitin-like oligomers. Although the role of GXM in cryptococcal infection has been well explored, there is no information on how chitooligomers affect fungal pathogenesis. In this study, surface chitooligomers of C. neoformans were blocked through the use of the wheat germ lectin (WGA) and the effects on animal pathogenesis, interaction with host cells, fungal growth and capsule formation were analyzed. Treatment of C. neoformans cells with WGA followed by infection of mice delayed mortality relative to animals infected with untreated fungal cells. This observation was associated with reduced brain colonization by lectin-treated cryptococci. Blocking chitooligomers also rendered yeast cells less efficient in their ability to associate with phagocytes. WGA did not affect fungal viability, but inhibited GXM release to the extracellular space and capsule formation. In WGA-treated yeast cells, genes that are involved in capsule formation and GXM traffic had their transcription levels decreased in comparison with untreated cells. Our results suggest that cellular pathways required for capsule formation and pathogenic mechanisms are affected by blocking chitin-derived structures at the cell surface of C. neoformans. Targeting chitooligomers with specific ligands may reveal new therapeutic alternatives to control cryptococcosis. PMID:23608320

  15. The Lipid Kinase PI5P4Kβ Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.

    PubMed

    Sumita, Kazutaka; Lo, Yu-Hua; Takeuchi, Koh; Senda, Miki; Kofuji, Satoshi; Ikeda, Yoshiki; Terakawa, Jumpei; Sasaki, Mika; Yoshino, Hirofumi; Majd, Nazanin; Zheng, Yuxiang; Kahoud, Emily Rose; Yokota, Takehiro; Emerling, Brooke M; Asara, John M; Ishida, Tetsuo; Locasale, Jason W; Daikoku, Takiko; Anastasiou, Dimitrios; Senda, Toshiya; Sasaki, Atsuo T

    2016-01-21

    While cellular GTP concentration dramatically changes in response to an organism's cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kβ, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kβ preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation, and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kβ is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kβ is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kβ. The critical role of the GTP-sensing activity of PI5P4Kβ in cancer signifies this lipid kinase as a cancer therapeutic target. PMID:26774281

  16. 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.

  17. Redistribution of the Lamin B1 genomic binding profile affects rearrangement of heterochromatic domains and SAHF formation during senescence

    PubMed Central

    Sadaie, Mahito; Salama, Rafik; Carroll, Thomas; Tomimatsu, Kosuke; Chandra, Tamir; Young, Andrew R.J.; Narita, Masako; Pérez-Mancera, Pedro A.; Bennett, Dorothy C.; Chong, Heung; Kimura, Hiroshi; Narita, Masashi

    2013-01-01

    Senescence is a stress-responsive form of stable cell cycle exit. Senescent cells have a distinct gene expression profile, which is often accompanied by the spatial redistribution of heterochromatin into senescence-associated heterochromatic foci (SAHFs). Studying a key component of the nuclear lamina lamin B1 (LMNB1), we report dynamic alterations in its genomic profile and their implications for SAHF formation and gene regulation during senescence. Genome-wide mapping reveals that LMNB1 is depleted during senescence, preferentially from the central regions of lamina-associated domains (LADs), which are enriched for Lys9 trimethylation on histone H3 (H3K9me3). LMNB1 knockdown facilitates the spatial relocalization of perinuclear H3K9me3-positive heterochromatin, thus promoting SAHF formation, which could be inhibited by ectopic LMNB1 expression. Furthermore, despite the global reduction in LMNB1 protein levels, LMNB1 binding increases during senescence in a small subset of gene-rich regions where H3K27me3 also increases and gene expression becomes repressed. These results suggest that LMNB1 may contribute to senescence in at least two ways due to its uneven genome-wide redistribution: first, through the spatial reorganization of chromatin and, second, through gene repression. PMID:23964094

  18. rRNA Pseudouridylation Defects Affect Ribosomal Ligand Binding and Translational Fidelity from Yeast to Human Cells

    PubMed Central

    Jack, Karen; Bellodi, Cristian; Landry, Dori M.; Niederer, Rachel; Meskauskas, Arturas; Musalgaonkar, Sharmishtha; Kopmar, Noam; Krasnykh, Olya; Dean, Alison M.; Thompson, Sunnie R.; Ruggero, Davide; Dinman, Jonathan D.

    2011-01-01

    Summary How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked Dyskeratosis Congenita (X-DC) and Hoyeraal-Hreidarsson syndrome (HH). Here we characterize ribosomes isolated from a yeast strain where Cbf5p, the yeast homologue of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A- and P-sites as well as the cricket paralysis virus IRES (Internal Ribosome Entry Site), which interacts with both the P- and E-sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans. PMID:22099312

  19. Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function

    PubMed Central

    D’Agostino, Vito Giuseppe; Lal, Preet; Mantelli, Barbara; Tiedje, Christopher; Zucal, Chiara; Thongon, Natthakan; Gaestel, Matthias; Latorre, Elisa; Marinelli, Luciana; Seneci, Pierfausto; Amadio, Marialaura; Provenzani, Alessandro

    2015-01-01

    Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells. PMID:26553968

  20. Mutation in the primer binding site of the type 1 human immunodeficiency virus genome affects virus production and infectivity.

    PubMed Central

    Nagashunmugam, T; Velpandi, A; Goldsmith, C S; Zaki, S R; Kalyanaraman, V S; Srinivasan, A

    1992-01-01

    In an effort to understand the contribution of the primer-binding site (PBS) region to human immunodeficiency virus (HIV) replication, we have constructed a mutant HIV proviral DNA with an alteration in the 5' end of the PBS. The PBS mutant proviral DNA was characterized by transfection of the viral DNA into CD4+ and non-CD4+ target cells. The results indicate that mutation in the PBS reduced the level of viral particles released into the medium of transfected cells in comparison to wild-type proviral DNA. The viral particles were noninfectious upon transmission to established CD4+ cell lines and phytohemagglutinin-stimulated peripheral blood lymphocytes. Electron microscopic analysis of the transfected cells revealed no abnormalities in the structure of the virion directed by the mutant proviral DNA. Also, the protein and RNA contents of the mutant virions were similar to the wild type. The quantitation of intracellular viral structural protein in the transfected cells, however, indicated that the PBS mutation may have an effect on the assembly of viral particles in addition to completely abolishing reverse transcription of viral RNA into DNA. These results provide evidence that the PBS region of the viral genome has multiple functions in HIV-1 replication. Images PMID:1373895

  1. Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein.

    PubMed

    Werner, Ernst R; Bahrami, Soheyl; Heller, Regine; Werner-Felmayer, Gabriele

    2002-03-22

    GTP cyclohydrolase I feedback regulatory protein (GFRP) is a 9.7-kDa protein regulating GTP cyclohydrolase I activity in dependence of tetrahydrobiopterin and phenylalanine concentrations, thus enabling stimulation of tetrahydrobiopterin biosynthesis by phenylalanine to ensure its efficient metabolism by phenylalanine hydroxylase. Here, we were interested in regulation of GFRP expression by proinflammatory cytokines and stimuli, which are known to induce GTP cyclohydrolase I expression. Recombinant human GFRP stimulated recombinant human GTP cyclohydrolase I in the presence of phenylalanine and mediated feedback inhibition by tetrahydrobiopterin. Levels of GFRP mRNA in human myelomonocytoma (THP-1) cells remained unaltered by treatment of cells with interferon-gamma or interleukin-1beta, but were significantly down-regulated by bacterial lipopolysaccharide (LPS, 1 microg/ml), without or with cotreatment by interferon-gamma, which strongly up-regulated GTP cyclohydrolase I expression and activity. GFRP expression was also suppressed in human umbilical vein endothelial cells treated with 1 microg/ml LPS, as well as in rat tissues 7 h post intraperitoneal injection of 10 mg/kg LPS. THP-1 cells stimulated with interferon-gamma alone showed increased pteridine synthesis by addition of phenylalanine to the culture medium. Cells stimulated with interferon-gamma plus LPS, in contrast, showed phenylalanine-independent pteridine synthesis. These results demonstrate that LPS down-regulates expression of GFRP, thus rendering pteridine synthesis independent of metabolic control by phenylalanine. PMID:11799107

  2. GTP/GDP exchange by Sec12p enables COPII vesicle bud formation on synthetic liposomes

    PubMed Central

    Futai, Eugene; Hamamoto, Susan; Orci, Lelio; Schekman, Randy

    2004-01-01

    The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12ΔCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12ΔCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12ΔCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane. PMID:15457212

  3. Mg2+ dependence of guanine nucleotide binding to tubulin.

    PubMed

    Correia, J J; Baty, L T; Williams, R C

    1987-12-25

    The relationship between the concentration of Mg2+ and the binding of GDP and GTP to tubulin dimers was investigated by measuring the displacement of the nucleotide bound at the exchangeable site (E-site) by radiolabeled GDP and GTP. A wide range of concentrations of GTP, GDP, and Mg2+ was explored. In the near absence of Mg2+, the affinity of tubulin for GDP was found to be much greater than its affinity for GTP. In the presence of 1.0 mM Mg2+, however, its affinity for GDP was slightly less than for GTP. The results could be quantitatively described in terms of a small number of reversible equilibria. Equilibrium constants, pertaining to measurements at 0 degrees C, in 0.1 M piperazine-N,N'-bis(2-ethanesulfonic acid), 0.2 mM dithioerythritol, 2 mM EGTA, pH 6.9, were obtained by nonlinear least squares fitting of the data. When the association constant of tubulin for GDP uncomplexed with Mg2+ was taken to be 1.6 X 10(7) M-1, that for uncomplexed GTP was found to be no larger than 1.4 x 10(4) M-1, at least 1100-fold smaller. The association constant of tubulin for the GDP.Mg2+ complex was found to be 2.5-2.7 x 10(7) M-1, while that for the GTP.Mg2+ complex is 6.4-9.0 x 10(7) M-1. PMID:2826416

  4. Tree species affect cation exchange capacity (CEC) and cation binding properties of organic matter in acid forest soils.

    PubMed

    Gruba, Piotr; Mulder, Jan

    2015-04-01

    Soil organic matter (SOM) in forest soil is of major importance for cation binding and acid buffering, but its characteristics may differ among soils under different tree species. We investigated acidity, cation exchange properties and Al bonding to SOM in stands of Scots pine, pedunculate oak, Norway spruce, European beech and common hornbeam in southern Poland. The content of total carbon (Ct) was by far the major contributor to total cation exchange capacity (CECt) even in loamy soils and a strong relationship between Ct and CECt was found. The slope of the regression of CECt to Ct increased in the order hornbeam≈oak

  5. Polymorphism rs11085226 in the Gene Encoding Polypyrimidine Tract-Binding Protein 1 Negatively Affects Glucose-Stimulated Insulin Secretion

    PubMed Central

    Heni, Martin; Ketterer, Caroline; Wagner, Robert; Linder, Katarzyna; Böhm, Anja; Herzberg-Schäfer, Silke A.; Machicao, Fausto; Knoch, Klaus-Peter; Fritsche, Andreas; Staiger, Harald; Häring, Hans-Ulrich; Solimena, Michele

    2012-01-01

    Objective Polypyrimidine tract-binding protein 1 (PTBP1) promotes stability and translation of mRNAs coding for insulin secretion granule proteins and thereby plays a role in β-cells function. We studied whether common genetic variations within the PTBP1 locus influence insulin secretion, and/or proinsulin conversion. Methods We genotyped 1,502 healthy German subjects for four tagging single nucleotide polymorphisms (SNPs) within the PTBP1 locus (rs351974, rs11085226, rs736926, and rs123698) covering 100% of genetic variation with an r2≥0.8. The subjects were metabolically characterized by an oral glucose tolerance test with insulin, proinsulin, and C-peptide measurements. A subgroup of 320 subjects also underwent an IVGTT. Results PTBP1 SNP rs11085226 was nominally associated with lower insulinogenic index and lower cleared insulin response in the OGTT (p≤0.04). The other tested SNPs did not show any association with the analyzed OGTT-derived secretion parameters. In the IVGTT subgroup, SNP rs11085226 was accordingly associated with lower insulin levels within the first ten minutes following glucose injection (p = 0.0103). Furthermore, SNP rs351974 was associated with insulin levels in the IVGTT (p = 0.0108). Upon interrogation of MAGIC HOMA-B data, our rs11085226 result was replicated (MAGIC p = 0.018), but the rs351974 was not. Conclusions We conclude that common genetic variation in PTBP1 influences glucose-stimulated insulin secretion. This underlines the importance of PTBP1 for beta cell function in vivo. PMID:23077502

  6. Lesions in two Escherichia coli type 1 pilus genes alter pilus number and length without affecting receptor binding.

    PubMed Central

    Russell, P W; Orndorff, P E

    1992-01-01

    We describe the characterization of two genes, fimF and fimG (also called pilD), that encode two minor components of type 1 pili in Escherichia coli. Defined, in-frame deletion mutations were generated in vitro in each of these two genes. A double mutation that had deletions identical to both single lesions was also constructed. Examination of minicell transcription and translation products of parental and mutant plasmids revealed that, as predicted from the nucleotide sequence and previous reports, the fimF gene product was a protein of ca. 16 kDa and that the fimG gene product was a protein of ca. 14 kDa. Each of the constructions was introduced, via homologous recombination, into the E. coli chromosome. All three of the resulting mutants produced type 1 pili and exhibited hemagglutination of guinea pig erythrocytes. The latter property was also exhibited by partially purified pili isolated from each of the mutants. Electron microscopic examination revealed that the fimF mutant had markedly reduced numbers of pili per cell, whereas the fimG mutant had very long pili. The double mutant displayed the characteristics of both single mutants. However, pili in the double mutant were even longer than those seen in the fimG mutant, and the numbers of pili were even fewer than those displayed by the fimF mutant. All three mutants could be complemented in trans with a single-copy-number plasmid bearing the appropriate parental gene or genes to give near-normal parental piliation. On the basis of the phenotypes exhibited by the single and double mutants, we believe that the fimF gene product may aid in initiating pilus assembly and that the fimG product may act as an inhibitor of pilus polymerization. In contrast to previous studies, we found that neither gene product was required for type 1 pilus receptor binding. Images PMID:1355769

  7. Mutations Affecting the BHLHA9 DNA-Binding Domain Cause MSSD, Mesoaxial Synostotic Syndactyly with Phalangeal Reduction, Malik-Percin Type

    PubMed Central

    Malik, Sajid; Percin, Ferda E.; Bornholdt, Dorothea; Albrecht, Beate; Percesepe, Antonio; Koch, Manuela C.; Landi, Antonio; Fritz, Barbara; Khan, Rizwan; Mumtaz, Sara; Akarsu, Nurten A.; Grzeschik, Karl-Heinz

    2014-01-01

    Mesoaxial synostotic syndactyly, Malik-Percin type (MSSD) (syndactyly type IX) is a rare autosomal-recessive nonsyndromic digit anomaly with only two affected families reported so far. We previously showed that the trait is genetically distinct from other syndactyly types, and through autozygosity mapping we had identified a locus on chromosome 17p13.3 for this unique limb malformation. Here, we extend the number of independent pedigrees from various geographic regions segregating MSSD to a total of six. We demonstrate that three neighboring missense mutations affecting the highly conserved DNA-binding region of the basic helix-loop-helix A9 transcription factor (BHLHA9) are associated with this phenotype. Recombinant BHLHA9 generated by transient gene expression is shown to be located in the cytoplasm and the cell nucleus. Transcription factors 3, 4, and 12, members of the E protein (class I) family of helix-loop-helix transcription factors, are highlighted in yeast two-hybrid analysis as potential dimerization partners for BHLHA9. In the presence of BHLHA9, the potential of these three proteins to activate expression of an E-box-regulated target gene is reduced considerably. BHLHA9 harboring one of the three substitutions detected in MSSD-affected individuals eliminates entirely the transcription activation by these class I bHLH proteins. We conclude that by dimerizing with other bHLH protein monomers, BHLHA9 could fine tune the expression of regulatory factors governing determination of central limb mesenchyme cells, a function made impossible by altering critical amino acids in the DNA binding domain. These findings identify BHLHA9 as an essential player in the regulatory network governing limb morphogenesis in humans. PMID:25466284

  8. Flow Cytometry for Real-Time Measurement of Guanine Nucleotide Binding and Exchange by Ras-like GTPases

    PubMed Central

    Schwartz, Samantha L.; Tessema, Mathewos; Buranda, Tione; Phlypenko, Olena; Rak, Alexey; Simons, Peter C.; Surviladze, Zurab; Sklar, Larry A.; Wandinger-Ness, Angela

    2008-01-01

    Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here, we report a bead-based, flow cytometric assay that quantitatively measures the nucleotide binding properties of GST-chimeras for prototypical Ras-family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg2+, with magnesium cations principally increasing affinity and slowing nucleotide dissociation rate 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for GDP relative to GTP that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used γ-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP-binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real-time and quantitatively assess differences between GTPases. PMID:18638444

  9. Protein synthesis in brine shrimp embryos and rabbit reticulocytes. The effect of Mg2+ on binary (eukaryotic initiation factor 2 X GDP) and ternary (eukaryotic initiation factor 2 X GTP X met-tRNAf) complex formation.

    PubMed

    Mehta, H B; Woodley, C L; Wahba, A J

    1983-03-25

    We have prepared eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes and Artemia embryos and studied the effect of Mg2+ on binary (eIF-2 X GDP) and ternary (eIF-2 X GTP X Met-tRNAf) complex formation. Under conditions where Mg2+ inhibits Met-tRNAf binding to reticulocyte eIF-2, ternary complex formation with Artemia eIF-2 is not inhibited. Similarly, the formation of eIF-2 X GDP with Artemia eIF-2 is stimulated by Mg2+, whereas the corresponding reticulocyte binary complex is strongly inhibited. In the presence of 1 mM Mg2+, the isolated Artemia eIF-2 X GDP complex is stable in the absence of any added nucleotide, but readily exchanges bound GDP for free GTP. However, the reticulocyte eIF-2 X GDP complex is significantly more stable in the presence of GTP, and nucleotide exchange is dependent upon the addition of a factor isolated from either the postribosomal supernatant or the high salt wash of rabbit reticulocyte ribosomes. This factor also stimulates Met-tRNAf binding to both Artemia and reticulocyte eIF-2. PMID:6550599

  10. [Effect of iron, actinomycin D and cycloheximide on the GTP-cyclohydrolase synthesis in flavinogenic yeasts].

    PubMed

    Logvinenko, E M; Shavlovskiĭ, G M; Zakal'skiĭ, A E; Zakhodylo, I V

    1982-01-01

    The effect of Fe on the GTP-cyclohydrolase activity of the yeasts Pichia guilliermondii ATCC 9058 and Torulopsis candida BKM 13 whose flavinogenesis is controlled by Fe was investigated. The GTP-cyclohydrolase activity of yeast cells grown in an iron-deficient medium was 40-50 times that of the cells grown in an iron-rich medium. In the latter case the incubation of cells with alpha, alpha'-dipyridyl or 8-oxyquinoline also increased the enzyme activity. Cycloheximide prevented the rise in the cyclohydrolase activity in both cases, thus suggesting the participation of Fe in the control of the enzyme synthesis. Actinomycin D inhibited the enzyme derepression induced by alpha, alpha1-dipyridyl or 8-oxyquinoline in the P. guilliermondii MS1-37 mutant possessing a high sensitivity to this antibiotic. It is assumed that Fe is involved in the control of GTP-cyclohydrolase synthesis in flavinogenic yeasts at the transcription level. PMID:7199939

  11. Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein.

    PubMed

    Maita, Nobuo; Hatakeyama, Kazuyuki; Okada, Kengo; Hakoshima, Toshio

    2004-12-01

    GTP cyclohydrolase I (GTPCHI) is the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin, a key cofactor necessary for nitric oxide synthase and for the hydroxylases that are involved in the production of catecholamines and serotonin. In animals, the GTPCHI feedback regulatory protein (GFRP) binds GTPCHI to mediate feed-forward activation of GTPCHI activity in the presence of phenylalanine, whereas it induces feedback inhibition of enzyme activity in the presence of biopterin. Here, we have reported the crystal structure of the biopterin-induced inhibitory complex of GTPCHI and GFRP and compared it with the previously reported phenylalanine-induced stimulatory complex. The structure reveals five biopterin molecules located at each interface between GTPCHI and GFRP. Induced fitting structural changes by the biopterin binding expand large conformational changes in GTPCHI peptide segments forming the active site, resulting in inhibition of the activity. By locating 3,4-dihydroxy-phenylalanine-responsive dystonia mutations in the complex structure, we found mutations that may possibly disturb the GFRP-mediated regulation of GTPCHI. PMID:15448133

  12. Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics

    PubMed Central

    Datta-Mannan, Amita; Thangaraju, Arunkumar; Leung, Donmienne; Tang, Ying; Witcher, Derrick R; Lu, Jirong; Wroblewski, Victor J

    2015-01-01

    Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition. PMID:25695748

  13. Do ATP-binding cassette transporters cause pharmacoresistance in epilepsy? Problems and approaches in determining which antiepileptic drugs are affected.

    PubMed

    Löscher, Wolfgang; Luna-Tortós, Carlos; Römermann, Kerstin; Fedrowitz, Maren

    2011-01-01

    Resistance to multiple antiepileptic drugs (AEDs) is a common problem in epilepsy, affecting at least 30% of patients. One prominent hypothesis to explain this resistance suggests an inadequate penetration or excess efflux of AEDs across the blood - brain barrier (BBB) as a result of overexpressed efflux transporters such as P-glycoprotein (Pgp), the encoded product of the multidrug resistance- 1 (MDR1, ABCB1) gene. Pgp and MDR1 are markedly increased in epileptogenic brain tissue of patients with AED-resistant partial epilepsy and following seizures in rodent models of partial epilepsy. In rodent models, AED-resistant rats exhibit higher Pgp levels than responsive animals; increased Pgp expression is associated with lower brain levels of AEDs; and, most importantly, co-administration of Pgp inhibitors reverses AED resistance. Thus, it is reasonable to conclude that Pgp plays a significant role in mediating resistance to AEDs in rodent models of epilepsy - however, whether this phenomenon extends to at least some human refractory epilepsy remains unclear, particularly because it is still a matter of debate which AEDs, if any, are transported by human Pgp. The difficulty in determining which AEDs are substrates of human Pgp is mainly a consequence of the fact that AEDs are highly permeable compounds, which are not easily identified as Pgp substrates in in vitro models of the BBB, such as monolayer (Transwell(®)) efflux assays. By using a modified assay (concentration equilibrium transport assay; CETA), which minimizes the influence of high transcellular permeability, two groups have recently demonstrated that several major AEDs are transported by human Pgp. Importantly, it was demonstrated in these studies that Pgp-mediated transport highly depends on the AED concentration and may not be identified if concentrations below or above the therapeutic range are used. In addition to the efflux transporters, seizure-induced alterations in BBB integrity and activity of

  14. 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

  15. Binding intensity and metal partitioning in soils affected by mining and smelting activities in Minas Gerais, Brazil.

    PubMed

    Lopes, G; Costa, E T S; Penido, E S; Sparks, D L; Guilherme, L R G

    2015-09-01

    Mining and smelting activities are potential sources of heavy metal contamination, which pose a threat to human health and ecological systems. This study investigated single and sequential extractions of Zn, Pb, and Cd in Brazilian soils affected by mining and smelting activities. Soils from a Zn mining area (soils A, B, C, D, E, and the control soil) and a tailing from a smelting area were collected in Minas Gerais state, Brazil. The samples were subjected to single (using Mehlich I solution) and sequential extractions. The risk assessment code (RAC), the redistribution index (U ts ), and the reduced partition index (I R ) have been applied to the sequential extraction data. Zinc and Cd, in soil samples from the mining area, were found mainly associated with carbonate forms. This same pattern did not occur for Pb. Moreover, the Fe-Mn oxides and residual fractions had important contributions for Zn and Pb in those soils. For the tailing, more than 70 % of Zn and Cd were released in the exchangeable fraction, showing a much higher mobility and availability of these metals at this site, which was also supported by results of RAC and I R . These differences in terms of mobility might be due to different chemical forms of the metals in the two sites, which are attributable to natural occurrence as well as ore processing. PMID:25940493

  16. Analysis of polyglutamine-coding repeats in the TATA-binding protein in different human populations and in patients with schizophrenia an bipolar affective disorder

    SciTech Connect

    Rubinsztein, D.C.; Leggo, J.; Crow, T.J.

    1996-09-20

    A new class of disease (including Huntington disease, Kennedy disease, and spinocerebellar ataxias types 1 and 3) results from abnormal expansions of CAG trinucleotides in the coding regions of genes. In all of these diseases the CAG repeats are thought to be translated into polyglutamine tracts. There is accumulating evidence arguing for CAG trinucleotide expansions as one of the causative disease mutations in schizophrenia and bipolar affective disorder. We and others believe that the TATA-binding protein (TBP) is an important candidate to investigate in these diseases as it contains a highly polymorphic stretch of glutamine codons, which are close to the threshold length where the polyglutamine tracts start to be associated with disease. Thus, we examined the lengths of this polyglutamine repeat in normal unrelated East Anglians, South African Blacks, sub-Saharan Africans mainly from Nigeria, and Asian Indians. We also examined 43 bipolar affective disorder patients and 65 schizophrenic patients. The range of polyglutamine tract-lengths that we found in humans was from 26-42 codons. No patients with bipolar affective disorder and schizophrenia had abnormal expansions at this locus. 22 refs., 1 tab.

  17. Structure of an archaeal heterotrimeric initiation factor 2 reveals a nucleotide state between the GTP and the GDP states

    PubMed Central

    Yatime, Laure; Mechulam, Yves; Blanquet, Sylvain; Schmitt, Emmanuelle

    2007-01-01

    Initiation of translation in eukaryotes and in archaea involves eukaryotic/archaeal initiation factor (e/aIF)1 and the heterotrimeric initiation factor e/aIF2. In its GTP-bound form, e/aIF2 provides the initiation complex with Met–tRNAiMet. After recognition of the start codon by initiator tRNA, e/aIF1 leaves the complex. Finally, e/aIF2, now in a GDP-bound form, loses affinity for Met–tRNAiMet and dissociates from the ribosome. Here, we report a 3D structure of an aIF2 heterotrimer from the archeon Sulfolobus solfataricus obtained in the presence of GDP. Our report highlights how the two-switch regions involved in formation of the tRNA-binding site on subunit γ exchange conformational information with α and β. The zinc-binding domain of β lies close to the guanine nucleotide and directly contacts the switch 1 region. As a result, switch 1 adopts a not yet described conformation. Moreover, unexpectedly for a GDP-bound state, switch 2 has the “ON” conformation. The stability of these conformations is accounted for by a ligand, most probably a phosphate ion, bound near the nucleotide binding site. The structure suggests that this GDP–inorganic phosphate (Pi) bound state of aIF2 may be proficient for tRNA binding. Recently, it has been proposed that dissociation of eIF2 from the initiation complex is closely coupled to that of Pi from eIF2γ upon start codon recognition. The nucleotide state of aIF2 shown here is indicative of a similar mechanism in archaea. Finally, we consider the possibility that release of Pi takes place after e/aIF2γ has been informed of e/aIF1 dissociation by e/aIF2β. PMID:18000047

  18. How Hinge Positioning in Cross-Country Ski Bindings Affect Exercise Efficiency, Cycle Characteristics and Muscle Coordination during Submaximal Roller Skiing

    PubMed Central

    Bolger, Conor M.; Sandbakk, Øyvind; Ettema, Gertjan; Federolf, Peter

    2016-01-01

    The purposes of the current study were to 1) test if the hinge position in the binding of skating skis has an effect on gross efficiency or cycle characteristics and 2) investigate whether hinge positioning affects synergistic components of the muscle activation in six lower leg muscles. Eleven male skiers performed three 4-min sessions at moderate intensity while cross-country ski-skating and using a klapskate binding. Three different positions were tested for the binding’s hinge, ranging from the front of the first distal phalange to the metatarsal-phalangeal joint. Gross efficiency and cycle characteristics were determined, and the electromyographic (EMG) signals of six lower limb muscles were collected. EMG signals were wavelet transformed, normalized, joined into a multi-dimensional vector, and submitted to a principle component analysis (PCA). Our results did not reveal any changes to gross efficiency or cycle characteristics when altering the hinge position. However, our EMG analysis found small but significant effects of hinge positioning on muscle coordinative patterns (P < 0.05). The changed patterns in muscle activation are in alignment with previously described mechanisms that explain the effects of hinge positioning in speed-skating klapskates. Finally, the within-subject results of the EMG analysis suggested that in addition to the between-subject effects, further forms of muscle coordination patterns appear to be employed by some, but not all participants. PMID:27203597

  19. TET2 Mutations Affect Non-CpG Island DNA Methylation at Enhancers and Transcription Factor-Binding Sites in Chronic Myelomonocytic Leukemia.

