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Sample records for acetyltransferase couples sup35

  1. The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

    PubMed Central

    Pezza, John A.; Langseth, Sara X.; Raupp Yamamoto, Rochele; Doris, Stephen M.; Ulin, Samuel P.; Salomon, Arthur R.

    2009-01-01

    Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI+], a prion form of the S. cerevisiae Sup35 protein (Sup35[PSI+]), and the three Nα-acetyltransferases, NatA, NatB, and NatC, which collectively modify ∼50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI+] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI+] NatA mutants continue to propagate heritable Sup35[PSI+]. This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI+] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35[PSI+] complexes and their phenotypic effects. PMID:19073888

  2. Fock-space multireference coupled-cluster calculations of the hyperfine structure of isoelectronic {sup 33}S{sup -} and {sup 35,37}Cl

    SciTech Connect

    Das, Madhulita; Chaudhuri, Rajat K.; Chattopadhyay, Sudip; Mahapatra, Uttam Sinha

    2011-10-15

    Due to its flexibility and possible systematic improvement, the Fock-space (FS) multireference coupled-cluster (MRCC) method remains a very important tool for the computation of energy differences of spectroscopic interest. In the present work, the FS MRCC method for the electron detachment process has been applied to determine the magnetic hyperfine constant A{sub J} and nuclear quadrupole moments Q (related to electric hyperfine constant B{sub J}) for the lowest multiplets of {sup 33}S{sup -}, {sup 35}Cl, and {sup 37}Cl with Dirac-Fock orbitals. In addition, we also report {sup 2}P{sub 3/2}([Ne]3s{sup 2}3p{sup 5}) {yields} {sup 2}P{sub 1/2}([Ne]3s{sup 2}3p{sup 5}) magnetic dipole transition matrix element and electron affinity of {sup 35}Cl (i.e., ionization energy of Cl{sup -}). Calculated properties are in very good agreement with the available new standard or reference values.

  3. The beta decay asymmetry parameter of /sup 35/Ar

    SciTech Connect

    Garnett, J.D.

    1987-11-01

    The beta decay asymmetry parameter for /sup 35/Ar = /sup 35/Cl + e/sup +/ + nu/sub e/ has been remeasured in order to resolve a long standing puzzle. Previous asymmetry measurements, when combined with the comparative half-life, yield a value for the vector coupling constant, G/sub v/, that is in serious disagreement with the accepted value. We produced polarized /sup 35/Ar by a (p,n) reaction on /sup 35/Cl using the polarized proton beam provided by Lawrence Berkeley Laboratory's 88-Inch Cyclotron. The polarization of the /sup 35/Ar was determined by measuring the asymmetry of the positrons produced in /sup 35/Ar decay to the first excited state in /sup 35/Cl (branching ratio = 1.3%) in coincidence with a 1219.4 keV gamma ray. Our result, A/sub 0/ = 0.49 +- 0.10, combined with the comparative half-life yields a value for G/sub v/ in agreement with the accepted value.

  4. Coupled action of γ-glutamyl transpeptidase-glutathione and keratinase effectively degrades feather keratin and surrogate prion protein, Sup 35NM.

    PubMed

    Sharma, Richa; Gupta, Rani

    2012-09-01

    Recombinant Escherichia coli HB101 harboring keratinase rKP2 from Pseudomonas aeruginosa KS-1 degraded 2% chicken feather in LB-Amp medium in 24h. SEM analysis and detailed studies revealed that bacterial colonization of feather was a pre-requisite for degradation of feather by keratinase. The mechanism of sulfitolysis revealed involvement of free cystinyl group as a source of redox during colonization as DTNB inhibited feather degradation by rKP2. Involvement of GGT-GSH system in contribution of free cystinyl group for redox was established by using GGT knockout recombinant E. coli strain that failed to degrade feather inspite of successful colonization and keratinase production. Short term experiments further confirmed enhanced protein release from feather keratin in presence of GGT-GSH redox. In the presence of similar redox, rKP2 also degraded surrogate prion protein, Sup 35NM in 15 min at 37°C, pH 7.0. PMID:22776236

  5. /sup 35/S-glycosaminoglycan and /sup 35/S-glycopeptide metabolism by diabetic glomeruli and aorta

    SciTech Connect

    Brown, D.M.; Klein, D.J.; Michael, A.F.; Oegema, T.R.

    1982-05-01

    /sup 35/S-glycosaminoglycan metabolism by glomeruli isolated from streptozotocin-diabetic and control rats was studied in vivo and in vitro. Total /sup 35/S-glycosaminoglycan synthesis and retention in the matrix by diabetic glomeruli was reduced while degradation was increased. /sup 35/S-glycosaminoglycan content of isolated GBM was similarly decreased. Whereas /sup 35/S-glycosaminoglycan content of glomeruli and GBM was decreased after in vitro incubation with /sup 35/SO/sub 4/, a larger proportion of total /sup 35/S-glycosaminoglycans was found in the incubation medium from diabetic glomeruli. Both control and diabetic glomeruli synthesize /sup 35/S-labeled glycopeptides, the quantity from diabetic glomeruli being reduced. Aorta from /sup 35/SO/sub 4/-injected diabetic rats also synthesized reduced quantities of /sup 35/S-glycosaminoglycans. There were no preferential metabolic alterations of species of /sup 35/S-glycosaminoglycans by diabetic glomeruli or aortas. These studies suggest that synthesis of /sup 35/S-glycosaminoglycans and /sup 35/S-glycopeptides by diabetic glomeruli are altered by disturbances of both synthetic as well as degradative pathways. An alteration of /sup 35/S-glycosaminoglycans interaction with matrix components in diabetes is postulated.

  6. Horizontal Transmission of Cytosolic Sup35 Prions by Extracellular Vesicles

    PubMed Central

    Liu, Shu; Hossinger, André; Hofmann, Julia P.; Denner, Philip

    2016-01-01

    ABSTRACT Prions are infectious protein particles that replicate by templating their aggregated state onto soluble protein of the same type. Originally identified as the causative agent of transmissible spongiform encephalopathies, prions in yeast (Saccharomyces cerevisiae) are epigenetic elements of inheritance that induce phenotypic changes of their host cells. The prototype yeast prion is the translation termination factor Sup35. Prions composed of Sup35 or its modular prion domain NM are heritable and are transmitted vertically to progeny or horizontally during mating. Interestingly, in mammalian cells, protein aggregates derived from yeast Sup35 NM behave as true infectious entities that employ dissemination strategies similar to those of mammalian prions. While transmission is most efficient when cells are in direct contact, we demonstrate here that cytosolic Sup35 NM prions are also released into the extracellular space in association with nanometer-sized membrane vesicles. Importantly, extracellular vesicles are biologically active and are taken up by recipient cells, where they induce self-sustained Sup35 NM protein aggregation. Thus, in mammalian cells, extracellular vesicles can serve as dissemination vehicles for protein-based epigenetic information transfer. PMID:27406566

  7. Preparation of sup 35 S-labeled polyphosphorothioate oligodeoxyribonucleotides by use of hydrogen phosphonate chemistry

    SciTech Connect

    Stein, A.; Iversen, P.L.; Subasinghe, C.; Cohen, J.S.; Stec, W.J.; Zon, G. )

    1990-07-01

    The title compounds were chemically synthesized as their 5'-dimethoxytrityl derivatives by base-catalyzed reaction of {sup 35}S-enriched elemental sulfur with support-bound hydrogen phosphonate oligomer. This was derived from adamantane carbonyl chloride-activated coupling of nucleotide hydrogen phosphonate monomers, and similarly activated capping with isopropyl phosphite. A convenient, disposable, reversed-phase cartridge was utilized to purify and isolate the 5'-dimethoxytrityl derivative for subsequent in situ detritylation and elution of the final product. The specific activity obtained for the title compounds was ca. 10(7) cpm/mumols-eq P(O)S-. The procedure should be readily adaptable to appropriate syntheses of other P-S containing analogs of DNA and RNA.

  8. In vivo evidence for the fibrillar structures of Sup35 prions in yeast cells

    PubMed Central

    Kawai-Noma, Shigeko; Pack, Chan-Gi; Kojidani, Tomoko; Asakawa, Haruhiko; Hiraoka, Yasushi; Kinjo, Masataka; Haraguchi, Tokuko; Hirata, Aiko

    2010-01-01

    Yeast prion [PSI+] is caused by aggregated structures of the Sup35 protein. Although Sup35 forms typical amyloid fibrils in vitro, there is no direct evidence for the fibrillar structures of Sup35 in vivo. We analyzed [PSI+] cells in which Sup35 fused with green fluorescent protein (GFP) formed aggregates visible by fluorescence microscopy using thin-section electron microscopy (EM). Rapid-freeze EM combined with an immunogold-labeling technique as well as correlative light EM, which allows high-resolution imaging by EM of the same structure observed by light (fluorescence) microscopy, shows that the aggregates contain bundled fibrillar structures of Sup35-GFP. Additional biochemical and fluorescent correlation spectroscopy results suggest that the Sup35 oligomers diffused in the [PSI+] lysates adopt fibril-like shapes. Our findings demonstrate that [PSI+] cells contain Sup35 fibrillar structures closely related to those formed in vitro and provide insight into the molecular mechanism by which Sup35 aggregates are assembled and remodeled in [PSI+] cells. PMID:20643880

  9. Structure of newly synthesized (/sup 35/S)-proteoglycans and (/sup 35/S)-proteoglycan turnover products of cartilage explant cultures from dogs with experimental osteoarthritis

    SciTech Connect

    Carney, S.L.; Billingham, M.E.; Muir, H.; Sandy, J.D.

    1985-01-01

    The structure of newly synthesized proteoglycans from explant cultures of cartilage from joints subjected to transection of the anterior cruciate ligament (osteoarthritic) and from normal (non- or sham-operated) joints was examined. The structure of the products of proteoglycan turnover was also examined using explants of normal and osteoarthritic cartilage maintained in culture for a 48 h chase period. The findings were as follows: Newly synthesized (/sup 35/S)-proteoglycans extracted from cartilage explants from osteoarthritic joints whether examined 3 weeks, 3 months, or 6 months after surgery were larger than those from corresponding normal cartilage. This can be explained by the synthesis in osteoarthritic cartilage of abnormally long chondroitin sulfate chains on newly synthesised proteoglycans. The extracts also contained a newly formed small proteoglycan species that was unable to interact with hyaluronic acid. The proportion of this species was higher in osteoarthritic cartilage compared with normal, examined 3 weeks after surgery, but was generally absent from cartilage obtained 3 and 6 months after surgery. Compared with controls, a smaller proportion of the (/sup 35/S)-proteoglycans released into the maintenance medium of explant cultures of osteoarthritic cartilage during a 48 h chase period was able to interact with hyaluronic acid. However, although furnished with longer (/sup 35/S)-glycosaminoglycan chains, these proteoglycans were smaller than those from control explants.

  10. Foliar leaching, translocation, and biogenic emission of sup 35 S in radiolabeled loblolly pines

    SciTech Connect

    Garten, C.T. Jr. )

    1990-02-01

    Foliar leaching, basipetal (downward) translocation, and biogenic emission of sulfur (S), as traced by {sup 35}S, were examined in a field study of loblolly pines. Four trees were radiolabeled by injection with amounts of {sup 35}S in the 6-8 MBq range, and concentrations in needle fall, stemflow, throughfall, and aboveground biomass were measured over a period of 15-20 wk after injection. The contribution of dry deposition to sulfate-sulfur (SO{sub 4}{sup 2{minus}}-S) concentrations in net throughfall (throughfall SO{sub 4}{sup 2{minus}}-S concentration minus that in incident precipitation) beneath all four trees was > 90%. Calculations indicated that about half of the summertime SO{sub 2} dry deposition flux to the loblolly pines was fixed in the canopy and not subsequently leached by rainfall. Based on mass balance calculations, {sup 35}S losses through biogenic emissions from girdled trees were inferred to be 25-28% of the amount injected. Estimates based on chamber methods and mass balance calculations indicated a range in daily biogenic S emission of 0.1-10 {mu}g/g dry needles. Translocation of {sup 35}S to roots in nongirdled trees was estimated to be between 14 and 25% of the injection. It is hypothesized that biogenic emission and basipetal translocation of S (and not foliar leaching) are important mechanisms by which forest trees physiologically adapt to excess S in the environment.

  11. Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-linking.

    PubMed

    Wong, Shenq-Huey; King, Chih-Yen

    2015-10-01

    Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-l-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1-61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine substitutions and two alanine substitutions were introduced at fixed positions in Sup(1-61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala-substituted Sup(1-61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and intermolecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amounts of (14)N- and (15)N-labeled protein. Fibers propagating the VL strain type exhibited intra- and intermolecular cross-linking between amino acid residues 3 and 28, as well as intra- and intermolecular linking between 32 and 55. Inter- and intramolecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain folds of different [PSI] strains. PMID:26265470

  12. W8, a new Sup35 prion strain, transmits distinctive information with a conserved assembly scheme.

    PubMed

    Huang, Yu-Wen; Chang, Yuan-Chih; Diaz-Avalos, Ruben; King, Chih-Yen

    2015-01-01

    Prion strains are different self-propagating conformers of the same infectious protein. Three strains of the [PSI] prion, infectious forms of the yeast Sup35 protein, have been previously characterized in our laboratory. Here we report the discovery of a new [PSI] strain, named W8. We demonstrate its robust cellular propagation as well as the protein-only transmission. To reveal strain-specific sequence requirement, mutations that interfered with the propagation of W8 were identified by consecutive substitution of residues 5-55 of Sup35 by proline and insertion of glycine at alternate sites in this segment. Interestingly, propagating W8 with single mutations at residues 5-7 and around residue 43 caused the strain to transmute. In contrast to the assertion that [PSI] existed as a dynamic cloud of sub-structures, no random drift in transmission characteristics was detected in mitotically propagated W8 populations. Electron diffraction and mass-per-length measurements indicate that, similar to the 3 previously characterized strains, W8 fibers are composed of about 1 prion molecule per 4.7-Å cross-β repeat period. Thus differently folded single Sup35 molecules, not dimeric and trimeric assemblies, form the basic repeating units to build the 4 [PSI] strains. PMID:26038983

  13. W8, a new Sup35 prion strain, transmits distinctive information with a conserved assembly scheme

    PubMed Central

    Huang, Yu-Wen; Chang, Yuan-Chih; Diaz-Avalos, Ruben; King, Chih-Yen

    2015-01-01

    ABSTRACT Prion strains are different self-propagating conformers of the same infectious protein. Three strains of the [PSI] prion, infectious forms of the yeast Sup35 protein, have been previously characterized in our laboratory. Here we report the discovery of a new [PSI] strain, named W8. We demonstrate its robust cellular propagation as well as the protein-only transmission. To reveal strain-specific sequence requirement, mutations that interfered with the propagation of W8 were identified by consecutive substitution of residues 5–55 of Sup35 by proline and insertion of glycine at alternate sites in this segment. Interestingly, propagating W8 with single mutations at residues 5–7 and around residue 43 caused the strain to transmute. In contrast to the assertion that [PSI] existed as a dynamic cloud of sub-structures, no random drift in transmission characteristics was detected in mitotically propagated W8 populations. Electron diffraction and mass-per-length measurements indicate that, similar to the 3 previously characterized strains, W8 fibers are composed of about 1 prion molecule per 4.7-Å cross-β repeat period. Thus differently folded single Sup35 molecules, not dimeric and trimeric assemblies, form the basic repeating units to build the 4 [PSI] strains. PMID:26038983

  14. Measurement of the deposition and fate of {sup 35}SO{sub 2} in a pine plantation

    SciTech Connect

    Gay, D.W.; Murphy, C.E. Jr.

    1988-12-31

    An experiment was carried out to determine the uptake rate and fate of SO{sub 2} absorbed by the forest canopy. The radioactive tracer, {sup 35}S was used. Branches of loblolly pine were exposed to {sup 35}SO{sub 2} in the field with a portable chamber. The exposed branches were excised and a sample of needles was immediately washed with distilled water. The needles and water collected in the field was analyzed for {sup 35}S content. The results indicated that a portion of the {sup 35}S could be washed from the needles and another portion was not removed with a water rinse. The portion that could not be removed by rinsing was absorbed at a rate, and followed patterns of absorption, that suggested that absorption was controlled by stomatal diffusion, and thus was internal. The {sup 35}S that could be removed by rinsing appeared to be surface absorption.

  15. NMR shielding and spin–rotation constants of {sup 175}LuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br, {sup 127}I) molecules

    SciTech Connect

    Demissie, Taye B.

    2015-12-31

    This presentation demonstrates the relativistic effects on the spin-rotation constants, absolute nuclear magnetic resonance (NMR) shielding constants and shielding spans of {sup 175}LuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br, {sup 127}I) molecules. The results are obtained from calculations performed using density functional theory (non-relativistic and four-component relativistic) and coupled-cluster calculations. The spin-rotation constants are compared with available experimental values. In most of the molecules studied, relativistic effects make an order of magnitude difference on the NMR absolute shielding constants.

  16. {sup 35}Cl+{sup 12}C asymmetrical fission excitation functions

    SciTech Connect

    Beck, C.; Mahboub, D.; Nouicer, R.; Matsuse, T.; Djerroud, B.; Freeman, R.M.; Haas, F.; Hachem, A.; Morsad, A.; Youlal, M.; Dayras, R.; Wieleczko, J.P.; Berthoumieux, E.; Legrain, R.; Pollacco, E.