    PubMed

    Yamazaki, Jumpei; Jelinek, Jaroslav; Lu, Yue; Cesaroni, Matteo; Madzo, Jozef; Neumann, Frank; He, Rong; Taby, Rodolphe; Vasanthakumar, Aparna; Macrae, Trisha; Ostler, Kelly R; Kantarjian, Hagop M; Liang, Shoudan; Estecio, Marcos R; Godley, Lucy A; Issa, Jean-Pierre J

    2015-07-15

    TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here, we report on DNA methylomes in TET2 wild-type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites [28,114 sites in CpG islands (CGI) and 57,020 in non-CpG islands (NCGI)]. TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. In contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and nonpromoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1 and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor-binding sites. PMID:25972343

  20. Clostridium botulinum type C hemagglutinin affects the morphology and viability of cultured mammalian cells via binding to the ganglioside GM3.

    PubMed

    Sugawara, Yo; Iwamori, Masao; Matsumura, Takuhiro; Yutani, Masahiro; Amatsu, Sho; Fujinaga, Yukako

    2015-09-01

    Botulinum neurotoxin is conventionally divided into seven serotypes, designated A-G, and is produced as large protein complexes through associations with non-toxic components, such as hemagglutinin (HA) and non-toxic non-HA. These non-toxic proteins dramatically enhance the oral toxicity of the toxin complex. HA is considered to have a role in toxin transport through the intestinal epithelium by carbohydrate binding and epithelial barrier-disrupting activity. Type A and B HAs disrupt E-cadherin-mediated cell adhesion, and, in turn, the intercellular epithelial barrier. Type C HA (HA/C) disrupts the barrier function by affecting cell morphology and viability, the mechanism of which remains unknown. In this study, we identified GM3 as the target molecule of HA/C. We found that sialic acid binding of HA is essential for the activity. It was abolished when cells were pre-treated with an inhibitor of ganglioside synthesis. Consistent with this, HA/C bound to a-series gangliosides in a glycan array. In parallel, we isolated clones resistant to HA/C activity from a susceptible mouse fibroblast strain. These cells lacked expression of ST-I, the enzyme that transfers sialic acid to lactosylceramide to yield GM3. These clones became sensitive to HA/C activity when GM3 was expressed by transfection with the ST-I gene. The sensitivity of fibroblasts to HA/C was reduced by expressing ganglioside synthesis genes whose products utilize GM3 as a substrate and consequently generate other a-series gangliosides, suggesting a GM3-specific mechanism. Our results demonstrate that HA/C affects cells in a GM3-dependent manner. PMID:26077172

  1. Specific binding sites for proadrenomedullin N-terminal 20 peptide (PAMP) in the rat.

    PubMed

    Iwasaki, H; Hirata, Y; Iwashina, M; Sato, K; Marumo, F

    1996-07-01

    Adrenomedullin (AM), a potent and novel vasodilator 52-residue peptide originally isolated from pheochromocytoma, is processed from a precursor molecule (preproAM) in which another unique 20-residue sequence, termed proadrenomedullin N-terminal 20 peptide (PAMP), exists. Using [125I Tyr0] rat PAMP as a radioligand, we have examined PAMP binding sites in various rat tissues and cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding sites for rat PAMP, although very low, were widely distributed in various rat tissues examined. The relatively more abundant sites were present in aorta and adrenal glands, followed by lung, kidney, brain, spleen, and heart. An equilibrium binding study using cultured rat VSMC revealed the presence of a single class of high-affinity [dissociation constant (Kd): 3.5 x 10(-8) M] binding sites for rat PAMP with a maximal binding capacity of 4.5 x 10(6) sites per cell. Binding studies revealed that synthetic rat PAMP(1-19)-NH2 was about 10-fold less potent, and rat PAMP(1-20)-OH and human PAMP were about 20-fold less potent than rat PAMP(1-20)-NH2. SDS-polyacylamide gel electrophoresis after affinity-labeling of membranes from various rat tissues (aorta, adrenal glands, lung) and VSMC revealed a distinct labeled band with the apparent molecular mass of 90 kDa, which was diminished by excess unlabeled rat PAMP. A nonhydrolyzable GTP analog (GTP-gammaS) dose-dependently reduced binding of [125I] rat PAMP to VSMC membranes, while ATP-gammaS had no effect. Neither cyclic AMP nor inositol-1,4,5-triphosphate formation was affected by rat PAMP in rat VSMC. The present study demonstrates for the first time that PAMP receptors are widely distributed in various rat tissues, among which aorta and adrenal glands have the most abundant sites. Our data suggest that PAMP receptors are functionally coupled to G-proteins, although its signal transduction remains obscure. The present study also shows that amidation of C-terminal residue

  2. A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants

    PubMed Central

    2013-01-01

    Background Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. Results RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. Conclusions Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation

  3. Sigma 1 receptor modulation of G-protein-coupled receptor signaling: potentiation of opioid transduction independent from receptor binding.

    PubMed

    Kim, Felix J; Kovalyshyn, Ivanka; Burgman, Maxim; Neilan, Claire; Chien, Chih-Cheng; Pasternak, Gavril W

    2010-04-01

    sigma Ligands modulate opioid actions in vivo, with agonists diminishing morphine analgesia and antagonists enhancing the response. Using human BE(2)-C neuroblastoma cells that natively express opioid receptors and human embryonic kidney (HEK) cells transfected with a cloned mu opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding, by sigma(1) receptors. sigma Ligands do not compete opioid receptor binding. Administered alone, neither sigma agonists nor antagonists significantly stimulated [(35)S]GTP gamma S binding. Yet sigma receptor selective antagonists, but not agonists, shifted the EC(50) of opioid-induced stimulation of [(35)S]GTP gamma S binding by 3- to 10-fold to the left. This enhanced potency was seen without a change in the efficacy of the opioid, as assessed by the maximal stimulation of [(35)S]GTP gamma S binding. sigma(1) Receptors physically associate with mu opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, sigma receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of sigma(1) in BE(2)-C cells also potentiated mu opioid-induced stimulation of [(35)S]GTP gamma S binding. These modulatory actions are not limited to mu and delta opioid receptors. In mouse brain membrane preparations, sigma(1)-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [(35)S]GTP gamma S binding, suggesting a broader role for sigma receptors in modulating G-protein-coupled receptor signaling. PMID:20089882

  4. Polypyrimidine tract-binding protein (PTB) inhibits Hepatitis C virus internal ribosome entry site (HCV IRES)-mediated translation, but does not affect HCV replication.

    PubMed

    Tischendorf, J J W; Beger, C; Korf, M; Manns, M P; Krüger, M

    2004-10-01

    Polypyrimidine tract-binding protein (PTB) has previously been shown to affect Hepatitis C virus (HCV) IRES-mediated translation. In the present study we investigated the functional role of PTB for HCV translation, replication and chronic HCV infection. Bicistronic HCV IRES reporter plasmids and two different subgenomic replicons (bicistronic: pHCVrep1bBB7 (s1179I); monocistronic: pFK1-389/hyg-ubi/NS3-3'/5.1) were used to analyze the effects of PTB. Following transfection of plasmids expressing PTB RNA in sense or antisense orientation, translational activity and HCV RNA were analyzed by luciferase assay, quantitative real-time RT-PCR and northern blot analysis. Additionally, in liver tissue (n = 53) intrahepatic PTB RNA levels were determined by quantitative real-time RT-PCR. Significant inhibition of HCV IRES activity up to 42.6% was observed upon PTB sense RNA expression for HCV IRES reporter plasmids, while translational activity was enhanced up to 63.8% for PTB antisense RNA expression. In the HCV replicons PTB did not affect replication and no correlation was found between intrahepatic PTB mRNA levels and serum HCV RNA or histological changes in liver tissue of HCV infected patients. Although PTB inhibits HCV IRES-mediated translation from bicistronic reporter constructs, data obtained from two subgenomic HCV replicons and liver tissue do not indicate a significant role of PTB for HCV replication and chronic HCV infection. PMID:15669107

  5. Mannose-Binding Lectin Inhibits the Motility of Pathogenic Salmonella by Affecting the Driving Forces of Motility and the Chemotactic Response

    PubMed Central

    Nakamura, Shuichi; Islam, Md. Shafiqul; Guo, Yijie; Ihara, Kohei; Tomioka, Rintaro; Masuda, Mizuki; Yoneyama, Hiroshi; Isogai, Emiko

    2016-01-01

    Mannose-binding lectin (MBL) is a key pattern recognition molecule in the lectin pathway of the complement system, an important component of innate immunity. MBL functions as an opsonin which enhances the sequential immune process such as phagocytosis. We here report an inhibitory effect of MBL on the motility of pathogenic bacteria, which occurs by affecting the energy source required for motility and the signaling pathway of chemotaxis. When Salmonella cells were treated with a physiological concentration of MBL, their motile fraction and free-swimming speed decreased. Rotation assays of a single flagellum showed that the flagellar rotation rate was significantly reduced by the addition of MBL. Measurements of the intracellular pH and membrane potential revealed that MBL affected a driving force for the Salmonella flagellum, the electrochemical potential difference of protons. We also found that MBL treatment increased the reversal frequency of Salmonella flagellar rotation, which interfered with the relative positive chemotaxis toward an attractive substrate. We thus propose that the motility inhibition effect of MBL may be secondarily involved in the attack against pathogens, potentially facilitating the primary role of MBL in the complement system. PMID:27104738

  6. Cyclic AMP-dependent protein kinase interferes with GTP. gamma. S stimulated IP sub 3 formation in differentiated HL-60 cell membranes

    SciTech Connect

    Misaki, Naoyuki; Imaizumi, Taro; Watanabe, Yashuiro )

    1989-01-01

    The effects of addition of activated cyclic AMP-dependent protein kinase (PKA) on the function of islet-activating protein (IAP)-sensitive GTP-binding (G) protein were studied in the plasma membranes of {sup 3}H-inositol-labeled differentiated human leukemic (HL-60) cells. Pretreatment of the membranes with activated PKA in the presence of MgATP for 15 min. at 37{degree}C decreased GTP {gamma}S-stimulated inositol trisphosphate (IP{sub 3}) formation by about 30%, but had no influence on Ca{sup 2+}-stimulated IP{sub 3} formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some {sup 32}P incorporated bands, and among them one of the major bands showed the migration at 40 kDa supporting that the G protein coupling with PI response was phosphorylated by PKA. These results showed that pretreatment with activated PKA inhibited the mediating function of the G protein between the fMLP receptor and phospholipase C by its phosphorylation.

  7. TBC-Domain GAPs for Rab GTPases Accelerate GTP Hydrolysis by a Dual-Finger Mechanism

    SciTech Connect

    Pan,X.; Eathiraj, S.; Lambright, D.

    2006-01-01

    Rab GTPases regulate membrane trafficking by cycling between inactive (GDP-bound) and active (GTP-bound) conformations. The duration of the active state is limited by GTPase-activating proteins (GAPs), which accelerate the slow intrinsic rate of GTP hydrolysis. Proteins containing TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotic organisms and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis. An exposed arginine residue is a critical determinant of GAP activity in vitro and in vivo. It has been expected that the catalytic mechanism of TBC domains would parallel that of Ras and Rho family GAPs. Here we report crystallographic, mutational and functional analyses of complexes between Rab GTPases and the TBC domain of Gyp1p. In the crystal structure of a TBC-domain-Rab-GTPase-aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase. The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain. Strong conservation of both catalytic fingers indicates that most TBC-domain GAPs may accelerate GTP hydrolysis by a similar dual-finger mechanism.

  8. Sensitive assay of GTP cyclohydrolase I activity in rat and human tissues using radioimmunoassay of neopterin

    SciTech Connect

    Sawada, M.; Horikoshi, T.; Masada, M.; Akino, M.; Sugimoto, T.; Matsuura, S.; Nagatsu, T.

    1986-04-01

    A highly sensitive and simple assay for the activity of GTP cyclohydrolase I (EC 3.5.4.16) was established using a newly developed radioimmunoassay. D-erythro-7,8-Dihydroneopterin triphosphate formed from GTP by GTP cyclohydrolase I was oxidized by iodine and dephosphorylated by alkaline phosphatase to D-erythro-neopterin, and quantified by a radioimmunoassay for D-erythro-neopterin. This method was highly sensitive and required only 0.2 mg of rat liver tissues for the measurement of the activity. It was reproducible and can be applied for the simultaneous assay of many samples. The activity of GTP cyclohydrolase I was measured in several rat tissues. For example, the enzyme activity in rat striatum (n = 5) was 13.7 +/- 1.5 pmol/mg protein per hour (mean +/- SE), and agreed well with those obtained by high-performance liquid chromatography with fluorescence detection. The activity in the autopsy human brains (caudate nucleus) was measured by this new method for the first time. The activity in the caudate nucleus from parkinsonian patients (n = 6) was 0.82 +/- 0.56 pmol/mg protein per hour which was significantly lower than the control value, 4.22 +/- 0.43 pmol/mg protein per hour (n = 10).

  9. Occurrence and Ecological Significance of GTP in the Ocean and in Microbial Cells

    PubMed Central

    Karl, D. M.

    1978-01-01

    A comparison between the ATP concentrations based on peak height light emission values (0 to 3 s) and integrated light flux determinations (15 to 75 s) for a variety of seawater samples revealed that the integrated method of light detection consistently yielded higher ATP concentrations, ranging from 1.38 to 2.35 times larger than the corresponding peak ATP values. A significant correlation (r = 0.923) was observed for a plot of ΔADP (i.e., integrated ATP - peak ATP) versus GTP + UTP, suggesting that the analytical interference on the ATP assay was the result of the presence of non-adenine nucleotide triphosphates. Size-fractionation studies revealed an enrichment of the non-adenine nucleotide triphosphates, relative to ATP, in the smallest size fraction analyzed (<10 μm). Investigations were conducted with 20 species of unicellular marine algae to determine their intracellular nucleotide concentrations, and these determinations were compared to the levels measured in lab cultures of the marine bacterium Serratia marinorubra. These results indicated that the intracellular GTP/ATP ratios in S. marinorubra increase in direct proportion to the rate of cell growth, and that the GTP/ATP ratios in bacteria are much greater than in growing algae, presumably due to the differences in rates of cellular biosynthesis. It is concluded that quantitative determinations of GTP/ATP ratios in environmental sample extracts may be useful for measuring microbial growth. PMID:16345313

  10. Evolution of New Function in the GTP Cyclohydrolase II Proteins of Streptomyces coelicolor†

    PubMed Central

    Spoonamore, James E.; Dahlgran, Annie L.; Jacobsen, Neil E.; Bandarian, Vahe

    2009-01-01

    The genome sequence of Streptomyces coelicolor contains three open reading frames (sco1441, sco2687, and sco6655) that encode proteins with significant (>40%) amino acid identity to GTP cyclohydrolase II (GCH II), which catalyzes the committed step in the biosynthesis of riboflavin. The physiological significance of the redundancy of these proteins in S. coelicolor is not known. However, the gene contexts of the three proteins are different, suggesting that they may serve alternate biological niches. Each of the three proteins was overexpressed in Escherichia coli and characterized to determine if their functions are biologically overlapping. As purified, each protein contains 1 molar equiv of zinc/ mol of protein and utilizes guanosine 5′-triphosphate (GTP) as substrate. Two of these proteins (SCO 1441 and SCO 2687) produce the canonical product of GCH II, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (APy). Remarkably, however, one of the three proteins (SCO 6655) converts GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate (FAPy), as shown by UV-visible spectrophotometry, mass spectrometry, and NMR. This activity has been reported for a GTP cyclohydrolase III protein from Methanocaldococcus jannaschii [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074–15084], which has no amino acid sequence homology to SCO 6655. Comparison of the sequences of these proteins and mapping onto the structure of the E. coli GCH II protein [Ren, J., Kotaka, M., Lockyer, M., Lamb, H. K., Hawkins, A. R., and Stammers, D. K. (2005) J. Biol. Chem. 280, 36912–36919] allowed identification of a switch residue, Met120, which appears to be responsible for the altered fate of GTP observed with SCO 6655; a Tyr is found in the analogous position of all proteins that have been shown to catalyze the conversion of GTP to APy. The Met120Tyr variant of SCO 6655 acquires the ability to catalyze the conversion of GTP to APy, suggesting

  11. Signal Transduction by BvgS Sensor Kinase: BINDING OF MODULATOR NICOTINATE AFFECTS THE CONFORMATION AND DYNAMICS OF THE ENTIRE PERIPLASMIC MOIETY.

    PubMed

    Dupré, Elian; Lesne, Elodie; Guérin, Jérémy; Lensink, Marc F; Verger, Alexis; de Ruyck, Jérôme; Brysbaert, Guillaume; Vezin, Hervé; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2015-09-18

    The two-component sensory transduction system BvgAS controls the virulence regulon of the whooping-cough agent Bordetella pertussis. The periplasmic moiety of the homodimeric sensor kinase BvgS is composed of four bilobed Venus flytrap (VFT) perception domains followed by α helices that extend into the cytoplasmic membrane. In the virulent phase, the default state of B. pertussis, the cytoplasmic enzymatic moiety of BvgS acts as kinase by autophosphorylating and transferring the phosphoryl group to the response regulator BvgA. Under laboratory conditions, BvgS shifts to phosphatase activity in response to modulators, notably nicotinate ions. Here we characterized the effects of nicotinate and related modulators on the BvgS periplasmic moiety by using site-directed mutagenesis and in silico and biophysical approaches. Modulators bind with low affinity to BvgS in the VFT2 cavity. Electron paramagnetic resonance shows that their binding globally affects the conformation and dynamics of the periplasmic moiety. Specific amino acid substitutions designed to slacken interactions within and between the VFT lobes prevent BvgS from responding to nicotinate, showing that BvgS shifts from kinase to phosphatase activity in response to this modulator via a tense transition state that involves a large periplasmic structural block. We propose that this transition enables the transmembrane helices to adopt a distinct conformation that sets the cytoplasmic enzymatic moiety in the phosphatase mode. The bona fide, in vivo VFT ligands that remain to be identified are likely to trigger similar effects on the transmembrane and cytoplasmic moieties. This mechanism may be relevant to the other VFT-containing sensor kinases homologous to BvgS. PMID:26203186

  12. The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.

    PubMed

    Miner, Gregory E; Starr, Matthew L; Hurst, Logan R; Sparks, Robert P; Padolina, Mark; Fratti, Rutilio A

    2016-08-19

    The yeast vacuole requires four SNAREs to trigger membrane fusion including the soluble Qc-SNARE Vam7. The N-terminal PX domain of Vam7 binds to the lipid phosphatidylinositol 3-phosphate (PI3P) and the tethering complex HOPS (homotypic fusion and vacuole protein sorting complex), whereas the C-terminal SNARE motif forms SNARE complexes. Vam7 also contains an uncharacterized middle domain that is predicted to be a coiled-coil domain with multiple helices. One helix contains a polybasic region (PBR) composed of Arg-164, Arg-168, Lys-172, Lys-175, Arg-179, and Lys-186. Polybasic regions are often associated with nonspecific binding to acidic phospholipids including phosphoinositides. Although the PX (phox homology) domain alone binds PI3P, we theorized that the Vam7 PBR could bind to additional acidic phospholipids enriched at fusion sites. Mutating each of the basic residues in the PBR to an alanine (Vam7-6A) led to attenuated vacuole fusion. The defective fusion of Vam7-6A was due in part to inefficient association with its cognate SNAREs and HOPS, yet the overall vacuole association of Vam7-6A was similar to wild type. Experiments testing the binding of Vam7 to specific signaling lipids showed that mutating the PBR to alanines augmented binding to PI3P. The increased binding to PI3P by Vam7-6A likely contributed to the observed wild type levels of vacuole association, whereas protein-protein interactions were diminished. PI3P binding was inhibited when the PX domain mutant Y42A was introduced into Vam7-6A to make Vam7-7A. Thus the Vam7 PBR affects PI3P binding by the PX domain and in turn affects binding to SNAREs and HOPS to support efficient fusion. PMID:27365394

  13. Specificity and affinity of binding of phosphate-containing compounds to CheY protein.

    PubMed Central

    Kar, L; De Croos, P Z; Roman, S J; Matsumura, P; Johnson, M E

    1992-01-01

    1H- and 31P-n.m.r. have been used to study the interaction of the bacterial chemotaxis protein, CheY, with ATP and a variety of other phosphates in the presence and absence of bivalent metal ions. In the metal-bound conformation, CheY will bind nucleotide phosphates and phosphates in general, while in the metal-free conformation CheY loses its affinity for phosphates. In the presence of low concentrations of nitroxide-spin-labelled ATP (SL-ATP), specific proton resonances of metal-bound CheY are suppressed, indicating that ATP binds to a specific site on this metal-bound form of the protein. These studies also show that the same resonances are affected by the binding of SL-ATP and Mn2+, indicating that the phosphate- and metal-binding sites are close to each other and to Asp-57 (the site of phosphorylation in CheY). 1H- and 31P-n.m.r. studies using ATP, GTP, TTP, UTP, ADP, AMP and inorganic phosphates show that the binding is not specific for adenine, and does not involve the base directly, but is mediated primarily by the phosphate groups. Experiments with a phosphorylation mutant (Asp-13-->Asn) suggest that the observed phosphate binding and activation of CheY by phosphorylation may be related. Our results indicate that the conformational change and charge interactions brought about by the binding of a metal ion at the active site are required for CheY to interact with a phosphate. These studies also demonstrate the utility of spin-label-induced relaxation in conjunction with two-dimensional-n.m.r. measurements for exploring ligand-binding sites. PMID:1332676

  14. High resolution microtubule structures reveal the structural transitions in αβ–tubulin upon GTP hydrolysis

    PubMed Central

    Alushin, Gregory M.; Lander, Gabriel C.; Kellogg, Elizabeth H.; Zhang, Rui; Baker, David; Nogales, Eva

    2014-01-01

    Summary Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice, and is inhibited by anticancer agents like Taxol. We provide new insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7–5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α–tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α– and β–tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces. PMID:24855948

  15. Use of 8-azidoguanosine 5'-(gamma-/sup 32/P)triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

    SciTech Connect

    Kohnken, R.E.; Mc Connell, D.G.

    1985-07-02

    In an in vitro incubation, 8-azidoguanosine 5'-(gamma-/sup 32/P)triphosphate ( (gamma-/sup 32/P)-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with (gamma-/sup 32/P)-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, (gamma-/sup 32/P)-8-azido-GTP also labeled G alpha (Mr 40 000). This labeling was ultraviolet light dependent. G beta (Mr 35 000) was also labeled dependent for the most part upon ultraviolet light, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G alpha and G beta was found to vary intricately with experimental conditions, especially prebleaching of rhodopsin, tonicity of the medium, and the presence or absence of 2-mercaptoethanol. Affinity labeling of G alpha and G beta by (gamma-/sup 32/P)-8-azido-GTP in competition with ATP or GTP was kinetically complex, consistent with possible multiple binding sites for GTP on both subunits. Independent evidence for two or more binding sites on G alpha has been offered by other laboratories, and recently, at least one binding site on G beta and its analogues among the N proteins of adenylate cyclases has been identified.

  16. The ARF-like 2 (ARL2)-binding protein, BART. Purification, cloning, and initial characterization.

    PubMed

    Sharer, J D; Kahn, R A

    1999-09-24

    ARF-like proteins (ARLs) comprise a functionally distinct group of incompletely characterized members in the ARF family of RAS-related GTPases. We took advantage of the GTP binding characteristics of human ARL2 to develop a specific, high affinity binding assay that allowed the purification of a novel ARL2-binding protein. A 19-kDa protein (BART, Binder of Arl Two) was identified and purified from bovine brain homogenate. BART binding is specific to ARL2.GTP with high affinity but does not interact with ARL2.GDP or activated ARF or RHO proteins. Based on peptide sequences of purified bovine BART, the human cDNA sequence was determined. The 489-base pair BART open reading frame encodes a novel 163-amino acid protein with a predicted molecular mass of 18,822 Da. Recombinant BART was found to bind ARL2.GTP in a manner indistinguishable from native BART. Northern and Western analyses indicated BART is expressed in all tissues sampled. The lack of detectable membrane association of ARL2 or BART upon activation of ARL2 is suggestive of actions quite distinct from those of the ARFs. The lack of ARL2 GTPase-activating protein activity in BART led us to conclude that the specific interaction with ARL2.GTP is most consistent with BART being the first identified ARL2-specific effector. PMID:10488091

  17. Mutations within the LINC-HELLP non-coding RNA differentially bind ribosomal and RNA splicing complexes and negatively affect trophoblast differentiation.

    PubMed

    van Dijk, Marie; Visser, Allerdien; Buabeng, Kwadwo M L; Poutsma, Ankie; van der Schors, Roel C; Oudejans, Cees B M

    2015-10-01

    LINC-HELLP, showing chromosomal linkage with the pregnancy-specific HELLP syndrome in Dutch families, reduces differentiation from a proliferative to an invasive phenotype of first-trimester extravillous trophoblasts. Here we show that mutations in LINC-HELLP identified in HELLP families negatively affect this trophoblast differentiation either by inducing proliferation rate or by causing cell cycle exit as shown by a decrease in both proliferation and invasion. As LincRNAs predominantly function through interactions with proteins, we identified the directly interacting proteins using chromatin isolation by RNA purification followed by protein mass spectrometry. We found 22 proteins predominantly clustering in two functional networks, i.e. RNA splicing and the ribosome. YBX1, PCBP1, PCBP2, RPS6 and RPL7 were validated, and binding to these proteins was influenced by the HELLP mutations carried. Finally, we show that the LINC-HELLP transcript levels are significantly upregulated in plasma of women in their first trimester of pregnancy compared with non-pregnant women, whereas this upregulation seems absent in a pilot set of patients later developing pregnancy complications, indicative of its functional significance in vivo. PMID:26173455

  18. The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A.

    PubMed

    Jousselin, Ambre; Manzano, Caroline; Biette, Alexandra; Reed, Patricia; Pinho, Mariana G; Rosato, Adriana E; Kelley, William L; Renzoni, Adriana

    2015-01-01

    Expression of the methicillin-resistant S. aureus (MRSA) phenotype results from the expression of the extra penicillin-binding protein 2A (PBP2A), which is encoded by mecA and acquired horizontally on part of the SCCmec cassette. PBP2A can catalyze dd-transpeptidation of peptidoglycan (PG) because of its low affinity for β-lactam antibiotics and can functionally cooperate with the PBP2 transglycosylase in the biosynthesis of PG. Here, we focus upon the role of the membrane-bound PrsA foldase protein as a regulator of β-lactam resistance expression. Deletion of prsA altered oxacillin resistance in three different SCCmec backgrounds and, more importantly, caused a decrease in PBP2A membrane amounts without affecting mecA mRNA levels. The N- and C-terminal domains of PrsA were found to be critical features for PBP2A protein membrane levels and oxacillin resistance. We propose that PrsA has a role in posttranscriptional maturation of PBP2A, possibly in the export and/or folding of newly synthesized PBP2A. This additional level of control in the expression of the mecA-dependent MRSA phenotype constitutes an opportunity to expand the strategies to design anti-infective agents. PMID:26711778

  19. The nucleotide-binding oligomerization domain-containing protein 1 (NOD1) polymorphism S7N does not affect receptor function

    PubMed Central

    2014-01-01

    Background Activation and signal transduction in the Nucleotide binding, leucine-rich repeat containing receptor (NLR) family needs to be tightly regulated in order to control the inflammatory response to exogenous and endogenous danger signals. Phosphorylation is a common cellular mechanism of regulation that has recently been shown to be important in signalling in another family of cytoplasmic pattern recognition receptors, the RIG-I like receptors. In addition, single nucleotide polymorphisms can alter receptor activity, potentially leading to dysfunction and/or a predisposition to inflammatory barrier diseases. Findings We have computationally analysed the N-terminus of NOD1 and found seven theoretical phosphorylation sites in, or immediately before, the NOD1 Caspase Activation Domain (CARD). Two of these, serine 7 and tyrosine 49 are also found as rare polymorphisms in the African-American population and European-American populations respectively. Mutating serine 7 to either an aspartic acid or an asparagine to mimic the potential impact of phosphorylation or the polymorphism respectively did not affect the response of NOD1 to ligand-mediated NFκB signalling. Conclusions The NOD1 polymorphism S7N does not interfere with receptor function in response to ligand stimulation. PMID:24598002

  20. Validating the GTP-cyclohydrolase 1-feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches

    PubMed Central

    Hussein, D; Starr, A; Heikal, L; McNeill, E; Channon, K M; Brown, P R; Sutton, B J; McDonnell, J M; Nandi, M

    2015-01-01

    Background and Purpose 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide biosynthesis. Substantial clinical evidence indicates that intravenous BH4 restores vascular function in patients. Unfortunately, oral BH4 has limited efficacy. Therefore, orally bioavailable pharmacological activators of endogenous BH4 biosynthesis hold significant therapeutic potential. GTP-cyclohydrolase 1 (GCH1), the rate limiting enzyme in BH4 synthesis, forms a protein complex with GCH1 feedback regulatory protein (GFRP). This complex is subject to allosteric feed-forward activation by L-phenylalanine (L-phe). We investigated the effects of L-phe on the biophysical interactions of GCH1 and GFRP and its potential to alter BH4 levels in vivo. Experimental Approach Detailed characterization of GCH1–GFRP protein–protein interactions were performed using surface plasmon resonance (SPR) with or without L-phe. Effects on systemic and vascular BH4 biosynthesis in vivo were investigated following L-phe treatment (100 mg·kg−1, p.o.). Key Results GCH1 and GFRP proteins interacted in the absence of known ligands or substrate but the presence of L-phe doubled maximal binding and enhanced binding affinity eightfold. Furthermore, the complex displayed very slow association and dissociation rates. In vivo, L-phe challenge induced a sustained elevation of aortic BH4, an effect absent in GCH1(fl/fl)-Tie2Cre mice. Conclusions and Implications Biophysical data indicate that GCH1 and GFRP are constitutively bound. In vivo, data demonstrated that L-phe elevated vascular BH4 in an endothelial GCH1 dependent manner. Pharmacological agents which mimic the allosteric effects of L-phe on the GCH1–GFRP complex have the potential to elevate endothelial BH4 biosynthesis for numerous cardiovascular disorders. PMID:26014146

  1. Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus.