    1996-07-01

    The fully energy-damped yields from the {sup 35}Cl+{sup 12}C reaction have been systematically investigated using particle-particle coincidence techniques at a {sup 35}Cl bombarding energy of {approximately}8 MeV/nucleon. The fragment-fragment correlation data show that the majority of events arises from a binary-decay process with rather large numbers of secondary light-charged particles emitted from the two excited exit fragments. No evidence is observed for ternary break-up events. The binary-process results of the present measurement, along with those of earlier, inclusive experimental data obtained at several lower bombarding energies are compared with predictions of two different kinds of statistical model calculations. These calculations are performed using the transition-state formalism and the extended Hauser-Feshbach method and are based on the available phase space at the saddle point and scission point of the compound nucleus, respectively. The methods give comparable predictions and are both in good agreement with the experimental results thus confirming the fusion-fission origin of the fully damped yields. The similarity of the predictions for the two models supports the claim that the scission point configuration is very close to that of the saddle point for the light {sup 47}V compound system. The results also give further support for the specific mass-asymmetry-dependent fission barriers needed in the transition-state calculation. {copyright} {ital 1996 The American Physical Society.}

  17. Sup35 methionine oxidation is a trigger for de novo [PSI+] prion formation

    PubMed Central

    Grant, Chris M

    2015-01-01

    ABSTRACT. The molecular basis by which fungal and mammalian prions arise spontaneously is poorly understood. A number of different environmental stress conditions are known to increase the frequency of yeast [PSI+] prion formation in agreement with the idea that conditions which cause protein misfolding may promote the conversion of normally soluble proteins to their amyloid forms. A recent study from our laboratory has shown that the de novo formation of the [PSI+] prion is significantly increased in yeast mutants lacking key antioxidants suggesting that endogenous reactive oxygen species are sufficient to promote prion formation. Our findings strongly implicate oxidative damage of Sup35 as an important trigger for the formation of the heritable [PSI+] prion in yeast. This review discusses the mechanisms by which the direct oxidation of Sup35 might lead to structural transitions favoring conversion to the transmissible amyloid-like form. This is analogous to various environmental factors which have been proposed to trigger misfolding of the mammalian prion protein (PrPC) into the aggregated scrapie form (PrPSc). PMID:26267336

  18. Biosynthesis of lutropin in ovine pituitary slices: incorporation of (/sup 35/S)sulfate in carbohydrate units

    SciTech Connect

    Anumula, K.R.; Bahl, O.P.

    1983-02-01

    Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H/sub 2/(/sup 35/)SO/sub 4/. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the alpha and beta subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, (/sup 35/S)sulfate was also incorporated into several other proteins in addition to LH. The location of /sup 35/SO/sub 2-(4)/ in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-(/sup 35/S)sulfo-N-acetylhexosaminyl-glycerols and 4-O-(/sup 35/S) sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of /sup 35/SO/sub 2-(4)/ by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.

  19. Importance of low-oligomeric-weight species for prion propagation in the yeast prion system Sup35/Hsp104

    PubMed Central

    Narayanan, Saravanakumar; Bösl, Benjamin; Walter, Stefan; Reif, Bernd

    2003-01-01

    The [PSI+] determinant of Saccharomyces cerevisiae, consisting of the cytosolic translation termination factor Sup35, is a prion-type genetic element that induces an inheritable conformational change and converts the Sup35 protein into amyloid fibers. The molecular chaperone Hsp104 is required to maintain self-replication of [PSI+]. We observe in vitro that addition of catalytic amounts of Hsp104 to the prion-determining region of the NM domain of Sup35, Sup355–26, results in the dissociation of oligomeric Sup35 into monomeric species. Several intermediates of Sup355–26 could be detected during this process. Strong interactions are found between Hsp104 and hexameric/tetrameric Sup355–26, whereas the intermediate and monomeric “release” forms show a decreased affinity with respect to Hsp104, as monitored by saturation transfer difference and diffusion-ordered NMR spectroscopic experiments. Interactions are mediated mostly by the side chains of Gln, Asn, and Tyr residues in Sup355–26. No interaction can be detected between Hsp104 and higher oligomeric states (≥8) of Sup355–26. Taking into account the fact that Hsp104 is required for maintenance of [PSI+], we suggest that low-oligomeric-weight species of Sup35 are important for prion propagation in yeast. PMID:12876196

  20. Determination of {sup 35}S in radioisotope wastes by a wet oxidation

    SciTech Connect

    Lee, Heung N.; Sang-Hoon Kang; Hong Joo Ahn; Kwang Yong Jee; Wook Hyun Sohn

    2007-07-01

    The oxidation studies of a sulfur to a sulfate ion by various oxy-halide oxidants in organic (thiourea, methionine) and inorganic (sulfate, thiophosphate) compounds were carried out in an acidic solution. The optimized result of the oxidation reaction was obtained when a bromate compound (BrO{sub 3}{sup -}) as an oxidant and a 3 M HNO{sub 3} solvent. The chemical yield for the oxidation of the organic and inorganic sulfur compounds to a sulfate ion was monitored as 80% for thiophosphate, 87% for methionine, and 100% for thiourea and sulfate within 5% RSD. The oxidation of thiourea required at least 1.6 equivalents of the bromate in an acidic solution. In the case of the oxidation of methionine and thiophosphate, the oxidation yield was above 80% if the bromate was used at 20 times that of the substrates. The chemical yield in the paper sample (WypAll) exceeded 100% because of its background sulfur contents (910 ppm). The sulfate ion was quantitatively measured by using GPC and/or LSC counting of 3 S followed by precipitates of BaSO{sub 4}. The interfering nuclides ({sup 14}C, {sup 32}P) were removed in an acidic condition. The minimum detectable activity (MDA) of {sup 35}S was found to be 0.1 Bq/g. (authors)

  1. Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils

    PubMed Central

    Sulatskaya, Anna I.; Kuznetsova, Irina M.; Belousov, Mikhail V.; Bondarev, Stanislav A.; Zhouravleva, Galina A.; Turoverov, Konstantin K.

    2016-01-01

    In this work two modes of binding of the fluorescent probe thioflavin T to yeast prion protein Sup35p amyloid fibrils were revealed by absorption spectrometry of solutions prepared by equilibrium microdialysis. These binding modes exhibited significant differences in binding affinity and stoichiometry. Moreover, the absorption spectrum and the molar extinction coefficient of the dye bound in each mode were determined. The fluorescence quantum yield of the dye bound in each mode was determined via a spectrofluorimetric study of the same solutions in which the recorded fluorescence intensity was corrected for the primary inner filter effect. As previously predicted, the existence of one of the detected binding modes may be due to the incorporation of the dye into the grooves along the fiber axis perpendicular to the β-sheets of the fibrils. It was assumed that the second type of binding with higher affinity may be due to the existence of ThT binding sites that are localized to areas where amyloid fibrils are clustered. PMID:27228180

  2. Endosperm protein synthesis and L-(/sup 35/S)methionine incorporation in maize kernels cultured in vitro

    SciTech Connect

    Cully, D.E.; Gengenbach, B.G.; Smith, J.A.; Rubenstein, I.; Connely, J.A.; Park, W.D.

    1984-02-01

    This study was conducted to examine protein synthesis and L-(/sup 35/S)methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two-day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries L-(/sup 35/S)methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endosperm for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages. Sodium, dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing patterns for endosperm proteins were similar for field and cultured kernels throughout development. By 15 days, over 70% of the L-(/sup 35/S)methionine taken up was present in endosperm proteins. Label incorporation visualized by fluorography generally followed the protein intensity of the stained gels. The high methionine content, low molecular weight zeins (i.e. 15 and 9 kilodaltons) were highly labeled. All of the radioactivity in hydrolyzed zein samples was recovered in the methionine peak indicating minimal conversion to L-(/sup 35/S)cysteine. The procedure described here is suitable for long term culture and labeling experiments in which continued kernel development is required.

  3. Effect of electrostatics on aggregation of prion protein Sup35 peptide

    NASA Astrophysics Data System (ADS)

    Portillo, Alexander M.; Krasnoslobodtsev, Alexey V.; Lyubchenko, Yuri L.

    2012-04-01

    Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. The GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI = 5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0), suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 h, whereas the lag time decreases ˜5 times when the ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 h under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. The ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics to dissociation of transient peptide dimers.

  4. CNS depressants accelerate the dissociation of /sup 35/S-TBPS binding and GABA enhances their displacing potencies

    SciTech Connect

    Maksay, G.; Ticku, M.K.

    1988-01-01

    The specific binding of /sup 35/S-t-butylbicyclophosphorothionate (TBPS) was studied in synaptosomal membranes of rat cerebral cortex. The displacing potencies of eleven CNS depressants and three convulsants were determined in the presence of 1 /sup +/M GABA and 10 nM R 5135. GABA enhanced the displacing potencies of depressants of most diverse chemical structures: diaryltriazine (LY 81067), pyrazolopyridine (etazolate), cinnamide, glutarimide, 2,3-benzodiazepine (tofizopam) and alcohol derivatives, barbiturates, (+)etomidate, methaqualone and meprobamate. In contrast, the IC/sub 50/ values of convulsants (picrotoxinin, pentetrazol and the barbiturate enantiomer S(+)MPPB) were not significantly affected. The depressants accelerated either basal or GABA-augmented dissociation of /sup 35/-TBPS mainly by increasing the contribution of its rapid first phase.

  5. Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of (/sup 35/S)methionine-labeled proteins

    SciTech Connect

    Tabaqchali, S.; O'Farrell, S.; Holland, D.; Silman, R.

    1986-01-01

    A typing method for Clostridium difficile based on the incorporation of (/sup 35/S)methionine into cellular proteins, their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their visualization by autoradiography is described. On analysis of the radiolabeled-protein profiles, nine distinct groups were observed (A to E and W to Z). The method, which is simple, reproducible, and readily expandable, has been applied in epidemiological studies to demonstrate cross-infection and hospital acquisition of C. difficile.

  6. High performance liquid chromatography (HPLC) study of (/sup 35/S)- and (/sup 3/H)-labeled mucus glycoproteins secreted by the isolated mucociliated gill epithelium of Mytilus edulis

    SciTech Connect

    Sabouni, A.; Ma, J.K.; Malanga, C.J.

    1986-03-05

    HPLC combined with (/sup 35/S)-sulfate/(/sup 3/H)-glucosamine radiolabeling were employed to study the synthesis and secretion of mucous glycoproteins. The radiolabeled secreted glycoproteins were separated from the medium by precipitation with a mixture of trichloroacetic-phosphotungstic acids (TCA/PTA). The redissolved glycoproteins were chromatographed on an anion exchange protein column at varying pH of the mobile phase and fractions were collected for liquid scintillation counting. Varying the pH of the mobile phase from pH 3 to 7 resulted in a decrease of glycoprotein bound (/sup 35/S) from 69.5 to 0.5% of the total recovered (/sup 35/S)-sulfate with the remainder recovered as free (/sup 35/S)-sulfate. The (/sup 3/H)-labeled glycoprotein recovered under the uV peaks at this pH range was 99.5%. When high performance size exclusion chromatography was performed the change in mobile phase pH did not affect the 100% recovery of either (/sup 35/S)- or (/sup 3/H)-labels under the uV peaks. No free (/sup 35/S)-sulfate was obtained when (/sup 35/S)-labeled glycoproteins were separated form the medium using dialysis. These data suggest that the standard method of TCA/PTA precipitation of (/sup 35/S)-labeled glycoproteins may cleave the (/sup 35/S)-sulfate ester linkages to the oligosaccharide chains. The (/sup 35/S)-sulfate may then rebind to the macromolecule by a relatively strong noncovalent bond. This may prove critical in anion exchange protein HPLC studies.

  7. Source of error in the chromatographic study of /sup 35/S-sulfate labeled mucous glycoproteins secreted by the gill epithelium of Mytilus edulis

    SciTech Connect

    Sabouni, A.H.; Ma, J.K.; Malanga, C.J.

    1986-01-01

    HPLC combined with (/sup 35/S)-sulfate/(/sup 3/H)-glucosamine radiolabeling were employed to study the synthesis and secretion of mucous glycoproteins. The secreted radiolabeled glycoproteins were separated from the medium by precipitation with a mixture of trichloroacetic-phosphotungstic acids (TCA/PTA). The redissolved glycoproteins were chromatographed on an anion exchange protein column at varying pH of the mobile phase and fractions were collected for liquid scintillation counting. Varying the pH of the mobile phase from pH 3 to 7 resulted in a decrease of glycoprotein bound (/sup 35/S) from 69.5 to 0.5% of the total recovered (/sup 35/S)-sulfate with the remainder recovered as free (/sup 35/S)-sulfate. The (/sup 3/H)-labeled glycoprotein recovered under the uV peaks at this pH range was 99.5%. When high performance size exclusion chromatography was performed the change in mobile phase pH did not affect the 100% recovery of either (/sup 35/S)-or (/sup 3/H)-labels under the uV peaks. No free (/sup 35/S)-sulfate was obtained when (/sup 35/S)-labeled glycoproteins were separated from the medium using dialysis. These data suggest that the standard method of TCA/PTA precipitation of (/sup 35/S)-labeled glycoproteins may cleave the (/sup 35/S)-sulfate ester linkages to the oligosaccharide chains. The (/sup 35/S)-sulfate may then rebind to the macromolecule by a relatively strong noncovalent bond. This may prove critical in anion exchange protein HPLC studies.

  8. Differential identification of Candida species and other yeasts by analysis of (/sup 35/S)methionine-labeled polypeptide profiles

    SciTech Connect

    Shen, H.D.; Choo, K.B.; Tsai, W.C.; Jen, T.M.; Yeh, J.Y.; Han, S.H.

    1988-12-01

    This paper describes a scheme for differential identification of Candida species and other yeasts based on autoradiographic analysis of protein profiles of (/sup 35/S)methionine-labeled cellular proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using ATCC strains as references, protein profile analysis showed that different Candida and other yeast species produced distinctively different patterns. Good agreement in results obtained with this approach and with other conventional systems was observed. Being accurate and reproducible, this approach provides a basis for the development of an alternative method for the identification of yeasts isolated from clinical specimens.

  9. Protein N-terminal acetyltransferases in cancer.

    PubMed

    Kalvik, T V; Arnesen, T

    2013-01-17

    The human N-terminal acetyltransferases (NATs) catalyze the transfer of acetyl moieties to the N-termini of 80-90% of all human proteins. Six NAT types are present in humans, NatA-NatF, each is composed of specific subunits and each acetylates a set of substrates defined by the N-terminal amino-acid sequence. NATs have been suggested to act as oncoproteins as well as tumor suppressors in human cancers, and NAT expression may be both elevated and decreased in cancer versus non-cancer tissues. Manipulation of NATs in cancer cells induced cell-cycle arrest, apoptosis or autophagy, implying that these enzymes target a variety of pathways. Of particular interest is hNaa10p (human ARD1), the catalytic subunit of the NatA complex, which was coupled to a number of signaling molecules including hypoxia inducible factor-1α, β-catenin/cyclin D1, TSC2/mammalian target of rapamycin, myosin light chain kinase , DNA methyltransferase1/E-cadherin and p21-activated kinase-interacting exchange factors (PIX)/Cdc42/Rac1. The variety of mechanistic links where hNaa10p acts as a NAT, a lysine acetyltransferase or displaying a non-catalytic role, provide insights to how hNaa10p may act as both a tumor suppressor and oncoprotein. PMID:22391571

  10. Thermostable keratinase from Bacillus pumilus KS12: production, chitin crosslinking and degradation of Sup35NM aggregates.

    PubMed

    Rajput, Rinky; Gupta, Rani

    2013-04-01

    Production of thermostable keratinase from Bacillus pumilus KS12 was enhanced up to seven fold by statistical methods. The enzyme was partially purified by ultrafiltration followed by thermal precipitation with purity of 3.2-fold and recovery of 89%. Keratinase was immobilized using covalent method by crosslinking 2 mg protein (688 U/mg) onto 1g chitin activated with 2.5% (v/v) glutaraldehyde for 60 min. Its comparative biochemical studies with that of free keratinase revealed the shift in optimum pH with increased stability towards pH from 9.0 to 10.0 and temperature. Also, it showed statistically significant improved hydrolysis of a number of soluble and insoluble substrates in comparison to free keratinase. Owing to improved catalytic efficiency of immobilized keratinase, its potential for degradation of Sup35NM was evaluated, where 100 μg of enzyme could degrade 60 μg Sup35NM after 60 min at pH 7.0 and 37°C. PMID:23425582

  11. Saturable binding of /sup 35/S-t-butylbicyclophosphorothionate to the sites linked to the GABA receptor and the interaction with gabaergic agents

    SciTech Connect

    Wong, D.T.; Threlkeld, P.G.; Bymaster, F.P.; Squires, R.F.

    1984-02-27

    /sup 35/S-t-Butylbicyclophosphorothionate (/sup 35/S-TBPS) binds in a concentration-saturable manner to specific sites on membranes from rat cerebral cortex. Using a filtration assay at 25/sup 0/C, in 250 mM NaCl, specific binding of /sup 35/S-TBPS constitutes about 84 to 94 percent of total binding, depending on radioligand concentrations. /sup 35/S-TBPS binding is optimal in the presence of NaCl or NaBr and substantially less in the presence of NaI or NaF. It is sensitive to the treatment with 0.05 percent Triton X-100 but not to repeated freezing and thawing, procedures which increase /sup 3/H-GABA binding. Pharmacological studies show that /sup 35/S-TBPS binding is strongly inhibited by GABA-A receptor agonists (e.g., GABA and muscimol) and by the noncompetitive antagonist, picrotoxin, but not the competitive antagonist, bicuculline. Compounds which enhance binding of radioactive GABA and benzodiazepines, such as the pyrazolopyridines, cartazolate and tracazolate, and a diaryltriazine, LY81067, are also potent inhibitors of /sup 35/S-TBPS binding, with LY81067 being the most effective. The effects of GABA, picrotoxin and LY81067 on the saturable binding of /sup 35/S-TBPS in cortical membranes are compared. The present findings are consistent with the interpretation that /sup 35/S-TBPS bind at or near the picrotoxin-sensitive anion recognition sites of the GABA/benzodiazepine/picrotoxin receptor complex.

  12. In Sup35p filaments (the [PSI+] prion), the globular C-terminal domains are widely offset from the amyloid fibril backbone

    SciTech Connect

    Baxa, U.; Wall, J.; Keller, P. W.; Cheng, N.; Steven, A. C.

    2011-01-01

    In yeast cells infected with the [PSI+] prion, Sup35p forms aggregates and its activity in translation termination is downregulated. Transfection experiments have shown that Sup35p filaments assembled in vitro are infectious, suggesting that they reproduce or closely resemble the prion. We have used several EM techniques to study the molecular architecture of filaments, seeking clues as to the mechanism of downregulation. Sup35p has an N-terminal 'prion' domain; a highly charged middle (M-)domain; and a C-terminal domain with the translation termination activity. By negative staining, cryo-EM and scanning transmission EM (STEM), filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse 65-nm-wide cloud of globular C-domains. In diameter ({approx}8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield -1 subunit per 0.47 nm for N-fibrils, NM-filaments and Sup35p filaments, further supporting the fibril backbone model. The 30 nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations, offering an explanation for the residual Sup35p activity in infected cells, whereby the C-domains remain free enough to interact with ribosomes.

  13. Saturable binding of /sup 35/S-t-butylbicyclophosphorothionate to the sites linked to the GABA receptor and the interaction with gabaergic agents

    SciTech Connect

    Wong, D.T.; Threlkeld, P.G.; Bymaster, F.P.; Squires, R.F.

    1984-02-27

    /sup 35/-S-t-Butylbicyclophosphorothionate (/sup 35/S-TBPS) binds in a concentration-saturable manner to specific sites on membranes from rat cerebral cortex. Using a filtration assay at 25/sup 0/C, in 250 mM NaCl, specific binding of /sup 35/S-TBPS constitutes about 84 to 94 percent of total binding, depending on radioligand concentrations. /sup 35/S-TBPS binding is optimal in the presence of NaCl or NaBr and substantially less in the presence of NaI or NaF. It is sensitive to the treatment with 0.05 percent Triton X-100 but not to repeated freezing and thawing, procedures which increase /sup 3/H-GABA binding. Pharmacological studies show that /sup 35/S-TBPS binding is strongly inhibited by GABA-A receptor agonists (e.g., GABA and muscimol) and by the noncompetitive antagonist, picrotoxin, but not the competitive antagonist, bicuculline. Compounds which enhance binding of radioactive GABA and benzodiazepines, such as the pyrazolopyridines, cartazolate and trazolate, and a diaryl-triazine, LY81067, are also potent inhibitors of /sup 35/S-TBPS binding, with LY81067 being the most effective. The effects of GABA, picrotoxin

  14. Spermidine induces autophagy by inhibiting the acetyltransferase EP300

    PubMed Central

    Pietrocola, F; Lachkar, S; Enot, D P; Niso-Santano, M; Bravo-San Pedro, J M; Sica, V; Izzo, V; Maiuri, M C; Madeo, F; Mariño, G; Kroemer, G

    2015-01-01

    Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors. PMID:25526088

  15. Effects of cysteamine administration on the in vivo incorporation of (/sup 35/S)cysteine into somatostatin-14, somatostatin-28, arginine vasopressin, and oxytocin in rat hypothalamus

    SciTech Connect

    Cameron, J.L.; Fernstrom, J.D.

    1986-09-01

    The effect of cysteamine injection on the in vivo incorporation of (/sup 35/S)cysteine into somatostatin-14 (SRIF-14), SRIF-28, arginine vasopressin (AVP), and oxytocin (OXT) in rat hypothalamus was studied. (/sup 35/S)Cysteine was injected into the third ventricle 1 h, 4 h, or 1 week after cysteamine (300 mg/kg, sc) injection; animals were killed 4 h later. The drug was found to substantially reduce immunoreactive SRIF levels, but not OXT or AVP, 4 h after its injection. Cysteamine also caused large reductions in label incorporation into SRIF-14, SRIF-28, and OXT 1 and 4 h after drug injection. However, (/sup 35/S)cysteine incorporation into AVP was increased substantially at these time points, while that into acid-precipitable protein was normal. One week after cysteamine injection, label incorporation into all hypothalamic peptides was normal. Cysteine specific activity was also measured after (/sup 35/S)cysteine injection and was found to be similar in treatment and control groups. The results suggest that cysteamine inhibits the syntheses of SRIF-14, SRIF-28, and OXT and stimulates that of AVP.

  16. Expression of. beta. -conglycinin gene driven by CaMV /sup 35/S promoter in transgenic plants

    SciTech Connect

    Nakamura, I.; Dube, P.H.; Beachy, R.N.

    1987-04-01

    ..beta..-conglycinin is a abundant protein stored in protein bodies of soybean seeds. This protein consists of three major subunits, ..cap alpha..' (76 kDa), ..cap alpha.. (72 kDa) and ..beta.. (53 kDa), and accumulates in developing soybean embryos during the mid- to late-maturation stages of seed development. Coding sequence of an ..cap alpha..'-subunit gene was expressed in transgenic petunia plants under control of the promoter from the CaMV (cauliflower mosaic virus) /sup 35/S transcript. Two different types of ..cap alpha..'-protein accumulated in tissues of the transgenic plant; seed-type ..cap alpha..'-protein accumulated only in seeds during mid- to late-maturation stages, while non-seed-type ..cap alpha..'-protein was found in non-seed tissues and in early stages of seed maturation. Seed-type ..cap alpha..'-protein was the same size as soybean ..cap alpha..'-subunit, while non-seed-type ..cap alpha..'-protein was larger by about 4 kDa. Seeds contained approximately 30-fold greater levels of ..cap alpha..'-protein than did non-seed tissues. This is presumably due to differences in protein stability because the amount of ..cap alpha..'-mRNA was equivalent in each of the tissues examined. The ..cap alpha..'-protein in leaves was localized in microsomal membrane fractions. Proteins solubilized from the membranes were sedimented by sucrose gradient centrifugation and analyzed by immuno blot technique. The results suggest that the protein assembles into multimeric forms in leaf membranes, as it does in seed protein bodies.

  17. Genetic study of interactions between the cytoskeletal assembly protein sla1 and prion-forming domain of the release factor Sup35 (eRF3) in Saccharomyces cerevisiae.

    PubMed Central

    Bailleul, P A; Newnam, G P; Steenbergen, J N; Chernoff, Y O

    1999-01-01

    Striking similarities between cytoskeletal assembly and the "nucleated polymerization" model of prion propagation suggest that similar or overlapping sets of proteins may assist in both processes. We show that the C-terminal domain of the yeast cytoskeletal assembly protein Sla1 (Sla1C) specifically interacts with the N-terminal prion-forming domain (Sup35N) of the yeast release factor Sup35 (eRF3) in the two-hybrid system. Sla1C and several other Sup35N-interacting proteins also exhibit two-hybrid interactions with the poly-Gln-expanded N-proximal fragment of human huntingtin, which promotes Huntington disease-associated aggregation. The Sup35N-Sla1C interaction is inhibited by Sup35N alterations that make Sup35 unable to propagate the [PSI(+)] state and by the absence of the chaperone protein Hsp104, which is essential for [PSI] propagation. In a Sla1(-) background, [PSI] curing by dimethylsulfoxide or excess Hsp104 is increased, while translational readthrough and de novo [PSI] formation induced by excess Sup35 or Sup35N are decreased. These data show that, in agreement with the proposed function of Sla1 during cytoskeletal formation, Sla1 assists in [PSI] formation and propagation, but is not required for these processes. Sla1(-) strains are sensitive to some translational inhibitors, and some sup35 mutants, obtained in a Sla1(-) background, are sensitive to Sla1, suggesting that the interaction between Sla1 and Sup35 proteins may play a role in the normal function of the translational apparatus. We hypothesize that Sup35N is involved in regulatory interactions with intracellular structural networks, and [PSI] prion may be formed as a by-product of this process. PMID:10471702

  18. Distributions of /sup 35/S-sulfate and /sup 3/H-glucosamine in the angular region of the hamster: light and electron microscopic autoradiography

    SciTech Connect

    Ohnishi, Y.; Taniguchi, Y.

    1983-06-01

    The distribution of /sup 35/S-sulfate and /sup 3/H-glucosamine in the angular region of the hamster was studied by light and electron microscopic autoradiography following intraperitoneal injection of these compounds to hamsters. Exposed silver grains of /sup 35/S-sulfate were concentrated in the trabecular meshwork, sclera, and cornea, and grains of /sup 3/H-glucosamine were localized in the trabecular region. The radioactivity of both isotopes was observed in the Golgi apparatuses of the endothelial cells of the angular aqueous plexus and the trabecular meshwork. The grains were noted over the entire cytoplasm, except for the nucleus, and then were incorporated into the amorphous substance and collagen fibers in the region adjacent to the angular aqueous sinus. These results suggest that endothelial cells in the angular region synthesize and secrete the sulfated glycosaminoglycans and hyaluronic acid.

  19. Formation of a necklike structure in {sup 35}Cl+{sup 12}C and {sup 197}Au reactions at 43 MeV/nucleon

    SciTech Connect

    Larochelle, Y.; Gingras, L.; Beaulieu, L.; Qian, X.; Saddiki, Z.; Djerroud, B.; Dore, D.; Laforest, R.; Roy, R.; Samri, M.; St-Pierre, C.; Ball, G.C.; Bowman, D.R.; Galindo-Uribarri, A.; Hagberg, E.; Horn, D.; Lopez, J.A.; Robinson, T.

    1997-04-01

    The experimental signature of the formation of a necklike structure, with a velocity between that of the projectilelike emitter and that of the targetlike emitter, is investigated with the same beam and experimental setup for targets lighter and heavier than the projectile. The reactions are {sup 35}Cl on {sup 12}C and on {sup 197}Au at 43 MeV/nucleon. Particle velocity distributions are compared with two-source statistical simulations and the presence of a necklike structure is inferred from the data. In the second part of the paper, dynamical model simulations with the formation of a necklike structure are presented for the {sup 35}Cl+{sup 12}C system at 43 MeV/nucleon. {copyright} {ital 1997} {ital The American Physical Society}

  20. Decoloration and solubilization of plant tissue prior to determination of /sup 3/H, /sup 14/C, and /sup 35/S by liquid scintillation

    SciTech Connect

    Smith, I.K.; Lang, A.L.

    1987-08-01

    A method is described for the decoloration and partial solubilization of plant tissue with 2% sodium hypochlorite. Following treatment of the digest with ammonia, the samples are suitable for the determination of /sup 3/H, /sup 14/C, and /sup 35/S by liquid scintillation counting. The color quenching is negligible and counting efficiencies are high: 30-40% for /sup 3/H and 90-95% for /sup 14/C.

  1. In vivo biosynthesis of L-(/sup 35/S)Cys-arginine vasopressin, -oxytocin, and -somatostatin: rapid estimation using reversed phase high pressure liquid chromatography. [Rats

    SciTech Connect

    Franco-Bourland, R.E.; Fernstrom, J.D.

    1981-01-01

    L(/sup 35/S)Cys-arginine vasopressin, -oxytocin, and -somatostatin were purified from hypothalami and neurohypophyses 4 h after rats received L(/sup 35/S)Cys via the third ventricle. After acetic acid extraction, Sephadex G-25 filtration, and chemoadsorption to C18-silica (Sep-Pak cartridges), the labeled peptides were rapidly separated by gradient elution, reversed phase, high pressure liquid chromatography (HPLC). The identity and isotopic purity of the labeled peptides were determined by several reversed phase HPLC procedures in conjunction with chemical modification. The labeled peptide fractions were at least 50% radiochemically pure. Using this HPLC isolation procedure, incorporation of L-(/sup 35/S)Cys into each peptide was determined in hydrated and dehydrated rats. Label incorporation into arginine vasopressin and oxytocin in the hypothalamus and the neurohypophysis of dehydrated rats was 2-3 times greater than that in hydrated rats. Incorporation of label into hypothalamic and neurohypophyseal somatostatin was unaffected by the hydration state of the animal. This procedure thus provides a very rapid, but sensitive, set of techniques for studying the control of small peptide biosynthesis in the brain.

  2. Neisseria meningitidis serogroup A capsular polysaccharide acetyltransferase, methods and compositions

    SciTech Connect

    Stephens, David S.; Gudlavalleti, Seshu K.; Tzeng, Yih-Ling; Datta, Anup K.; Carlson, Russell W.

    2011-02-08

    Provided are methods for recombinant production of an O-acetyltransferase and methods for acetylating capsular polysaccharides, especially those of a Serogroup A Neisseria meningitidis using the recombinant O-acetyltransferase, and immunogenic compositions comprising the acetylated capsular polysaccharide.

  3. Insights into the Specificity of Lysine Acetyltransferases*

    PubMed Central

    Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; Rayment, Ivan; Escalante-Semerena, Jorge C.

    2014-01-01

    Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. Here we report the structure of a GNAT in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs. PMID:25381442

  4. Defining the Orphan Functions of Lysine Acetyltransferases

    PubMed Central

    2016-01-01

    Long known for their role in histone acetylation, recent studies have demonstrated that lysine acetyltransferases also carry out distinct “orphan” functions. These activities impact a wide range of biological phenomena including metabolism, RNA modification, nuclear morphology, and mitochondrial function. Here, we review the discovery and characterization of orphan lysine acetyltransferase functions. In addition to highlighting the evidence and biological role for these functions in human disease, we discuss the part emerging chemical tools may play in investigating this versatile enzyme superfamily. PMID:25591746

  5. Decay studies of nuclei near the proton drip line: /sup 35/Ca, /sup 31/Ar, /sup 69/Br, /sup 65/As

    SciTech Connect

    Reiff, J.E.

    1989-06-01

    Studies of new beta-delayed two-proton emitters and a search for ground state proton radioactivity in medium mass nuclei were performed using various experimental techniques in conjunction with several detection systems. A helium-jet transport system and three-element silicon telescopes were used to discover the existence and detect the decay of the first T/sub Z/ = /minus/5/2 nuclide, /sup 35/Ca. Two-proton emission from the T = 5/2 isobaric analog state in /sup 35/K at an excitation energy of 9.053 /plus minus/ 0.045 MeV, fed by the superallowed beta decay of /sup 35/Ca, resulted in transitions to both the ground state and first excited state of /sup 33/Cl. The corresponding two-proton sum energies were 4.089 /plus minus/ 0.030 MeV and 3.287 /plus minus/ 0.030 MeV. Measurements of the individual proton energies indicated the prevalence of a sequential decay mechanism. Using the isobaric multiplet mass equation, the mass excess of /sup 35/Ca was calculated to be 4.453 /plus minus/ 0.060 MeV. In order to study whose half-lives were too short for the helium-jet system, an in-beam recoil catcher wheel was constructed. The wheel speed can be varied to study nuclides whose half-lives range from 100 /mu/s to /approximately/250 ms. The first new decay observed with the wheel system and traditional /Delta/E-E telescopes was the beta-delayed two-proton emission from /sup 31/Ar. The two-proton sum energy of /approximately/7.5 MeV corresponds to a transition from the isobaric analog state in /sup 31/Cl to the ground state of /sup 29/P. The search for proton radioactivity required the development of low energy, particle identification detector telescopes. These telescopes, comprised of a gas /Delta/E and silicon E, were used in conjunction with the in-beam recoil catcher wheel to search for ground state proton emission from /sup 69/Br and /sup 65/As. 90 refs., 24 figs., 8 tabs.

  6. Effects of recombinant eel growth hormone on the uptake of ( sup 35 S)sulfate by ceratobranchial cartilages of the Japanese eel, Anguilla japonica

    SciTech Connect

    Duan, C.M.; Inui, Y. )

    1990-08-01

    Effects of growth hormone (GH) on the synthesis of mucopolysaccharide by ceratobranchial cartilages of the Japanese eel, Anguilla japonica, were examined by monitoring the in vitro uptake of ({sup 35}S)sulfate. The ({sup 35}S)sulfate uptake decreased rapidly to one-third of the initial level during the first 3 days after hypophysectomy, and decreased gradually thereafter. When hypophysectomized eels were injected intramuscularly with recombinant eel GH (2 micrograms/g), the plasma GH concentrations increased maximally after 6 hr, and declined rapidly thereafter. On the other hand, the sulfate uptake increased significantly after 12 hr, and high levels were maintained until 48 hr. The stimulating effect of GH was dose dependent (0.02-2 micrograms/g). However, the addition of eel GH (0.05-5 micrograms/ml) to the culture medium did not affect the sulfate uptake by hypophysectomized eel cartilages, suggesting that the stimulative action of GH on the sulfate uptake by the cartilages is indirect.