    PubMed

    Schindler, Bryan D; Seo, Susan M; Birukou, Ivan; Brennan, Richard G; Kaatz, Glenn W

    2015-03-01

    The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of various mepA operator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and an in vivo confirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for the mepA operator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under which mepA expression is advantageous likely contributed to the accumulation of mutations in the mepA operator, resulting in the current sequence from which MepR is readily induced by MepA substrates. PMID:25583977

  2. The Role of Gln61 in HRas GTP Hydrolysis: A Quantum Mechanics/Molecular Mechanics Study

    PubMed Central

    Martín-García, Fernando; Mendieta-Moreno, Jesús Ignacio; López-Viñas, Eduardo; Gómez-Puertas, Paulino; Mendieta, Jesús

    2012-01-01

    Activation of the water molecule involved in GTP hydrolysis within the HRas⋅RasGAP system is analyzed using a tailored approach based on hybrid quantum mechanics/molecular mechanics (QM/MM) simulation. A new path emerges: transfer of a proton from the attacking water molecule to a second water molecule, then a different proton is transferred from this second water molecule to the GTP. Gln61 will stabilize the transient OH− and H3O+ molecules thus generated. This newly proposed mechanism was generated by using, for the first time to our knowledge, the entire HRas-RasGAP protein complex in a QM/MM simulation context. It also offers a rational explanation for previous experimental results regarding the decrease of GTPase rate found in the HRas Q61A mutant and the increase exhibited by the HRas Q61E mutant. PMID:22225809

  3. Nitric oxide formation in acutely rejecting cardiac allografts correlates with GTP cyclohydrolase I activity

    PubMed Central

    2005-01-01

    Inducible nitric oxide synthase (iNOS) is a prominent component of the complex array of mediators in acute graft rejection. While NO production is determined by iNOS expression, BH4 (tetrahydrobiopterin), a cofactor of iNOS synthesized by GTP cyclohydrolase I, has been considered critical in sustaining NO production. In the present study, we examined time-dependent changes in iNOS and GTP cyclohydrolase I in rat cardiac allografts. The increase in iNOS protein and mRNA in allografts was similar at POD4 (post-operative day 4) and POD6. However, the peak increase in intragraft NO level at POD4 was not sustained at POD6. This disparity could not be explained by any decrease in iNOS enzyme activity measured ex vivo with optimal amounts of substrate and cofactors. Lower iNOS activity could be explained by changes in total biopterin levels in allografts at POD4 that was decreased to baseline at POD6. Changes in biopterin production correlated with lower GTP cyclohydrolase I protein levels but not by any change in GTP cyclohydrolase I mRNA. Functionally, allografts displayed bradycardia and distended diastolic and systolic dimensions at POD6 but not at POD4. Likewise, histological rejection scores were increased at POD4 but with a secondary increased stage at POD6. It is hypothesized that the dissimilar amounts of NO at early and later stages of rejection is due to uncoupling of iNOS arising from disproportionate synthesis of BH4. These findings provide insight into a potential pathway regulating NO bioactivity in graft rejection. Such knowledge may potentially assist in the design of newer strategies to prevent acute graft rejection. PMID:16000090

  4. Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

    PubMed Central

    Li, Wen; Liu, Zheng; Koripella, Ravi Kiran; Langlois, Robert; Sanyal, Suparna; Frank, Joachim

    2015-01-01

    During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome. PMID:26229983

  5. De novo GTP Biosynthesis Is Critical for Virulence of the Fungal Pathogen Cryptococcus neoformans

    PubMed Central

    Morrow, Carl A.; Valkov, Eugene; Stamp, Anna; Chow, Eve W. L.; Lee, I. Russel; Wronski, Ania; Williams, Simon J.; Hill, Justine M.; Djordjevic, Julianne T.; Kappler, Ulrike; Kobe, Bostjan; Fraser, James A.

    2012-01-01

    We have investigated the potential of the GTP synthesis pathways as chemotherapeutic targets in the human pathogen Cryptococcus neoformans, a common cause of fatal fungal meningoencephalitis. We find that de novo GTP biosynthesis, but not the alternate salvage pathway, is critical to cryptococcal dissemination and survival in vivo. Loss of inosine monophosphate dehydrogenase (IMPDH) in the de novo pathway results in slow growth and virulence factor defects, while loss of the cognate phosphoribosyltransferase in the salvage pathway yielded no phenotypes. Further, the Cryptococcus species complex displays variable sensitivity to the IMPDH inhibitor mycophenolic acid, and we uncover a rare drug-resistant subtype of C. gattii that suggests an adaptive response to microbial IMPDH inhibitors in its environmental niche. We report the structural and functional characterization of IMPDH from Cryptococcus, revealing insights into the basis for drug resistance and suggesting strategies for the development of fungal-specific inhibitors. The crystal structure reveals the position of the IMPDH moveable flap and catalytic arginine in the open conformation for the first time, plus unique, exploitable differences in the highly conserved active site. Treatment with mycophenolic acid led to significantly increased survival times in a nematode model, validating de novo GTP biosynthesis as an antifungal target in Cryptococcus. PMID:23071437

  6. Polymorphisms at positions -22 and -348 in the promoter of the BAT1 gene affect transcription and the binding of nuclear factors.

    PubMed

    Price, Patricia; Wong, Agnes M-L; Williamson, David; Voon, Dominic; Baltic, Svetlana; Allcock, Richard J N; Boodhoo, Alvin; Christiansen, Frank T

    2004-05-01

    BAT1 (D6S81E, UAP56) lies in the central MHC between TNF and HLA-B, a region containing genes that affect susceptibility to immunopathologic disorders. BAT1 protein may be directly responsible for the genetic association, as antisense studies show it can down-regulate inflammatory cytokines. Here we investigate polymorphisms at positions -22 and -348 relative to the BAT1 transcription start site. DNA samples from healthy donors were used to confirm haplotypic associations with the type 1 diabetes-susceptible 8.1 ancestral haplotype (AH; HLA-A1,B8,BAT1-22*C,BAT1-348*C,DR3 ) and the diabetes-resistant 7.1 AH (HLA-A3,B7,BAT1-22*G,BAT1-348*T,DR15). Alleles carried at BAT1-22 and -348 were in linkage disequilibrium. Electrophoretic mobility shift assays using nuclear proteins from T-cells (Jurkat and HT2), monocytes (THP1, U937) and epithelial cells (HeLa and MDA468) demonstrated DNA : protein complexes binding oligonucleotides spanning positions -22 and -348 on the 7.1 AH only. Competition assays, supershifts and molecular weight determinations suggest the complexes include the transcription factors YY1 (at -348) and Oct1 (at -22). Promoter activity was demonstrated using 520 bp and 336 bp fragments cloned from immediately upstream of the transcription start site and carrying all combinations of -22 and -348 alleles, suggesting an unidentified non-polymorphic sequence within 336 bp of the start site drives transcription. The 520 bp fragment of the BAT1 promoter cloned from the 8.1 AH was slightly less efficient than the equivalent from the 7.1 AH, whilst the reverse was observed with 336 bp fragments. This suggests BAT1 transcription on the 7.1 AH is modified by interactions involving DNA flanking positions -22 and -348. PMID:15028669

  7. Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion.

    PubMed Central

    Prinsen, C F; Veerkamp, J H

    1998-01-01

    We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette. Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs. The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells. In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity. Uptake rates of palmitate were equal for all clone types, in spite of different FABP content and composition. Palmitate oxidation over a 3 h period was similar in all clones from growth medium. After being cultured in differentiation medium, mock- and H-FABP-cDNA-transfected cells showed a lower fatty acid-oxidation rate, in contrast with A-FABP-cDNA-transfected clones. The ratio of [14C]palmitic acid incorporation into phosphatidylcholine and phosphatidylethanolamine of A-FABP-cDNA-transfected clones changed in the opposite direction in differentiation medium from that of mock- and H-FABP-cDNA-transfected clones. In conclusion, transfection of L6 myoblasts with A-FABP cDNA does not affect H-FABP content and fatty acid uptake, but changes fatty acid metabolism. The latter changes may be related to the observed fusion defect. PMID:9425108

  8. RanGTP is required for meiotic spindle organization and the initiation of embryonic development in Drosophila

    PubMed Central

    Cesario, J.; McKim, K. S.

    2011-01-01

    RanGTP is important for chromosome-dependent spindle assembly in Xenopus extracts. Here we report on experiments to determine the role of the Ran pathway on microtubule dynamics in Drosophila oocytes and embryos. Females expressing a dominant-negative form of Ran have fertility defects, suggesting that RanGTP is required for normal fertility. This is not, however, because of a defect in acentrosomal meiotic spindle assembly. Therefore, RanGTP does not appear to be essential or sufficient for the formation of the acentrosomal spindle. Instead, the most important function of the Ran pathway in spindle assembly appears to be in the tapering of microtubules at the spindle poles, which might be through regulation of proteins such as TACC and the HURP homolog, Mars. One consequence of this spindle organization defect is an increase in the nondisjunction of achiasmate chromosomes. However, the meiotic defects are not severe enough to cause the decreased fertility. Reductions in fertility occur because RanGTP has a role in microtubule assembly that is not directly nucleated by the chromosomes. This includes microtubules nucleated from the sperm aster, which are required for pronuclear fusion. We propose that following nuclear envelope breakdown, RanGTP is released from the nucleus and creates a cytoplasm that is activated for assembling microtubules, which is important for processes such as pronuclear fusion. Around the chromosomes, however, RanGTP might be redundant with other factors such as the chromosome passenger complex. PMID:22100918

  9. Construction and Characterization of Mutations within the Klebsiella mrkD1P Gene That Affect Binding to Collagen Type V

    PubMed Central

    Sebghati, Tricia A.; Clegg, Steven

    1999-01-01

    The fimbria-associated MrkD1P protein mediates adherence of type 3 fimbriate strains of Klebsiella pneumoniae to collagen type V. Currently, three different MrkD adhesins have been described in Klebsiella species, and each possesses a distinctive binding pattern. Therefore, the binding abilities of mutants possessing defined mutations within the mrkD1P gene were examined in order to determine whether specific regions of the adhesin molecule were responsible for collagen binding. Both site-directed and chemically induced mutations were constructed within mrkD1P, and the ability of the gene products to be incorporated into fimbrial appendages or bind to collagen was determined. Binding to type V collagen was not associated solely with one particular region of the MrkD1P protein, and two classes of nonadhesive mutants were isolated. In one class of mutants, the MrkD adhesin was not assembled into the fimbrial shaft, whereas in the second class of mutants, the adhesin was associated with fimbriae but did not bind to collagen. Both hemagglutinating and collagen-binding activities were associated with the MrkD1P molecule, since P pili and type 3 fimbriae carrying adhesive MrkD proteins exhibited identical binding properties. PMID:10085002

  10. Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

    SciTech Connect

    Feltner, D.E.; Marasco, W.A.

    1989-06-01

    The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of (3H)FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM (3H)FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. (3H)FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of (3H)FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM (3H)FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state.

  11. Modification of opiate agonist binding by pertussis toxin

    SciTech Connect

    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. 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.

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

    SciTech Connect

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

    1989-05-01

    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 increased 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.

  13. Cysteine-to-Serine Mutants Dramatically Reorder the Active Site of Human ABO(H) Blood Group B Glycosyltransferase without Affecting Activity: Structural Insights into Cooperative Substrate Binding

    PubMed Central

    Schuman, Brock; Persson, Mattias; Landry, Roxanne C.; Polakowski, Robert; Weadge, Joel T.; Seto, Nina O. L.; Borisova, Svetlana N.; Palcic, Monica M.; Evans, Stephen V.

    2011-01-01

    A common feature in the structures of GT-A-fold-type glycosyltransferases is a mobile polypeptide loop that has been observed to participate in substrate recognition and enclose the active site upon substrate binding. This is the case for the human ABO(H) blood group B glycosyltransferase GTB, where amino acid residues 177–195 display significantly higher levels of disorder in the unliganded state than in the fully liganded state. Structural studies of mutant enzymes GTB/C80S/C196S and GTB/C80S/C196S/C209S at resolutions ranging from 1.93 to 1.40 Å display the opposite trend, where the unliganded structures show nearly complete ordering of the mobile loop residues that is lost upon substrate binding. In the liganded states of the mutant structures, while the UDP moiety of the donor molecule is observed to bind in the expected location, the galactose moiety is observed to bind in a conformation significantly different from that observed for the wild-type chimeric structures. Although this would be expected to impede catalytic turnover, the kinetics of the transfer reaction are largely unaffected. These structures demonstrate that the enzymes bind the donor in a conformation more similar to the dominant solution rotamer and facilitate its gyration into the catalytically competent form. Further, by preventing active-site closure, these structures provide a basis for recently observed cooperativity in substrate binding. Finally, the mutation of C80S introduces a fully occupied UDP binding site at the enzyme dimer interface that is observed to be dependent on the binding of H antigen acceptor analog. PMID:20655926

  14. Reconstitution of high-affinity opioid agonist binding in brain membranes

    SciTech Connect

    Remmers, A.E.; Medzihradsky, F. )

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  15. Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion.

    PubMed Central

    Augustin, L B; Jacobson, B A; Fuchs, J A

    1994-01-01

    The Escherichia coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. The regulation of the nrd operon has been observed to be very complex. The specific binding of two proteins to the nrd regulatory region and expression of mutant nrd-lac fusions that do not bind these proteins are described. A partially purified protein from an E. coli cell extract was previously shown to bind to the promoter region and to regulate transcription of the nrd operon (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990). We have purified this protein to homogeneity by affinity chromatography and identified it as the E. coli factor for inversion stimulation (Fis). Cu-phenanthroline footprinting experiments showed that Fis binds to a site centered 156 bp upstream of the start of nrd transcription. Mutants with deletion and site-directed mutations that do not bind Fis at this site have two- to threefold-lower expression of an nrd-lac fusion. The previously reported negative regulatory nature of this site (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990) was found to be due to a change in polarity in the vectors used to construct promoter fusions. Two nine-base sequences with homology to the DnaA consensus binding sequence are located immediately upstream of the nrd putative -35 RNA polymerase binding site. Binding of DnaA to these sequences on DNA fragments containing the nrd promoter region was confirmed by in vitro Cu-phenanthroline footprinting. Footprinting experiments on fragments with each as well as both of the mutated 9-mers suggests cooperativity between the two sites in binding DnaA. Assay of in vivo expression from wild-type and DnaA box-mutated nrd promoter fragments fused to lacZ on single-copy plasmids indicates a positive effect of DnaA binding on expression of nrd. Images PMID:8288532

  16. 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

  17. Folate synthesis in plants: The first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I

    PubMed Central

    Basset, Gilles; Quinlivan, Eoin P.; Ziemak, Michael J.; Díaz de la Garza, Rocío; Fischer, Markus; Schiffmann, Susi; Bacher, Adelbert; Gregory, Jesse F.; Hanson, Andrew D.

    2002-01-01

    GTP cyclohydrolase I (GCHI) mediates the first and committing step of the pterin branch of the folate-synthesis pathway. In microorganisms and mammals, GCHI is a homodecamer of ≈26-kDa subunits. Genomic approaches identified tomato and Arabidopsis cDNAs specifying ≈50-kDa proteins containing two GCHI-like domains in tandem and indicated that such bimodular proteins occur in other plants. Neither domain of these proteins has a full set of the residues involved in substrate binding and catalysis in other GCHIs. The tomato and Arabidopsis cDNAs nevertheless encode functional enzymes, as shown by complementation of a yeast fol2 mutant and by assaying GCHI activity in extracts of complemented yeast cells. Neither domain expressed separately had GCHI activity. Recombinant tomato GCHI formed dihydroneopterin triphosphate as reaction product, as do other GCHIs, but unlike these enzymes it did not show cooperative behavior and was inhibited by its substrate. Denaturing gel electrophoresis verified that the bimodular GCHI polypeptide is not cleaved in vivo into its component domains, and size-exclusion chromatography indicated that the active enzyme is a dimer. The deduced tomato and Arabidopsis GCHI polypeptides lack overt targeting sequences and thus are presumably cytosolic, in contrast to other plant folate-synthesis enzymes, which are mitochondrial proteins with typical signal peptides. GCHI mRNA and protein are strongly in expressed unripe tomato fruits, implying that fruit folate is made in situ rather than imported. As ripening advances, GCHI expression declines sharply, and folate content drops, suggesting that folate synthesis fails to keep pace with turnover. PMID:12221287

  18. Specific binding of nucleotides and NAD+ to Clostridium difficile toxin A.

    PubMed

    Lobban, M D; Borriello, S P

    1992-02-24

    Binding of nucleotides, a tetrapolyphosphate, and NAD+ to purified toxin A of Clostridium difficile was determined by monitoring changes in intrinsic fluorescence following excitation at 280 nm, and recording emissions at 340 nm. Binding was specific for concentrations over the range 5 to 100 microM for ATP, GTP, and their respective non-hydrolysable analogues AMP-PNP and Gpp(NH)p, tetrapolyphosphate and NAD+. PMID:1544441

  19. Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

    PubMed Central

    Koch, Miriam; Flür, Sara; Kreutz, Christoph; Ennifar, Eric; Micura, Ronald; Polacek, Norbert

    2015-01-01

    Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases. PMID:25941362

  20. Klenow Fragment Discriminates against the Incorporation of the Hyperoxidized dGTP Lesion Spiroiminodihydantoin into DNA.

    PubMed

    Huang, Ji; Yennie, Craig J; Delaney, Sarah

    2015-12-21

    Defining the biological consequences of oxidative DNA damage remains an important and ongoing area of investigation. At the foundation of understanding the repercussions of such damage is a molecular-level description of the action of DNA-processing enzymes, such as polymerases. In this work, we focus on a secondary, or hyperoxidized, oxidative lesion of dG that is formed by oxidation of the primary oxidative lesion, 2'-deoxy-8-oxo-7,8-dihydroguanosine (8-oxodG). In particular, we examine incorporation into DNA of the diastereomers of the hyperoxidized guanosine triphosphate lesion spiroiminodihydantoin-2'-deoxynucleoside-5'-triphosphate (dSpTP). Using kinetic parameters, we describe the ability of the Klenow fragment of Escherichia coli DNA polymerase I lacking 3' → 5' exonuclease activity (KF(-)) to utilize (S)-dSpTP and (R)-dSpTP as building blocks during replication. We find that both diastereomers act as covert lesions, similar to a Trojan horse: KF(-) incorporates the lesion dNTP opposite dC, which is a nonmutagenic event; however, during the subsequent replication, it is known that dSp is nearly 100% mutagenic. Nevertheless, using kpol/Kd to define the nucleotide incorporation specificity, we find that the extent of oxidation of the dGTP-derived lesion correlates with its ability to be incorporated into DNA. KF(-) has the highest specificity for incorporation of dGTP opposite dC. The selection factors for incorporating 8-oxodGTP, (S)-dSpTP, and (R)-dSpTP are 1700-, 64000-, and 850000-fold lower, respectively. Thus, KF(-) is rigorous in its discrimination against incorporation of the hyperoxidized lesion, and these results suggest that the specificity of cellular polymerases provides an effective mechanism to avoid incorporating dSpTP lesions into DNA from the nucleotide pool. PMID:26572218

  1. The integrated global temperature change potential (iGTP) and relationships between emission metrics

    NASA Astrophysics Data System (ADS)

    Peters, Glen P.; Aamaas, Borgar; Berntsen, Terje; Fuglestvedt, Jan S.

    2011-12-01

    The Kyoto Protocol compares greenhouse gas emissions (GHGs) using the global warming potential (GWP) with a 100 yr time-horizon. The GWP was developed, however, to illustrate the difficulties in comparing GHGs. In response, there have been many critiques of the GWP and several alternative emission metrics have been proposed. To date, there has been little focus on understanding the linkages between, and interpretations of, different emission metrics. We use an energy balance model to mathematically link the absolute GWP, absolute global temperature change potential (AGTP), absolute ocean heat perturbation (AOHP), and integrated AGTP. For pulse emissions, energy conservation requires that AOHP = AGWP - iAGTP/λ and hence AGWP and iAGTP are closely linked and converge as AOHP decays to zero. When normalizing the metrics with CO2 (GWP, GTP, and iGTP), we find that the iGTP and GWP are similar numerically for a wide range of GHGs and time-horizons, except for very short-lived species. The similarity between the iGTPX and GWPX depends on how well a pulse emission of CO2 can substitute for a pulse emission of X across a range of time-horizons. The ultimate choice of emission metric(s) and time-horizon(s) depends on policy objectives. To the extent that limiting integrated temperature change over a specific time-horizon is consistent with the broader objectives of climate policy, our analysis suggests that the GWP represents a relatively robust, transparent and policy-relevant emission metric.

  2. Topological relations embodied in a generalized tri-prism (GTP) model for a 3D geoscience modeling system

    NASA Astrophysics Data System (ADS)

    Wu, Lixin

    2004-05-01

    3D geoscience modeling system (3D GMS) embodied with topological relations is of extreme importance for Geosciences. This paper presents a universal 3D model, generalized tri-prism (GTP) for 3D GMS and real-3D GIS, which is a modification and improvement of former presented analogous tri-prism (ATP) model and is the common model of pyramid model, tetrahedron model and tri-prism (TP) model. The GTP model takes the divergent drill holes, rather than triangulation network after interpolation or vertical parallel drill holes after projection transformation, as its direct data source. Hence, the reliability and quality of the 3D model is maximatily ensured. The GTP component is comprised of six primitives as node, TIN-edge, side-edge, TIN-face, side-face and GTP. Besides, three intermediary diagonal lines in each GTP component are temporary applied for spatial operations. Six groups of topological relations between the six primitives are carefully designed for geo-spatial inquiry and geo-spatial analysis. The mechanisms of chipping, dynamic updating and local refining operations of so constructed 3D geological model are introduced. A real-3D software platform, GeoMo 3D@, developed with VC ++, OPGL and SQL server, demonstrates most of the 3D geo-spatial operations including clipping, separating, uncovering and geo-fence diagram generating based on an actual 3D geological model of a coal mine, Tangshan, P.R. China.

  3. Structural basis for the binding of succinate to succinyl-CoA synthetase.

    PubMed

    Huang, Ji; Fraser, Marie E

    2016-08-01

    Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate. PMID:27487822

  4. How Do Structure and Charge Affect Metal-Complex Binding to DNA? An Upper-Division Integrated Laboratory Project Using Cyclic Voltammetry

    ERIC Educational Resources Information Center

    Kulczynska, Agnieszka; Johnson, Reed; Frost, Tony; Margerum, Lawrence D.

    2011-01-01

    An advanced undergraduate laboratory project is described that integrates inorganic, analytical, physical, and biochemical techniques to reveal differences in binding between cationic metal complexes and anionic DNA (herring testes). Students were guided to formulate testable hypotheses based on the title question and a list of different metal…

  5. Changes in ganglioside content affect the binding of Clostridium perfringens epsilon-toxin to detergent-resistant membranes of Madin-Darby canine kidney cells.

    PubMed

    Shimamoto, Seiko; Tamai, Eiji; Matsushita, Osamu; Minami, Junzaburo; Okabe, Akinobu; Miyata, Shigeru

    2005-01-01

    Epsilon-toxin (ET) of Clostridium perfringens, which causes fatal enterotoxemia in ungulates, was previously shown to bind to and form a heptameric pore within the detergent-resistant membranes (DRMs) of MDCK cells. Depletion of cholesterol has also been shown to decrease the cytotoxicity of ET and its heptamerization. In this study, we investigated the effects of changes in sphingolipids, other DRM components of MDCK cells, on the cells' susceptibility to ET. Treatment with fumonisin B1 and PDMP, inhibitors of sphingolipid and glycosphingolipid syntheses, respectively, increased the susceptibility, while D609, a sphingomyelin synthesis inhibitor, had the opposite effect. The exogenous addition of ganglioside G(M1) dramatically decreased the ET binding, heptamerization and cytotoxicity. These effects were shown not to be due to ET binding to G(M1) or to denaturation of ET. We also found that the ET cytotoxicity towards MDCK cells decreased with an increase in culture time. In accordance with the resistance observed for prolonged cultured cells, G(M3), a major ganglioside component, increased and sialidase treatment increased their susceptibility. These results suggest that membrane-anchored sialic acid of G(M3) within DRMs inhibits ET binding, leading to prevention of the heptamerization of ET and cell death. It is also suggested that sialidase produced by this organism aids the targeting of ET to MDCK cells. PMID:15781998

  6. Regulation of K-Ras4B Membrane Binding by Calmodulin.

    PubMed

    Sperlich, Benjamin; Kapoor, Shobhna; Waldmann, Herbert; Winter, Roland; Weise, Katrin

    2016-07-12

    K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca(2+)-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca(2+)-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca(2+)/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca(2+)/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca(2+)/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca(2+)/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδ exhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell. PMID:27410739

  7. Further characterization of the red beet plasma membrane Ca sup 2+ -ATPase using GTP as an alternative substrate

    SciTech Connect

    Williams, L.E.; Schueler, S.B.; Briskin, D.P. )

    1990-03-01

    The GTP-driven component of Ca{sup 2+} uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca{sup 2+}-translocating ATPase and assess its utility as a probe for this transport system. Uptake of {sup 45}Ca{sup 2+} in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca{sup 2+} concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca{sup 2+}-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of {sup 45}Ca{sup 2+}-uptake by exogeneous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven {sup 45}Ca{sup 2+} uptake represents the capacity of the plasma membrane Ca{sup 2+}-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with ({gamma}-{sup 32}P)GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca{sup 2+}-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca{sup 2+}-ATPases present in animal cells.

  8. Characterization of Specific Nucleotide Substitutions in DtxR-Specific Operators of Corynebacterium diphtheriae That Dramatically Affect DtxR Binding, Operator Function, and Promoter Strength

    PubMed Central

    Lee, John H.; Holmes, Randall K.

    2000-01-01

    The diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae uses Fe2+ as a corepressor. Holo-DtxR inhibits transcription from the iron-regulated promoters (IRPs) designated IRP1 through IRP5 as well as from the promoters for the tox and hmuO genes. DtxR binds to 19-bp operators with the consensus sequence 5′-TTAGGTTAGCCTAACCTAA-3′, a perfect 9-bp palindrome interrupted by a single C · G base pair. Among the seven known DtxR-specific operators, IRP3 exhibits the weakest binding to DtxR. The message (sense) strand of the IRP3 operator (5′-TTAGGTGAGACGCACCCAT-3′ [nonconsensus nucleotides underlined]) overlaps by 2 nucleotides at its 5′ end with the putative −10 sequence of the IRP3 promoter. The underlined C at position +7 from the center of the IRP3 operator [C(+7)] is unique, because T is conserved at that position in other DtxR-specific operators. The present study examined the effects of nucleotide substitutions at position +7 or −7 in the IRP3 operator. In gel mobility shift assays, only the change of C(+7) to the consensus nucleotide T caused a dramatic increase in the binding of DtxR, whereas other nucleotide substitutions for C(+7) or replacements for A(−7) had only small positive or negative effects on DtxR binding. All substitutions for C(+7) or A(−7) except for A(−7)C dramatically decreased IRP3 promoter strength. In contrast, the A(−7)C variant caused increased promoter strength at the cost of nearly eliminating repressibility by DtxR. The message (sense) strand of the IRP1 operator (5′-TTAGGTTAGCCAAACCTTT-3′) includes the −35 region of the IRP3 promoter. A T(+7)C variant of the IRP1 operator was also constructed, and it was shown to exhibit decreased binding to DtxR, decreased repressibility by DtxR, and increased promoter strength. The nucleotides at positions +7 and −7 in DtxR-specific operators are therefore important determinants of DtxR binding and repressibility of transcription by DtxR, and they also have

  9. Purification, crystallization and preliminary X-ray characterization of the human GTP fucose pyrophosphorylase

    SciTech Connect

    Quirk, Stephen; Seley-Radtke, Katherine L.

    2006-04-01

    The human GTP fucose pyrophosphohydrolase protein has been crystallized via the hanging-drop technique over a reservoir of polyethylene glycol (MW 8000) and ethylene glycol. The orthorhombic crystals diffract to 2.8 Å resolution. The human nucleotide-sugar metabolizing enzyme GTP fucose pyrophosphorylase (GFPP) has been purified to homogeneity by an affinity chromatographic procedure that utilizes a novel nucleoside analog. This new purification regime results in a protein preparation that produces significantly better crystals than traditional purification methods. The purified 66.6 kDa monomeric protein has been crystallized via hanging-drop vapor diffusion at 293 K. Crystals of the native enzyme diffract to 2.8 Å and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. There is a single GFPP monomer in the asymmetric unit, giving a Matthews coefficient of 2.38 Å{sup 3} Da{sup −1} and a solvent content of 48.2%. A complete native data set has been collected as a first step in determining the three-dimensional structure of this enzyme.