  7. Regulation of a Protein Acetyltransferase in Myxococcus xanthus by the Coenzyme NADP+

    PubMed Central

    Liu, Xin-Xin

    2015-01-01

    ABSTRACT NADP+ is a vital cofactor involved in a wide variety of activities, such as redox potential and cell death. Here, we show that NADP+ negatively regulates an acetyltransferase from Myxococcus xanthus, Mxan_3215 (MxKat), at physiologic concentrations. MxKat possesses an NAD(P)-binding domain fused to the Gcn5-type N-acetyltransferase (GNAT) domain. We used isothermal titration calorimetry (ITC) and a coupled enzyme assay to show that NADP+ bound to MxKat and that the binding had strong effects on enzyme activity. The Gly11 residue of MxKat was confirmed to play an important role in NADP+ binding using site-directed mutagenesis and circular dichroism spectrometry. In addition, using mass spectrometry, site-directed mutagenesis, and a coupling enzymatic assay, we demonstrated that MxKat acetylates acetyl coenzyme A (acetyl-CoA) synthetase (Mxan_2570) at Lys622 in response to changes in NADP+ concentration. Collectively, our results uncovered a mechanism of protein acetyltransferase regulation by the coenzyme NADP+ at physiological concentrations, suggesting a novel signaling pathway for the regulation of cellular protein acetylation. IMPORTANCE Microorganisms have developed various protein posttranslational modifications (PTMs), which enable cells to respond quickly to changes in the intracellular and extracellular milieus. This work provides the first biochemical characterization of a protein acetyltransferase (MxKat) that contains a fusion between a GNAT domain and NADP+-binding domain with Rossmann folds, and it demonstrates a novel signaling pathway for regulating cellular protein acetylation in M. xanthus. We found that NADP+ specifically binds to the Rossmann fold of MxKat and negatively regulates its acetyltransferase activity. This finding provides novel insight for connecting cellular metabolic status (NADP+ metabolism) with levels of protein acetylation, and it extends our understanding of the regulatory mechanisms underlying PTMs. PMID:26598367

  8. Absolute NMR shielding scales and nuclear spin–rotation constants in {sup 175}LuX and {sup 197}AuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br and {sup 127}I)

    SciTech Connect

    Demissie, Taye B. Komorovsky, Stanislav; Repisky, Michal; Ruud, Kenneth; Jaszuński, Michał

    2015-10-28

    We present nuclear spin–rotation constants, absolute nuclear magnetic resonance (NMR) shielding constants, and shielding spans of all the nuclei in {sup 175}LuX and {sup 197}AuX (X = {sup 19}F, {sup 35}Cl, {sup 79}Br, {sup 127}I), calculated using coupled-cluster singles-and-doubles with a perturbative triples (CCSD(T)) correction theory, four-component relativistic density functional theory (relativistic DFT), and non-relativistic DFT. The total nuclear spin–rotation constants determined by adding the relativistic corrections obtained from DFT calculations to the CCSD(T) values are in general in agreement with available experimental data, indicating that the computational approach followed in this study allows us to predict reliable results for the unknown spin–rotation constants in these molecules. The total NMR absolute shielding constants are determined for all the nuclei following the same approach as that applied for the nuclear spin–rotation constants. In most of the molecules, relativistic effects significantly change the computed shielding constants, demonstrating that straightforward application of the non-relativistic formula relating the electronic contribution to the nuclear spin–rotation constants and the paramagnetic contribution to the shielding constants does not yield correct results. We also analyze the origin of the unusually large absolute shielding constant and its relativistic correction of gold in AuF compared to the other gold monohalides.

  9. Detection of {sup 14}N and {sup 35}Cl in cocaine base and hydrochloride using NQR, NMR, and SQUID techniques

    SciTech Connect

    Yesinowski, J.P.; Buess, M.L.; Garroway, A.N.; Ziegeweid, M.; Pines, A. |

    1995-07-01

    Results from {sup 14}N pure NQR of cocaine in the free base form (cocaine base) yield a nuclear quadrupole coupling constant (NQCC) e{sup 2}Qq/h of 5.0229 ({+-}0.0001) MHz and an asymmetry parameter {eta} of 0.0395 ({+-}0.0001) at 295 K, with corresponding values of 5.0460 ({+-}0.0013) MHz and 0.0353 ({+-}0.0008) at 77 K. Both pure NQR (at 295-77 K) and a superconducting quantum interference device (SQUID) detector (at 4.2 K) were used to measure the very low (<1 MHz) {sup 14}N transition frequencies in cocaine hydrochloride; at 295 K the NQCC is 1.1780 ({+-}0.0014) MHz and the asymmetry parameter is 0.2632 ({+-}0.0034). Stepping the carrier frequency enables one to obtain a powder pattern without the severe intensity distortions that otherwise arise from finite pulse power. A powder pattern simulation using an NQCC value of 5.027 MHz and an asymmetry parameter {eta} of 0.2 agrees reasonably well with the experimental stepped-frequency spectrum. The use of pure NQR for providing nondestructive, quantitative, and highly specific detection of crystalline compounds is discussed, as are experimental strategies. 31 refs., 8 figs., 1 tab.

  10. Complete and incomplete fusion in the reaction {sup 35}Cl+{sup 12}C at the energy range 70{endash}154 MeV

    SciTech Connect

    Pirrone, S.; Aiello, S.; Arena, N.; Cavallaro, S.; Femino, S.; Lanzalone, G.; Politi, G.; Porto, F.; Romano, S.; Sambataro, S.

    1997-05-01

    Velocity spectra of evaporation residues produced in the {sup 35}Cl+{sup 12}C reaction have been measured at bombarding energies of 125, 140, and 154 MeV using time-of-flight techniques. These distributions were used to identify evaporation residues and to separate complete fusion and incomplete fusion components. The results show the presence of small contributions of incomplete fusion components which appear to be due to a cluster transfer reaction mechanism. Angular distributions and total and complete fusion evaporation residue cross sections were extracted at 70, 90, 110, 125, 140, and 154 MeV. The complete fusion cross sections and the deduced critical angular momenta are compared with other experimental data and the predictions of existing models. {copyright} {ital 1997} {ital The American Physical Society}

  11. Ex vivo binding of t-( sup 35 S) butylbicyclophosphorothionate: A biochemical tool to study the pharmacology of ethanol at the gamma-aminobutyric acid-coupled chloride channel

    SciTech Connect

    Sanna, E.; Concas, A.; Serra, M.; Santoro, G.; Biggio, G. )

    1991-03-01

    The effects of acute administration of ethanol on t-(35S)Butylbiclophosphorothionate (35S-TBPS) binding measured ex vivo in unwashed membrane preparations of rat cerebral cortex were investigated. Ethanol, given i.g., decreased in a dose-related (0.5-4 g/kg) and time-dependent manner the binding of 35S-TBPS. This effect was similar to that induced by the administration of diazepam (0.5-4 mg/kg i.p.). Scatchard plot analysis of this radioligand binding revealed that ethanol, differently from diazepam, decreased the apparent affinity of 35S-TBPS recognition sites whereas it failed to change the density of these binding sites. The effect of ethanol on 35S-TBPS binding could not be reversed by the previous administration to rats of the benzodiazepine receptor antagonist, Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H- imidazo (1,5a) (1,4) benzodiazepine-3-carboxylate). Vice versa, the benzodiazepine receptor partial inverse agonist, Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H- imidazo (1,5a) (4,4) benzodiazepine-3-carboxylate) (8 mg/kg i.p.), prevented completely ethanol-induced decrease of 35S-TBPS binding. The ability of Ro 15-4513 to prevent the action of ethanol was shared by the anxiogenic and proconvulsant beta-carboline derivatives, FG 7142 (N-methyl-beta-carboline-3-carboxamide) (12.5 mg/kg i.p.) and ethyl-beta-carboline-3-carboxylate (0.6 mg/kg i.v.), which, per se, enhanced this parameter. Moreover, ethanol (0.5-4 g/kg) was able to reverse the increase of 35S-TBPS binding elicited by the s.c. injection of isoniazid (350 mg/kg) and to clearly attenuate the severity of tonic-clonic seizures produced by this inhibitor of the GABAergic transmission.

  12. Regulation and function of histone acetyltransferase MOF.

    PubMed

    Yang, Yang; Han, Xiaofei; Guan, Jingyun; Li, Xiangzhi

    2014-03-01

    The mammalian MOF (male absent on the first), a member of the MYST (MOZ, YBF2, SAS2, and Tip60) family of histone acetyltransferases (HATs), is the major enzyme that catalyzes the acetylation of histone H4 on lysine 16. Acetylation of K16 is a prevalent mark associated with chromatin decondensation. MOF has recently been shown to play an essential role in maintaining normal cell functions. In this study, we discuss the important roles of MOF in DNA damage repair, apoptosis, and tumorigenesis. We also analyze the role of MOF as a key regulator of the core transcriptional network of embryonic stem cells. PMID:24452550

  13. Mechanism of hydrodesulfurization of dibenzothiophene on Co-Mo/Al[sub 2]O[sub 3] and Co/Al[sub 2]O[sub 3] catalyst by the use of radioisotope [sup 35]S tracer

    SciTech Connect

    Kabe, T.; Qian, W.; Ogawa, S.; Ishihara, A. )

    1993-09-01

    In order to estimate the behavior of sulfur on hydrodesulfurization catalyst, the [sup 35]S-labelled dibenzothiophene ([sup 35]S-DBT) was hydrodesulfurized on sulfided Co-Mo/Al[sub 2]O[sub 3] and Co/Al[sub 2]O[sub 3] in a fixed-bed pressurized flow reactor. During the reaction, the radioactivities of unreacted [sup 35]S-DBT and formed [sup 35]S-H[sub 2]S were monitored. The rate of the release of sulfur on the catalyst was estimated from the rates of an increase and a decrease of the radioactivity in formed [sup 35]S-H[sub 2]S. The activation energies of the release of sulfur were 3.7 and 9.9 kcal/mol for sulfided Co-Mo/Al[sub 2]O[sub 3] and Co/Al[sub 2]O[sub 3], respectively. The amount of labile sulfur (S[sub 0]), which could be calculated from the maximum amount of [sup 35]S accomodated on the catalyst, increased with increasing temperature. When it was assumed that sulfur in sulfided Co-Mo/Al[sub 2]O[sub 3] existed in the form of Co[sub 9]S[sub 8]-MoS[sub 2]/Al[sub 2]O[sub 3], the ratio of labile sulfur to total sulfur did not exceed 50%, even under conditions where the rate of DBT HDS is relatively high. On the contrary, the ratio of labile sulfur to total sulfur for Co/Al[sub 2]O[sub 3] exceeded 80%. 34 refs., 7 figs., 2 tabs.

  14. The role of side-chain interactions in the early steps of aggregation: Molecular dynamics simulations of an amyloid-forming peptide from the yeast prion Sup35

    NASA Astrophysics Data System (ADS)

    Gsponer, Jörg; Haberthür, Urs; Caflisch, Amedeo

    2003-04-01

    Understanding the early steps of aggregation at atomic detail might be crucial for the rational design of therapeutics preventing diseases associated with amyloid deposits. In this paper, aggregation of the heptapeptide GNNQQNY, from the N-terminal prion-determining domain of the yeast protein Sup35, was studied by 20 molecular dynamics runs for a total simulation time of 20 μs. The simulations generate in-register parallel packing of GNNQQNY -strands that is consistent with x-ray diffraction and Fourier transform infrared data. The statistically preferred aggregation pathway does not correspond to a purely downhill profile of the energy surface because of the presence of enthalpic barriers that originate from out-of-register interactions. The parallel -sheet arrangement is favored over the antiparallel because of side-chain contacts; in particular, stacking interactions of the tyrosine rings and hydrogen bonds between amide groups. No ordered aggregation was found in control simulations with the mutant sequence SQNGNQQRG in accord with experimental data and the strong sequence dependence of aggregation.

  15. Synthesis of proteins from ( sup 35 S)methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets

    SciTech Connect

    Schick, B.P. )

    1990-09-01

    The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of ({sup 35}S)methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS-PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.

  16. Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies.

    PubMed

    Krasnoslobodtsev, Alexey V; Deckert-Gaudig, Tanja; Zhang, Yuliang; Deckert, Volker; Lyubchenko, Yuri L

    2016-06-01

    Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have β-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of β-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface. PMID:27060278

  17. Global Profiling of Acetyltransferase Feedback Regulation.

    PubMed

    Montgomery, David C; Garlick, Julie M; Kulkarni, Rhushikesh A; Kennedy, Steven; Allali-Hassani, Abdellah; Kuo, Yin-Ming; Andrews, Andrew J; Wu, Hong; Vedadi, Masoud; Meier, Jordan L

    2016-05-25

    Lysine acetyltransferases (KATs) are key mediators of cell signaling. Methods capable of providing new insights into their regulation thus constitute an important goal. Here we report an optimized platform for profiling KAT-ligand interactions in complex proteomes using inhibitor-functionalized capture resins. This approach greatly expands the scope of KATs, KAT complexes, and CoA-dependent enzymes accessible to chemoproteomic methods. This enhanced profiling platform is then applied in the most comprehensive analysis to date of KAT inhibition by the feedback metabolite CoA. Our studies reveal that members of the KAT superfamily possess a spectrum of sensitivity to CoA and highlight NAT10 as a novel KAT that may be susceptible to metabolic feedback inhibition. This platform provides a powerful tool to define the potency and selectivity of reversible stimuli, such as small molecules and metabolites, that regulate KAT-dependent signaling. PMID:27149119

  18. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

    SciTech Connect

    Gorman, C.M.; Moffat, L.F.; Howard, B.H.

    1982-09-01

    The authors constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV 40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. They also constructed a recombinant, pSVO-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

  19. Exploring the role of hydration and confinement in the aggregation of amyloidogenic peptides Aβ(16-22) and Sup35(7-13) in AOT reverse micelles.

    PubMed

    Martinez, Anna Victoria; Małolepsza, Edyta; Rivera, Eva; Lu, Qing; Straub, John E

    2014-12-14

    Knowledge of how intermolecular interactions of amyloid-forming proteins cause protein aggregation and how those interactions are affected by sequence and solution conditions is essential to our understanding of the onset of many degenerative diseases. Of particular interest is the aggregation of the amyloid-β (Aβ) peptide, linked to Alzheimer's disease, and the aggregation of the Sup35 yeast prion peptide, which resembles the mammalian prion protein linked to spongiform encephalopathies. To facilitate the study of these important peptides, experimentalists have identified small peptide congeners of the full-length proteins that exhibit amyloidogenic behavior, including the KLVFFAE sub-sequence, Aβ16-22, and the GNNQQNY subsequence, Sup357-13. In this study, molecular dynamics simulations were used to examine these peptide fragments encapsulated in reverse micelles (RMs) in order to identify the fundamental principles that govern how sequence and solution environment influence peptide aggregation. Aβ16-22 and Sup357-13 are observed to organize into anti-parallel and parallel β-sheet arrangements. Confinement in the sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles is shown to stabilize extended peptide conformations and enhance peptide aggregation. Substantial fluctuations in the reverse micelle shape are observed, in agreement with earlier studies. Shape fluctuations are found to facilitate peptide solvation through interactions between the peptide and AOT surfactant, including direct interaction between non-polar peptide residues and the aliphatic surfactant tails. Computed amide I IR spectra are compared with experimental spectra and found to reflect changes in the peptide structures induced by confinement in the RM environment. Furthermore, examination of the rotational anisotropy decay of water in the RM demonstrates that the water dynamics are sensitive to the presence of peptide as well as the peptide sequence. Overall, our results

  20. Inhibition of Histone Acetyltransferase by Glycosaminoglycans

    PubMed Central

    Buczek-Thomas, Jo Ann; Hsia, Edward; Rich, Celeste B.; Foster, Judith A.; Nugent, Matthew A.

    2008-01-01

    Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin-derived oligosaccharides (> 8 sugars) and N-desulfated or O-desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive-like inhibitor causing an ~50-fold increase in the apparent Km of pCAF for histone H4. Heparan sulfate proteoglycans isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous glycosaminoglycans might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an ~50% reduction of histone H3 acetylation after 6 hours of treatment. In addition, Chinese hamster ovary cells deficient in glycosaminoglycan synthesis showed increased levels of acetylated histone H3 compared to wild-type parent cells. Glycosaminoglycans represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation. PMID:18459114

  1. Autoacetylation of the Histone Acetyltransferase Rtt109*

    PubMed Central

    Albaugh, Brittany N.; Arnold, Kevin M.; Lee, Susan; Denu, John M.

    2011-01-01

    Rtt109 is a yeast histone acetyltransferase (HAT) that associates with histone chaperones Asf1 and Vps75 to acetylate H3K56, H3K9, and H3K27 and is important in DNA replication and maintaining genomic integrity. Recently, mass spectrometry and structural studies of Rtt109 have shown that active site residue Lys-290 is acetylated. However, the functional role of this modification and how the acetyl group is added to Lys-290 was unclear. Here, we examined the mechanism of Lys-290 acetylation and found that Rtt109 catalyzes intramolecular autoacetylation of Lys-290 ∼200-times slower than H3 acetylation. Deacetylated Rtt109 was prepared by reacting with a sirtuin protein deacetylase, producing an enzyme with negligible HAT activity. Autoacetylation of Rtt109 restored full HAT activity, indicating that autoacetylation is necessary for HAT activity and is a fully reversible process. To dissect the mechanism of activation, biochemical, and kinetic analyses were performed with Lys-290 variants of the Rtt109-Vps75 complex. We found that autoacetylation of Lys-290 increases the binding affinity for acetyl-CoA and enhances the rate of acetyl-transfer onto histone substrates. This study represents the first detailed investigation of a HAT enzyme regulated by single-site intramolecular autoacetylation. PMID:21606491

  2. COUPLING

    DOEpatents

    Hawke, B.C.

    1963-02-26

    This patent relates to a releasable coupling connecting a control rod to a control rod drive. This remotely operable coupling mechanism can connect two elements which are laterally and angviarly misaligned, and provides a means for sensing the locked condition of the elements. The coupling utilizes a spherical bayonet joint which is locked against rotation by a ball detent lock. (AEC)

  3. Rapid quantitative assay for chloramphenicol acetyltransferase

    SciTech Connect

    Neumann, J.R.; Morency, C.A.; Russian, K.O.

    1987-05-01

    Measuring the expression of exogenous genetic material in mammalian cells is commonly done by fusing the DNA of interest to a gene encoding an easily-detected enzyme. Chloramphenicol acetyltransferase(CAT) is a convenient marker because it is not normally found in eukaryotes. CAT activity has usually been detected using a thin-layer chromatographic separation followed by autoradiography. An organic solvent extraction-based method for CAT detection has also been described, as well as a procedure utilizing HPLC analysis. Building on the extraction technique, they developed a rapid sensitive kinetic method for measuring CAT activity in cell homogenates. The method exploits the differential organic solubility of the substrate ((/sup 3/H) or (/sup 14/C)acetyl CoA) and the product (labeled acetylchloramphenicol). The assay is a simple one-vial, two-phase procedure and requires no tedious manipulations after the initial setup. Briefly, a 0.25 ml reaction with 100mM Tris-HCL, 1mM chloramphenicol, 0.1mM (/sup 14/C)acetyl CoA and variable amounts of cell homogenate is pipetted into a miniscintillation vial, overlaid with 5 ml of a water-immiscible fluor, and incubated at 37/sup 0/C. At suitable intervals the vial is counted and the CAT level is quantitatively determined as the rate of increase in counts/min of the labeled product as it diffuses into the fluor phase, compared to a standard curve. When used to measure CAT in transfected Balb 3T3 cells the method correlated well with the other techniques.

  4. Structure of a putative acetyltransferase (PA1377) from Pseudomonas aeruginosa

    SciTech Connect

    Davies, Anna M.; Tata, Renée; Chauviac, François-Xavier; Sutton, Brian J.; Brown, Paul R.

    2008-05-01

    The crystal structure of an acetyltransferase encoded by the gene PA1377 from Pseudomonas aeruginosa has been determined at 2.25 Å resolution. Comparison with a related acetyltransferase revealed a structural difference in the active site that was taken to reflect a difference in substrate binding and/or specificity between the two enzymes. Gene PA1377 from Pseudomonas aeruginosa encodes a 177-amino-acid conserved hypothetical protein of unknown function. The structure of this protein (termed pitax) has been solved in space group I222 to 2.25 Å resolution. Pitax belongs to the GCN5-related N-acetyltransferase family and contains all four sequence motifs conserved among family members. The β-strand structure in one of these motifs (motif A) is disrupted, which is believed to affect binding of the substrate that accepts the acetyl group from acetyl-CoA.

  5. Structure and Biochemical Characterization of Protein Acetyltransferase from Sulfolobus solfataricus

    SciTech Connect

    Brent, Michael M.; Iwata, Ayaka; Carten, Juliana; Zhao, Kehao; Marmorstein, Ronen

    2009-09-02

    The Sulfolobus solfataricus protein acetyltransferase (PAT) acetylates ALBA, an abundant nonspecific DNA-binding protein, on Lys{sup 16} to reduce its DNA affinity, and the Sir2 deacetylase reverses the modification to cause transcriptional repression. This represents a 'primitive' model for chromatin regulation analogous to histone modification in eukaryotes. We report the 1.84-{angstrom} crystal structure of PAT in complex with coenzyme A. The structure reveals homology to both prokaryotic GNAT acetyltransferases and eukaryotic histone acetyltransferases (HATs), with an additional 'bent helix' proximal to the substrate binding site that might play an autoregulatory function. Investigation of active site mutants suggests that PAT does not use a single general base or acid residue for substrate deprotonation and product reprotonation, respectively, and that a diffusional step, such as substrate binding, may be rate-limiting. The catalytic efficiency of PAT toward ALBA is low relative to other acetyltransferases, suggesting that there may be better, unidentified substrates for PAT. The structural similarity of PAT to eukaryotic HATs combined with its conserved role in chromatin regulation suggests that PAT is evolutionarily related to the eukaryotic HATs.

  6. COUPLING

    DOEpatents

    Frisch, E.; Johnson, C.G.

    1962-05-15

    A detachable coupling arrangement is described which provides for varying the length of the handle of a tool used in relatively narrow channels. The arrangement consists of mating the key and keyhole formations in the cooperating handle sections. (AEC)

  7. Coenzyme A Binding to the Aminoglycoside Acetyltransferase (3)-IIIb Increases Conformational Sampling of Antibiotic Binding Site

    SciTech Connect

    Hu, Xiaohu; Norris, Adrianne; Baudry, Jerome Y; Serpersu, Engin H

    2011-01-01

    NMR spectroscopy experiments and molecular dynamics simulations were performed to describe the dynamic properties of the aminoglycoside acetyltransferase (3)-IIIb (AAC) in its apo and coenzyme A (CoASH) bound forms. The {sup 15}N-{sup 1}H HSQC spectra indicate a partial structural change and coupling of the CoASH binding site with another region in the protein upon the CoASH titration into the apo enzyme. Molecular dynamics simulations indicate a significant structural and dynamic variation of the long loop in the antibiotic binding domain in the form of a relatively slow (250 ns), concerted opening motion in the CoASH enzyme complex and that binding of the CoASH increases the structural flexibility of the loop, leading to an interchange between several similar equally populated conformations.

  8. Radioenzymatic assays for aminoglycosides with kanamycin 6'- acetyltransferase

    SciTech Connect

    Weber, A.; Smith, A.L.; Opheim, K.E.

    1985-03-01

    To facilitate the rapid and accurate quantitation of parenterally administered aminoglycosides, the optimum conditions (pH, duration of incubation, and cofactor concentrations) were defined to permit radioenzymatic assays with kanamycin acetyltransferase. The accuracy in quantitating tobramycin, netilmicin, kanamycin, and amikacin at concentrations in the therapeutic range was greater than 90%, with a mean recovery of 102.8%. The mean of the interassay coefficient of variation was 7.8%. Typical standard curves at six different concentrations resulted in a correlation coefficient (r value) of greater than 0.99 for each aminoglycoside. The radioenzymatic assay correlates well with the bioassay (tobramycin and netilmicin) and radioimmunoassay (amikacin and kanamycin); the correlation coefficient is greater than 0.90 for all. The authors conclude that the radioenzymatic assay utilizing kanamycin 6'-acetyltransferase is feasible for all commercially available parenterally administered aminoglycosides.

  9. Paramecium bursaria Chlorella Virus 1 Encodes a Polyamine Acetyltransferase*

    PubMed Central

    Charlop-Powers, Zachary; Jakoncic, Jean; Gurnon, James R.; Van Etten, James L.; Zhou, Ming-Ming

    2012-01-01

    Paramecium bursaria chlorella virus 1 (PBCV-1), a large DNA virus that infects green algae, encodes a histone H3 lysine 27-specific methyltransferase that functions in global transcriptional silencing of the host. PBCV-1 has another gene a654l that encodes a protein with sequence similarity to the GCN5 family histone acetyltransferases. In this study, we report a 1.5 Å crystal structure of PBCV-1 A654L in a complex with coenzyme A. The structure reveals a unique feature of A654L that precludes its acetylation of histone peptide substrates. We demonstrate that A654L, hence named viral polyamine acetyltransferase (vPAT), acetylates polyamines such as putrescine, spermidine, cadaverine, and homospermidine present in both PBCV-1 and its host through a reaction dependent upon a conserved glutamate 27. Our study suggests that as the first virally encoded polyamine acetyltransferase, vPAT plays a possible key role in the regulation of polyamine catabolism in the host during viral replication. PMID:22277659

  10. Rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase

    SciTech Connect

    Nordeen, S.K.; Green, P.P. III; Fowlkes, D.M.

    1987-04-01

    We present a rapid, sensitive enzymatic assay for chloramphenicol acetyltransferase (CAT) that does not require chromatography, HPLC, or autoradiography. The assay is based on the use of an inexpensive substrate, tritiated acetate, instead of (/sup 14/C)chloramphenicol. The method is adapted from one originally used by de Crombrugghe et al. and by Shaw, but with simplifications appropriate for routine use. In our hands, the method is as sensitive as the customary thin-layer chromatography assay and is far more efficient for the performance of many assays, both in terms of labor and expense.

  11. Enzyme kinetics and inhibition of histone acetyltransferase KAT8.

    PubMed

    Wapenaar, Hannah; van der Wouden, Petra E; Groves, Matthew R; Rotili, Dante; Mai, Antonello; Dekker, Frank J

    2015-11-13

    Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of anacardic acid (AA) was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT. PMID:26505788

  12. Enzyme kinetics and inhibition of histone acetyltransferase KAT8

    PubMed Central

    Wapenaar, Hannah; van der Wouden, Petra E.; Groves, Matthew R.; Rotili, Dante; Mai, Antonello; Dekker, Frank J.

    2016-01-01

    Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of AA was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng-Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT. PMID:26505788

  13. Molecular mechanism underlying promiscuous polyamine recognition by spermidine acetyltransferase.

    PubMed

    Sugiyama, Shigeru; Ishikawa, Sae; Tomitori, Hideyuki; Niiyama, Mayumi; Hirose, Mika; Miyazaki, Yuma; Higashi, Kyohei; Murata, Michio; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Kashiwagi, Keiko; Igarashi, Kazuei; Matsumura, Hiroyoshi

    2016-07-01

    Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5Å resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13Å. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT. PMID:27163532

  14. A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

    PubMed Central

    Lee, Amy Huei-Yi; Hurley, Brenden; Felsensteiner, Corinna; Yea, Carmen; Ckurshumova, Wenzislava; Bartetzko, Verena; Wang, Pauline W.; Quach, Van; Lewis, Jennifer D.; Liu, Yulu C.; Börnke, Frederik; Angers, Stephane; Wilde, Andrew

    2012-01-01

    The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption. PMID:22319451

  15. Biochemical evidence for relaxed substrate specificity of Nα-acetyltransferase (Rv3420c/rimI) of Mycobacterium tuberculosis

    PubMed Central

    Pathak, Deepika; Bhat, Aadil Hussain; Sapehia, Vandana; Rai, Jagdish; Rao, Alka

    2016-01-01

    Nα-acetylation is a naturally occurring irreversible modification of N-termini of proteins catalyzed by Nα-acetyltransferases (NATs). Although present in all three domains of life, it is little understood in bacteria. The functional grouping of NATs into six types NatA - NatF, in eukaryotes is based on subunit requirements and stringent substrate specificities. Bacterial orthologs are phylogenetically divergent from eukaryotic NATs, and only a couple of them are characterized biochemically. Accordingly, not much is known about their substrate specificities. Rv3420c of Mycobacterium tuberculosis is a NAT ortholog coding for RimIMtb. Using in vitro peptide-based enzyme assays and mass-spectrometry methods, we provide evidence that RimIMtb is a protein Nα-acetyltransferase of relaxed substrate specificity mimicking substrate specificities of eukaryotic NatA, NatC and most competently that of NatE. Also, hitherto unknown acetylation of residues namely, Asp, Glu, Tyr and Leu by a bacterial NAT (RimIMtb) is elucidated, in vitro. Based on in vivo acetylation status, in vitro assay results and genetic context, a plausible cellular substrate for RimIMtb is proposed. PMID:27353550

  16. Biochemical evidence for relaxed substrate specificity of Nα-acetyltransferase (Rv3420c/rimI) of Mycobacterium tuberculosis.

    PubMed

    Pathak, Deepika; Bhat, Aadil Hussain; Sapehia, Vandana; Rai, Jagdish; Rao, Alka

    2016-01-01

    Nα-acetylation is a naturally occurring irreversible modification of N-termini of proteins catalyzed by Nα-acetyltransferases (NATs). Although present in all three domains of life, it is little understood in bacteria. The functional grouping of NATs into six types NatA - NatF, in eukaryotes is based on subunit requirements and stringent substrate specificities. Bacterial orthologs are phylogenetically divergent from eukaryotic NATs, and only a couple of them are characterized biochemically. Accordingly, not much is known about their substrate specificities. Rv3420c of Mycobacterium tuberculosis is a NAT ortholog coding for RimI(Mtb). Using in vitro peptide-based enzyme assays and mass-spectrometry methods, we provide evidence that RimI(Mtb) is a protein Nα-acetyltransferase of relaxed substrate specificity mimicking substrate specificities of eukaryotic NatA, NatC and most competently that of NatE. Also, hitherto unknown acetylation of residues namely, Asp, Glu, Tyr and Leu by a bacterial NAT (RimI(Mtb)) is elucidated, in vitro. Based on in vivo acetylation status, in vitro assay results and genetic context, a plausible cellular substrate for RimI(Mtb) is proposed. PMID:27353550

  17. Atomic resolution structure of human α-tubulin acetyltransferase bound to acetyl-CoA

    PubMed Central

    Taschner, Michael; Vetter, Melanie; Lorentzen, Esben

    2012-01-01

    Acetylation of lysine residues is an important posttranslational modification found in all domains of life. α-tubulin is specifically acetylated on lysine 40, a modification that serves to stabilize microtubules of axons and cilia. Whereas histone acetyltransferases have been extensively studied, there is no structural and mechanistic information available on α-tubulin acetyltransferases. Here, we present the structure of the human α-tubulin acetyltransferase catalytic domain bound to its cosubstrate acetyl-CoA at 1.05 Å resolution. Compared with other lysine acetyltransferases of known structure, α-tubulin acetyltransferase displays a relatively well-conserved cosubstrate binding pocket but is unique in its active site and putative α-tubulin binding site. Using acetylation assays with structure-guided mutants, we map residues important for acetyl-CoA binding, substrate binding, and catalysis. This analysis reveals a basic patch implicated in substrate binding and a conserved glutamine residue required for catalysis, demonstrating that the family of α-tubulin acetyltransferases uses a reaction mechanism different from other lysine acetyltransferases characterized to date. PMID:23071318

  18. Biochemical pathways that regulate acetyltransferase and deacetylase activity in mammalian cells

    PubMed Central

    Mellert, Hestia S.; McMahon, Steven B.

    2009-01-01

    Protein phosphorylation is dynamically regulated in eukaryotic cells via modulation of the enzymatic activity of kinases and phosphatases. Like phosphorylation, acetylation has emerged as a critical regulatory protein modification that is dynamically altered in response to diverse cellular cues. Moreover, acetyltransferases and deacetylases are tightly linked to cellular signaling pathways. Recent studies provide clues about the mechanisms utilized to regulate acetyltransferases and deacetylases. The therapeutic value of deacetylase inhibitors suggests that understanding acetylation pathways will directly impact our ability to rationally target these enzymes in patients. Recently discovered mechanisms which directly regulate the catalytic activity of acetyltransferases and deacetylases provide exciting new insights about these enzymes. PMID:19819149

  19. KATching-Up on Small Molecule Modulators of Lysine Acetyltransferases.

    PubMed

    Simon, Roman P; Robaa, Dina; Alhalabi, Zayan; Sippl, Wolfgang; Jung, Manfred

    2016-02-25

    The reversible acetylation of lysines is one of the best characterized epigenetic modifications. Its involvement in many key physiological and pathological processes has been documented in numerous studies. Lysine deacetylases (KDACs) and acetyltransferases (KATs) maintain the acetylation equilibrium at histones but also many other proteins. Besides acetylation, also other acyl groups are reversibly installed at the side chain of lysines in proteins. Because of their involvement in disease, KDACs and KATs were proposed to be promising drug targets, and for KDACs, indeed, five inhibitors are now approved for human use. While there is a similar level of evidence for the potential of KATs as drug targets, no inhibitor is in clinical trials. Here, we review the evidence for the diverse roles of KATs in disease pathology, provide an overview of structural features and the available modulators, including those targeting the bromodomains of KATs, and present an outlook. PMID:26701186

  20. [The biological role of prokaryotic and eukaryotic N-acetyltransferase].