  10. Tubulin exchanges divalent cations at both guanine nucleotide-binding sites.

    PubMed

    Correia, J J; Beth, A H; Williams, R C

    1988-08-01

    The tubulin heterodimer binds a molecule of GTP at the nonexchangeable nucleotide-binding site (N-site) and either GDP or GTP at the exchangeable nucleotide-binding site (E-site). Mg2+ is known to be tightly linked to the binding of GTP at the E-site (Correia, J. J., Baty, L. T., and Williams, R. C., Jr. (1987) J. Biol. Chem. 262, 17278-17284). Measurements of the exchange of Mn2+ for bound Mg2+ (as monitored by atomic absorption and EPR) demonstrate that tubulin which has GDP at the E-site possesses one high affinity metal-binding site and that tubulin which has GTP at the E-site possesses two such sites. The apparent association constants are 0.7-1.1 x 10(6) M-1 for Mg2+ and approximately 4.1-4.9 x 10(7) M-1 for Mn2+. Divalent cations do bind to GDP at the E-site, but with much lower affinity (2.0-2.3 x 10(3) M-1 for Mg2+ and 3.9-6.6 x 10(3) M-1 for Mn2+). These data suggest that divalent cations are involved in GTP binding to both the N- and E-sites of tubulin. The N-site metal exchanges slowly (kapp = 0.020 min-1), suggesting a mechanism involving protein "breathing" or heterodimer dissociation. The N-site metal exchange rate is independent of the concentration of protein and metal, an observation consistent with the possibility that a dynamic breathing process is the rate-limiting step. The exchange of Mn2+ for Mg2+ has no effect on the secondary structure of tubulin at 4 degrees C or on the ability of tubulin to form microtubules. These results have important consequences for the interpretation of distance measurements within the tubulin dimer using paramagnetic ions. They are also relevant to the detailed mechanism of divalent cation release from microtubules after GTP hydrolysis. PMID:3392036

  11. Loss of Calmodulin Binding to Bax Inhibitor-1 Affects Pseudomonas-mediated Hypersensitive Response-associated Cell Death in Arabidopsis thaliana*

    PubMed Central

    Kawai-Yamada, Maki; Hori, Zenta; Ogawa, Taro; Ihara-Ohori, Yuri; Tamura, Katsunori; Nagano, Minoru; Ishikawa, Toshiki; Uchimiya, Hirofumi

    2009-01-01

    Bax inhibitor-1 (BI-1) is a cell death suppressor protein conserved across a variety of organisms. The Arabidopsis atbi1-1 plant is a mutant in which the C-terminal 6 amino acids of the expressed BI-1 protein have been replaced by T-DNA insertion. This mutant BI-1 protein (AtBI-CM) produced in Escherichia coli can no longer bind to calmodulin. A promoter-reporter assay demonstrated compartmentalized expression of BI-1 during hypersensitive response, introduced by the inoculation of Pseudomonas syringae possessing the avrRTP2 gene, Pst(avrRPT2). In addition, both BI-1 knockdown plants and atbi1-1 showed increased sensitivity to Pst(avrRPT2)-induced cell death. The results indicated that the loss of calmodulin binding reduces the cell death suppressor activity of BI-1 in planta. PMID:19674971

  12. RNA-binding protein Hermes/RBPMS inversely affects synapse density and axon arbor formation in retinal ganglion cells in vivo.

    PubMed

    Hörnberg, Hanna; Wollerton-van Horck, Francis; Maurus, Daniel; Zwart, Maarten; Svoboda, Hanno; Harris, William A; Holt, Christine E

    2013-06-19

    The RNA-binding protein Hermes [RNA-binding protein with multiple splicing (RBPMS)] is expressed exclusively in retinal ganglion cells (RGCs) in the CNS, but its function in these cells is not known. Here we show that Hermes protein translocates in granules from RGC bodies down the growing axons. Hermes loss of function in both Xenopus laevis and zebrafish embryos leads to a significant reduction in retinal axon arbor complexity in the optic tectum, and expression of a dominant acting mutant Hermes protein, defective in RNA-granule localization, causes similar defects in arborization. Time-lapse analysis of branch dynamics reveals that the decrease in arbor complexity is caused by a reduction in new branches rather than a decrease in branch stability. Surprisingly, Hermes depletion also leads to enhanced early visual behavior and an increase in the density of presynaptic puncta, suggesting that reduced arborization is accompanied by increased synaptogenesis to maintain synapse number. PMID:23785151

  13. The size, shape and specificity of the sugar-binding site of the jacalin-related lectins is profoundly affected by the proteolytic cleavage of the subunits.

    PubMed Central

    Houlès Astoul, Corinne; Peumans, Willy J; van Damme, Els J M; Barre, Annick; Bourne, Yves; Rougé, Pierre

    2002-01-01

    Mannose-specific lectins with high sequence similarity to jacalin and the Maclura pomifera agglutinin have been isolated from species belonging to the families Moraceae, Convolvulaceae, Brassicaceae, Asteraceae, Poaceae and Musaceae. Although these novel mannose-specific lectins are undoubtedly related to the galactose-specific Moraceae lectins there are several important differences. Apart from the obvious differences in specificity, the mannose- and galactose-specific jacalin-related lectins differ in what concerns their biosynthesis and processing, intracellular location and degree of oligomerization of the protomers. Taking into consideration that the mannose-specific lectins are widely distributed in higher plants, whereas their galactose-specific counterparts are confined to a subgroup of the Moraceae sp. one can reasonably assume that the galactose-specific Moraceae lectins are a small-side group of the main family. The major change that took place in the structure of the binding site of the diverging Moraceae lectins concerns a proteolytic cleavage close to the N-terminus of the protomer. To corroborate the impact of this change, the specificity of jacalin was re-investigated using surface plasmon resonance analysis. This approach revealed that in addition to galactose and N -acetylgalactosamine, the carbohydrate-binding specificity of jacalin extends to mannose, glucose, N -acetylmuramic acid and N -acetylneuraminic acid. Owing to this broad carbohydrate-binding specificity, jacalin is capable of recognizing complex glycans from plant pathogens or predators. PMID:12169094

  14. The RNA-binding protein quaking maintains endothelial barrier function and affects VE-cadherin and β-catenin protein expression

    PubMed Central

    de Bruin, Ruben G.; van der Veer, Eric P.; Prins, Jurriën; Lee, Dae Hyun; Dane, Martijn J. C.; Zhang, Huayu; Roeten, Marko K.; Bijkerk, Roel; de Boer, Hetty C.; Rabelink, Ton J.; van Zonneveld, Anton Jan; van Gils, Janine M.

    2016-01-01

    Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis, and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells, and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and β-catenin and can induce mRNA translation mediated by the 3′UTR of these genes. Reduced quaking levels attenuated VE-cadherin and β-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity. PMID:26905650

  15. The RNA-binding protein quaking maintains endothelial barrier function and affects VE-cadherin and β-catenin protein expression.

    PubMed

    de Bruin, Ruben G; van der Veer, Eric P; Prins, Jurriën; Lee, Dae Hyun; Dane, Martijn J C; Zhang, Huayu; Roeten, Marko K; Bijkerk, Roel; de Boer, Hetty C; Rabelink, Ton J; van Zonneveld, Anton Jan; van Gils, Janine M

    2016-01-01

    Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis, and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells, and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and β-catenin and can induce mRNA translation mediated by the 3'UTR of these genes. Reduced quaking levels attenuated VE-cadherin and β-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity. PMID:26905650

  16. Strategic mutations in the class I major histocompatibility complex HLA-A2 independently affect both peptide binding and T cell receptor recognition.

    PubMed

    Baxter, Tiffany K; Gagnon, Susan J; Davis-Harrison, Rebecca L; Beck, John C; Binz, Anne-Kathrin; Turner, Richard V; Biddison, William E; Baker, Brian M

    2004-07-01

    Mutational studies of T cell receptor (TCR) contact residues on the surface of the human class I major histocompatibility complex (MHC) molecule HLA-A2 have identified a "functional hot spot" that comprises Arg(65) and Lys(66) and is involved in recognition by most peptide-specific HLA-A2-restricted TCRs. Although there is a significant amount of functional data on the effects of mutations at these positions, there is comparatively little biochemical information that could illuminate their mode of action. Here, we have used a combination of fluorescence anisotropy, functional assays, and Biacore binding experiments to examine the effects of mutations at these positions on the peptide-MHC interaction and TCR recognition. The results indicate that mutations at both position 65 and position 66 influence peptide binding by HLA-A2 to various extents. In particular, mutations at position 66 result in significantly increased peptide dissociation rates. However, these effects are independent of their effects on TCR recognition, and the Arg(65)-Lys(66) region thus represents a true "hot spot" for TCR recognition. We also made the observation that in vitro T cell reactivity does not scale with the half-life of the peptide-MHC complex, as is often assumed. Finally, position 66 is implicated in the "dual recognition" of both peptide and TCR, emphasizing the multiple roles of the class I MHC peptide-binding domain. PMID:15131131

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

    PubMed

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

    2016-03-01

    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

  18. 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

  19. Neisseria gonorrhoeae MutS Affects Pilin Antigenic Variation through Mismatch Correction and Not by pilE Guanine Quartet Binding

    PubMed Central

    Rotman, Ella

    2015-01-01

    ABSTRACT Many pathogens use homologous recombination to vary surface antigens to avoid immune surveillance. Neisseria gonorrhoeae achieves this in part by changing the properties of its surface pili in a process called pilin antigenic variation (AV). Pilin AV occurs by high-frequency gene conversion reactions that transfer silent pilS sequences into the expressed pilE locus and requires the formation of an upstream guanine quartet (G4) DNA structure to initiate this process. The MutS and MutL proteins of the mismatch correction (MMC) system act to correct mismatches after replication and prevent homeologous (i.e., partially homologous) recombination, but MutS orthologs can also bind to G4 structures. A previous study showed that mutation of MutS resulted in a 3-fold increase in pilin AV, which could be due to the loss of MutS antirecombination properties or loss of G4 binding. We tested two site-directed separation-of-function MutS mutants that are both predicted to bind to G4s but are not able to perform MMC. Pilus phase variation assays and DNA sequence analysis of pilE variants produced in these mutants showed that all three mutS mutants and a mutL mutant had similar increased frequencies of pilin AV. Moreover, the mutS mutants all showed similar increased levels of pilin AV-dependent synthetic lethality. These results show that antirecombination by MMC is the reason for the effect that MutS has on pilin AV and is not due to pilE G4 binding by MutS. IMPORTANCE Neisseria gonorrhoeae continually changes its outer surface proteins to avoid recognition by the immune system. N. gonorrhoeae alters the antigenicity of the pilus by directed recombination between partially homologous pilin copies in a process that requires a guanine quartet (G4) structure. The MutS protein of the mismatch correction (MMC) system prevents recombination between partially homologous sequences and can also bind to G4s. We confirmed that loss of MMC increases the frequency of pilin antigenic

  20. GTP cyclohydrolase I and tyrosine hydroxylase gene mutations in familial and sporadic dopa-responsive dystonia patients.

    PubMed

    Cai, Chunyou; Shi, Wentao; Zeng, Zheng; Zhang, Meiyun; Ling, Chao; Chen, Lei; Cai, Chunquan; Zhang, Benshu; Li, Wei-Dong

    2013-01-01

    Dopa-responsive dystonia (DRD) is a rare inherited dystonia that responds very well to levodopa treatment. Genetic mutations of GTP cyclohydrolase I (GCH1) or tyrosine hydroxylase (TH) are disease-causing mutations in DRD. To evaluate the genotype-phenotype correlations and diagnostic values of GCH1 and TH mutation screening in DRD patients, we carried out a combined study of familial and sporadic cases in Chinese Han subjects. We collected 23 subjects, 8 patients with DRD, 5 unaffected family members, and 10 sporadic cases. We used PCR to sequence all exons and splicing sites of the GCH1 and TH genes. Three novel heterozygous GCH1 mutations (Tyr75Cys, Ala98Val, and Ile135Thr) were identified in three DRD pedigrees. We failed to identify any GCH1 or TH mutation in two affected sisters. Three symptom-free male GCH1 mutation carriers were found in two DRD pedigrees. For those DRD siblings that shared the same GCH1 mutation, symptoms and age of onset varied. In 10 sporadic cases, only two heterozygous TH mutations (Ser19Cys and Gly397Arg) were found in two subjects with unknown pathogenicity. No GCH1 and TH mutation was found in 40 unrelated normal Han Chinese controls. GCH1 mutation is the main etiology of familial DRD. Three novel GCH1 mutations were identified in this study. Genetic heterogeneity and incomplete penetrance were quite common in DRD patients, especially in sporadic cases. Genetic screening may help establish the diagnosis of DRD; however, a negative GCH1 and TH mutation test would not exclude the diagnosis. PMID:23762320

  1. Aspartic Acid 397 in Subunit B of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae Forms Part of a Sodium-binding Site, Is Involved in Cation Selectivity, and Affects Cation-binding Site Cooperativity

    PubMed Central

    Shea, Michael E.; Juárez, Oscar; Cho, Jonathan; Barquera, Blanca

    2013-01-01

    The Na+-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC). PMID:24030824

  2. The mechanism of potent GTP cyclohydrolase I inhibition by 2,4-diamino-6-hydroxypyrimidine: requirement of the GTP cyclohydrolase I feedback regulatory protein.

    PubMed

    Kolinsky, Monica A; Gross, Steven S

    2004-09-24

    Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) in a biological system. Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor. Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH. Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP). Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4. Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system. We further demonstrate that L-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP. These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of L-Phe. PMID:15292175

  3. The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding

    PubMed Central

    Ejendal, Karin F.K.; Diop, Ndeye Khady; Schweiger, Linda C.; Hrycyna, Christine A.

    2006-01-01

    Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding. PMID:16815914

  4. Binding of sFRP-3 to EGF in the Extra-Cellular Space Affects Proliferation, Differentiation and Morphogenetic Events Regulated by the Two Molecules

    PubMed Central

    Tosoni, Daniela; Borello, Ugo; Sampaolesi, Maurilio; Sciorati, Clara; Cannata, Stefano; Clementi, Emilio; Brunelli, Silvia; Cossu, Giulio

    2008-01-01

    Background sFRP-3 is a soluble antagonist of Wnts, widely expressed in developing embryos. The Wnt gene family comprises cysteine-rich secreted ligands that regulate cell proliferation, differentiation, organogenesis and oncogenesis of different organisms ranging from worms to mammals. In the canonical signal transduction pathway Wnt proteins bind to the extracellular domain of Frizzled receptors and consequently recruit Dishevelled (Dsh) to the cell membrane. In addition to Wnt membrane receptors belonging to the Frizzled family, several other molecules have been described which share homology in the CRD domain and lack the putative trans-membrane domain, such as sFRP molecules (soluble Frizzled Related Protein). Among them, sFRP-3 was originally isolated from bovine articular cartilage and also as a component of the Spemann organizer. sFRP-3 blocks Wnt-8 induced axis duplication in Xenopus embryos and binds to the surface of cells expressing a membrane-anchored form of Wnt-1. Injection of sFRP-3 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Methodology/Principal Findings Here we report that sFRP-3 specifically blocks EGF-induced fibroblast proliferation and foci formation. Over-expression of sFRP-3 reverts EGF-mediated inhibition of hair follicle development in the mouse ectoderm while its ablation in Xenopus maintains EGF-mediated inhibition of ectoderm differentiation. Conversely, over-expression of EGF reverts the inhibition of somitic myogenesis and axis truncation in Xenopus and mouse embryos caused by sFRP-3. In vitro experiments demonstrated a direct binding of EGF to sFRP-3 both on heparin and on the surface of CHO cells where the molecule had been membrane anchored. Conclusions/Significance sFRP-3 and EGF reciprocally inhibit their effects on cell proliferation, differentiation and morphogenesis and indeed are expressed in contiguous domains of the embryo, suggesting that in addition to their

  5. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    SciTech Connect

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

    1991-07-01

    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 bound 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.

  6. Potassium Acts as a GTPase-Activating Element on Each Nucleotide-Binding Domain of the Essential Bacillus subtilis EngA

    PubMed Central

    Foucher, Anne-Emmanuelle; Reiser, Jean-Baptiste; Ebel, Christine; Housset, Dominique; Jault, Jean-Michel

    2012-01-01

    EngA proteins form a unique family of bacterial GTPases with two GTP-binding domains in tandem, namely GD1 and GD2, followed by a KH (K-homology) domain. They have been shown to interact with the bacterial ribosome and to be involved in its biogenesis. Most prokaryotic EngA possess a high GTPase activity in contrast to eukaryotic GTPases that act mainly as molecular switches. Here, we have purified and characterized the GTPase activity of the Bacillus subtilis EngA and two shortened EngA variants that only contain GD1 or GD2-KH. Interestingly, the GTPase activity of GD1 alone is similar to that of the whole EngA, whereas GD2-KH has a 150-fold lower GTPase activity. At physiological concentration, potassium strongly stimulates the GTPase activity of each protein construct. Interestingly, it affects neither the affinities for nucleotides nor the monomeric status of EngA or the GD1 domain. Thus, potassium likely acts as a chemical GTPase-activating element as proposed for another bacterial GTPase like MnmE. However, unlike MnmE, potassium does not promote dimerization of EngA. In addition, we solved two crystal structures of full-length EngA. One of them contained for the first time a GTP-like analogue bound to GD2 while GD1 was free. Surprisingly, its overall fold was similar to a previously solved structure with GDP bound to both sites. Our data indicate that a significant structural change must occur upon K+ binding to GD2, and a comparison with T. maritima EngA and MnmE structures allowed us to propose a model explaining the chemical basis for the different GTPase activities of GD1 and GD2. PMID:23056455

  7. The Era GTPase recognizes the GAUCACCUCC sequence and binds helix 45 near the 3; end of 16S rRNA

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tarasov, Sergey G.; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    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, whether 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.

  8. The Actin-binding Domain of Cortactin is Dynamic and Unstructured and Affects Lateral and Longitudinal Contacts in F-actin

    PubMed Central

    Shvetsov, Alexander; Berkane, Emir; Chereau, David; Dominguez, Roberto; Reisler, Emil

    2011-01-01

    Cortactin is an F-actin- and Arp2/3 complex-binding protein, implicated in the regulation of cytoskeleton dynamics and cortical actin-assembly. The actin-binding domain of cortactin consists of a 6.5 tandem repeat of a 37-amino acid sequence known as the cortactin repeat (residues 80-325). Using a combination of structure prediction, circular dichroism and cysteine crosslinking, we tested a recently published three-dimensional model of the cortactin molecule in which the cortactin repeat is folded as a globular helical domain (Zhang et al., 2007). We show that the cortactin repeat is unstructured in solution. Thus, wild type and mutant constructs of the cortactin repeat, containing pairs of cysteines at positions 112 and 246, 83 and 112, 83 and 246, and 83 and 306, could be readily crosslinked with reagents of varying lengths (0–9.6 Å). Using yeast actin cysteine mutants, we also show that cortactin inhibits disulfide and dibromobimane crosslinking across the lateral and longitudinal interfaces of actin subunits in the filament, suggesting a weakening of inter-subunits contacts. Our results are in disagreement with the proposed model of the cortactin molecule and have important implications for our understanding of cortactin regulation of cytoskeleton dynamics. PMID:19089942

  9. A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

    NASA Technical Reports Server (NTRS)

    Lu, C.; Fedoroff, N.

    2000-01-01

    Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

  10. Loss of the Otx2-Binding Site in the Nanog Promoter Affects the Integrity of Embryonic Stem Cell Subtypes and Specification of Inner Cell Mass-Derived Epiblast.

    PubMed

    Acampora, Dario; Omodei, Daniela; Petrosino, Giuseppe; Garofalo, Arcomaria; Savarese, Marco; Nigro, Vincenzo; Di Giovannantonio, Luca Giovanni; Mercadante, Vincenzo; Simeone, Antonio

    2016-06-21

    Mouse embryonic stem cells (ESCs) and the inner cell mass (ICM)-derived epiblast exhibit naive pluripotency. ESC-derived epiblast stem cells (EpiSCs) and the postimplantation epiblast exhibit primed pluripotency. Although core pluripotency factors are well-characterized, additional regulators, including Otx2, recently have been shown to function during the transition from naive to primed pluripotency. Here we uncover a role for Otx2 in the control of the naive pluripotent state. We analyzed Otx2-binding activity in ESCs and EpiSCs and identified Nanog, Oct4, and Sox2 as direct targets. To unravel the Otx2 transcriptional network, we targeted the strongest Otx2-binding site in the Nanog promoter, finding that this site modulates the size of specific ESC-subtype compartments in cultured cells and promotes Nanog expression in vivo, predisposing ICM differentiation to epiblast. Otx2-mediated Nanog regulation thus contributes to the integrity of the ESC state and cell lineage specification in preimplantation development. PMID:27292645

  11. The actin-binding domain of cortactin is dynamic and unstructured and affects lateral and longitudinal contacts in F-actin.

    PubMed

    Shvetsov, Alexander; Berkane, Emir; Chereau, David; Dominguez, Roberto; Reisler, Emil

    2009-02-01

    Cortactin is an F-actin- and Arp2/3 complex-binding protein, implicated in the regulation of cytoskeleton dynamics and cortical actin-assembly. The actin-binding domain of cortactin consists of a 6.5 tandem repeat of a 37-amino acid sequence known as the cortactin repeat (residues 80-325). Using a combination of structure prediction, circular dichroism, and cysteine crosslinking, we tested a recently published three-dimensional model of the cortactin molecule in which the cortactin repeat is folded as a globular helical domain [Zhang et al., 2007, Mol Cell 27:197-213]. We show that the cortactin repeat is unstructured in solution. Thus, wild type and mutant constructs of the cortactin repeat, containing pairs of cysteines at positions 112 and 246, 83 and 112, 83 and 246, and 83 and 306, could be readily crosslinked with reagents of varying lengths (0-9.6 A). Using yeast actin cysteine mutants, we also show that cortactin inhibits disulfide and dibromobimane crosslinking across the lateral and longitudinal interfaces of actin subunits in the filament, suggesting a weakening of intersubunits contacts. Our results are in disagreement with the proposed model of the cortactin molecule and have important implications for our understanding of cortactin regulation of cytoskeleton dynamics. PMID:19089942

  12. Removal of 8-oxo-GTP by MutT hydrolase is not a major contributor to transcriptional fidelity.

    PubMed

    Gordon, Alasdair J E; Satory, Dominik; Wang, Mengyu; Halliday, Jennifer A; Golding, Ido; Herman, Christophe

    2014-10-29

    Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage DNA, RNA, protein and lipids. The MutT protein in Escherichia coli removes 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-GTP) from the nucleotide pools precluding incorporation into DNA and RNA. While 8-oxo-dGTP incorporation into DNA is mutagenic, it is not clear if 8-oxo-GTP incorporation into RNA can have phenotypic consequences for the cell. We use a bistable epigenetic switch sensitive to transcription errors in the Escherichia coli lacI transcript to monitor transient RNA errors. We do not observe any increase in epigenetic switching in mutT cells. We revisit the original observation of partial phenotypic suppression of a lacZamber allele in a mutT background that was attributed to RNA errors. We find that Lac+ revertants can completely account for the increase in β-galactosidase levels in mutT lacZamber cultures, without invoking participation of transient transcription errors. Moreover, we observe a fluctuation type of distribution of β-galactosidase appearance in a growing culture, consistent with Lac+ DNA revertant events. We conclude that the absence of MutT produces a DNA mutator but does not equally create an RNA mutator. PMID:25294823

  13. Molecular Mechanism for Conformational Dynamics of Ras·GTP Elucidated from In-Situ Structural Transition in Crystal.

    PubMed

    Matsumoto, Shigeyuki; Miyano, Nao; Baba, Seiki; Liao, Jingling; Kawamura, Takashi; Tsuda, Chiemi; Takeda, Azusa; Yamamoto, Masaki; Kumasaka, Takashi; Kataoka, Tohru; Shima, Fumi

    2016-01-01

    Ras•GTP adopts two interconverting conformational states, state 1 and state 2, corresponding to inactive and active forms, respectively. However, analysis of the mechanism for state transition was hampered by the lack of the structural information on wild-type Ras state 1 despite its fundamental nature conserved in the Ras superfamily. Here we solve two new crystal structures of wild-type H-Ras, corresponding to state 1 and state 2. The state 2 structure seems to represent an intermediate of state transition and, intriguingly, the state 1 crystal is successfully derived from this state 2 crystal by regulating the surrounding humidity. Structural comparison enables us to infer the molecular mechanism for state transition, during which a wide range of hydrogen-bonding networks across Switch I, Switch II and the α3-helix interdependently undergo gross rearrangements, where fluctuation of Tyr32, translocation of Gln61, loss of the functional water molecules and positional shift of GTP play major roles. The NMR-based hydrogen/deuterium exchange experiments also support this transition mechanism. Moreover, the unveiled structural features together with the results of the biochemical study provide a new insight into the physiological role of state 1 as a stable pool of Ras•GTP in the GDP/GTP cycle of Ras. PMID:27180801

  14. Molecular Mechanism for Conformational Dynamics of Ras·GTP Elucidated from In-Situ Structural Transition in Crystal

    PubMed Central

    Matsumoto, Shigeyuki; Miyano, Nao; Baba, Seiki; Liao, Jingling; Kawamura, Takashi; Tsuda, Chiemi; Takeda, Azusa; Yamamoto, Masaki; Kumasaka, Takashi; Kataoka, Tohru; Shima, Fumi

    2016-01-01

    Ras•GTP adopts two interconverting conformational states, state 1 and state 2, corresponding to inactive and active forms, respectively. However, analysis of the mechanism for state transition was hampered by the lack of the structural information on wild-type Ras state 1 despite its fundamental nature conserved in the Ras superfamily. Here we solve two new crystal structures of wild-type H-Ras, corresponding to state 1 and state 2. The state 2 structure seems to represent an intermediate of state transition and, intriguingly, the state 1 crystal is successfully derived from this state 2 crystal by regulating the surrounding humidity. Structural comparison enables us to infer the molecular mechanism for state transition, during which a wide range of hydrogen-bonding networks across Switch I, Switch II and the α3-helix interdependently undergo gross rearrangements, where fluctuation of Tyr32, translocation of Gln61, loss of the functional water molecules and positional shift of GTP play major roles. The NMR-based hydrogen/deuterium exchange experiments also support this transition mechanism. Moreover, the unveiled structural features together with the results of the biochemical study provide a new insight into the physiological role of state 1 as a stable pool of Ras•GTP in the GDP/GTP cycle of Ras. PMID:27180801

  15. Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme.

    PubMed

    Khrenova, Maria G; Grigorenko, Bella L; Kolomeisky, Anatoly B; Nemukhin, Alexander V

    2015-10-01

    Molecular mechanisms of the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by the Ras·GAP protein complex are fully investigated by using modern modeling tools. The previously hypothesized stages of the cleavage of the phosphorus-oxygen bond in GTP and the formation of the imide form of catalytic Gln61 from Ras upon creation of Pi are confirmed by using the higher-level quantum-based calculations. The steps of the enzyme regeneration are modeled for the first time, providing a comprehensive description of the catalytic cycle. It is found that for the reaction Ras·GAP·GTP·H2O → Ras·GAP·GDP·Pi, the highest barriers correspond to the process of regeneration of the active site but not to the process of substrate cleavage. The specific shape of the energy profile is responsible for an interesting kinetic mechanism of the GTP hydrolysis. The analysis of the process using the first-passage approach and consideration of kinetic equations suggest that the overall reaction rate is a result of the balance between relatively fast transitions and low probability of states from which these transitions are taking place. Our theoretical predictions are in excellent agreement with available experimental observations on GTP hydrolysis rates. PMID:26374425

  16. Multiple receptors mobilize calcium through a pertussis toxin (PT) sensitive GTP-binding protein in human neutrophils (PMN's)

    SciTech Connect

    Lad, P.M.; Olson, C.V.; Grewal, I.S.; Frolich, M.; Scott, S.J.

    1986-03-05

    Treatment of PMN's with PT causes an abolition of chemotaxis, enzyme release, superoxide generation and aggregation caused by f-met-leu-phe (FMLP),C5a and platelet activating factor (PAF). Lectin (Con-A) induced capping and receptor induced shape change are abolished, but phagocytosis is unaltered. In whole cells, calcium mobilization induced by FMLP, PAF and Con-A inhibited by PT although the FMLP-mediated effect is more susceptible to PT's effects. Treatment of PMN's with phorbol 12-myristate 13 acetate (PMA) causes an abolition of calcium mobilization by all agents in a range which also inhibits cap formation. Investigation of calcium uptake reveals PT sensitive and insensitive components. Reciprocal interactions between Ns and Ni proteins are also observed since pretreatment with FMLP and PAF causes a stimulation of Ns-mediated cyclic AMP enhancement while pretreatment with Ns linked receptors (PGE/sub 1/ and beta receptor agonists) inhibits calcium mobilization. Comparative peptide mapping studies indicate substantial similarity between Ni proteins in PMN's, platelets and human erythrocytes. The authors results suggest that the Ni linked calcium mobilization sensitive to PMA is important to the regulation of the human neutrophil.