    PubMed

    Zabost, Anna; Zwolska, Zofia; Augustynowicz-Kopeć, Ewa

    2013-01-01

    The N-acetyltransferases (NAT; E.C.2.3.1.5) are involved in the metabolism of drugs and environmental toxins. They catalyse the acetyl transfer from acetyl coenzyme A to an aromatic amine, heterocyclic amine, or hydrazine compound. NAT homologues are present in numerous species from bacteria to human. Sequence variations in the human NAT1 and NAT2 result in the production of NAT proteins with variable enzyme activity or stability, leading to slow or rapid acetylation. Therefore, genetic polymorphisms in NAT1 and NAT2 influence drug metabolism and drug-related toxicity. Epidemiological studies suggest that the NAT1 and NAT2 acetylation polymorphisms modify the risk of developing cancers of the urinary bladder, colorectal, breast, head and neck, and lung. PMID:23420430

  1. The Role of Histone Acetyltransferases in Normal and Malignant Hematopoiesis

    PubMed Central

    Sun, Xiao-Jian; Man, Na; Tan, Yurong; Nimer, Stephen D.; Wang, Lan

    2015-01-01

    Histone, and non-histone, protein acetylation plays an important role in a variety of cellular events, including the normal and abnormal development of blood cells, by changing the epigenetic status of chromatin and regulating non-histone protein function. Histone acetyltransferases (HATs), which are the enzymes responsible for histone and non-histone protein acetylation, contain p300/CBP, MYST, and GNAT family members. HATs are not only protein modifiers and epigenetic factors but also critical regulators of cell development and carcinogenesis. Here, we will review the function of HATs such as p300/CBP, Tip60, MOZ/MORF, and GCN5/PCAF in normal hematopoiesis and the pathogenesis of hematological malignancies. The inhibitors that have been developed to target HATs will also be reviewed here. Understanding the roles of HATs in normal/malignant hematopoiesis will provide the potential therapeutic targets for the hematological malignancies. PMID:26075180

  2. The Role of Histone Acetyltransferases in Normal and Malignant Hematopoiesis.

    PubMed

    Sun, Xiao-Jian; Man, Na; Tan, Yurong; Nimer, Stephen D; Wang, Lan

    2015-01-01

    Histone, and non-histone, protein acetylation plays an important role in a variety of cellular events, including the normal and abnormal development of blood cells, by changing the epigenetic status of chromatin and regulating non-histone protein function. Histone acetyltransferases (HATs), which are the enzymes responsible for histone and non-histone protein acetylation, contain p300/CBP, MYST, and GNAT family members. HATs are not only protein modifiers and epigenetic factors but also critical regulators of cell development and carcinogenesis. Here, we will review the function of HATs such as p300/CBP, Tip60, MOZ/MORF, and GCN5/PCAF in normal hematopoiesis and the pathogenesis of hematological malignancies. The inhibitors that have been developed to target HATs will also be reviewed here. Understanding the roles of HATs in normal/malignant hematopoiesis will provide the potential therapeutic targets for the hematological malignancies. PMID:26075180

  3. The MOZ histone acetyltransferase in epigenetic signaling and disease.

    PubMed

    Carlson, Samuel; Glass, Karen C

    2014-11-01

    The monocytic leukemic zinc finger (MOZ) histone acetyltransferase (HAT) plays a role in acute myeloid leukemia (AML). It functions as a quaternary complex with the bromodomain PHD finger protein 1 (BRPF1), the human Esa1-associated factor 6 homolog (hEAF6), and the inhibitor of growth 5 (ING5). Each of these subunits contain chromatin reader domains that recognize specific post-translational modifications (PTMs) on histone tails, and this recognition directs the MOZ HAT complex to specific chromatin substrates. The structure and function of these epigenetic reader modules has now been elucidated, and a model describing how the cooperative action of these domains regulates HAT activity in response to the epigenetic landscape is proposed. The emerging role of epigenetic reader domains in disease, and their therapeutic potential for many types of cancer is also highlighted. PMID:24633655

  4. Crystal structure of homoserine O-acetyltransferase from Leptospira interrogans

    SciTech Connect

    Wang Mingzhu; Liu Lin; Wang Yanli; Wei Zhiyi; Zhang Ping; Li Yikun; Jiang Xiaohua; Xu Hang Gong Weimin

    2007-11-30

    Homoserine O-acetyltransferase (HTA, EC 2.3.1.31) initiates methionine biosynthesis pathway by catalyzing the transfer of acetyl group from acetyl-CoA to homoserine. This study reports the crystal structure of HTA from Leptospira interrogans determined at 2.2 A resolution using selenomethionyl single-wavelength anomalous diffraction method. HTA is modular and consists of two structurally distinct domains-a core {alpha}/{beta} domain containing the catalytic site and a helical bundle called the lid domain. Overall, the structure fold belongs to {alpha}/{beta} hydrolase superfamily with the characteristic 'catalytic triad' residues in the active site. Detailed structure analysis showed that the catalytic histidine and serine are both present in two conformations, which may be involved in the catalytic mechanism for acetyl transfer.

  5. Structure of Mesorhizobium loti arylamine N-acetyltransferase 1

    SciTech Connect

    Holton, Simon J.; Dairou, Julien; Sandy, James; Rodrigues-Lima, Fernando; Dupret, Jean-Marie; Noble, Martin E. M.; Sim, Edith

    2005-01-01

    The crystal structure of a M. loti arylamine N-acetyltransferase 1 has been determined at 2.0 Å resolution. The arylamine N-acetyltransferase (NAT) enzymes have been found in a broad range of both eukaryotic and prokaryotic organisms. The NAT enzymes catalyse the transfer of an acetyl group from acetyl Co-enzyme A onto the terminal nitrogen of a range of arylamine, hydrazine and arylhydrazine compounds. Recently, several NAT structures have been reported from different prokaryotic sources including Salmonella typhimurium, Mycobacterium smegmatis and Pseudomonas aeruginosa. Bioinformatics analysis of the Mesorhizobium loti genome revealed two NAT paralogues, the first example of multiple NAT isoenzymes in a eubacterial organism. The M. loti NAT 1 enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme was crystallized in 0.5 M Ca(OAc){sub 2}, 16% PEG 3350, 0.1 M Tris–HCl pH 8.5 using the sitting-drop vapour-diffusion method. A data set diffracting to 2.0 Å was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.2, b = 97.3, c = 114.3 Å. The structure was refined to a final free-R factor of 24.8%. The structure reveals that despite low sequence homology, M. loti NAT1 shares the common fold as reported in previous NAT structures and exhibits the same catalytic triad of residues (Cys-His-Asp) in the active site.

  6. Mass and charge distributions in chlorine-induced nuclear reactions. [[sup 35]Cl at E/A = 15 MeV, [sup 37]Cl at E/A = 7. 3 MeV

    SciTech Connect

    Marchetti, A.A.

    1991-01-01

    Projectile-like fragments were detected and characterized in terms of A, Z, and energy for the reactions [sup 37]Cl on [sup 40]Ca and [sup 209]Bi at E/A = 7.3 MeV, and [sup 35]Cl, on [sup 209]Bi at E/A = 15 MeV, at angles close to the grazing angle. Mass and charge distributions were generated in the N-Z plane as a function of energy loss, and have been parameterized in terms of their centroids, variances, and coefficients of correlation. Due to experimental problems, the mass resolution corresponding to the [sup 31]Cl on [sup 209]Bi reaction was very poor. This prompted the study and application of a deconvolution technique for peak enhancement. The drifts of the charge and mass centroids for the system [sup 37]Cl on [sup 40]Ca are consistent with a process of mass and charge equilibration mediated by nucleon exchange between the two partners, followed by evaporation. The asymmetric systems show a strong drift towards larger asymmetry, with the production of neutron-rich nuclei. It was concluded that this is indicative of a net transfer of protons from the light to the heavy partner, and a net flow of neutrons in the opposite direction. The variances for all systems increase with energy loss, as it would be expected from a nucleon exchange mechanism; however, the variances for the reaction [sup 37]Cl on [sup 40]Ca are higher than those expected from that mechanism. The coefficients of correlation indicate that the transfer of nucleons between projectile and target is correlated. The results were compared to the predictions of two current models based on a stochastic nucleon exchange mechanism. In general, the comparisons between experimental and predicted variances support this mechanism; however, the need for more realistic driving forces in the model calculations is indicated by the disagreement between predicted and experimental centroids.

  7. Study of hydrosulfurization of dibenzothiophene on Ni-Mo/Al{sub 2}O{sub 3}, Mo/Al{sub 2}O{sub 3}, and Ni/Al{sub 2}O{sub 3} catalysts by the use of radioisotope {sup 35}S tracer

    SciTech Connect

    Kabe, Toshiaki; Qian, Weihua; Ishihara, Atsushi

    1994-09-01

    The radioisotope tracer method has been used to quantify the behavior of sulfur on sulfided Ni-Mo/Al{sub 2}O{sub 3}, Mo/Al{sub 2}O{sub 3}, and Ni/Al{sub 2}O{sub 3}. The apparent activation energies of HDS reaction for DBT for the three catalysts were 20{+-}1 kcal/mol. The formation rate constants of {sup 35}S-H{sub 2}S were determined and the amount of labile sulfur on the sulfided catalysts were estimated by tracing the changes in radioactivities of the unreacted {sup 35}S-DBT and the formed {sup 35}S-H{sub 2}S during the HDS reaction of {sup 35}S-labeled dibenzothiophene ({sup 35}S-DBT). It was deduced that ca. 75% of sulfur in the sulfided Mo/Al{sub 2}O{sub 3} was related to HDS reaction at infinite rate of HDS. Compared with the amounts of labile sulfur in the sulfided Ni-Mo/Al{sub 2}O{sub 3}, Mo/Al{sub 2}O{sub 3}, and Ni/Al{sub 2}O{sub 3}, it was determined that the amounts of labile sulfur were 1.6, 9.8, and 18.4 mg sulfur/g catalyst at 280{degrees}C for the three catalysts, respectively. It was suggested that the sulfur in the form of NiS on the sulfided Ni-Mo/Al{sub 2}O{sub 3} was not labile and that the sulfur attached to both Mo and Ni atom were more labile and related to HDS. The promotion of Ni for Mo-based catalysts was attributed to the sulfur bonded to both Mo and Ni in the MoS{sub 2} phase being more labile. 35 refs., 12 figs., 2 tabs.

  8. In vitro inhibition of choline acetyltransferase by a series of 2-benzylidene-3-quinuclidinones

    SciTech Connect

    Capacio, B.R.

    1988-01-01

    Ten substituted 2-benzylidene-3-quinuclidinones were synthesized and evaluated for their relative potency as in vitro inhibitors of choline acetyltransferase (ChAT). Acetylcholine (ACh) synthesis was followed radiometrically by the incorporation of labeled acetate originating from {sup 14}C-acetyl-CoA. Woolf-Augustinsson-Hofstee data analysis was used to calculate Vmax, Km, and Ki values. The inhibition was found to be noncompetitive or uncompetitive with respect to choline. Quantitative structure activity relationship correlations demonstrated a primary dependence on {kappa}-{sigma}, as well as steric properties of the substituted benzene ring. Additional radiometric and spectrophotometric were performed with 2-(3{prime}-methyl)-benzylidene-3-quinuclidinone, one of the more potent analogs, to further elucidate the inhibitory mechanism. ChAT-mediated cleavage of ACh was measured spectrophotometrically by following the appearance of NADH at 340 nanometers in an enzyme coupled assay. Lineweaver-Burk analysis indicated mixed or uncompetitive inhibition with respect to both substrates of the forward reaction, suggesting interference with a rate limiting step.

  9. Comparative genomic and phylogenetic investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes characterized in several bacteria and eukaryotic organisms. We report a comprehensive phylogenetic analysis employing an exhaustive dataset of NAT-homologous sequences recovered through inspection of 2445 genomes. We describe ...

  10. Enhancer of Acetyltransferase Chameau (EAChm) Is a Novel Transcriptional Co-Activator

    PubMed Central

    Imamura, Yuko; Higashi, Miki; Yoneda, Mitsuhiro; Ito, Takashi

    2015-01-01

    Acetylation of nucleosomal histones by diverse histone acetyltransferases (HAT) plays pivotal roles in many cellular events. Discoveries of novel HATs and HAT related factors have provided new insights to understand the roles and mechanisms of histone acetylation. In this study, we identified prominent Histone H3 acetylation activity in vitro and purified its activity, showing that it is composed of the MYST acetyltransferase Chameau and Enhancer of the Acetyltransferase Chameau (EAChm) family. EAChm is a negatively charged acidic protein retaining aspartate and glutamate. Furthermore, we identified that Chameau and EAChm stimulate transcription in vitro together with purified general transcription factors. In addition, RNA-seq analysis of Chameu KD and EAChm KD S2 cells suggest that Chameau and EAChm regulate transcription of common genes in vivo. Our results suggest that EAChm regulates gene transcription in Drosophila embryos by enhancing Acetyltransferase Chameau activity. PMID:26555228

  11. Reconstruction of N-acetyltransferase 2 haplotypes using PHASE.

    PubMed

    Golka, Klaus; Blaszkewicz, Meinolf; Samimi, Mirabutaleb; Bolt, Hermann M; Selinski, Silvia

    2008-04-01

    The genotyping of N-acetyltransferase 2 (NAT2) by PCR/RFLP methods yields in a considerable percentage ambiguous results. To resolve this methodical problem a statistical approach was applied. PHASE v2.1.1, a statistical program for haplotype reconstruction was used to estimate haplotype pairs from NAT2 genotyping data, obtained by the analysis of seven single nucleotide polymorphisms relevant for Caucasians. In 1,011 out of 2,921 (35%) subjects the haplotype pairs were clearcut by the PCR/RFLP data only. For the majority of the data the applied method resulted in a multiplicity (2-4) of possible haplotype pairs. Haplotype reconstruction using PHASE v2.1.1 cleared this ambiguity in all cases but one, where an alternative haplotype pair was considered with a probability of 0.029. The estimation of the NAT2 haplotype is important because the assignment of the NAT2 alleles *12A, *12B, *12C or *13 to the rapid or slow NAT2 genotype has been discussed controversially. A clear assignment is indispensable in surveys of human bladder cancer caused by aromatic amine exposures. In conclusion, PHASE v2.1.1 software allowed an unambiguous haplotype reconstruction in 2,920 of 2,921 cases (>99.9%). PMID:17879084

  12. Small molecule modulators of histone acetyltransferase p300.

    PubMed

    Balasubramanyam, Karanam; Swaminathan, V; Ranganathan, Anupama; Kundu, Tapas K

    2003-05-23

    Histone acetyltransferases (HATs) are a group of enzymes that play a significant role in the regulation of gene expression. These enzymes covalently modify the N-terminal lysine residues of histones by the addition of acetyl groups from acetyl-CoA. Dysfunction of these enzymes is often associated with the manifestation of several diseases, predominantly cancer. Here we report that anacardic acid from cashew nut shell liquid is a potent inhibitor of p300 and p300/CBP-associated factor histone acetyltranferase activities. Although it does not affect DNA transcription, HAT-dependent transcription from a chromatin template was strongly inhibited by anacardic acid. Furthermore, we describe the design and synthesis of an amide derivative N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) using anacardic acid as a synthon, which remarkably activates p300 HAT activity but not that of p300/CBP-associated factor. Although CTPB does not affect DNA transcription, it enhances the p300 HAT-dependent transcriptional activation from in vitro assembled chromatin template. However, it has no effect on histone deacetylase activity. These compounds would be useful as biological switching molecules for probing into the role of p300 in transcriptional studies and may also be useful as new chemical entities for the development of anticancer drugs. PMID:12624111

  13. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis

    PubMed Central

    Su, Jiaming; Wang, Fei; Cai, Yong; Jin, Jingji

    2016-01-01

    Changes in chromatin structure and heritably regulating the gene expression by epigenetic mechanisms, such as histone post-translational modification, are involved in most cellular biological processes. Thus, abnormal regulation of epigenetics is implicated in the occurrence of various diseases, including cancer. Human MOF (males absent on the first) is a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs). As a catalytic subunit, MOF can form at least two distinct multiprotein complexes (MSL and NSL) in human cells. Both complexes can acetylate histone H4 at lysine 16 (H4K16); however, the NSL complex possesses broader substrate specificity and can also acetylate histone H4 at lysines 5 and 8 (H4K5 and H4K8), suggesting the complexity of the intracellular functions of MOF. Silencing of MOF in cells leads to genomic instability, inactivation of gene transcription, defective DNA damage repair and early embryonic lethality. Unbalanced MOF expression and its corresponding acetylation of H4K16 have been found in certain primary cancer tissues, including breast cancer, medulloblastoma, ovarian cancer, renal cell carcinoma, colorectal carcinoma, gastric cancer, as well as non-small cell lung cancer. In this review, we provide a brief overview of MOF and its corresponding histone acetylation, introduce recent research findings that link MOF functions to tumorigenesis and speculate on the potential role that may be relevant to tumorigenic pathways. PMID:26784169

  14. N-acetyltransferase 2 activity and folate levels

    PubMed Central

    Cao, Wen; Strnatka, Diana; McQueen, Charlene A.; Hunter, Robert J.; Erickson, Robert P.

    2010-01-01

    Aims To determine whether increased N-acetyltransferase (NAT) activity might have a toxic effect during development and an influence on folate levels since previous work has shown that only low levels of exogenous NAT can be achieved in constitutionally transgenic mice (Cao, et al, 2005) Main Methods A human NAT1 tet-inducible construct was used that would not be expressed until the inducer was delivered. Human NAT1 cDNA was cloned into pTRE2 and injected into mouse oocytes. Two transgenic lines were crossed to mouse line TgN(rtTahCMV)4Uh containing the CMV promoted “teton.”Measurements of red blood cell folate levels in inbred strains of mice were performed. Key findings Only low levels of human NAT1 could be achieved in kidney (highly responsive in other studies) whether the inducer, doxycycline, was given by gavage or in drinking water.An inverse correlation of folate levels with Nat2 enzyme activity was found. Significance Since increasing NAT1 activity decrease folate in at least one tissue, the detrimental effect of expression of human NAT1 in combination with endogenous mouse Nat2 may be a consequence of increased catabolism of folate. PMID:19932120

  15. The histone acetyltransferase hMOF suppresses hepatocellular carcinoma growth.

    PubMed

    Zhang, Jin; Liu, Hui; Pan, Hao; Yang, Yuan; Huang, Gang; Yang, Yun; Zhou, Wei-Ping; Pan, Ze-Ya

    2014-09-26

    Males absent on the first (MOF) is a histone acetyltransferase belongs to the MYST (MOZ, Ybf2/Sas3, Sas2 and TIP60) family. In mammals, MOF plays critical roles in transcription activation by acetylating histone H4K16, a prevalent mark associated with chromatin decondensation. MOF can also acetylate transcription factor p53 on K120, which is important for activation of pro-apoptotic genes; and TIP5, the largest subunit of NoRC, on K633. However, the role of hMOF in hepatocellular carcinoma remains unknown. Here we find that the expression of hMOF is significantly down-regulated in human hepatocellular carcinoma and cell lines. Furthermore, our survival analysis indicates that low hMOF expression predicts poor overall and disease-free survival. We demonstrate that hMOF knockdown promotes hepatocellular carcinoma growth in vitro and in vivo, while hMOF overexpression reduces hepatocellular carcinoma growth in vitro and in vivo. Mechanically, we show that hMOF regulates the expression of SIRT6 and its downstream genes. In summary, our findings demonstrate that hMOF participates in human hepatocellular carcinoma by targeting SIRT6, and hMOF activators may serve as potential drug candidates for hepatocellular carcinoma therapy. PMID:25181338

  16. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis.

    PubMed

    Su, Jiaming; Wang, Fei; Cai, Yong; Jin, Jingji

    2016-01-01

    Changes in chromatin structure and heritably regulating the gene expression by epigenetic mechanisms, such as histone post-translational modification, are involved in most cellular biological processes. Thus, abnormal regulation of epigenetics is implicated in the occurrence of various diseases, including cancer. Human MOF (males absent on the first) is a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs). As a catalytic subunit, MOF can form at least two distinct multiprotein complexes (MSL and NSL) in human cells. Both complexes can acetylate histone H4 at lysine 16 (H4K16); however, the NSL complex possesses broader substrate specificity and can also acetylate histone H4 at lysines 5 and 8 (H4K5 and H4K8), suggesting the complexity of the intracellular functions of MOF. Silencing of MOF in cells leads to genomic instability, inactivation of gene transcription, defective DNA damage repair and early embryonic lethality. Unbalanced MOF expression and its corresponding acetylation of H4K16 have been found in certain primary cancer tissues, including breast cancer, medulloblastoma, ovarian cancer, renal cell carcinoma, colorectal carcinoma, gastric cancer, as well as non-small cell lung cancer. In this review, we provide a brief overview of MOF and its corresponding histone acetylation, introduce recent research findings that link MOF functions to tumorigenesis and speculate on the potential role that may be relevant to tumorigenic pathways. PMID:26784169

  17. Obesity and lipid stress inhibit carnitine acetyltransferase activity.

    PubMed

    Seiler, Sarah E; Martin, Ola J; Noland, Robert C; Slentz, Dorothy H; DeBalsi, Karen L; Ilkayeva, Olga R; An, Jie; Newgard, Christopher B; Koves, Timothy R; Muoio, Deborah M

    2014-04-01

    Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes. PMID:24395925

  18. Substrate Binding and Catalytic Mechanism of Human Choline Acetyltransferase

    SciTech Connect

    Kim,A.; Rylett, J.; Shilton, B.

    2006-01-01

    Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 {angstrom} along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the K{sub M} for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.

  19. Inhibition of aminoglycoside acetyltransferase resistance enzymes by metal salts.

    PubMed

    Li, Yijia; Green, Keith D; Johnson, Brooke R; Garneau-Tsodikova, Sylvie

    2015-07-01

    Aminoglycosides (AGs) are clinically relevant antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria, as well as Mycobacteria. As with all current antibacterial agents, resistance to AGs is an increasing problem. The most common mechanism of resistance to AGs is the presence of AG-modifying enzymes (AMEs) in bacterial cells, with AG acetyltransferases (AACs) being the most prevalent. Recently, it was discovered that Zn(2+) metal ions displayed an inhibitory effect on the resistance enzyme AAC(6')-Ib in Acinetobacter baumannii and Escherichia coli. In this study, we explore a wide array of metal salts (Mg(2+), Cr(3+), Cr(6+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Au(3+) with different counter ions) and their inhibitory effect on a large repertoire of AACs [AAC(2')-Ic, AAC(3)-Ia, AAC(3)-Ib, AAC(3)-IV, AAC(6')-Ib', AAC(6')-Ie, AAC(6')-IId, and Eis]. In addition, we determine the MIC values for amikacin and tobramycin in combination with a zinc pyrithione complex in clinical isolates of various bacterial strains (two strains of A. baumannii, three of Enterobacter cloacae, and four of Klebsiella pneumoniae) and one representative of each species purchased from the American Type Culture Collection. PMID:25941215

  20. Inhibition of Aminoglycoside Acetyltransferase Resistance Enzymes by Metal Salts

    PubMed Central

    Li, Yijia; Green, Keith D.; Johnson, Brooke R.

    2015-01-01

    Aminoglycosides (AGs) are clinically relevant antibiotics used to treat infections caused by both Gram-negative and Gram-positive bacteria, as well as Mycobacteria. As with all current antibacterial agents, resistance to AGs is an increasing problem. The most common mechanism of resistance to AGs is the presence of AG-modifying enzymes (AMEs) in bacterial cells, with AG acetyltransferases (AACs) being the most prevalent. Recently, it was discovered that Zn2+ metal ions displayed an inhibitory effect on the resistance enzyme AAC(6′)-Ib in Acinetobacter baumannii and Escherichia coli. In this study, we explore a wide array of metal salts (Mg2+, Cr3+, Cr6+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Au3+ with different counter ions) and their inhibitory effect on a large repertoire of AACs [AAC(2′)-Ic, AAC(3)-Ia, AAC(3)-Ib, AAC(3)-IV, AAC(6′)-Ib′, AAC(6′)-Ie, AAC(6′)-IId, and Eis]. In addition, we determine the MIC values for amikacin and tobramycin in combination with a zinc pyrithione complex in clinical isolates of various bacterial strains (two strains of A. baumannii, three of Enterobacter cloacae, and four of Klebsiella pneumoniae) and one representative of each species purchased from the American Type Culture Collection. PMID:25941215

  1. Histone acetyltransferase inhibitors block neuroblastoma cell growth in vivo

    PubMed Central

    Gajer, J M; Furdas, S D; Gründer, A; Gothwal, M; Heinicke, U; Keller, K; Colland, F; Fulda, S; Pahl, H L; Fichtner, I; Sippl, W; Jung, M

    2015-01-01

    We have previously described novel histone acetyltransferase (HAT) inhibitors that block neuroblastoma cell growth in vitro. Here we show that two selected pyridoisothiazolone HAT inhibitors, PU139 and PU141, induce cellular histone hypoacetylation and inhibit growth of several neoplastic cell lines originating from different tissues. Broader in vitro selectivity profiling shows that PU139 blocks the HATs Gcn5, p300/CBP-associated factor (PCAF), CREB (cAMP response element-binding) protein (CBP) and p300, whereas PU141 is selective toward CBP and p300. The pan-inhibitor PU139 triggers caspase-independent cell death in cell culture. Both inhibitors block growth of SK-N-SH neuroblastoma xenografts in mice and the PU139 was shown to synergize with doxorubicin in vivo. The latter also reduces histone lysine acetylation in vivo at concentrations that block neoplastic xenograft growth. This is one of the very few reports on hypoacetylating agents with in vivo anticancer activity. PMID:25664930

  2. Energy level properties of 4p{sup 6}4d{sup 3}, 4p{sup 6}4d{sup 2}4f, and 4p{sup 5}4d{sup 4} configurations of the W{sup 35+} ion

    SciTech Connect

    Bogdanovich, P. Kisielius, R.

    2014-11-15

    The ab initio quasirelativistic Hartree–Fock method developed specifically for the calculation of spectroscopic parameters of heavy atoms and highly charged ions was used to derive spectral data for the multicharged tungsten ion W{sup 35+}. The configuration interaction method was applied to include the electron-correlation effects. The relativistic effects were taken into account in the Breit–Pauli approximation for quasirelativistic Hartree–Fock radial orbitals. The energy level spectra, radiative lifetimes τ, and Lande g-factors have been calculated for the 4p{sup 6}4d{sup 3}, 4p{sup 6}4d{sup 2}4f, and 4p{sup 5}4d{sup 4} configurations of the W{sup 35+} ion.

  3. N-Alpha-Acetyltransferases and Regulation of CFTR Expression

    PubMed Central

    Patrick, Anna E.; Hudson, Henry; Thomas, Philip J.

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein. PMID:27182737

  4. Polymorphisms of human N-acetyltransferases and cancer risk.

    PubMed

    Agúndez, José A G

    2008-07-01

    Human arylamine N-acetyltransferases (CoASAc; NAT, EC 2.3.1.5) NAT1 and NAT2 play a key role in the metabolism of drugs and environmental chemicals and in the metabolic activation and detoxification of procarcinogens. Phenotyping analyses have revealed an association between NAT enzyme activities and the risk of developing several forms of cancer. As genotyping procedures have become available for NAT1 and NAT2 gene variations, hundreds of association studies on NAT polymorphisms and cancer risk have been conducted. Here we review the findings obtained from these studies. Evidence for a putative association of NAT1 polymorphism and myeloma, lung and bladder cancer, as well as association of NAT2 polymorphisms with non-Hodgkin lymphoma, liver, colorectal and bladder cancer have been reported. In contrast, no consistent evidence for a relevant association of NAT polymorphisms with brain, head & neck, breast, gastric, pancreatic or prostate cancer have been described. Although preliminary data are available, further well-powered studies are required to fully elucidate the role of NAT1 in most human cancers, and that of NAT2 in astrocytoma, meningioma, esophageal, renal, cervical and testicular cancers, as well as in leukaemia and myeloma. This review discusses controversial findings on cancer risk and putative causes of heterogeneity in the proposed associations, and it identifies topics that require further investigation, particularly mechanisms underlying association of NAT polymorphisms and risk for subsets of cancer patients with specific exposures, putative epistatic contribution of polymorphism for other xenobiotic-metabolising enzymes such as glutathione S-transferases of Cytochrome P450 enzymes, and genetic plus environmental interaction. PMID:18680472

  5. N-Alpha-Acetyltransferases and Regulation of CFTR Expression.

    PubMed

    Vetter, Ali J; Karamyshev, Andrey L; Patrick, Anna E; Hudson, Henry; Thomas, Philip J

    2016-01-01

    The majority of cystic fibrosis (CF)-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) lead to the misfolding, mistrafficking, and degradation of the mutant protein. Inhibition of degradation does not effectively increase the amount of trafficking competent CFTR, but typically leads to increased ER retention of misfolded forms. Thus, the initial off pathway steps occur early in the processing of the protein. To identify proteins that interact with these early forms of CFTR, in vitro crosslink experiments identified cotranslational partners of the nascent chain of the severe misfolded mutant, G85E CFTR. The mutant preferentially interacts with a subunit of an N-alpha-acetyltransferase A. Based on recent reports that acetylation of the N-termini of some N-end rule substrates control their ubiquitination and subsequent degradation, a potential role for this modification in regulation of CFTR expression was assessed. Knockdown experiments identified two complexes, which affect G85E CFTR proteins levels, NatA and NatB. Effects of the knockdowns on mRNA levels, translation rates, and degradation rates established that the two complexes regulate G85E CFTR through two separate mechanisms. NatA acts indirectly by regulating transcription levels and NatB acts through a previously identified, but incompletely understood posttranslational mechanism. This regulation did not effect trafficking of G85E CFTR, which remains retained in the ER, nor did it alter the degradation rate of CFTR. A mutation predicted to inhibit N-terminal acetylation of CFTR, Q2P, was without effect, suggesting neither system acts directly on CFTR. These results contradict the prediction that N-terminal acetylation of CFTR determines its fitness as a proteasome substrate, but rather NatB plays a role in the conformational maturation of CFTR in the ER through actions on an unidentified protein. PMID:27182737

  6. An Acetyltransferase Conferring Tolerance to Toxic Aromatic Amine Chemicals

    PubMed Central

    Martins, Marta; Rodrigues-Lima, Fernando; Dairou, Julien; Lamouri, Aazdine; Malagnac, Fabienne; Silar, Philippe; Dupret, Jean-Marie

    2009-01-01

    Aromatic amines (AA) are a major class of environmental pollutants that have been shown to have genotoxic and cytotoxic potentials toward most living organisms. Fungi are able to tolerate a diverse range of chemical compounds including certain AA and have long been used as models to understand general biological processes. Deciphering the mechanisms underlying this tolerance may improve our understanding of the adaptation of organisms to stressful environments and pave the way for novel pharmaceutical and/or biotechnological applications. We have identified and characterized two arylamine N-acetyltransferase (NAT) enzymes (PaNAT1 and PaNAT2) from the model fungus Podospora anserina that acetylate a wide range of AA. Targeted gene disruption experiments revealed that PaNAT2 was required for the growth and survival of the fungus in the presence of toxic AA. Functional studies using the knock-out strains and chemically acetylated AA indicated that tolerance of P. anserina to toxic AA was due to the N-acetylation of these chemicals by PaNAT2. Moreover, we provide proof-of-concept remediation experiments where P. anserina, through its PaNAT2 enzyme, is able to detoxify the highly toxic pesticide residue 3,4-dichloroaniline in experimentally contaminated soil samples. Overall, our data show that a single xenobiotic-metabolizing enzyme can mediate tolerance to a major class of pollutants in a eukaryotic species. These findings expand the understanding of the role of xenobiotic-metabolizing enzyme and in particular of NATs in the adaptation of organisms to their chemical environment and provide a basis for new systems for the bioremediation of contaminated soils. PMID:19416981

  7. Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase.

    PubMed

    Dopkins, Brandon J; Tipton, Peter A; Thoden, James B; Holden, Hazel M

    2016-08-16

    Glucosamine/glucosaminide N-acetyltransferase or GlmA catalyzes the transfer of an acetyl group from acetyl CoA to the primary amino group of glucosamine. The enzyme from Clostridium acetobutylicum is thought to be involved in cell wall rescue. In addition to glucosamine, GlmA has been shown to function on di- and trisaccharides of glucosamine as well. Here we present a structural and kinetic analysis of the enzyme. For this investigation, eight structures were determined to resolutions of 2.0 Å or better. The overall three-dimensional fold of GlmA places it into the tandem GNAT superfamily. Each subunit of the dimer folds into two distinct domains which exhibit high three-dimensional structural similarity. Whereas both domains bind acetyl CoA, it is the C-terminal domain that is catalytically competent. On the basis of the various structures determined in this investigation, two amino acid residues were targeted for further study: Asp 287 and Tyr 297. Although their positions in the active site suggested that they may play key roles in catalysis by functioning as active site bases and acids, respectively, this was not borne out by characterization of the D287N and Y297F variants. The kinetic properties revealed that both residues were important for substrate binding but had no critical roles as acid/base catalysts. Kinetic analyses also indicated that GlmA follows an ordered mechanism with acetyl CoA binding first followed by glucosamine. The product N-acetylglucosamine is then released prior to CoA. The investigation described herein provides significantly new information on enzymes belonging to the tandem GNAT superfamily. PMID:27348258

  8. Characterization of a Trypanosoma cruzi acetyltransferase: cellular location, activity and structure.

    PubMed

    Ochaya, Stephen; Respuela, Patricia; Simonsson, Maria; Saraswathi, Abhiman; Branche, Carole; Lee, Jennifer; Búa, Jacqueline; Nilsson, Daniel; Aslund, Lena; Bontempi, Esteban J; Andersson, Björn

    2007-04-01

    Trypanosomatids are widespread parasites that cause three major tropical diseases. In trypanosomatids, as in most other organisms, acetylation is a common protein modification that is important in multiple, diverse processes. This paper describes a new member of the Trypanosoma cruzi acetyltransferase family. The gene is single copy and orthologs are also present in the other two sequenced trypanosomatids, Trypanosoma brucei and Leishmania major. This protein (TcAT-1) has the essential motifs present in members of the GCN5-related acetyltransferase (GNAT) family, as well as an additional motif also found in some enzymes from plant and animal species. The protein is evolutionarily more closely related to this group of enzymes than to histone acetyltransferases. The native protein has a cytosolic cellular location and is present in all three life-cycle stages of the parasite. The recombinant protein was shown to have autoacetylation enzymatic activity. PMID:17270289

  9. Entrainment of the circadian rhythm in the rat pineal N-acetyltransferase activity by prolonged periods of light.

    PubMed

    Illnerová, H; Vanĕcek, J

    1987-08-01

    Entertainment of the circadian rhythm in the pineal N-acetyltranferase activity by prolonged periods of light was studied in rats synchronized with a light:dark regime of 12:12 h by observing phase-shifts in rhythm after delays in switching off the light in the evening or after bringing forward of the morning onset of light. When rats were subjected to delays in switching off the light of up to 10 h and then were released into darkness, phase-delays of the evening N-acetyltransferase rise during the same night corresponded roughly to delays in the light switch off. However, phase-delays of the morning decline were much smaller. After a delay in the evening switch off of 11 h, no N-acetyltransferase rhythm was found in the subsequent darkness. The evening N-acetyltransferase rise was phase-delayed by 6.2 h at most 1 day after delays. Phase-delays of the morning N-acetyltransferase decline were shorter than phase-delays of the N-acetyltransferase rise by only 0.7 h to 0.9 h at most. Hence, 1 day after delays in the evening switch off, the period of the high night N-acetyltransferase activity may be shortened only slightly. The N-acetyltransferase rhythm was abolished only after a 12 h delay in switching off the light. Rats were subjected to a bringing forward of the morning light onset and then were released into darkness 4 h before the usual switch off of light. In the following night, the morning N-acetyltransferase decline, but not the evening rise, was phase advanced considerably. Moreover, when the onset of light was brought forward to before midnight, the N-acetyltransferase rise was even phase-delayed. Hence, 1 day after bringing forward the morning onset of light, the period of the high night N-acetyltransferase activity may be drastically reduced. When rats were subjected to a 4 h light pulse around midnight and then released into darkness, the N-acetyltransferase rhythm in the next night was abolished. The data are discussed in terms of a two

  10. AAC(3)-XI, a New Aminoglycoside 3-N-Acetyltransferase from Corynebacterium striatum

    PubMed Central

    Galimand, Marc; Fishovitz, Jennifer; Lambert, Thierry; Barbe, Valérie; Zajicek, Jaroslav

    2015-01-01

    Corynebacterium striatum BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related N-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other Corynebacterium spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in Actinomycetales. The gene was cloned in Escherichia coli, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-N-acetyltransferase type XI. PMID:26149994

  11. Comparative investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated. The NAT1 gene of Gibberella moniliformis was the first NAT cloned and characterized from fun...