  17. The GTP binding protein-dependent activation and deactivation of cGMP phosphodiesterase in rod photoreceptors

    SciTech Connect

    Yamazaki, Akio.

    1989-01-01

    Cyclic GMP (cGMP) has a crucial role in visual transduction. Recent electrophysiological studies clearly indicate the existence of cGMP-activated conductance in photoreceptor plasma membranes. In darkness, Na{sup +}, Ca{sup ++}, and Mg{sup ++} enter rod outer segments (ROS) through cGMP-activated channels while light closes channels by lowering cGMP concentrations through activation of cGMP phosphodiesterase (PDE). Many excellent reviews reference the mechanism of PDE activation in photoreceptors. However, recent progress in understanding the mechanisms regulating cGMP hydrolysis has raised an important question in the PDE-regulation: how does the three-dimensional movement of a subunit of transducin (retinal G protein) relate to the PDE activation Associated with that question, the mechanism of PDE regulation appears to vary at different stages of evolution, for example, frog and bovine photoreceptors. This review examines recent progress of the cGMP hydrolysis mechanism by focusing on the subunit interactions between transducin and PDE. 36 refs., 2 figs.

  18. 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.

  19. AMPD2 Regulates GTP Synthesis and is Mutated in a Potentially-Treatable Neurodegenerative Brainstem Disorder

    PubMed Central

    Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K.; Vleet, Jeremy Van; Fenstermaker, Ali G.; Silhavy, Jennifer L.; Scheliga, Judith S.; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma Mujgan; Celep, Figen; Oraby, Azza; Zaki, Maha S.; Al-Baradie, Raidah; Faqeih, Eissa; Saleh, Mohammad; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W.; Gleeson, Joseph G.

    2013-01-01

    Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acids synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a new distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH), due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a new, potentially treatable early-onset neurodegenerative disease. PMID:23911318

  20. Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome.

    PubMed

    Kumar, Veerendra; Ero, Rya; Ahmed, Tofayel; Goh, Kwok Jian; Zhan, Yin; Bhushan, Shashi; Gao, Yong-Gui

    2016-06-17

    Elongation factor 4 (EF4) is a member of the family of ribosome-dependent translational GTPase factors, along with elongation factor G and BPI-inducible protein A. Although EF4 is highly conserved in bacterial, mitochondrial, and chloroplast genomes, its exact biological function remains controversial. Here we present the cryo-EM reconstitution of the GTP form of EF4 bound to the ribosome with P and E site tRNAs at 3.8-Å resolution. Interestingly, our structure reveals an unrotated ribosome rather than a clockwise-rotated ribosome, as observed in the presence of EF4-GDP and P site tRNA. In addition, we also observed a counterclockwise-rotated form of the above complex at 5.7-Å resolution. Taken together, our results shed light on the interactions formed between EF4, the ribosome, and the P site tRNA and illuminate the GTPase activation mechanism at previously unresolved detail. PMID:27137929

  1. Reduction of GTP cyclohydrolase I feedback regulating protein expression by hydrogen peroxide in vascular endothelial cells.

    PubMed

    Ishii, Masakazu; Shimizu, Shunichi; Wajima, Teruaki; Hagiwara, Tamio; Negoro, Takaharu; Miyazaki, Akira; Tobe, Takashi; Kiuchi, Yuji

    2005-02-01

    We examined the effect of H(2)O(2) on the expression of GTP cyclohydrolase I (GTPCH) feedback regulating protein (GFRP). Addition of H(2)O(2) to endothelial cells decreased GFRP mRNA levels, in contrast to the increase of tetrahydrobiopterin (BH(4)) content and GTPCH mRNA levels. The inhibitors of nitric oxide (NO) synthase and GTPCH had no influence on the decrease of GFRP mRNA levels in H(2)O(2)-treated cells. It is suggested that H(2)O(2) induces BH(4) synthesis through not only induction of GTPCH but also reduction of GFRP. The decrease of GFRP mRNA level appears to be independent of the produced NO and BH(4). PMID:15699573

  2. Enhanced enzymatic saccharification of pretreated biomass using glycerol thermal processing (GTP).

    PubMed

    Zhang, Wei; Sathitsuksanoh, Noppadon; Barone, Justin R; Renneckar, Scott

    2016-01-01

    Biomass was heated (200-240°C) in the presence of glycerol, for 4-12 min, under shear to disrupt the native cell wall architecture. The impact of this method, named glycerol thermal processing (GTP), on saccharification efficiency of the hardwood Liquidambar styraciflua, and a control cellulose sample was studied as a function of treatment severity. Furthermore, the enzymatic conversion of samples with varying compositions was studied after extraction of the structural polymers. Interestingly, the sweet gum processed materials crystallinity index increased by 10% of the initial value. The experiments revealed that the residual lignin was not a barrier to limiting the digestibility of cellulose after pretreatment yielding up to 70% glucose based on the starting wood material. Further xylan removal greatly improved the cellulose hydrolysis rate, converting nearly 70% of the cellulose into glucose within 24h, and reaching 78% of ultimate glucan digestibility after 72 h. PMID:26384086

  3. Root-induced changes in pH and dissolved organic matter binding capacity affect copper dynamic speciation in the rhizosphere

    NASA Astrophysics Data System (ADS)

    Bravin, Matthieu N.; Garnier, Cédric; Lenoble, Véronique; Gérard, Frédéric; Dudal, Yves; Hinsinger, Philippe

    2012-05-01

    Due to only few experimental evidences, the importance of root-induced alteration of metal dynamic speciation in the rhizosphere in the determination of metal bioavailability to plants is still a matter for debate. The present study thus investigated how root-induced changes in pH and dissolved organic matters (DOM) altered copper (Cu) dynamic speciation in the rhizosphere of durum wheat (Triticum turgidum durum L.). Plants were exposed to a Cu-contaminated soil previously alkalised by liming to cover soil pH values ranging from 4.8 to 7.5. A range of analytical techniques was deployed on soil exposed (i.e. in the rhizosphere) or not (i.e. in the bulk soil) to plant roots, including the measurement and the modelling (using the Humic Ion-Binding Model VI) of Cu2+ activity, the measurement of labile Cu concentration and Cu lability by Differential Pulse Anodic Stripping Voltammetry (DPASV) and Diffusive Gradients in Thin films (DGT). Due to root-induced alkalisation, pH reached about 7.3 in the rhizosphere whatever the initial bulk soil pH. Compared to the most acidic bulk soil (pH ≈ 4.8), Cu2+ activity decreased by three orders of magnitude in the rhizosphere while DPASV-Cu concentration decreased by 6-fold. DOM became the key driver of Cu dynamic speciation in the rhizosphere, where roots induced up to an order of magnitude increase in DOM concentration compared to bulk soils. This resulted in an increase in labile-Cu (both DPASV and DGT) concentrations, in spite of a decrease in Cu2+ activity. Model VI calculations supported a decrease in DOM binding capacity towards Cu in the rhizosphere. DPASV measurements unequivocally demonstrated that the increase in Cu lability in the rhizosphere solution can be attributed to a greater lability of organically-bound Cu. Collectively, our data introduce a consistent picture of root-induced changes of Cu dynamic speciation in the rhizosphere that were notably related to substantial alterations of DOM binding capacity.

  4. RNA-binding protein Hermes/RBPMS inversely affects synapse density and axon arbor formation in retinal ganglion cells in vivo

    PubMed Central

    Hörnberg, Hanna; Horck, Francis Wollerton-van; Maurus, Daniel; Zwart, Maarten; Svoboda, Hanno; Harris, William A.; Holt, Christine E.

    2015-01-01

    The RNA-binding protein, Hermes (RBPMS), is expressed exclusively in retinal ganglion cells (RGCs) in the CNS, but its function in these cells is not known. Here we show that Hermes protein translocates in granules from RGC bodies down the growing axons. Hermes loss-of-function in both Xenopus laevis and zebrafish embryos leads to a significant reduction in retinal axon arbor complexity in the optic tectum, and expression of a dominant acting mutant Hermes protein, defective in RNA-granule localisation, causes similar defects in arborisation. Time-lapse analysis of branch dynamics reveals that the decrease in arbor complexity is caused by a reduction in new branches rather than a decrease in branch stability. Surprisingly, Hermes depletion also leads to enhanced early visual behaviour and an increase in the density of pre-synaptic puncta suggesting that reduced arborisation is accompanied by increased synaptogenesis to maintain synapse number. PMID:23785151

  5. Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

    PubMed Central

    Narayan, Monisha; Chou, Ching-Shan; Park, Hay-Oak

    2013-01-01

    Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model. PMID:23437206

  6. X-linked thrombocytopenia with thalassemia from a mutation in the amino finger of GATA-1 affecting DNA binding rather than FOG-1 interaction

    PubMed Central

    Yu, Channing; Niakan, Kathy K.; Matsushita, Mark; Stamatoyannopoulos, George; Orkin, Stuart H.; Raskind, Wendy H.

    2010-01-01

    Transcription factor GATA-1 is essential for the development of erythroid cells and megakaryocytes. Each of its 2 zinc fingers is critical for normal function. The C-terminal finger is necessary for DNA binding. The N finger mediates interaction with FOG-1, a cofactor for GATA-1, and also modulates DNA-binding affinity, notably at complex or palindromic GATA sites. Residues of the N finger–mediating interaction with FOG-1 lie on the surface of the N finger facing away from DNA. Strong sequence conservation of residues facing DNA suggests that this other surface may also have an important role. We report here that a syndrome of X-linked thrombocytopenia with thalassemia in humans is caused by a missense mutation (Arg216Gln) in the GATA-1 N finger. To investigate the functional consequences of this substitution, we used site-directed mutagenesis to alter the corresponding residue in GATA-1. Compared with wild-type GATA-1, Arg216Gln GATA-1 shows comparable affinity to single GATA sites but decreased affinity to palindromic sites. Arg216Gln GATA-1 interacts with FOG-1 similarly with wild-type GATA-1. Arg216Gln GATA-1 supports erythroid maturation of GATA-1 erythroid cells, albeit at reduced efficiency compared with wild-type GATA-1. Together, these findings suggest that residues of the N finger of GATA-1–facing DNA contribute to GATA-1 function apart from interaction with the cofactor FOG-1. This is also the first example of β-thalassemia in humans caused by a mutation in an erythroid transcription factor. PMID:12200364

  7. Point mutations of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid(A) receptor affecting modulation of the channel by ligands of the benzodiazepine binding site.

    PubMed

    Buhr, A; Baur, R; Malherbe, P; Sigel, E

    1996-06-01

    Clinically relevant benzodiazepines allosterically stimulate neurotransmitter-evoked chloride currents at the gamma-aminobutyric acid type A(GABAA) receptor. Rat wild-type or mutated alpha 1, beta 2, and gamma 2S subunits were coexpressed in Xenopus oocytes and investigated with electrophysiological techniques. Point mutations in two subunits were identified that affect the response of gamma-aminobutyric acid (GABA)-induced currents by benzodiazepines. Mutation of one of three amino acid residues to alanine (alpha Tyr161 and alpha Thr206) or leucine (gamma Phe77) resulted in a approximately 3-fold increase in potentiation by diazepam. The response to zolpidem was increased in two mutant channels containing the mutated alpha subunit but was nearly absent in channels containing the mutated gamma subunit. In the former cases, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) acted as a negative allosteric modulator of the channel, much stronger than in the wild-type channel, whereas there was no significant difference to the wild-type channel in the latter case. Thus, the mutant gamma subunit has different functional consequences for the various types of ligand of the benzodiazepine binding site. All three amino acid residues, alpha Tyr161, alpha Thr206, and gamma Phe77, are close or identical to homologous residues that are implicated in GABA binding. If the residues binding the channel agonist GABA are located at subunit interfaces, the residues influencing the benzodiazepine effects must also be located at subunit interfaces. PMID:8649346

  8. The role of GTP in transient splitting of 70S ribosomes by RRF (ribosome recycling factor) and EF-G (elongation factor G)

    PubMed Central

    Hirokawa, Go; Iwakura, Nobuhiro; Kaji, Akira; Kaji, Hideko

    2008-01-01

    Ribosome recycling factor (RRF), elongation factor G (EF-G) and GTP split 70S ribosomes into subunits. Here, we demonstrated that the splitting was transient and the exhaustion of GTP resulted in re-association of the split subunits into 70S ribosomes unless IF3 (initiation factor 3) was present. However, the splitting was observed with sucrose density gradient centrifugation (SDGC) without IF3 if RRF, EF-G and GTP were present in the SDGC buffer. The splitting of 70S ribosomes causes the decrease of light scattering by ribosomes. Kinetic constants obtained from the light scattering studies are sufficient to account for the splitting of 70S ribosomes by RRF and EF-G/GTP during the lag phase for activation of ribosomes for the log phase. As the amount of 70S ribosomes increased, more RRF, EF-G and GTP were necessary to split 70S ribosomes. In the presence of a physiological amount of polyamines, GTP and factors, even 0.6 μM 70S ribosomes (12 times higher than the 70S ribosomes for routine assay) were split. Spermidine (2 mM) completely inhibited anti-association activity of IF3, and the RRF/EF-G/GTP-dependent splitting of 70S ribosomes. PMID:18948280

  9. Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules

    PubMed Central

    Kedersha, Nancy; Chen, Samantha; Gilks, Natalie; Li, Wei; Miller, Ira J.; Stahl, Joachim; Anderson, Paul

    2002-01-01

    Environmental stress-induced phosphorylation of eIF2α inhibits protein translation by reducing the availability of eIF2-GTP-tRNAiMet, the ternary complex that joins initiator tRNAMet to the 43S preinitiation complex. The resulting untranslated mRNA is dynamically routed to discrete cytoplasmic foci known as stress granules (SGs), a process requiring the related RNA-binding proteins TIA-1 and TIAR. SGs appear to be in equilibrium with polysomes, but the nature of this relationship is obscure. We now show that most components of the 48S preinitiation complex (i.e., small, but not large, ribosomal subunits, eIF3, eIF4E, eIF4G) are coordinately recruited to SGs in arsenite-stressed cells. In contrast, eIF2 is not a component of newly assembled SGs. Cells expressing a phosphomimetic mutant (S51D) of eIF2α assemble SGs of similar composition, confirming that the recruitment of these factors is a direct consequence of blocked translational initiation and not due to other effects of arsenite. Surprisingly, phospho-eIF2α is recruited to SGs that are disassembling in cells recovering from arsenite-induced stress. We discuss these results in the context of a translational checkpoint model wherein TIA and eIF2 are functional antagonists of translational initiation, and in which lack of ternary complex drives SG assembly. PMID:11809833

  10. GTP cyclohydrolase feedback regulatory protein controls cofactor 6-tetrahydrobiopterin synthesis in the cytosol and in the nucleus of epidermal keratinocytes and melanocytes.

    PubMed

    Chavan, Bhaven; Gillbro, Johanna M; Rokos, Hartmut; Schallreuter, Karin U

    2006-11-01

    (6R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH4) is crucial in the hydroxylation of L-phenylalanine-, L-tyrosine-, and L-tryptophan-regulating catecholamine and serotonin synthesis as well as tyrosinase in melanogenesis. The rate-limiting step of 6BH4 de novo synthesis is controlled by guanosine triphosphate (GTP) cyclohydrolase I (GTPCHI) and its feedback regulatory protein (GFRP), where binding of L-phenylalanine to GFRP increases enzyme activities, while 6BH4 exerts the opposite effect. Earlier it was demonstrated that the human epidermis holds the full capacity for autocrine 6BH4 de novo synthesis and recycling. However, besides the expression of epidermal mRNA for GFRP, the presence of a functioning GFRP feedback has never been shown. Therefore, it was tempting to investigate whether this important mechanism is present in epidermal cells. Our results identified indeed a functioning GFRP/GTPCHI axis in epidermal keratinocytes and melanocytes in the cytosol, adding the missing link for 6BH4 de novo synthesis which in turn controls cofactor supply for catecholamine and serotonin biosynthesis as well as melanogenesis in the human epidermis. Moreover, GFRP expression and GTPCHI activities have been found in the nucleus of both cell types. The significance of this result warrants further investigation. PMID:16778797

  11. Modification of Loop 1 Affects the Nucleotide-Binding Properties of Myo1c, The Adaptation Motor in the Inner Ear†

    PubMed Central

    Adamek, Nancy; Lieto-Trivedi, Alena; Geeves, Michael A.; Coluccio, Lynne M.

    2010-01-01

    Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner ear, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N-terminal motor domain, which contains the nucleotide- and actin-binding sites, some differences include the length and composition of surface loops, including loop 1, which lies near the nucleotide-binding domain. To investigate the role of loop 1, we expressed in insect cells mutants of a truncated form of Myo1c, Myo1c1IQ, as well as chimeras of Myo1c1IQ with the analogous loop from other myosins. We found that replacement of the charged residues in loop 1 with alanines or the whole loop with a series of alanines did not alter the ATPase activity, transient kinetics properties and Ca2+-sensitivity of Myo1c1IQ. Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c1IQ-tonic) or replacement with a single glycine (Myo1c1IQ-G) accelerated ADP release from A.M 2-3-fold in Ca2+, whereas substitution with loop 1 from phasic muscle myosin II (Myo1c1IQ-phasic) accelerated ADP release 35-fold. Motility assays with chimeras containing a single α-helix, or SAH, domain showed that Myo1cSAH-tonic translocated actin in vitro twice as fast as Myo1cSAH-WT and 3-fold faster than Myo1cSAH-G. The studies show that changes induced in Myo1c by modifying loop 1 showed no resemblance to the behaviour of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras. PMID:20039646

  12. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

    PubMed

    Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

    2016-02-12

    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. PMID:26668315

  13. Phospholipase C treatment of certain human target cells reduces their susceptibility to NK lysis without affecting binding or sensitivity to lytic granules.

    PubMed

    Une, C; Grönberg, A; Axberg, I; Jondal, M; Orn, A

    1991-03-01

    Phosphatidylinositol-specific phospholipase C (PI-PLC) is an enzyme that has the capacity to release glycosyl-phosphatidyl inositol (G-PI)-anchored proteins from the cells surface. Pretreatment of the human T-cell leukemia cell line Molt-4 with PI-PLC resulted in a decrease in the susceptibility to lysis by natural killer (NK) cells. Treatment of the erythroleukemia cell line K562 with PI-PLC had no effect on its NK susceptibility. PI-PLC-treated and untreated Molt-4 bound equally well to lymphocytes in target-binding studies with effector cell preparations enriched for NK cells. Susceptibility to cytolytic granules isolated from rat LGL tumor cells remained the same after treatment of Molt-4 or K562 with PI-PLC. Combined treatment of Molt-4 with PI-PLC and rlFN-alpha or rlFN-gamma resulted in additive reductions of the NK susceptibility, suggesting that PI-PLC and interferons act on different mechanisms to protect cells from NK lysis. When expression of a number of antigens on Molt-4 and K562 was analyzed in flow cytometry, only the expression of CD58 was reduced after PI-PLC treatment. The susceptibility of Con A blasts to MLR derived cytotoxic T-cells was not altered by treatment with phospholipase. These data suggest that PI-PLC treatment reduces the capacity of some target cells to activate NK cells upon contract. The mechanism behind this phenomenon is presently unclear. PMID:1703925

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

    PubMed

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

    2015-01-01

    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. PMID:26558346

  15. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26558346

  17. Small glutamine-rich protein/viral protein U–binding protein is a novel cochaperone that affects heat shock protein 70 activity

    PubMed Central

    Angeletti, Peter C.; Walker, Doriann; Panganiban, Antonito T.

    2002-01-01

    Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together. These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex. Here we provide evidence that small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70. By “Far-Western” and pull-down assays, SGT/UBP was shown to interact directly with Hsp70 and weakly with Hsp90. The interaction of SGT/UBP with both these protein chaperones was mapped to 3 TPRs in SGT/UBP (amino acids 95–195) that are flanked by charged residues. Moreover, SGT/UBP caused an approximately 30% reduction in both the intrinsic ATPase activity of Hsc70 and the ability of Hsc70 to refold denatured luciferase in vitro. This negative effect of SGT/UBP on Hsc70 is similar in magnitude to that observed for the cochaperone CHIP. A role for SGT/UBP in protein folding is also supported by evidence that a yeast strain containing a deletion in the yeast homolog to SGT/UBP (ΔSGT/UBP) displays a 50-fold reduction in recovery from heat shock compared with the wild type parent. Together, these results are consistent with a regulatory role for SGT/UBP in the chaperone complex. PMID:12482202

  18. A functional polymorphism in lnc-LAMC2-1:1 confers risk of colorectal cancer by affecting miRNA binding.

    PubMed

    Gong, Jing; Tian, Jianbo; Lou, Jiao; Ke, Juntao; Li, Lu; Li, Jiaoyuan; Yang, Yang; Gong, Yajie; Zhu, Ying; Zhang, Yi; Zhong, Rong; Chang, Jiang; Miao, Xiaoping

    2016-05-01

    Genome-wide association studies (GWASs) have identified multiple susceptibility loci of colorectal cancer (CRC), however, causative polymorphisms have not been fully elucidated. Long non-coding RNAs (lncRNAs) are a recently discovered class of non-protein coding RNAs that involved in a wide variety of biological processes. We hypothesized that single nucleotide polymorphisms (SNPs) in lncRNA may associate with the CRC risk by influencing lncRNA functions. To evaluate the effects of SNPs on CRC susceptibility in Chinese populations, we first screened out all potentially functional SNPs in exons of lncRNAs located in CRC susceptibility loci identified by GWAS. Eight SNPs were selected and genotyped in 875 CRC cases and 855 controls and replicated in an independent case-control study consisting of 768 CRC cases and 768 controls. Analyses showed that CG and GG genotypes of the rs2147578 were significantly associated with increased risk for CRC occurrence in both case-control studies [combined analysis OR = 1.29; 95% confidence interval (CI) = 1.11-1.51, P = 0.001] compared to the rs2147578 CC genotype. Bioinformatics analyses showed that rs2147578 is located in the transcript of lnc-LAMC2-1:1 and could influence the binding of lnc-LAMC2-1:1/miR-128-3p. Further luciferase reporter assays demonstrated that the construct with the risk rs2147578G allele had relatively high expression activity compared with that of the rs2147578C allele. Expression quantitative trait loci analyses also showed that rs2147578 is correlated with the expression of a well established oncogene LAMC2 (laminin subunit gamma 2). These findings indicated that rs2147578 in lnc-LAMC2-1:1 might be a genetic modifier for the development of CRC. PMID:26905585

  19. 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

  20. Antibiotic Binding Drives Catalytic Activation of Aminoglycoside Kinase APH(2″)-Ia.

    PubMed

    Caldwell, Shane J; Huang, Yue; Berghuis, Albert M

    2016-06-01

    APH(2″)-Ia is a widely disseminated resistance factor frequently found in clinical isolates of Staphylococcus aureus and pathogenic enterococci, where it is constitutively expressed. APH(2″)-Ia confers high-level resistance to gentamicin and related aminoglycosides through phosphorylation of the antibiotic using guanosine triphosphate (GTP) as phosphate donor. We have determined crystal structures of the APH(2″)-Ia in complex with GTP analogs, guanosine diphosphate, and aminoglycosides. These structures collectively demonstrate that aminoglycoside binding to the GTP-bound kinase drives conformational changes that bring distant regions of the protein into contact. These changes in turn drive a switch of the triphosphate cofactor from an inactive, stabilized conformation to a catalytically competent active conformation. This switch has not been previously reported for antibiotic kinases or for the structurally related eukaryotic protein kinases. This catalytic triphosphate switch presents a means by which the enzyme can curtail wasteful hydrolysis of GTP in the absence of aminoglycosides, providing an evolutionary advantage to this enzyme. PMID:27161980

  1. Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

    PubMed Central

    Leberer, E; Wu, C; Leeuw, T; Fourest-Lieuvin, A; Segall, J E; Thomas, D Y

    1997-01-01

    Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways. Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis. We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p. The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling. However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth. Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation. Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth. These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins. PMID:9009270

  2. A New Use for a Familiar Fold: the X-Ray Crystal Structure of GTP-Bound GTP Cyclohydrolase III From Methanocaldococcus Jannaschii Reveals a Two Metal Ion Catalytic Mechanism

    SciTech Connect

    Morrison, S.D.; Roberts, S.A.; Zegeer, A.M.; Montfort, W.R.; Bandarian, V.

    2009-05-26

    GTP cyclohydrolase (GCH) III from Methanocaldococcus jannaschii, which catalyzes the conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), has been shown to require Mg{sup 2+} for catalytic activity and is activated by monovalent cations such as K{sup +} and ammonium [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084]. The reaction is formally identical to that catalyzed by a GCH II ortholog (SCO 6655) from Streptomyces coelicolor; however, SCO 6655, like other GCH II proteins, is a zinc-containing protein. The structure of GCH III complexed with GTP solved at 2 {angstrom} resolution clearly shows that GCH III adopts a distinct fold that is closely related to the palm domains of phosphodiesterases, such as DNA polymerase I. GCH III is a tetramer of identical subunits; each monomer is composed of an N- and a C-terminal domain that adopt nearly superimposible structures, suggesting that the protein has arisen by gene duplication. Three metal ions were located in the active site, two of which occupy positions that are analogous to those occupied by divalent metal ions in the structures of a number of palm domain containing proteins, such as DNA polymerase I. Two conserved Asp residues that coordinate the metal ions, which are also found in palm domain containing proteins, are observed in GCH III. Site-directed variants (Asp{yields}Asn) of these residues in GCH III are less active than wild-type. The third metal ion, most likely a potassium ion, is involved in substrate recognition through coordination of O6 of GTP. The arrangement of the metal ions in the active site suggests that GCH III utilizes two metal ion catalysis. The structure of GCH III extends the repertoire of possible reactions with a palm fold to include cyclohydrolase chemistry.

  3. Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the gamma phosphate of GTP.

    PubMed Central

    Bigay, J; Deterre, P; Pfister, C; Chabre, M

    1987-01-01

    Fluoride activation of G proteins requires the presence of aluminium or beryllium and it has been suggested that AIF4- acts as an analogue of the gamma-phosphate of GTP in the nucleotide site. We have investigated the action of AIF4- or of BeF3- on transducin (T), the G protein of the retinal rods, either indirectly through the activation of cGMP phosphodiesterase, or more directly through their effects on the conformation of transducin itself. In the presence of AIF4- or BeF3-, purified T alpha subunit of transducin activates purified cyclic GMP phosphodiesterase (PDE) in the absence of photoactivated rhodopsin. Activation is totally reversed by elution of fluoride or partially reversed by addition of excess T beta gamma. Activation requires that GDP or a suitable analogue be bound to T alpha: T alpha-GDP and T alpha-GDP alpha S are activable by fluorides, but not T alpha-GDP beta S, nor T alpha that has released its nucleotide upon binding to photoexcited rhodopsin. Analysis of previous works on other G proteins and with other nucleotide analogues confirm that in all cases fluoride activation requires that a GDP unsubstituted at its beta phosphate be bound in T alpha. By contrast with alumino-fluoride complexes, which can adopt various coordination geometries, all beryllium fluoride complexes are tetracoordinated, with a Be-F bond length of 1.55 A, and strictly isomorphous to a phosphate group. Our study confirms that fluoride activation of transducin results from a reversible binding of the metal-fluoride complex in the nucleotide site of T alpha, next to the beta phosphate of GDP, as an analogue of the gamma phosphate.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2826123

  4. GTP cyclohydrolase I feedback regulatory protein is expressed in serotonin neurons and regulates tetrahydrobiopterin biosynthesis.

    PubMed

    Kapatos, G; Hirayama, K; Shimoji, M; Milstien, S

    1999-02-01

    Tetrahydrobiopterin, the coenzyme required for hydroxylation of phenylalanine, tyrosine, and tryptophan, regulates its own synthesis through feedback inhibition of GTP cyclohydrolase I (GTPCH) mediated by a regulatory subunit, the GTP cyclohydrolase feedback regulatory protein (GFRP). In the liver, L-phenylalanine specifically stimulates tetrahydrobiopterin synthesis by displacing tetrahydrobiopterin from the GTPCH-GFRP complex. To explore the role of this regulatory system in rat brain, we examined the localization of GFRP mRNA using double-label in situ hybridization. GFRP mRNA expression was abundant in serotonin neurons of the dorsal raphe nucleus but was undetectable in dopamine neurons of the midbrain or norepinephrine neurons of the locus coeruleus. Simultaneous nuclease protection assays for GFRP and GTPCH mRNAs showed that GFRP mRNA is most abundant within the brainstem and that the ratio of GFRP to GTPCH mRNA is much higher than in the ventral midbrain. Two species of GFRP mRNA differing by approximately 20 nucleotides in length were detected in brainstem but not in other tissues, with the longer, more abundant form being common to other brain regions. It is interesting that the pineal and adrenal glands did not contain detectable levels of GFRP mRNA, although GTPCH mRNA was abundant in both. Primary neuronal cultures were used to examine the role of GFRP-mediated regulation of GTPCH on tetrahydrobiopterin synthesis within brainstem serotonin neurons and midbrain dopamine neurons. L-Phenylalanine increased tetrahydrobiopterin levels in serotonin neurons to a maximum of twofold in a concentration-dependent manner, whereas D-phenylalanine and L-tryptophan were without effect. In contrast, tetrahydrobiopterin levels within cultured dopamine neurons were not altered by L-phenylalanine. The time course of this effect was very rapid, with a maximal response observed within 60 min. Inhibitors of tetrahydrobiopterin biosynthesis prevented the L

  5. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    different classes of protein pocket dynamics: (1) appearance/disappearance of a subpocket in an existing pocket; (2) appearance/disappearance of an adjacent pocket on the protein surface in the direct vicinity of an already existing pocket; (3) pocket breathing, which may be caused by side-chain fluctuations or backbone or interdomain vibrational motion; (4) opening/closing of a channel or tunnel, connecting a pocket inside the protein with solvent, including lid motion; and (5) the appearance/disappearance of an allosteric pocket at a site on a protein distinct from an already existing pocket with binding of a ligand to the allosteric binding site affecting the original pocket. We suggest that the class of pocket dynamics, as well as the type and extent of protein motion affecting the binding pocket, should be factors considered in choosing the most appropriate computational approach to study a given binding pocket. Furthermore, we examine the relationship between pocket dynamics classes and induced fit, conformational selection, and gating models of ligand binding on binding kinetics and thermodynamics. We discuss the implications of protein binding pocket dynamics for drug design and conclude with potential future directions for computational analysis of protein binding pocket dynamics. PMID:27110726

  6. Formation of. beta. ,. gamma. -methylene-7,8-dihydroneopterin 3'-triphosphate from. beta. ,. gamma. -methyleneguanosine 5'-triphosphate by GTP cyclohydrolase I of Escherichia coli

    SciTech Connect

    Ferre, J.; Jacobson, K.B.

    1984-01-01

    GTP cyclohydrolase I of Escherichia coli converts (..beta..,..gamma..-methylene)GTP to a fluorescent product that is characterized as (..beta..,..gamma..-methylene)dihydroneopterin triphosphate. Interaction between the GTP analog and the enzyme gave a K/sub i/ of 3.0 ..mu..M, which may be compared to the K/sub m/ of 0.1 ..mu..M for GTP. This new analog of dihydroneopterin triphosphate may, in turn, be converted to the same greenish-yellow pteridines (compounds X, X1, and X2) that are obtained from dihydroneopterin triphosphate. Because of its stability to phosphatase action, this analog may be useful for studies in pteridine metabolism. 14 references, 5 figures.

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

    SciTech Connect

    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 binding 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.

  8. Preparation and crystallization of the stimulatory and inhibitory complexes of GTP cyclohydrolase I and its feedback regulatory protein GFRP.

    PubMed

    Maita, N; Okada, K; Hirotsu, S; Hatakeyama, K; Hakoshima, T

    2001-08-01

    Mammalian GTP cyclohydrolase I is a decameric enzyme in the first and rate-limiting step in the biosynthesis of tetrahydrobiopterin, which is an essential cofactor for enzymes producing neurotransmitters such as catecholamines and for nitric oxide synthases. The enzyme is dually regulated by its feedback regulatory protein GFRP in the presence of its stimulatory effector phenylalanine and its inhibitory effector biopterin. Here, both the stimulatory and inhibitory complexes of rat GTP cyclohydrolase I bound to GFRP were crystallized by vapour diffusion. Diffraction data sets at resolutions of 3.0 and 2.64 A were collected for the stimulatory and inhibitory complexes, respectively. Each complex consists of two GTPCHI pentamer rings and two GFRP pentamer rings, with pseudo-52 point-group symmetry. PMID:11468403

  9. Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli.

    PubMed Central

    Richter, G; Ritz, H; Katzenmeier, G; Volk, R; Kohnle, A; Lottspeich, F; Allendorf, D; Bacher, A

    1993-01-01

    GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis. PMID:8320220

  10. 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

  11. Conserved arginine residues in the carboxyl terminus of the equine arteritis virus E protein may play a role in heparin binding but may not affect viral infectivity in equine endothelial cells.

    PubMed

    Lu, Zhengchun; Sarkar, Sanjay; Zhang, Jianqiang; Balasuriya, Udeni B R

    2016-04-01

    Equine arteritis virus (EAV), the causative agent of equine viral arteritis, has relatively broad cell tropism in vitro. In horses, EAV primarily replicates in macrophages and endothelial cells of small blood vessels. Until now, neither the cellular receptor(s) nor the mechanism(s) of virus attachment and entry have been determined for this virus. In this study, we investigated the effect of heparin on EAV infection in equine endothelial cells (EECs). Heparin, but not other glycosaminoglycans, could reduce EAV infection up to 93 %. Sequence analysis of the EAV E minor envelope protein revealed a conserved amino acid sequence (52 RSLVARCSRGARYR 65) at the carboxy terminus of the E protein, which was predicted to be the heparin-binding domain. The basic arginine (R) amino acid residues were subsequently mutated to glycine by site-directed mutagenesis of ORF2a in an E protein expression vector and an infectious cDNA clone of EAV. Two single mutations in E (R52G and R57G) did not affect the heparin-binding capability, whereas the E double mutation (R52,60G) completely eliminated the interaction between the E protein and heparin. Although the mutant R52,60G EAV did not bind heparin, the mutations did not completely abolish infectivity, indicating that heparin is not the only critical factor for EAV infection. This also suggested that other viral envelope protein(s) might be involved in attachment through heparin or other cell-surface molecules, and this warrants further investigation. PMID:26739582

  12. GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits. Purification, cDNA cloning, and bacterial expression.

    PubMed

    Yoneyama, T; Brewer, J M; Hatakeyama, K

    1997-04-11

    GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated Mr of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinity-purified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average Mr of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes. PMID:9092499

  13. The R215W mutation in NBS1 impairs {gamma}-H2AX binding and affects DNA repair: molecular bases for the severe phenotype of 657del5/R215W Nijmegen breakage syndrome patients

    SciTech Connect

    Masi, Alessandra di Viganotti, Mara; Polticelli, Fabio; Ascenzi, Paolo; Tanzarella, Caterina; Antoccia, Antonio

    2008-05-09

    Nijmegen breakage syndrome (NBS) is a genetic disorder characterized by chromosomal instability and hypersensitivity to ionising radiation. Compound heterozygous 657del5/R215W NBS patients display a clinical phenotype more severe than the majority of NBS patients homozygous for the 657del5 mutation. The NBS1 protein, mutated in NBS patients, contains a FHA/BRCT domain necessary for the DNA-double strand break (DSB) damage response. Recently, a second BRCT domain has been identified, however, its role is still unknown. Here, we demonstrate that the R215W mutation in NBS1 impairs histone {gamma}-H2AX binding after induction of DNA damage, leading to a delay in DNA-DSB rejoining. Molecular modelling reveals that the 215 residue of NBS1 is located between the two BRCT domains, affecting their relative orientation that appears critical for {gamma}-H2AX binding. Present data represent the first evidence for the role of NBS1 tandem BRCT domains in {gamma}-H2AX recognition, and could explain the severe phenotype observed in 657del5/R215W NBS patients.

  14. Occupation of nucleotide in the binding pocket is critical to the stability of Rab11A.

    PubMed

    Shin, Young-Cheul; Kim, Chang Min; Choi, Jae Young; Jeon, Ju-Hong; Park, Hyun Ho

    2016-04-01

    The Ras superfamily of small G proteins is a family of guanosine triphosphatases (GTPases) and each GTPase has conserved amino acid sequences in the enzymatic active site that are responsible for specific interactions with GDP and GTP molecules. Rab GTPases, which belong to the Ras superfamily, are key regulators of intracellular vesicle trafficking via the recruitment of effector molecules. Here, we purified wild type, active mutant and inactive mutant of Rab11A. In this process, we found that the inactive mutant (Rab11A S25N) had low stability compared with wild type and other mutants. Further analysis revealed that the stability of Rab11A S25N is dependent on the occupation of GDP in the nucleotide binding pocket of the protein. We found that the stability of Rab11A S25N is affected by the presence of GDP, not other nucleotides, and is independent of pH or salt in FPLC buffer. Our results provide a better understanding of how GTPase can be stable under in vitro conditions without effector proteins and how proper substrate/cofactor coordination is crucial to the stability of Rab11A. Successful purification and proposed purification methods will provide a valuable guide for investigation of other small GTPase proteins. PMID:26767484

  15. GTP cyclohydrolase I expression and enzymatic activity are present in caveolae of endothelial cells

    PubMed Central

    Peterson, Timothy E.; d’Uscio, Livius V.; Cao, Sheng; Wang, Xiao-Li; Katusic, Zvonimir S.

    2009-01-01

    Tetrahydrobiopterin is an essential cofactor required for the synthesis of nitric oxide. GTP cyclohydrolase I (GTPCH I) is the rate limiting enzyme for tetrahydrobiopterin production in endothelial cells, yet little is known about the subcellular localization of this enzyme. In this study, we demonstrate that GTPCH I is localized to caveolar membrane microdomains along with caveolin-1 and endothelial nitric oxide synthase. GTPCH I activity was detected in isolated caveolar membranes from cultured endothelial cells. Confocal and electron microscopy analyses confirmed GTPCH I colocalization with caveolin-1. Consistent with in vitro studies, GTPCH I activity was evident in isolated caveolar microdomains from lung homogenates of wild-type mice. Importantly, a two-fold increase in GTPCH I activity was detected in the aortas of caveolin-1 deficient mice suggesting that caveolin-1 may be involved in the control of GTPCH I enzymatic activity. Indeed, overexpression of caveolin-1 inhibits GTPCH I activity, and tetrahydrobiopterin biosynthesis is activated by disruption of caveolae structure. These studies demonstrate that GTPCH I is targeted to caveolae microdomains in vascular endothelial cells and tetrahydrobiopterin production occurs in close proximity to endothelial nitric oxide synthase. Additionally, our findings provide new insights into the regulation of GTPCH I activity by the caveolar coat protein, caveolin-1. PMID:19104007

  16. Phenylalanine improves dilation and blood pressure in GTP cyclohydrolase inhibition-induced hypertensive rats.

    PubMed

    Mitchell, Brett M; Dorrance, Anne M; Webb, R Clinton

    2004-06-01

    GTP cyclohydrolase (GTPCH), the rate-limiting enzyme in the production of the nitric oxide synthase cofactor tetrahydrobiopterin (BH4), is partly regulated by the GTPCH feedback regulatory protein (GFRP). GFRP can inhibit GTPCH by end-product negative feedback, and L-phenylalanine (L-Phe) reverses this inhibition and increases BH4 biosynthesis in vitro. We hypothesized that L-Phe would increase endothelium-dependent relaxation and decrease blood pressure in rats made hypertensive by GTPCH inhibition. Di-amino-hydroxypyrimidine (DAHP, 10 mmol/L), a known inhibitor of GTPCH, was given with or without L-Phe or D-Phe (2 mmol/L) in the drinking water of rats for 3 days and blood pressure was measured via tail-cuff. Endothelium-intact aortic segments were hung in organ chambers for measurement of isometric force generation. Systolic blood pressure was increased significantly in DAHP-treated rats compared with controls. The addition of L-Phe attenuated the hypertensive effect, whereas D-Phe had no effect. Acetylcholine- and A23187-induced relaxation was decreased in aortas from DAHP-treated rats compared with controls, but was restored in aortas from DAHP+L-Phe-treated rats. Following NOS inhibition, sensitivity to sodium nitroprusside was increased in aortas from DAHP-treated rats, but restored in DAHP+L-Phe-treated rats. These results suggest that L-Phe can reverse GTPCH inhibition in vivo leading to increased vasodilation and decreased blood pressure. PMID:15167268

  17. From GTP and G proteins to TRPC channels: a personal account.

    PubMed

    Birnbaumer, Lutz

    2015-09-01

    By serendipity and good fortune, as a postdoctoral fellow in 1967, I landed at the right place at the right time, as I was allowed to investigate the mechanism by which hormones activate the enzyme adenylyl cyclase (then adenyl cyclase) in Martin Rodbell's Laboratory at the NIH in Bethesda, Maryland. The work uncovered first, the existence of receptors separate from the enzyme and then, the existence of transduction mechanisms requiring guanosine-5'-triphosphate (GTP) and Mg(2+). With my laboratory colleagues first and postdoctoral fellows after leaving NIH, I participated in the development of the field "signal transduction by G proteins," uncovered by molecular cloning several G-protein-coupled receptors (GPCRs) and became interested in both the molecular makeup of voltage-gated Ca channels and Ca2+ homeostasis downstream of activation of phospholipase C (PLC) by the Gq/11 signaling pathway. We were able to confirm the hypothesis that there would be mammalian homologues of the Drosophila "transient receptor potential" channel and discovered the existence of six of the seven mammalian genes, now called transient receptor potential canonical (TRPC) channels. In the present article, I summarize from a bird's eye view of what I feel were key findings along this path, not only from my laboratory but also from many others, that allowed for the present knowledge of cell signaling involving G proteins to evolve. Towards the end, I summarize roles of TRPC channels in health and disease. PMID:26377676

  18. Cdc42p GDP/GTP Cycling Is Necessary for Efficient Cell Fusion during Yeast Mating

    PubMed Central

    Barale, Sophie; McCusker, Derek

    2006-01-01

    The highly conserved small Rho G-protein, Cdc42p plays a critical role in cell polarity and cytoskeleton organization in all eukaryotes. In the yeast Saccharomyces cerevisiae, Cdc42p is important for cell polarity establishment, septin ring assembly, and pheromone-dependent MAP-kinase signaling during the yeast mating process. In this study, we further investigated the role of Cdc42p in the mating process by screening for specific mating defective cdc42 alleles. We have identified and characterized novel mating defective cdc42 alleles that are unaffected in vegetative cell polarity. Replacement of the Cdc42p Val36 residue with Met resulted in a specific cell fusion defect. This cdc42[V36M] mutant responded to mating pheromone but was defective in cell fusion and in localization of the cell fusion protein Fus1p, similar to a previously isolated cdc24 (cdc24-m6) mutant. Overexpression of a fast cycling Cdc42p mutant suppressed the cdc24-m6 fusion defect and conversely, overexpression of Cdc24p suppressed the cdc42[V36M] fusion defect. Taken together, our results indicate that Cdc42p GDP–GTP cycling is critical for efficient cell fusion. PMID:16571678

  19. Erythropoietin prevents endothelial dysfunction in GTP-cyclohydrolase I-deficient hph1 mice

    PubMed Central

    d'Uscio, Livius V.; Santhanam, Anantha V.R.; Katusic, Zvonimir S.

    2014-01-01

    In the present study, we used the mutant hph-1 mouse model, that has deficiency in GTP-cyclohydrolase I (GTPCH I) activity, to test the hypothesis that erythropoietin (EPO) protects aortic wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and tetrahydrobiopterin (BH4) levels were reduced in hph-1 mice while 7,8-dihydrobiopterin (7,8-BH2) levels were significantly increased. Furthermore, BH4 deficiency caused increased production of superoxide anion and hydrogen peroxide in the aorta thus resulting in impairment of endothelium-dependent relaxations to acetylcholine. Treatment of hph1 mice with recombinant human EPO (1000 U/kg, s.c. for three days) significantly decreased superoxide anion production by eNOS and improved BH4 to 7,8-BH2 ratio in aortas. EPO also significantly decreased production of hydrogen peroxide and improved endothelium-dependent relaxations in aortas of hph1 mice. In addition, EPO treatment increased protein expressions of copper-/zinc-superoxide dismutase, manganese-superoxide dismutase, and catalase in the aorta of hph1 mice. Our findings demonstrate that treatment with EPO prevented oxidative stress and endothelial dysfunction caused by eNOS uncoupling. Increased vascular expressions of antioxidants appear to be an important molecular mechanism underlying vascular protection by EPO during chronic BH4 deficiency. PMID:25490417

  20. Cleanrooms and tissue banking how happy I could be with either GMP or GTP?

    PubMed

    Klykens, J; Pirnay, J-P; Verbeken, G; Giet, O; Baudoux, E; Jashari, R; Vanderkelen, A; Ectors, N

    2013-12-01

    The regulatory framework of tissue banking introduces a number of requirements for monitoring cleanrooms for processing tissue or cell grafts. Although a number of requirements were clearly defined, some requirements are open for interpretation. This study aims to contribute to the interpretation of GMP or GTP guidelines for tissue banking. Based on the experience of the participating centers, the results of the monitoring program were evaluated to determine the feasibility of a cleanroom in tissue banking and the monitoring program. Also the microbial efficacy of a laminar airflow cabinet and an incubator in a cleanroom environment was evaluated. This study indicated that a monitoring program of a cleanroom at rest in combination with (final) product testing is a feasible approach. Although no statistical significance (0.90 < p < 0.95) was found there is a strong indication that a Grade D environment is not the ideal background environment for a Grade A obtained through a laminar airflow cabinet. The microbial contamination of an incubator in a cleanroom is limited but requires closed containers for tissue and cell products. PMID:23288450

  1. Exceptionally large entropy contributions enable the high rates of GTP hydrolysis on the ribosome

    PubMed Central

    Åqvist, Johan; Kamerlin, Shina C.L.

    2015-01-01

    Protein synthesis on the ribosome involves hydrolysis of GTP in several key steps of the mRNA translation cycle. These steps are catalyzed by the translational GTPases of which elongation factor Tu (EF-Tu) is the fastest GTPase known. Here, we use extensive computer simulations to explore the origin of its remarkably high catalytic rate on the ribosome and show that it is made possible by a very large positive activation entropy. This entropy term (TΔS‡) amounts to more than 7 kcal/mol at 25 °C. It is further found to be characteristic of the reaction mechanism utilized by the translational, but not other, GTPases and it enables these enzymes to attain hydrolysis rates exceeding 500 s−1. This entropy driven mechanism likely reflects the very high selection pressure on the speed of protein synthesis, which drives the rate of each individual GTPase towards maximal turnover rate of the whole translation cycle. PMID:26497916

  2. A Filter Binding Assay to Quantify the Association of Cyclic di-GMP to Proteins

    PubMed Central

    Srivastava, Disha; Waters, Christopher M.

    2016-01-01

    Cyclic di-GMP (c-di-GMP) is a ubiquitous second messenger that regulates many processes in bacteria including biofilm formation, motility, and virulence (Hengge, 2009). Analysis of c-di-GMP binding properties of bacterial proteins is an important step to characterize c-di-GMP signaling pathways. C-di-GMP binds numerous proteins such as transcription factors, enzymes, and multimeric protein complexes (Hickman and Harwood, 2008, Ryjenkov et al., 2006, Weinhouse et al., 1997). The c-di-GMP binding assay described here is a relatively simple and cost effective method to characterize c-di-GMP binding to a protein using [32P]-labeled c-di-GMP. Radiolabeled c-di-GMP is readily synthesized with a purified GGDEF enzyme [such as WspR from Pseudomonas aeruginosa (P. aeruginosa)] and [a-32P]-GTP (Srivastava et al., 2013). After incubation of the labeled c-di-GMP with the protein of interest in solution, the resulting mixture is filtered through a nitrocellulose protein binding membrane. The amount of labeled c-di-GMP that is retained on the membrane indicates the interaction between the signal and protein. The specificity of c-di-GMP binding can be tested by competing with unlabeled c-di-GMP or other nucleotides such as GTP in the reaction. By examining binding of a fixed protein concentration to increasing concentrations of c-di-GMP, this method is able to determine the dissociation constant of c-di-GMP-protein interaction.

  3. Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding

    PubMed Central

    Canault, Matthias; Ghalloussi, Dorsaf; Grosdidier, Charlotte; Guinier, Marie; Perret, Claire; Chelghoum, Nadjim; Germain, Marine; Raslova, Hana; Peiretti, Franck; Morange, Pierre E.; Saut, Noemie; Pillois, Xavier; Nurden, Alan T.; Cambien, François; Pierres, Anne; van den Berg, Timo K.; Kuijpers, Taco W.; Tregouet, David-Alexandre

    2014-01-01

    The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbβ3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet’s ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis. PMID:24958846

  4. PRESERVING MITOCHONDRIAL FUNCTION PREVENTS THE PROTEASOMAL DEGRADATION OF GTP CYCLOHYDROLASE I

    PubMed Central

    SHARMA, SHRUTI; SUN, XUTONG; KUMAR, SANJIV; RAFIKOV, RUSLAN; ARAMBURO, ANGELA; KALKAN, GOKHAN; TIAN, JING; REHMANI, IMRAN; KALLARACKAL, SUPHIN; FINEMAN, JEFFERY R.; BLACK, STEPHEN M.

    2012-01-01

    The development of pulmonary hypertension is a common accompaniment of congenital heart disease (CHD) with increased pulmonary blood flow. Our recent evidence suggests that asymmetric dimethylarginine (ADMA)-induced mitochondrial dysfunction causes endothelial nitric oxide synthase (eNOS) uncoupling secondary to a proteasome-dependent degradation of GTP cyclohydrolase I (GCH1) that results in a decrease in the NOS co-factor, tetrahydrobiopterin (BH4). Decreases in NO signaling are thought to be an early hallmark of endothelial dysfunction. As L-carnitine plays an important role in maintaining mitochondrial function in this study we examined the protective mechanisms and the therapeutic potential of L-carnitine on NO signaling in pulmonary arterial endothelial cells (PAEC) and in a lamb model of CHD and increased pulmonary blood flow (Shunt). Acetyl L-carnitine (ALC) attenuated the ADMA-mediated proteasomal degradation of GCH1. This preservation was associated with a decrease in the association of GCH1 with the Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) and a decrease in its ubiquitination. This in turn prevented the decrease in BH4 levels induced by ADMA and preserved NO signaling. Treatment of Shunt lambs with L-carnitine also reduced GCH1/CHIP interactions, attenuated the ubiquitination and degradation of GCH1, and increased BH4 levels compared to vehicle treated Shunt lambs. The increases in BH4 were associated with decreased NOS uncoupling and enhanced NO generation. Thus, we conclude that L-carnitine may have a therapeutic potential in the treatment of pulmonary hypertension in children with CHD with increased pulmonary blood flow. PMID:22583703

  5. Identification and Functional Characterization of Phosphorylation Sites on GTP Cyclohydrolase I

    PubMed Central

    Du, Jianhai; Wei, Na; Xu, Hao; Ge, Ying; Vásquez-Vivar, Jeannette; Guan, Tongju; Oldham, Keith T.; Pritchard, Kirkwood A.; Shi, Yang

    2009-01-01

    Objective The post-translational regulation of GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, remains elusive. Here, we identified specific phosphorylation sites on GCH-1 and characterized the function of these sites. Methods and Results Mass spectrometry studies showed overexpressed rat GCH-1 was phosphorylated at serine (S) 51, S167 and threonine (T) 231 in HEK293 cells whereas a computational analysis of GCH-1 revealed 8 potential phosphorylation sites [S51, S72, T85, T91, T103, S130, S167 and T231]. GCH-1 activity and BH4 were significantly decreased in cells transfected with the phospho-defective mutants (S72A, T85A, T91A, T103A or S130A) and increased in cells transfected with the T231A mutant. BH4 and BH2 were increased in cells transfected with S51E, S72E, T85E, T91E, T103D or T130D mutants, but decreased in cells transfected with the T231D mutant, while cells transfected with the S167A or the S167E mutant had increased BH2. Additionally, cells transfected with the T231A mutant had reduced GCH-1 nuclear localization and nuclear GCH-1 activity. Conclusion Our data suggest GCH-1 activity is regulated either positively by phosphorylation S51, S72, T85, T91, T103 and S130, or negatively at T231. Such information might be useful in designing new therapies aiming at improving BH4 bioavailability. PMID:19762783

  6. Preserving mitochondrial function prevents the proteasomal degradation of GTP cyclohydrolase I.

    PubMed

    Sharma, Shruti; Sun, Xutong; Kumar, Sanjiv; Rafikov, Ruslan; Aramburo, Angela; Kalkan, Gokhan; Tian, Jing; Rehmani, Imran; Kallarackal, Suphin; Fineman, Jeffrey R; Black, Stephen M

    2012-07-15

    The development of pulmonary hypertension is a common accompaniment of congenital heart disease (CHD) with increased pulmonary blood flow. Our recent evidence suggests that asymmetric dimethylarginine (ADMA)-induced mitochondrial dysfunction causes endothelial nitric oxide synthase (eNOS) uncoupling secondary to a proteasome-dependent degradation of GTP cyclohydrolase I (GCH1) that results in a decrease in the NOS cofactor tetrahydrobiopterin (BH(4)). Decreases in NO signaling are thought to be an early hallmark of endothelial dysfunction. As l-carnitine plays an important role in maintaining mitochondrial function, in this study we examined the protective mechanisms and the therapeutic potential of l-carnitine on NO signaling in pulmonary arterial endothelial cells and in a lamb model of CHD and increased pulmonary blood flow (Shunt). Acetyl-l-carnitine attenuated the ADMA-mediated proteasomal degradation of GCH1. This preservation was associated with a decrease in the association of GCH1 with Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) and a decrease in its ubiquitination. This in turn prevented the decrease in BH(4) levels induced by ADMA and preserved NO signaling. Treatment of Shunt lambs with l-carnitine also reduced GCH1/CHIP interactions, attenuated the ubiquitination and degradation of GCH1, and increased BH(4) levels compared to vehicle-treated Shunt lambs. The increases in BH(4) were associated with decreased NOS uncoupling and enhanced NO generation. Thus, we conclude that L-carnitine may have a therapeutic potential in the treatment of pulmonary hypertension in children with CHD with increased pulmonary blood flow. PMID:22583703

  7. Functional Interactions between BM88/Cend1, Ran-Binding Protein M and Dyrk1B Kinase Affect Cyclin D1 Levels and Cell Cycle Progression/Exit in Mouse Neuroblastoma Cells

    PubMed Central

    Tsioras, Konstantinos; Papastefanaki, Florentia; Politis, Panagiotis K.; Matsas, Rebecca; Gaitanou, Maria

    2013-01-01

    BM88/Cend1 is a neuronal-lineage specific modulator with a pivotal role in coordination of cell cycle exit and differentiation of neuronal precursors. In the current study we identified the signal transduction scaffolding protein Ran-binding protein M (RanBPM) as a BM88/Cend1 binding partner and showed that BM88/Cend1, RanBPM and the dual specificity tyrosine-phosphorylation regulated kinase 1B (Dyrk1B) are expressed in mouse brain as well as in cultured embryonic cortical neurons while RanBPM can form complexes with either of the two other proteins. To elucidate a potential mechanism involving BM88/Cend1, RanBPM and Dyrk1B in cell cycle progression/exit, we transiently co-expressed these proteins in mouse neuroblastoma Neuro 2a cells. We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM. More specifically, functional interaction of RanBPM with either BM88/Cend1 or Dyrk1B stabilizes cyclin D1 in the nucleus and promotes 5-bromo-2'-deoxyuridine (BrdU) incorporation as a measure of enhanced cell proliferation. However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization. Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation. Our results suggest that functional interactions between BM88/Cend1, RanBPM and Dyrk1B affect the balance between cellular proliferation and differentiation in Neuro 2a cells and indicate that a potentially similar mechanism may influence cell cycle progression/exit and differentiation of neuronal precursors. PMID:24312406

  8. A protein required for nuclear-protein import, Mog1p, directly interacts with GTP-Gsp1p, the Saccharomyces cerevisiae ran homologue.

    PubMed

    Oki, M; Nishimoto, T

    1998-12-22

    We previously isolated 25 temperature-sensitive gsp1 alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other. We report here isolation of three multicopy suppressors-PDE2, NTF2, and a gene designated MOG1-all of which rescued a growth defect of these gsp1 strains. The gsp1 suppression occurred even in the absence of GSP2, another S. cerevisiae GSP1-like gene. Previously, NTF2 was reported to suppress gsp1 but not PDE2. Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W. Both MOG1 and NTF2 suppressed a series of gsp1 alleles with similar efficiency, and both suppressed gsp1 even with a single gene dose. Consistent with the high efficiency of gsp1 suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p. The disruption of MOG1 made yeast temperature-sensitive for growth. Deltamog1, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export. Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p. Furthermore, the finding that PDE2 suppressed both gsp1 and rna1-1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway. PMID:9860978

  9. GTP cyclohydrolase I mRNA: novel splice variants in the slime mould Physarum polycephalum and in human monocytes (THP-1) indicate conservation of mRNA processing.

    PubMed Central

    Golderer, G; Werner, E R; Heufler, C; Strohmaier, W; Gröbner, P; Werner-Felmayer, G

    2001-01-01

    GTP cyclohydrolase I (EC 3.5.4.16) is the first enzyme in the biosynthesis of tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)-biopterin] in mammals and of folic acid in bacteria. Here we have characterized the GTP cyclohydrolase I gene structure and two mRNA species from Physarum polycephalum, an acellular slime mould that synthesizes H(4)-biopterin and metabolites of the folic acid biosynthetic pathway. Its GTP cyclohydrolase I gene consists of seven exons, and the two GTP cyclohydrolase I cDNA species isolated from Physarum encode for proteins with 228 (25.7 kDa) and 195 (22.1 kDa) amino acids. Furthermore, we identified two previously undescribed mRNA species in interferon-gamma-treated human myelomonocytoma cells (THP-1) in addition to the cDNA coding for the fully functional 250-residue (27.9 kDa) protein, which is identical with that in human phaeochromocytoma cells. One of the new splice variants codes for a 233-residue (25.7 kDa) protein, whereas the other codes for the full-length protein but is alternatively spliced within the 3'-untranslated region. In heterologous expression, the shorter proteins of Physarum as well as of THP-1 cells identified here are degraded by proteolysis. Accordingly, only the 27.9 kDa protein was detectable in Western blots from THP-1 cell extracts. Quantification of GTP cyclohydrolase I mRNA species in different human cell types with and without cytokine treatment showed that in addition to the correct mRNA the two splice variants isolated here, as well as the two splice variants known from human liver, are strongly induced by cytokines in cell types with inducible GTP cyclohydrolase I (THP-1, dermal fibroblasts), but not in cell types with constitutive GTP cyclohydrolase I expression (SK-N-SH, Hep-G2). As in human liver, splicing of the new mRNA variant found in THP-1 cells occurs at the boundary of exons 5 and 6. Strikingly, the 195-residue protein from Physarum is alternatively spliced at a homologous position

  10. Galectin-3 Enhances Migration of Minature Pig Bone Marrow Mesenchymal Stem Cells Through Inhibition of RhoA-GTP Activity

    PubMed Central

    Gao, Qian; Xia, Ying; Liu, Lan; Huang, Lei; Liu, Yang; Zhang, Xue; Xu, Kui; Wei, Jingliang; Hu, Yanqing; Mu, Yulian; Li, Kui

    2016-01-01

    Bone marrow mesenchymal stem cells (BM-MSCs) are used in tissue engineering because of their migration characters. However, BM-MSCs have limitations in terms of reaching injuries and self-renewal. Therefore, enhancement of BM-MSC migration is important for therapeutic applications. Here, we assessed whether galectin-3 (Gal-3) increases the migration of minature pig BM-MSCs. Gal-3 was knocked down by short hairpin RNA (shRNA) or overexpressed using a lentiviral vector in Wuzhishan minature pig BM-MSCs. Proliferation and migration assays showed that knockdown of Gal-3 impaired BM-MSC proliferation and migration, whereas Gal-3 overexpression promoted these behaviors. RhoA-GTP activity was upregulated in Gal-3 shRNA-transfected BM-MSCs, while Rac-1- and Cdc42-GTP showed no changes. Western blotting indicated downregulation of p-AKT (ser473) and p-Erk1/2 after serum starvation for 12 h in Gal-3-knockdown BM-MSCs. p-AKT (ser473) expression was upregulated after serum starvation for 6 h, and p-Erk1/2 expression was unchanged in Gal-3-overexpressing BM-MSCs. Treatment with C3 transferase or Y27632 enhanced migration, whereas Gal-3 knockdown impaired migration in treated cells. These results demonstrate that Gal-3 may enhance BM-MSC migration, mainly through inhibiting RhoA-GTP activity, increasing p-AKT (ser473) expression, and regulating p-Erk1/2 levels. Our study suggests a novel function of Gal-3 in regulating minature pig BM-MSC migration, which may be beneficial for therapeutic applications. PMID:27215170

  11. Galectin-3 Enhances Migration of Minature Pig Bone Marrow Mesenchymal Stem Cells Through Inhibition of RhoA-GTP Activity.

    PubMed

    Gao, Qian; Xia, Ying; Liu, Lan; Huang, Lei; Liu, Yang; Zhang, Xue; Xu, Kui; Wei, Jingliang; Hu, Yanqing; Mu, Yulian; Li, Kui

    2016-01-01

    Bone marrow mesenchymal stem cells (BM-MSCs) are used in tissue engineering because of their migration characters. However, BM-MSCs have limitations in terms of reaching injuries and self-renewal. Therefore, enhancement of BM-MSC migration is important for therapeutic applications. Here, we assessed whether galectin-3 (Gal-3) increases the migration of minature pig BM-MSCs. Gal-3 was knocked down by short hairpin RNA (shRNA) or overexpressed using a lentiviral vector in Wuzhishan minature pig BM-MSCs. Proliferation and migration assays showed that knockdown of Gal-3 impaired BM-MSC proliferation and migration, whereas Gal-3 overexpression promoted these behaviors. RhoA-GTP activity was upregulated in Gal-3 shRNA-transfected BM-MSCs, while Rac-1- and Cdc42-GTP showed no changes. Western blotting indicated downregulation of p-AKT (ser473) and p-Erk1/2 after serum starvation for 12 h in Gal-3-knockdown BM-MSCs. p-AKT (ser473) expression was upregulated after serum starvation for 6 h, and p-Erk1/2 expression was unchanged in Gal-3-overexpressing BM-MSCs. Treatment with C3 transferase or Y27632 enhanced migration, whereas Gal-3 knockdown impaired migration in treated cells. These results demonstrate that Gal-3 may enhance BM-MSC migration, mainly through inhibiting RhoA-GTP activity, increasing p-AKT (ser473) expression, and regulating p-Erk1/2 levels. Our study suggests a novel function of Gal-3 in regulating minature pig BM-MSC migration, which may be beneficial for therapeutic applications. PMID:27215170

  12. Binding Procurement

    NASA Technical Reports Server (NTRS)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

    This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

  13. Role of Nucleotide Binding and GTPase Domain Dimerization in Dynamin-like Myxovirus Resistance Protein A for GTPase Activation and Antiviral Activity*

    PubMed Central

    Dick, Alexej; Graf, Laura; Olal, Daniel; von der Malsburg, Alexander; Gao, Song; Kochs, Georg; Daumke, Oliver

    2015-01-01

    Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action. PMID:25829498

  14. Role of nucleotide binding and GTPase domain dimerization in dynamin-like myxovirus resistance protein A for GTPase activation and antiviral activity.

    PubMed

    Dick, Alexej; Graf, Laura; Olal, Daniel; von der Malsburg, Alexander; Gao, Song; Kochs, Georg; Daumke, Oliver

    2015-05-15

    Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action. PMID:25829498

  15. Genome sequence of Bacillus subtilis subsp. spizizenii gtP20b, isolated from the Indian ocean.

    PubMed

    Fan, Longjiang; Bo, Shiping; Chen, Huan; Ye, Wanzhi; Kleinschmidt, Katrin; Baumann, Heike I; Imhoff, Johannes F; Kleine, Michael; Cai, Daguang

    2011-03-01

    Bacillus subtilis is an aerobic spore-forming Gram-positive bacterium that is a model organism and of great industrial significance as the source of diverse novel functional molecules. Here we present, to our knowledge, the first genome sequence of Bacillus subtilis strain gtP20b isolated from the marine environment. A subset of candidate genes and gene clusters were identified, which are potentially involved in production of diverse functional molecules, like novel ribosomal and nonribosomal antimicrobial peptides. The genome sequence described in this paper is due to its high strain specificity of great importance for basic as well as applied researches on marine organisms. PMID:21183663

  16. Cell cycle-dependent nuclear accumulation of the p94fer tyrosine kinase is regulated by its NH2 terminus and is affected by kinase domain integrity and ATP binding.

    PubMed

    Ben-Dor, I; Bern, O; Tennenbaum, T; Nir, U

    1999-02-01

    p94fer and p51ferT are two tyrosine kinases that are encoded by differentially spliced transcripts of the FER locus in the mouse. The two tyrosine kinases share identical SH2 and kinase domains but differ in their NH2-terminal amino acid sequence. Unlike p94fer, the presence of which has been demonstrated in most mammalian cell lines analyzed, the expression of p51ferT is restricted to meiotic cells. Here, we show that the two related tyrosine kinases also differ in their subcellular localization profiles. Although p51ferT accumulates constitutively in the cell nucleus, p94fer is cytoplasmic in quiescent cells and enters the nucleus concomitantly with the onset of S phase. The nuclear translocation of the FER proteins is driven by a nuclear localization signal (NLS), which is located within the kinase domain of these enzymes. The functioning of that NLS depends on the integrity of the kinase domain but was not affected by inactivation of the kinase activity. The NH2 terminus of p94fer dictated the cell cycle-dependent functioning of the NLS of FER kinase. This process was governed by coiled-coil forming sequences that are present in the NH2 terminus of the kinase. The regulatory effect of the p94fer NH2-terminal sequences was not affected by kinase activity but was perturbed by mutations in the kinase domain ATP binding site. Ectopic expression of the constitutively nuclear p51ferT in CHO cells interfered with S-phase progression in these cells. This was not seen in p94fer-overexpressing cells. The FER tyrosine kinases seem, thus, to be regulated by novel mechanisms that direct their different subcellular distribution profiles and may, consequently, control their cellular functioning. PMID:10074905

  17. Movement of newly imported light-harvesting chlorophyll-binding protein from unstacked to stacked thylakoid membranes is not affected by light treatment or absence of amino-terminal threonines

    SciTech Connect

    Kohorn, B.D.; Yakir, D. )

    1990-02-05

    In higher plants and algae, the transduction of captured light energy is highly regulated as excess excitation of photosystem II (PSII) reaction centers can be redirected to photosystem I (PSI) reaction centers. Models that attempt to explain this phenomenon involve light-harvesting chlorophyll-protein complexes (LHCII) that capture light energy and migrate between PSII and PSI. This report shows that in pea chloroplasts, the major protein component of LHCII, light-harvesting chlorophyll-binding protein (LHCP), can indeed migrate within the thylakoid membrane. We show, however, that although newly imported LHCP inserts into both stacked and unstacked thylakoid membranes, it then moves only from the unstacked, PSI-rich membranes to the stacked, PSII-rich membranes. The observed migration is not affected by light treatment that induces a redistribution of captured light energy (state I-state II transition) that previously was thought to induce LHCP to migrate in the opposite direction, from stacked to unstacked membranes. A mutation that removes the site of LHCP phosphorylation, the proposed trigger of state transitions, also has no effect on the integration and movement of LHCP, but does render LHCP more susceptible to proteolytic degradation. These results are not consistent with current models that deal with the short-term change in the distribution of light energy.

  18. Supervillin Binds the Rac/Rho-GEF Trio and Increases Trio-mediated Rac1 Activation

    PubMed Central

    Son, Kyonghee; Smith, Tara C.; Luna, Elizabeth J.

    2015-01-01

    We investigated cross-talk between the membrane-associated, myosin II-regulatory protein supervillin and the actin-regulatory small GTPases Rac1, RhoA, and Cdc42. Supervillin knockdown reduced Rac1-GTP loading, but not the GTP loading of RhoA or Cdc42, in HeLa cells with normal levels of the Rac1-activating protein Trio. No reduction in Rac1-GTP loading was observed when supervillin levels were reduced in Trio-depleted cells. Conversely, overexpression of supervillin isoform 1 (SV1) or, especially, isoform 4 (SV4) increased Rac1 activation. Inhibition of the Trio-mediated Rac1 guanine nucleotide exchange (GEF) activity with ITX3 partially blocked the SV4-mediated increase in Rac1-GTP. Both SV4 and SV1 co-localized with Trio at or near the plasma membrane in ruffles and cell surface projections. Two sequences within supervillin bound directly to Trio spectrin repeats 4–7: SV1-171, which contains N-terminal residues found in both SV1 and SV4 and the SV4-specific differentially spliced coding exons 3, 4, and 5 within SV4 (SV4-E345; SV4 amino acids 276 – 669). In addition, SV4-E345 interacted with the homologous sequence in rat kalirin (repeats 4–7, amino acids 531 – 1101). Overexpressed SV1-174 and SV4-E345 affected Rac1-GTP loading, but only in cells with endogenous levels of Trio. Trio residues 771 – 1057, which contain both supervillin-interaction sites, exerted a dominant-negative effect on cell spreading. Supervillin and Trio knockdowns, separately or together, inhibited cell spreading, suggesting that supervillin regulates the Rac1 guanine nucleotide exchange activity of Trio, and potentially also kalirin, during cell spreading and lamellipodia extension. PMID:25655724

  19. Improvements in the methodology for analyzing receptor subtypes and neuronal populations affected by anticholinesterase exposure. Annual summary report, 15 November 1983-14 November 1984

    SciTech Connect

    Wamsley, J.K.

    1984-11-14

    Conditions were defined that provide a means of selectively labeling subtypes of muscarinic receptors. The so-called M1 receptor population can be labeled with tritiated pirenzepine, while the receptor population labeled with tritiated quinuclidinyl benzilate (QNB) but not labeled with pirenzepine represents M2 receptor population. High- and low-affinity states of the receptors were also defined on the basis of agonist displacement of antagonist binding. Both the M1 and M2 receptor populations undergo axonal transport and the affinity states of these receptors are altered by neurochemical and neurosurgical lesions. Radioactive standards were developed that provide a means of quantitating the femtomoles of receptor bound with each ligand in microscopic regions of the brain. The technology was also devised to directly localize nicotinic cholinergic receptors using tritiated nicotine. It is now possible to localize several peptide receptors associated with cholinergic function including receptors for thyrotropin-releasing hormone (TRH) and somatostatin. The receptor autoradiographic technique was also carried beyond the receptor level of localization by using compounds to label adenylate cyclase and the GTP binding protein. This methodology should provide an elegant means of determining how anticholinesterase exposure has affected these many parameters of cholinergic nerve function.

  20. Cloning, Expression, Mutagenesis Library Construction of Glycerol Dehydratase, and Binding Mode Simulation of Its Reactivase with Ligands.

    PubMed

    Jiang, Wei; Li, Wenjun; Hong, Yan; Wang, Shizhen; Fang, Baishan

    2016-02-01

    The production of 1, 3-propanediol (1, 3-PD) and 3-hydroxypropionaldehyde (3-HPA) by enzyme reaction has been a hot field, and glycerol dehydratase (GDHt) is the key and rate-limiting enzyme involved in their biosynthesis. The gldABC gene encoding GDHt was cloned from Klebsiella pneumoniae, and the activity of the corresponding proteins expressed extracellularly and intracellularly was 6.8 and 3.2 U/mg, respectively, about six and three times higher than that of the wild strain. The change of amino acids for the β subunit can adjust the length of the Co-N bond and affect the homolysis rate of the Co-C bond to change GDHt activity. The expression plasmid, pET-32a-gldAC (containing no gldB which encodes the β subunit of GDHt), was constructed to build the mutagenesis library to improve the GDHt activity. The binding models of glycerol dehydratase reactivation factor (GDHtR) with ATP, CTP, or GTP were simulated by semi-flexible docking, respectively, and there was almost no difference between them. This research provided the basis for studying the quantitative structure-activity relationships between GDHtR and its ligands, as well as searching inexpensive ligands to replace ATP. These results and methods are of great use in economical and highly efficient production of 3-HPA and 1, 3-PD by the enzyme method. PMID:26547853

  1. 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. PMID:27298341

  2. Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms.

    PubMed

    Chou, Shan-Ho; Galperin, Michael Y

    2016-01-01

    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

  3. 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

  4. Synchronous tRNA movements during translocation on the ribosome are orchestrated by elongation factor G and GTP hydrolysis.

    PubMed

    Holtkamp, Wolf; Wintermeyer, Wolfgang; Rodnina, Marina V

    2014-10-01

    The translocation of tRNAs through the ribosome proceeds through numerous small steps in which tRNAs gradually shift their positions on the small and large ribosomal subunits. The most urgent questions are: (i) whether these intermediates are important; (ii) how the ribosomal translocase, the GTPase elongation factor G (EF-G), promotes directed movement; and (iii) how the energy of GTP hydrolysis is coupled to movement. In the light of recent advances in biophysical and structural studies, we argue that intermediate states of translocation are snapshots of dynamic fluctuations that guide the movement. In contrast to current models of stepwise translocation, kinetic evidence shows that the tRNAs move synchronously on the two ribosomal subunits in a rapid reaction orchestrated by EF-G and GTP hydrolysis. EF-G combines the energy regimes of a GTPase and a motor protein and facilitates tRNA movement by a combination of directed Brownian ratchet and power stroke mechanisms. PMID:25118068

  5. Inositol-1,4,5-trisphosphate and non-hydrolysable GTP analogue induced calcium release from intracellular stores of the Helix pomatia neurons.

    PubMed

    Belan, P V; Osipenko, O N; Tepikin, A V

    1990-01-01

    1. Cytoplasmic Ca2+ concentration ICaIin and membrane potential of single Helix pomatia neurons was studied by Fura-2 fluorescence measurement and conventional current clamp methods. 2. Intracellular injection of inositol-1,4,5-trisphosphate (IP3) and nonhydrolysable GTP analogue (Gpp/NH/p) led to a rise of ICaIin; in contrast, GTP injection did not cause significant ICaIin changes. 3. We suggest that both IP3 and Gpp/NH/p directly activated Ca release from intracellular stores. PMID:1980882

  6. Two novel mutations of the GTP cyclohydrolase 1 gene and genotype-phenotype correlation in Chinese Dopa-responsive dystonia patients.

    PubMed

    Yu, Lihua; Zhou, Huayong; Hu, Fayun; Xu, Yanming

    2013-07-01

    The most common form of Dopa-responsive dystonia (DRD) is caused by heterozygous mutations in the GTP cyclohydrolase I (GCH1) gene. We screened two unrelated, DRD-symptomatic Chinese Han individuals, for GCH1 gene mutations by direct sequencing. As the clinical manifestations of DRD are highly variable, we also explored the association between genotype and phenotype in all Chinese DRD patients reported so far in the literature, comprising 62 DRD-affected patients from 36 Chinese families. Two novel missense mutations (T94M, L145F) and a novel variant (c. 453+6 G>T) were identified in our two new patients. None of these variants was detected in 200 healthy controls. On the basis of this and other reports, heterozygous mutations were detected in 90.3% of Chinese Han subjects with DRD. Seeming the age of onset for males and females, the mean age was 13 years older in males than in females (P=0.006). Different mutation types did not show any significant differences in age of onset, gender composition, initial symptoms, or the L-dopa dose that abolished the symptoms. Among DRD patients lacking missense or exon-intron boundary mutations, 68.4% were found to possess a large deletion in GCH1, which were detected by multiplex ligation-dependent probe amplification. Most GCH1 mutations were found to cluster in two regions of the coding sequence, suggesting the probable existence of mutation hotspot for the first time. The genotype-phenotype correlation described here may improve our understanding of DRD in Chinese individuals. PMID:23211702

  7. Specific residues of the GDP/GTP exchange factor Bud5p are involved in establishment of the cell type-specific budding pattern in yeast.

    PubMed

    Kang, Pil Jung; Lee, Bongyong; Park, Hay-Oak

    2004-07-01

    Cells of the budding yeast undergo oriented cell division by choosing a specific site for growth depending on their cell type. Haploid a and alpha cells bud in an axial pattern whereas diploid a/alpha cells bud in a bipolar pattern. The Ras-like GTPase Rsr1p/Bud1p, its GDP-GTP exchange factor Bud5p, and its GTPase-activating protein Bud2p are essential for selecting the proper site for polarized growth in all cell types. Here we showed that specific residues at the N terminus and the C terminus of Bud5p were important for bipolar budding, while some residues were involved in both axial and bipolar budding. These bipolar-specific mutations of BUD5 disrupted proper localization of Bud5p in diploid a/alpha cells without affecting Bud5p localization in haploid alpha cells. In contrast, Bud5p expressed in the bud5 mutants defective in both budding patterns failed to localize in all cell types. Thus, these results identify specific residues of Bud5p that are likely to be involved in direct interaction with spatial landmarks, which recruit Bud5p to the proper bud site. Finally, we found a new start codon of BUD5, which extends the open reading frame to 210 bp upstream of the previously estimated start site, thus encoding a polypeptide of 608 amino acid residues. Bud5p with these additional N-terminal residues interacted with Bud8p, a potential bipolar landmark, suggesting that the N-terminal region is necessary for recognition of the spatial cues. PMID:15136576

  8. Protein synthesis in brine shrimp embryos. Regulation of the formation of the ternary complex (Met-tRNAf X eIF-2 X GTP) by two purified protein factors and phosphorylation of Artemia eIF-2.

    PubMed

    Woodley, C L; Roychowdhury, M; MacRae, T H; Olsen, K W; Wahba, A J

    1981-07-01

    We have purified from the ribosomal wash of dormant and developing embryos of Artemia two proteins, Co-eIF-2(A) and Co-eIF-2(B). These factors are essential for ternary complex formation and binding of [35S]-Met-tRNAf to 40-S ribosomal subunits with 15-30 microgram eIF-2/ml of reaction mixture. On polyacrylamide gel electrophoresis in dodecylsulfate, Co-eIF-2(A) is composed of a single polypeptide of Mr 65 000, whereas Co-eIF-2(B) contains polypeptides of Mr 105000 and 112000. Co-eIF-2(A) is sensitive to 4.5 microM aurintricarboxylic acid but Co-eIF-2(B) requires approximately 15 microM aurintricarboxylic acid to give 50% inhibition of ternary complex formation. The stimulatory activity of both factors is abolished by pretreatment of the proteins with N-ethylmaleimide. Artemia eIF-2 rapidly bonds [3H]GDP or [3H]GTP and at 15 degrees C the initiation factor rapidly equilibrates bound nucleotides with free GDP or GTP. Both Co-eIF-2(A) and Co-eIF-2(B) have no effect on the exchange or the amount of nucleotide bound. The small subunit (Mr 43 000) of Artemia eIF-2 is phosphorylated in the presence of the rabbit reticulocyte heme-repressible kinase. Tryptic digestion of [32P]phosphorylated eIF-2 produces a single major phosphopeptide and several minor ones. Acid hydrolysis of these phosphopeptides, as well as of [32P]phosphorylated eIF-2, demonstrates that the radioactivity is predominantly associated with phosphoserine. Phosphorylated Artemia eIF-2 is active in ternary complex formation, in AUG-dependent binding of [35S]Met-tRNAf to 40-S ribosomal subunits and in cell-free protein synthesis. Both Co-eIF-2(A) and Co-eIF-2(B) stimulate ternary complex formation with phosphorylated eIF-2. A kinase that phosphorylates the small subunit of eIF-2 is present in the post-ribosomal supernatant as well as in the ribosomal wash of developing Artemia embryos. PMID:6912815

  9. H(2)O(2) increases de novo synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin via GTP cyclohydrolase I and its feedback regulatory protein in vitiligo.

    PubMed

    Chavan, B; Beazley, W; Wood, J M; Rokos, H; Ichinose, H; Schallreuter, K U

    2009-02-01

    Patients with vitiligo accumulate up to 10(-3) mol/L concentrations of H(2)O(2) in their epidermis, which in turn affects many metabolic pathways in this compartment, including the synthesis and recycling of the cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). De novo synthesis of 6BH(4) is dependent on the rate-limiting enzyme GTP cyclohydrolase I (GTPCHI) together with its feedback regulatory protein (GFRP). This step is controlled by 6BH(4) and the essential amino acid L-phenylalanine. In the study presented here we wanted to investigate whether H(2)O(2) affects the GTPCHI/GFRP cascade in these patients. Our results demonstrated concentration-dependent regulation of rhGTPCHI where 100 micromol/L H(2)O(2) was the optimum concentration for the activation of the enzyme and >300 micromol/L resulted in a decrease in activity. Oxidation of GFRP and GTPCHI does not affect feedback regulation via L-phenylalanine and 6BH(4). In vitiligo a constant upregulation of 6BH(4) de novo synthesis results from epidermal build up of L-phenylalanine that is not controlled by H(2)O(2). Taking the results together, 6BH(4) de novo synthesis is controlled by H(2)O(2) in a concentration-dependent manner, but H(2)O(2)-mediated oxidation does not affect the functionality of the GTPCHI/GFRP complex. PMID:19101819

  10. Tolerance to LSD and DOB induced shaking behaviour: differential adaptations of frontocortical 5-HT(2A) and glutamate receptor binding sites.

    PubMed

    Buchborn, Tobias; Schröder, Helmut; Dieterich, Daniela C; Grecksch, Gisela; Höllt, Volker

    2015-03-15

    Serotonergic hallucinogens, such as lysergic acid diethylamide (LSD) and dimethoxy-bromoamphetamine (DOB), provoke stereotype-like shaking behaviour in rodents, which is hypothesised to engage frontocortical glutamate receptor activation secondary to serotonin2A (5-HT2A) related glutamate release. Challenging this hypothesis, we here investigate whether tolerance to LSD and DOB correlates with frontocortical adaptations of 5-HT2A and/or overall-glutamate binding sites. LSD and DOB (0.025 and 0.25 mg/kg, i.p.) induce a ketanserin-sensitive (0.5 mg/kg, i.p., 30-min pretreatment) increase in shaking behaviour (including head twitches and wet dog shakes), which with repeated application (7× in 4 ds) is undermined by tolerance. Tolerance to DOB, as indexed by DOB-sensitive [(3)H]spiroperidol and DOB induced [(35)S]GTP-gamma-S binding, is accompanied by a frontocortical decrease in 5-HT2A binding sites and 5-HT2 signalling, respectively; glutamate-sensitive [(3)H]glutamate binding sites, in contrast, remain unchanged. As to LSD, 5-HT2 signalling and 5-HT2A binding, respectively, are not or only marginally affected, yet [(3)H]glutamate binding is significantly decreased. Correlation analysis interrelates tolerance to DOB to the reduced 5-HT2A (r=.80) as well as the unchanged [(3)H]glutamate binding sites (r=.84); tolerance to LSD, as opposed, shares variance with the reduction in [(3)H]glutamate binding sites only (r=.86). Given that DOB and LSD both induce tolerance, one correlating with 5-HT2A, the other with glutamate receptor adaptations, it might be inferred that tolerance can arise at either level. That is, if a hallucinogen (like LSD in our study) fails to induce 5-HT2A (down-)regulation, glutamate receptors (activated postsynaptic to 5-HT2A related glutamate release) might instead adapt and thus prevent further overstimulation of the cortex. PMID:25513973

  11. Evidence that a single monolayer tubulin-GTP cap is both necessary and sufficient to stabilize microtubules.

    PubMed Central

    Caplow, M; Shanks, J

    1996-01-01

    Evidence that 13 or 14 contiguous tubulin-GTP subunits are sufficient to cap and stabilize a microtubule end and that loss of only one of these subunits results in the transition to rapid disassembly(catastrophe) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules assembled with GTP was transiently stabilized against dilution-induced disassembly by reaction with tubulin-GMPCPP subunits for a time sufficient to cap the end with an average 40 subunits. The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated from an observed 25- to 2000-s lifetime of the GMPCPP-stabilized microtubules following dilution with buffer and from the time required for loss of a single tubulin-GMPCPP subunit from the microtubule end (found to be 15 s). Rather than assuming that the 25- to 2000-s dispersion in cap lifetime results from an unlikely 80-fold range in the number of tubulin-GMPCpP subunits added in the 25-s incubation, it is proposed that this results because the minimum stable cap contains 13 to 14 tubulin-GMPCPP subunits. As a consequence, a microtubule capped with 13-14 tubulin-GMPCPP subunits switches to disassembly after only one dissociation event (in about 15 s), whereas the time required for catastrophe of a microtubule with only six times as many subunits (84 subunits) corresponds to 71 dissociation events (84-13). The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was also estimated with microtubules in which a GMPCPP-cap was formed by allowing chance to result in the accumulation of multiple contiguous tubulin-GMPCPP subunits at the end, during the disassembly of microtubules containing both GDP and GMPCPP. Our observation that the disassembly rate was inhibited in proportion to the 13-14th power of the fraction of subunits containing GMPCPP again suggests that a minimum cap contains 13-14 tubulin-GMPCPP subunits. A remeasurement of

  12. Crystal structures of 7-methylguanosine 5'-triphosphate (m(7)GTP)- and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)-bound human full-length eukaryotic initiation factor 4E: biological importance of the C-terminal flexible region.

    PubMed Central

    Tomoo, Koji; Shen, Xu; Okabe, Koumei; Nozoe, Yoshiaki; Fukuhara, Shoichi; Morino, Shigenobu; Ishida, Toshimasa; Taniguchi, Taizo; Hasegawa, Hiroshi; Terashima, Akira; Sasaki, Masahiro; Katsuya, Yoshio; Kitamura, Kunihiro; Miyoshi, Hiroshi; Ishikawa, Masahide; Miura, Kin-ichiro

    2002-01-01

    The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation. PMID:11879179

  13. Activation of SIRT1 by resveratrol represses transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) by deacetylating hepatic nuclear factor 4alpha

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cytosolic isoform of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) is a key enzyme of gluconeogenesis and glyceroneogenesis. While this enzyme is often over-expressed in diabetes and obesity, studies showed that decrease in its expression results in lessening the diseases condition in animal...

  14. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

    SciTech Connect

    Park, Su Hyun; Park, Tae Jun; Lim, In Kyoung

    2011-04-15

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H{sub 2}O{sub 2}, rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  15. Living cell imaging and Rac1-GTP levels of CXCL12-treated migrating neural progenitor cells in stripe assay.

    PubMed

    Zhang, Min; Song, Aihong; Lai, Siqiang; Qiu, Lisha; Huang, Yunlong; Chen, Qiang; Zhu, Bing; Xu, Dongsheng; Zheng, Jialin C

    2015-12-01

    This data article contains three figures and three videos related to the research article entitled "Applications of Stripe Assay in the Study of CXCL12-mediated Neural Progenitor Cell Migration and Polarization" Zhang et al. (2015) [1], which uses stripe assay to study mouse neural progenitor cell (NPC) migration and polarization. The current article describes the neurosphere method used to culture NPCs. NPCs in neurospheres and monolayer were characterized using immunocytochemistry method with antibodies against two classic NPC markers: nestin and SOX2. The article also describes method to obtain sufficient protein lysates from NPCs in the stripe assay. When protein lysates were subjected to Rac1 affinity precipitation, Rac1-GTP was detected in the pull-down samples. In addition, the articles provides live cell imaging data to better understand CXCL12-mediated cellular migration and polarization. PMID:26693502

  16. GTP depletion synergizes the anti-proliferative activity of chemotherapeutic agents in a cell type-dependent manner

    SciTech Connect

    Lin, Tao; Meng, Lingjun; Tsai, Robert Y.L.

    2011-10-22

    Highlights: {yields} Strong synergy between mycophenolic acid (MPA) and 5-FU in MDA-MB-231 cells. {yields} Cell type-dependent synergy between MPA and anti-proliferative agents. {yields} The synergy of MPA on 5-FU is recapitulated by RNA polymerase-I inhibition. {yields} The synergy of MPA on 5-FU requires the expression of nucleostemin. -- Abstract: Mycophenolic acid (MPA) depletes intracellular GTP by blocking de novo guanine nucleotide synthesis. GTP is used ubiquitously for DNA/RNA synthesis and as a signaling molecule. Here, we made a surprising discovery that the anti-proliferative activity of MPA acts synergistically with specific chemotherapeutic agents in a cell type-dependent manner. In MDA-MB-231 cells, MPA shows an extremely potent synergy with 5-FU but not with doxorubicin or etoposide. The synergy between 5-FU and MPA works most effectively against the highly tumorigenic mammary tumor cells compared to the less tumorigenic ones, and does not work in the non-breast cancer cell types that we tested, with the exception of PC3 cells. On the contrary, MPA shows the highest synergy with paclitaxel but not with 5-FU in SCC-25 cells, derived from oral squamous cell carcinomas. Mechanistically, the synergistic effect of MPA on 5-FU in MDA-MB-231 cells can be recapitulated by inhibiting the RNA polymerase-I activity and requires the expression of nucleostemin. This work reveals that the synergy between MPA and anti-proliferative agents is determined by cell type-dependent factors.

  17. Global discovery of protein kinases and other nucleotide-binding proteins by mass spectrometry.

    PubMed

    Xiao, Yongsheng; Wang, Yinsheng

    2016-09-01

    Nucleotide-binding proteins, such as protein kinases, ATPases and GTP-binding proteins, are among the most important families of proteins that are involved in a number of pivotal cellular processes. However, global study of the structure, function, and expression level of nucleotide-binding proteins as well as protein-nucleotide interactions can hardly be achieved with the use of conventional approaches owing to enormous diversity of the nucleotide-binding protein family. Recent advances in mass spectrometry (MS) instrumentation, coupled with a variety of nucleotide-binding protein enrichment methods, rendered MS-based proteomics a powerful tool for the comprehensive characterizations of the nucleotide-binding proteome, especially the kinome. Here, we review the recent developments in the use of mass spectrometry, together with general and widely used affinity enrichment approaches, for the proteome-wide capture, identification and quantification of nucleotide-binding proteins, including protein kinases, ATPases, GTPases, and other nucleotide-binding proteins. The working principles, advantages, and limitations of each enrichment platform in identifying nucleotide-binding proteins as well as profiling protein-nucleotide interactions are summarized. The perspectives in developing novel MS-based nucleotide-binding protein detection platform are also discussed. © 2014 Wiley Periodicals, Inc. Mass Spec Rev 35:601-619, 2016. PMID:25376990

  18. Control of 6-(D-threo-1',2'-dihydroxypropyl) pterin (dictyopterin) synthesis during aggregation of Dictyostelium discoideum. Involvement of the G-protein-linked signalling pathway in the regulation of GTP cyclohydrolase I activity.

    PubMed Central

    Gütlich, M; Witter, K; Bourdais, J; Veron, M; Rödl, W; Ziegler, I

    1996-01-01

    6-(D-threo-1',2'-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium dicoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665-669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1',2',3'-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4-5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5'-[gamma-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum. PMID:8660315

  19. Mitogenic signaling pathways of growth factors can be distinguished by the involvement of pertussis toxin-sensitive guanosine triphosphate-binding protein and of protein kinase C.

    PubMed Central

    Nishizawa, N; Okano, Y; Chatani, Y; Amano, F; Tanaka, E; Nomoto, H; Nozawa, Y; Kohno, M

    1990-01-01

    We have examined the possible involvements of pertussis toxin (PT)-sensitive guanosine triphosphate (GTP)-binding protein (Gp) and protein kinase C (PKC) in the mitogenic signaling pathways of various growth factors by the use of PT-pretreated and/or 12-O-tetradecanoyl phorbol-13-acetate (TPA)-pretreated mouse fibroblasts. Effects of PT pretreatment (inactivation of PT-sensitive Gp) and TPA pretreatment (depletion of PKC) on mitogen-induced DNA synthesis varied significantly and systematically in response to growth factors: mitogenic responses of cells to thrombin, bombesin, and bradykinin were almost completely abolished both in PT- and TPA-pretreated cells; responses to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vanadate were reduced to approximately 50% both in PT- and TPA-pretreated cells compared with native cells; response to basic fibroblast growth factor (bFGF) was not affected in PT-pretreated cells but was inhibited to some extent in TPA-pretreated cells. Thus, growth factors examined have been classified into three groups with regard to the involvements of PT-sensitive Gp and PKC in their signal transduction pathways. Binding of each growth factor to its receptor was not affected significantly by pretreatment of cells with PT or TPA. Inhibitory effects of PT and TPA pretreatment on each mitogen-induced DNA synthesis were not additive, suggesting that the functions of PT-sensitive Gp and PKC lie on an identical signal transduction pathway. Although all three groups of mitogens activated PKC, signaling of each growth factor depends to a varying extent on the function of PKC. Our results indicate that a single peptide growth factor such as EGF, PDGF, or bFGF acts through multiple signaling pathways to induce cell proliferation. Images PMID:2129194

  20. The R6A-1 peptide binds to switch II of G{alpha}{sub i1} but is not a GDP-dissociation inhibitor

    SciTech Connect

    Willard, Francis S. . E-mail: fwillard@med.unc.edu; Siderovski, David P.

    2006-01-27

    Heterotrimeric G-proteins are molecular switches that convert signals from membrane receptors into changes in intracellular physiology. Recently, several peptides that bind heterotrimeric G-protein {alpha} subunits have been isolated including the novel G{alpha}{sub i1} . GDP binding peptides R6A and KB-752. The R6A peptide and its minimized derivative R6A-1 interact with G{alpha}{sub i1} . GDP. Based on spectroscopic analysis of BODIPYFL-GTP{gamma}S binding to G{alpha}{sub i1}, it has been reported that R6A-1 has guanine nucleotide dissociation inhibitor (GDI) activity against G{alpha}{sub i1} [W.W. Ja, R.W. Roberts, Biochemistry 43 (28) (2004) 9265-9275]. Using radioligand binding, we show that R6A-1 is not a GDI for G{alpha}{sub i1} subunits. Furthermore, we demonstrate that R6A-1 reduces the fluorescence quantum yield of the G{alpha}{sub i1}-BODIPYFL-GTP{gamma}S complex, thus explaining the previously reported GDI activity as a fluorescence artifact. We further show that R6A-1 has significant sequence similarity to the guanine nucleotide exchange factor peptide KB-752 that binds to switch II of G{alpha}{sub i1}. We use competitive binding analysis to show that R6A-1 also binds to switch II of G{alpha} subunits.

  1. Activated G-protein releases cGMP from high affinity binding sites on PDE from toad rod outer segments (ROS)

    SciTech Connect

    Yuen, P.S.T.; Walseth, T.F.; Panter, S.S.; Sundby, S.R.; Graeff, R.M.; Goldberg, N.D.

    1987-05-01

    cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with /sup 32/P-cGMP and quantified by retention of complexes on nitrocellulose filters. By PAL, high affinity sites were present on the ..cap alpha.. and ..beta.. subunits of the cGMP-specific phosphodiesterase (PDE) which have MW/sub app/ of 94 and 90 kDa. A doublet was deduced from its photolabeling properties to represent PDE/sub ..gamma../ photocrosslinked with PDE/sub ..cap alpha../ or PDE/sub ..beta../, respectively. cGMP prebound to these high affinity sites was released by light-activated G-protein or its ..cap alpha.. subunit complexed with GTP..gamma..S; this inhibition of cGMP binding to PDE did not result from decreased cGMP availability due to enhanced hydrolysis. A low affinity cGMP binding component identified by PAL is tightly associated with ROS membranes. Apparent ATP/light-dependent stimulation of cGMP binding was shown to result from light activated cGMP hydrolysis in conjunction with ATP-promoted conversion of GMP to GDP/GTP and increased GDP/GTP binding. These findings coincide with a model for light-related regulation of cGMP binding and metabolism predicted from intact and cellfree kinetic measurements: in the dark state the cGMP hydrolic rate is constrained by the availability of cGMP because of its binding to high affinity sites on PDE. Light activated G-protein releases cGMP from these sites and allows for its redistribution to lower affinity sites represented by PDE catalytic site(s) and possible cGMP-dependent membrane cation channels.

  2. Changes to gonadotropin-releasing hormone (GnRH) receptor extracellular loops differentially affect GnRH analog binding and activation: evidence for distinct ligand-stabilized receptor conformations.

    PubMed

    Pfleger, Kevin D G; Pawson, Adam J; Millar, Robert P

    2008-06-01

    GnRH and its structural variants bind to GnRH receptors from different species with different affinities and specificities. By investigating chimeric receptors that combine regions of mammalian and nonmammalian GnRH receptors, a greater understanding of how different domains influence ligand binding and receptor activation can be achieved. Using human-catfish and human-chicken chimeric receptors, we demonstrate the importance of extracellular loop conformation for ligand binding and agonist potency, providing further evidence for GnRH and GnRH II stabilization of distinct active receptor conformations. We demonstrate examples of GnRH receptor gain-of-function mutations that apparently improve agonist potency independently of affinity, implicating a role for extracellular loops in stabilizing the inactive receptor conformation. We also show that entire extracellular loop substitution can overcome the detrimental effects of localized mutations, thereby demonstrating the importance of considering the conformation of entire domains when drawing conclusions from point-mutation studies. Finally, we present evidence implicating the configuration of extracellular loops 2 and 3 in combination differentiating GnRH analog binding modes. Because there are two endogenous forms of GnRH ligand but only one functional form of full-length GnRH receptor in humans, understanding how GnRH and GnRH II can elicit distinct functional effects through the same receptor is likely to provide important insights into how these ligands can have differential effects in both physiological and pathological situations. PMID:18356273

  3. Rats with minimal hepatic encephalopathy due to portacaval shunt show differential increase of translocator protein (18 kDa) binding in different brain areas, which is not affected by chronic MAP-kinase p38 inhibition.

    PubMed

    Agusti, Ana; Dziedzic, Jennifer L; Hernandez-Rabaza, Vicente; Guilarte, Tomas R; Felipo, Vicente

    2014-12-01

    Neuroinflammation plays a main role in neurological deficits in rats with minimal hepatic encephalopathy (MHE) due to portacaval shunt (PCS). Treating PCS rats with SB239063, an inhibitor of MAP-kinase-p38, reduces microglial activation and brain inflammatory markers and restores cognitive and motor function. The translocator protein-(18-kDa) (TSPO) is considered a biomarker of neuroinflammation. TSPO is increased in brain of PCS rats and of cirrhotic patients that died in hepatic coma. Rats with MHE show strong microglial activation in cerebellum and milder in other areas when assessed by MHC-II immunohistochemistry. This work aims were assessing: 1) whether binding of TSPO ligands is selectively increased in cerebellum in PCS rats; 2) whether treatment with SB239063 reduces binding of TSPO ligands in PCS rats; 3) which cell type (microglia, astrocytes) increases TSPO expression. Quantitative autoradiography was used to assess TSPO-selective (3)H-(R)-PK11195 binding to different brain areas. TSPO expression increased differentially in PCS rats, reaching mild expression in striatum or thalamus and very high levels in cerebellum. TSPO was expressed in astrocytes and microglia. Treatment with SB239063 did not reduces (3)[H]-PK11195 binding in PCS rats. SB239063 reduces microglial activation and levels of inflammatory markers, but not binding of TSPO ligands. This indicates that SB239063-induced neuroinflammation reduction in PCS rats is not mediated by effects on TSPO. Also, enhanced TSPO expression is not always associated with cognitive or motor deficits. If enhanced TSPO expression plays a role in mechanisms leading to neurological alterations in MHE, SB239063 would interfere these mechanisms at a later step. PMID:24307181

  4. Rats with minimal hepatic encephalopathy due to portacaval shunt show differential increase of translocator protein (18 kDa) binding in different brain areas, which is not affected by chronic MAP-kinase p38 inhibition

    PubMed Central

    Agusti, Ana; Dziedzic, Jennifer L.; Hernandez-Rabaza, Vicente; Guilarte, Tomas R.; Felipo, Vicente

    2014-01-01

    Neuroinflammation plays a main role in neurological deficits in rats with minimal hepatic encephalopathy (MHE) due to portacaval shunt (PCS). Treating PCS rats with SB239063, an inhibitor of MAP-kinase-p38, reduces microglial activation and brain inflammatory markers and restores cognitive and motor function. The translocator protein-(18-kDa) (TSPO) is considered a biomarker of neuro-inflammation. TSPO is increased in brain of PCS rats and of cirrhotic patients that died in hepatic coma. Rats with MHE show strong microglial activation in cerebellum and milder in other areas when assessed by MHC-II immunohistochemistry. This work aims were assessing: 1) whether binding of TSPO ligands is selectively increased in cerebellum in PCS rats; 2) whether treatment with SB239063 reduces binding of TSPO ligands in PCS rats; 3) which cell type (microglia, astrocytes) increases TSPO expression. Quantitative autoradiography was used to assess TSPO-selective 3H-(R)-PK11195 binding to different brain areas. TSPO expression increased differentially in PCS rats, reaching mild expression in striatum or thalamus and very high levels in cerebellum. TSPO was expressed in astrocytes and microglia. Treatment with SB239063 did not reduces 3[H]-PK11195 binding in PCS rats. SB239063 reduces microglial activation and levels of inflammatory markers, but not binding of TSPO ligands. This indicates that SB239063-induced neuroinflammation reduction in PCS rats is not mediated by effects on TSPO. Also, enhanced TSPO expression is not always associated with cognitive or motor deficits. If enhanced TSPO expression plays a role in mechanisms leading to neurological alterations in MHE, SB239063 would interfere these mechanisms at a later step. PMID:24307181

  5. 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

  6. CiMutT, an asidian MutT homologue, has a 7, 8-dihydro-8-oxo-dGTP pyrophosphohydrolase activity responsible for sanitization of oxidized nucleotides in Ciona intestinalis.

    PubMed

    Yonekura, Shin-Ichiro; Sanada, U; Zhang-Akiyama, Qiu-Mei

    2010-01-01

    The oxidized nucleotide precursors 7, 8-dihydro-8-oxo-dGTP (8-oxo-dGTP) and 1, 2-dihydro-2-oxo-dATP (2-oxo-dATP) are readily incorporated into nascent DNA strands during replication, which would cause base substitution mutations. E. coli MutT and human homologue hMTH1 hydrolyze 8-oxo-dGTP, thereby preventing mutations. In this study, we searched for hMTH1 homologues in the ascidian Ciona intestinalis using the NCBI-BLAST database. Among several candidates, we focused on one open reading frame, designated as CiMutT, because of its high degree of identity (41.7%) and similarity (58.3%) to the overall amino acid sequence of hMTH1, including the Nudix box. CiMutT significantly suppressed the mutator activity of E. coli mutT mutant. Purified CiMutT had a pyrophosphohydrolase activity that hydrolyzed 8-oxo-dGTP to 8-oxo-dGMP and inorganic pyrophosphate. It had a pH optimum of 9.5 and Mg(++) requirement with optimal activity at 5 mM. The activity of CiMutT for 8-oxo-dGTP was comparable to that of hMTH1, while it was 100-fold lower for 2-oxo-dATP than that of hMTH1. These facts indicate that CiMutT is a functional homologue of E. coli MutT. In addition, the enzyme hydrolyzed all four of the unoxidized nucleoside triphosphates, with a preference for dATP. The specific activity for 8-oxo-dGTP was greater than that for unoxidized dATP and dGTP. These results suggest that CiMutT has the potential to prevent mutations by 8-oxo-dGTP in C. intestinalis. PMID:21178309

  7. A Competitive Nucleotide Binding Inhibitor: In vitro Characterization of Rab7 GTPase Inhibition

    PubMed Central

    Agola, Jacob O.; Hong, Lin; Surviladze, Zurab; Ursu, Oleg; Waller, Anna; Strouse, J. Jacob; Simpson, Denise S.; Schroeder, Chad E.; Oprea, Tudor I.; Golden, Jennifer E.; Aubé, Jeffrey; Buranda, Tione; Sklar, Larry A.; Wandinger-Ness, Angela

    2012-01-01

    Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (Ki) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase as well as serving as a model for other small molecular weight GTPase inhibitors. PMID:22486388

  8. Bacterial-induced expression of RAB18 protein in Orzya sativa salinity stress and insights into molecular interaction with GTP ligand.

    PubMed

    Jha, Yachana; Sablok, Gaurav; Subbarao, Naidu; Sudhakar, Raja; Fazil, M H U Turabe; Subramanian, R B; Squartini, Andrea; Kumar, Sunil

    2014-09-01

    In the present research, we have studied the inoculation effects of two root-associated plant growth-promoting rhizobacteria (PGPR) in rice and provide the pieces of evidence that the inoculation of the PGPR could potentially result in inducing the expression of the salt stress-related RAB18 plant gene under varying degrees of salinity stress. The sequenced putative gene of RAB18 of Oryza sativa in this study is 740 bp long, has a content of 44.4%, and a molecular weight of 492 102.00 Da. BLAST homology patterns revealed sequence similarity with the previously sequenced RAB in model plant species. We demonstrate the mode of action of this stress-related protein by performing comparative modeling of Q10RT8 (Os03g0146000 protein, homolog of the sequenced RAB18; O. sativa subsp. japonica) using energy minimization, molecular dynamic simulations, and molecular docking of a guanosine triphosphate (GTP) ligand with the protein. The docking results indicated that Ser21, Ala22, Lys25, Asp68, Ala70, Glu73, and Arg74 are important determinant residues for functional interaction with the GTP ligand. The present research contributes to the understanding of the PGPR inoculation in salinity stress. Additionally, it provides the layout of the understanding of the molecular interactions between RAB and GTP ligand. PMID:25042706

  9. Sliding of a 43S ribosomal complex from the recognized AUG codon triggered by a delay in eIF2-bound GTP hydrolysis

    PubMed Central

    Terenin, Ilya M.; Akulich, Kseniya A.; Andreev, Dmitry E.; Polyanskaya, Sofya A.; Shatsky, Ivan N.; Dmitriev, Sergey E.

    2016-01-01

    During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon–anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to Pi release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and Pi release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining. PMID:26717981

  10. Minimal Determinants for Binding Activated G alpha from the Structure of a G alpha i1-Peptide Dimer

    SciTech Connect

    Johnston,C.; Lobanova, E.; Shavkunov, A.; Low, J.; Ramer, J.; Blasesius, R.; Fredericks, Z.; willard, F.; Kuhlman, B.; et al.

    2006-01-01

    G-Proteins cycle between an inactive GDP-bound state and an 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 G{alpha}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{center_dot}AlF{sub 4{sup -}}- and GTP{gamma}S-bound states of G{alpha}{sup i} subunits. KB-1753 blocks interaction of G{alpha}{sub transducin} 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 G{alpha} in vitro. The crystal structure of KB-1753 bound to G{alpha}{sub i1}-GDP{center_dot}AlF{sub 4{sup -}} reveals binding to a conserved hydrophobic groove between switch II and 3 helices and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for G{alpha}i subunits.

  11. A presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP exchange factor msec7-1

    PubMed Central

    Ashery, Uri; Koch, Henriette; Scheuss, Volker; Brose, Nils; Rettig, Jens

    1999-01-01

    ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane. PMID:9927699

  12. Activated RhoA Binds to the Pleckstrin Homology (PH) Domain of PDZ-RhoGEF, a Potential Site for Autoregulation

    SciTech Connect

    Chen, Zhe; Medina, Frank; Liu, Mu-ya; Thomas, Celestine; Sprang, Stephen R.; Sternweis, Paul C.

    2010-07-19

    Guanine nucleotide exchange factors (GEFs) catalyze exchange of GDP for GTP by stabilizing the nucleotide-free state of the small GTPases through their Dbl homology/pleckstrin homology (DH {center_dot} PH) domains. Unconventionally, PDZ-RhoGEF (PRG), a member of the RGS-RhoGEFs, binds tightly to both nucleotide-free and activated RhoA (RhoA {center_dot} GTP). We have characterized the interaction between PRG and activated RhoA and determined the structure of the PRG-DH {center_dot} PH-RhoA {center_dot} GTP{gamma}S (guanosine 5{prime}-O-[{gamma}-thio]triphosphate) complex. The interface bears striking similarity to a GTPase-effector interface and involves the switch regions in RhoA and a hydrophobic patch in PRG-PH that is conserved among all Lbc RhoGEFs. The two surfaces that bind activated and nucleotide-free RhoA on PRG-DH {center_dot} PH do not overlap, and a ternary complex of PRG-DH {center_dot} PH bound to both forms of RhoA can be isolated by size-exclusion chromatography. This novel interaction between activated RhoA and PH could play a key role in regulation of RhoGEF activity in vivo.

  13. Microgravity and Signaling Molecules in Rat Osteoblasts: Downstream of Receptor Tyrosine Kinase, G-Protein-Coupled Receptor, and Small GTP-Binding Proteins

    NASA Technical Reports Server (NTRS)

    Kumel, Yasuhiro; Shimokawa, Hitoyata; Morita, Sadao; Katano, Hisako; Akiyama, Hideo; Hirano, Masahiko; Ohya, Keiichi; Sams, Clarence F.; Whitson, Peggy A.

    2005-01-01

    Rat osteoblasts were cultured for 4 and 5 days aboard Space Shuttle and solubilized on board. The mRNA levels of the post-receptor signaling molecules were analyzed by quantitative RT-PCR. The G-protein alpha subunit G(alpha)q mRNA levels were elevated 3-fold by microgravity. G(alpha)q stimulates PLC(beta), and then PKC. PKC(delta) and PKC(theta) mRNA levels were increased 2- to 5-fold by microgravity The mRNA levels of SOS and Ras GRF were increased 4 to 5-fold by microgravity, while Ras GAP was not altered. Spaceflight-induced bone loss might be attributed to microgravity modulation of the signaling pathway in osteoblasts.

  14. Contribution of the R-Ras2 GTP-binding protein to primary breast tumorigenesis and late-stage metastatic disease

    NASA Astrophysics Data System (ADS)

    Larive, Romain M.; Moriggi, Giulia; Menacho-Márquez, Mauricio; Cañamero, Marta; Álava, Enrique De; Alarcón, Balbino; Dosil, Mercedes; Bustelo, Xosé R.

    2014-05-01

    R-Ras2 is a transforming GTPase that shares downstream effectors with Ras subfamily proteins. However, little information exists about the function of this protein in tumorigenesis and its signalling overlap with classical Ras GTPases. Here we show, by combining loss- and gain-of-function studies in breast cancer cells, mammary epithelial cells and mouse models, that endogenous R-Ras2 has a role in both primary breast tumorigenesis and the late metastatic steps of cancer cells in the lung parenchyma. R-Ras2 drives tumorigenesis in a phosphatidylinostiol-3 kinase (PI3K)-dependent and signalling autonomous manner. By contrast, its prometastatic role requires other priming oncogenic signals and the engagement of several downstream elements. R-Ras2 function is required even in cancer cells exhibiting constitutive activation of classical Ras proteins, indicating that these GTPases are not functionally redundant. Our results also suggest that application of long-term R-Ras2 therapies will result in the development of compensatory mechanisms in breast tumours.

  15. Increased levels of p21ras-GTP and enhanced DNA synthesis accompany elevated tyrosyl phosphorylation of GAP-associated proteins, p190 and p62, in c-src overexpressors.

    PubMed

    Chang, J H; Wilson, L K; Moyers, J S; Zhang, K; Parsons, S J

    1993-04-01

    While examining the role of pp60c-src in cellular proliferation, we found that overexpression of c-src in C3H10T1/2 murine fibroblasts results in an augmented mitogenic response to epidermal growth factor (EGF) [Luttrell, D.K., Luttrell, L.M. & Parsons, S.J. (1988). Mol. Cell. Biol., 8, 497-501; Wilson, L.K., Luttrell, D.K., Parsons, S.J. (1989). Mol. Cell. Biol., 9, 1536-1544] and enhanced tyrosyl phosphorylation of specific cellular proteins [Wilson, L.K. & Parsons, S.J. (1990). 5, 1471-1480]. Here we identify two of these proteins as the GAP (GTPase-activating protein of p21ras)-associated proteins, p190 and p62. Evidence is presented to support the notion that, in 10T1/2 fibroblasts, p190 is a preferred substrate of pp60c-src, while p62 is preferentially phosphorylated by the EGF receptor. First, the phosphotyrosine content of p190 in quiescent cells is three- to fivefold higher in c-src overexpressors than in control cells and is not altered by growth factor treatment. In contrast, tyrosyl phosphorylation of p62 is undetectable in quiescent cells and transiently observable upon EGF addition. Second, the phosphotyrosine content of p190 in cells overexpressing defective pp60c-src is reduced in comparison with wild-type (wt) c-src overexpressors, while that of p62 is significantly less affected. Further studies revealed that tyrosyl phosphorylation of p190 and p62 is not required for GAP complex formation, as equal amounts of p190 and p62 proteins could be detected in GAP complexes from wt and variant c-src overexpressors both before and after EGF stimulation. However, analysis of GTP-bound p21ras revealed higher basal and EGF-stimulated levels in c-src overexpressors than in control cells. Taken together, these results suggest that one mechanism by which pp60c-src may contribute to early events in the EGF-induced mitogenic pathway in 10T1/2 fibroblasts is by increasing the level of GAP-associated p190 and p62 tyrosyl phosphorylation, which in turn results in

  16. Unraveling determinants of transcription factor binding outside the core binding site.

    PubMed

    Levo, Michal; Zalckvar, Einat; Sharon, Eilon; Dantas Machado, Ana Carolina; Kalma, Yael; Lotam-Pompan, Maya; Weinberger, Adina; Yakhini, Zohar; Rohs, Remo; Segal, Eran

    2015-07-01

    Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs, we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences serving as promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences and allows the characterization of TF binding determinants within and outside of core binding sites. PMID:25762553

  17. Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation.

    PubMed Central

    Haberland, J; Gerke, V

    1999-01-01

    GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man. All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins. The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated. In order to gain a better understanding of this interaction we generated a number of mutant RanGAPs carrying amino acid substitutions in the LRR domain and analysed their complex formation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein. We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficient Ran binding and GAP activity. These separating regions contain three conserved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases. However, mutations in two of these arginines do not affect the GAP activity and replacement of the third conserved arginine (Arg91 in human RanGAP) severely interferes not only with GAP activity but also with Ran binding. This indicates that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalytic arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction. PMID:10527945

  18. Cap-binding activity of an eIF4E homolog from Leishmania

    PubMed Central

    YOFFE, YAEL; ZUBEREK, JOANNA; LEWDOROWICZ, MAGDALENA; ZEIRA, ZIV; KEASAR, CHEN; ORR-DAHAN, IRIT; JANKOWSKA-ANYSZKA, MARZENA; STEPINSKI, JANUSZ; DARZYNKIEWICZ, EDWARD; SHAPIRA, MICHAL

    2004-01-01

    All eukaryotic mRNAs possess a 5′-cap (m7GpppN) that is recognized by a family of cap-binding proteins. These participate in various processes, such as RNA transport and stabilization, as well as in assembly of the translation initiation complex. The 5′-cap of trypanosomatids is complex; in addition to 7-methyl guanosine, it includes unique modifications on the first four transcribed nucleotides, and is thus denoted cap-4. Here we analyze a cap-binding protein of Leishmania, in an attempt to understand the structural features that promote its binding to this unusual cap. LeishIF4E-1, a homolog of eIF4E, contains the conserved cap-binding pocket, similar to its mouse counterpart. The mouse eIF4E has a higher Kas for all cap analogs tested, as compared with LeishIF4E-1. However, whereas the mouse eIF4E shows a fivefold higher affinity for m7GTP than for a chemically synthesized cap-4 structure, LeishIF4E-1 shows similar affinities for both