  12. Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...

  13. Unintended Consequences: High phosphinothricin acetyltransferase activity causes reduced fitness in barley

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Selectable markers used in plant transformation, such as phosphinothricin acetyltransferase (PAT) derived from the bar gene, have been chosen for selection efficacy as well as for the absence of pleiotropic effects. Recent research has suggested that expression of bar in Arabidopsis affects the tran...

  14. AAC(3)-XI, a new aminoglycoside 3-N-acetyltransferase from Corynebacterium striatum.

    PubMed

    Galimand, Marc; Fishovitz, Jennifer; Lambert, Thierry; Barbe, Valérie; Zajicek, Jaroslav; Mobashery, Shahriar; Courvalin, Patrice

    2015-09-01

    Corynebacterium striatum BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related N-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other Corynebacterium spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in Actinomycetales. The gene was cloned in Escherichia coli, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-N-acetyltransferase type XI. PMID:26149994

  15. Phylogenetic and biological investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and eukaryotic organisms. The role of NATs in fungal biology has only recently been investigated. The NAT1 (FDB2) gene of Fusarium verticillioides was the first NAT cloned and character...

  16. Genetic Variation at the N-acetyltransferase (NAT) Genes in Global Populations

    EPA Science Inventory

    Functional variability at the N-acetyltransferase (NAT) genes is associated with adverse drug reactions and cancer susceptibility in humans. Previous studies of small sets of ethnic groups have indicated that the NAT genes have high levels of amino acid variation that differ in f...

  17. Severe congenital myasthenia gravis of the presynaptic type with choline acetyltransferase mutation in a Chinese infant with respiratory failure.

    PubMed

    Yeung, Wai L; Lam, Ching W; Fung, Lai W E; Hon, Kam L E; Ng, Pak C

    2009-01-01

    We report a severe case of congenital myasthenia gravis in a Chinese newborn who presented with complete ptosis, severe hypotonia, dysphagia and respiratory insufficiency with recurrent apnea that required mechanical ventilatory support since birth. Routine neurophysiologic studies, including the 3-Hz repetitive stimulation test and electromyogram were normal. Neostigmine and edrophonium tests were also negative. However, decremental response to 3-Hz stimulation became apparent after depleting the muscles with trains of 10-Hz stimuli for 10 min. The infant was subsequently confirmed to have heterozygous mutations in the choline acetyltransferase genes, p.T553N and p.S704P. Both missense mutations are novel mutations. The child remained on positive pressure ventilation at 3 years of age despite treatment with high-dose anticholinesterase. This case highlights the difficulty of making an early diagnosis based on clinical presentation and routine electrophysiologic tests, especially when neonatologists are not familiar with this condition. Further, as there are different genetic defects causing different types of congenital myasthenia gravis, anticholinesterase therapy may be beneficial to some but detrimental to others. Therefore, the exact molecular diagnosis is an important guide to therapy. A high index of suspicion coupled with extended electrodiagnostic tests in clinically suspected patients will ensure the selection of appropriate genetic molecular study for confirming the diagnosis. PMID:18797171

  18. Nickel and cobalt resistance engineered in Escherichia coli by overexpression of serine acetyltransferase from the nickel hyperaccumulator plant Thlaspi goesingense.

    PubMed

    Freeman, John L; Persans, Michael W; Nieman, Ken; Salt, David E

    2005-12-01

    The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-L-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel. PMID:16332856

  19. Nickel and Cobalt Resistance Engineered in Escherichia coli by Overexpression of Serine Acetyltransferase from the Nickel Hyperaccumulator Plant Thlaspi goesingense

    PubMed Central

    Freeman, John L.; Persans, Michael W.; Nieman, Ken; Salt, David E.

    2005-01-01

    The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproduction of O-acetyl-l-serine, the product of serine acetyltransferase and a positive regulator of the cysteine regulon. Nickel in the serine acetyltransferase-overexpressing strains is not detoxified by coordination or precipitation with sulfur, suggesting that glutathione is involved in reducing the oxidative damage imposed by nickel. PMID:16332856

  20. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    SciTech Connect

    Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory {Greg} B; Escalante-Semerena, Jorge C

    2012-01-01

    Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

  1. Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145.

    PubMed Central

    Rende-Fournier, R; Leclercq, R; Galimand, M; Duval, J; Courvalin, P

    1993-01-01

    Enterococcus faecium BM4145, a clinical isolate from urine, was resistant to streptogramin group A antibiotics by inactivation. The strain harbored a plasmid containing a gene, satA, responsible for this resistance; this gene was cloned and sequenced. It encoded SatA, a protein deduced to be 23,634 Da in mass and homologous with a new family of chloramphenicol acetyltransferases described in Agrobacterium tumefaciens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The similarity of SatA to other acetyltransferases, LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) from E. coli, and to two putative acetyltransferases, NodL from Rhizobium leguminosarum and Urf1 from E. coli, was also observed in a region considered to be the enzyme's active site. Acetylation experiments indicated that acetyl coenzyme A was necessary for SatA activity and that a single acetylated derivative of pristinamycin IIA was produced. Other members of the streptogramin A group such as virginiamycin M and RP54476 were also substrates for the enzyme. We conclude that resistance to the streptogramin A group of antibiotics in E. faecium BM4145 is due to acetylation by an enzyme related to the novel chloramphenicol acetyltransferase family. Images PMID:8257133

  2. Kinesin-II Is Required for Axonal Transport of Choline Acetyltransferase in Drosophila

    PubMed Central

    Ray, Krishanu; Perez, Sharon E.; Yang, Zhaohuai; Xu, Jenny; Ritchings, Bruce W.; Steller, Hermann; Goldstein, Lawrence S.B.

    1999-01-01

    KLP64D and KLP68D are members of the kinesin-II family of proteins in Drosophila. Immunostaining for KLP68D and ribonucleic acid in situ hybridization for KLP64D demonstrated their preferential expression in cholinergic neurons. KLP68D was also found to accumulate in cholinergic neurons in axonal obstructions caused by the loss of kinesin light chain. Mutations in the KLP64D gene cause uncoordinated sluggish movement and death, and reduce transport of choline acetyltransferase from cell bodies to the synapse. The inviability of KLP64D mutations can be rescued by expression of mammalian KIF3A. Together, these data suggest that kinesin-II is required for the axonal transport of a soluble enzyme, choline acetyltransferase, in a specific subset of neurons in Drosophila. Furthermore, the data lead to the conclusion that the cargo transport requirements of different classes of neurons may lead to upregulation of specific pathways of axonal transport. PMID:10545496

  3. Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

    SciTech Connect

    Maes, Dominique Crabeel, Marjolaine; Van de Weerdt, Cécile; Martial, Joseph; Peeters, Eveline; Charlier, Daniël; Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid

    2006-12-01

    A study on the crystallization of ornithine acetyltransferase from yeast, catalysing the fifth step in microbial arginine synthesis, is presented. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either batch or hanging-drop techniques. A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å.

  4. Application of a High-throughput Fluorescent Acetyltransferase Assay to Identify Inhibitors of Homocitrate Synthase

    PubMed Central

    Bulfer, Stacie L.; McQuade, Thomas J.; Larsen, Martha J.; Trievel, Raymond C.

    2011-01-01

    Homocitrate synthase (HCS) catalyzes the first step of L-lysine biosynthesis in fungi by condensing acetyl-Coenzyme A and 2-oxoglutarate to form 3R-homocitrate and Coenzyme A. Due to its conservation in pathogenic fungi, HCS has been proposed as a candidate for antifungal drug design. Here we report the development and validation of a robust, fluorescent assay for HCS that is amenable to high-throughput screening for inhibitors in vitro. Using this assay, Schizosaccharomyces pombe HCS was screened against a diverse library of ~41,000 small molecules. Following confirmation, counter screens, and dose-response analysis, we prioritized over 100 compounds for further in vitro and in vivo analysis. This assay can be readily adapted to screen for small molecule modulators of other acyl-CoA-dependent acyltransferases or enzymes that generate a product with a free sulfhydryl group, including histone acetyltransferases, aminoglycoside N-acetyltransferases, thioesterases and enzymes involved in lipid metabolism. PMID:21073853

  5. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

    SciTech Connect

    Bame, K.J.

    1986-01-01

    Acetyl-CoA:..cap alpha..-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal ..cap alpha..-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from (/sup 3/H)CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with (/sup 3/H)acetyl-CoA. The acetyl group can be transferred to glucosamine, forming (/sup 3/H)N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism.

  6. The histone acetyltransferase MOF overexpression blunts cardiac hypertrophy by targeting ROS in mice.

    PubMed

    Qiao, Weiwei; Zhang, Weili; Gai, Yusheng; Zhao, Lan; Fan, Juexin

    2014-06-13

    Imbalance between histone acetylation/deacetylation critically participates in the expression of hypertrophic fetal genes and development of cardiac hypertrophy. While histone deacetylases play dual roles in hypertrophy, current evidence reveals that histone acetyltransferase such as p300 and PCAF act as pro-hypertrophic factors. However, it remains elusive whether some histone acetyltransferases can prevent the development of hypertrophy. Males absent on the first (MOF) is a histone acetyltransferase belonging to the MYST (MOZ, Ybf2/Sas3, Sas2 and TIP60) family. Here in this study, we reported that MOF expression was down-regulated in failing human hearts and hypertrophic murine hearts at protein and mRNA levels. To evaluate the roles of MOF in cardiac hypertrophy, we generated cardiac-specific MOF transgenic mice. MOF transgenic mice did not show any differences from their wide-type littermates at baseline. However, cardiac-specific MOF overexpression protected mice from transverse aortic constriction (TAC)-induced cardiac hypertrophy, with reduced radios of heart weight (HW)/body weight (BW), lung weight/BW and HW/tibia length, decreased left ventricular wall thickness and increased fractional shortening. We also observed lower expression of hypertrophic fetal genes in TAC-challenged MOF transgenic mice compared with that of wide-type mice. Mechanically, MOF overexpression increased the expression of Catalase and MnSOD, which blocked TAC-induced ROS and ROS downstream c-Raf-MEK-ERK pathway that promotes hypertrophy. Taken together, our findings identify a novel anti-hypertrophic role of MOF, and MOF is the first reported anti-hypertrophic histone acetyltransferase. PMID:24802406

  7. RNA Cytidine Acetyltransferase of Small-Subunit Ribosomal RNA: Identification of Acetylation Sites and the Responsible Acetyltransferase in Fission Yeast, Schizosaccharomyces pombe

    PubMed Central

    Taoka, Masato; Ishikawa, Daisuke; Nobe, Yuko; Ishikawa, Hideaki; Yamauchi, Yoshio; Terukina, Goro; Nakayama, Hiroshi; Hirota, Kouji; Takahashi, Nobuhiro; Isobe, Toshiaki

    2014-01-01

    The eukaryotic small-subunit (SSU) ribosomal RNA (rRNA) has two evolutionarily conserved acetylcytidines. However, the acetylation sites and the acetyltransferase responsible for the acetylation have not been identified. We performed a comprehensive MS-based analysis covering the entire sequence of the fission yeast, Schizosaccharomyces pombe, SSU rRNA and identified two acetylcytidines at positions 1297 and 1815 in the 3′ half of the rRNA. To identify the enzyme responsible for the cytidine acetylation, we searched for an S. pombe gene homologous to TmcA, a bacterial tRNA N-acetyltransferase, and found one potential candidate, Nat10. A temperature-sensitive strain of Nat10 with a mutation in the Walker A type ATP-binding motif abolished the cytidine acetylation in SSU rRNA, and the wild-type Nat10 supplemented to this strain recovered the acetylation, providing evidence that Nat10 is necessary for acetylation of SSU rRNA. The Nat10 mutant strain showed a slow-growth phenotype and was defective in forming the SSU rRNA from the precursor RNA, suggesting that cytidine acetylation is necessary for ribosome assembly. PMID:25402480

  8. Conformational Flexibility and Subunit Arrangement of the Modular Yeast Spt-Ada-Gcn5 Acetyltransferase Complex*

    PubMed Central

    Setiaputra, Dheva; Ross, James D.; Lu, Shan; Cheng, Derrick T.; Dong, Meng-Qiu; Yip, Calvin K.

    2015-01-01

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility. PMID:25713136

  9. Conformational flexibility and subunit arrangement of the modular yeast Spt-Ada-Gcn5 acetyltransferase complex.

    PubMed

    Setiaputra, Dheva; Ross, James D; Lu, Shan; Cheng, Derrick T; Dong, Meng-Qiu; Yip, Calvin K

    2015-04-17

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is a highly conserved, 19-subunit histone acetyltransferase complex that activates transcription through acetylation and deubiquitination of nucleosomal histones in Saccharomyces cerevisiae. Because SAGA has been shown to display conformational variability, we applied gradient fixation to stabilize purified SAGA and systematically analyzed this flexibility using single-particle EM. Our two- and three-dimensional studies show that SAGA adopts three major conformations, and mutations of specific subunits affect the distribution among these. We also located the four functional modules of SAGA using electron microscopy-based labeling and transcriptional activator binding analyses and show that the acetyltransferase module is localized in the most mobile region of the complex. We further comprehensively mapped the subunit interconnectivity of SAGA using cross-linking mass spectrometry, revealing that the Spt and Taf subunits form the structural core of the complex. These results provide the necessary restraints for us to generate a model of the spatial arrangement of all SAGA subunits. According to this model, the chromatin-binding domains of SAGA are all clustered in one face of the complex that is highly flexible. Our results relate information of overall SAGA structure with detailed subunit level interactions, improving our understanding of its architecture and flexibility. PMID:25713136

  10. MOZ and MORF acetyltransferases: Molecular interaction, animal development and human disease.

    PubMed

    Yang, Xiang-Jiao

    2015-08-01

    Lysine residues are subject to many forms of covalent modification and one such modification is acetylation of the ε-amino group. Initially identified on histone proteins in the 1960s, lysine acetylation is now considered as an important form of post-translational modification that rivals phosphorylation. However, only about a dozen of human lysine acetyltransferases have been identified. Among them are MOZ (monocytic leukemia zinc finger protein; a.k.a. MYST3 and KAT6A) and its paralog MORF (a.k.a. MYST4 and KAT6B). Although there is a distantly related protein in Drosophila and sea urchin, these two enzymes are vertebrate-specific. They form tetrameric complexes with BRPF1 (bromodomain- and PHD finger-containing protein 1) and two small non-catalytic subunits. These two acetyltransferases and BRPF1 play key roles in various developmental processes; for example, they are important for development of hematopoietic and neural stem cells. The human KAT6A and KAT6B genes are recurrently mutated in leukemia, non-hematologic malignancies, and multiple developmental disorders displaying intellectual disability and various other abnormalities. In addition, the BRPF1 gene is mutated in childhood leukemia and adult medulloblastoma. Therefore, these two acetyltransferases and their partner BRPF1 are important in animal development and human disease. PMID:25920810

  11. Assay for peptidoglycan O-acetyltransferase: a potential new antibacterial target.

    PubMed

    Moynihan, Patrick J; Clarke, Anthony J

    2013-08-15

    The O-acetylation of peptidoglycan occurs at the C-6 hydroxyl group of muramoyl residues in many human pathogens, both gram positive and gram negative, such as Staphylococcus aureus and species of Campylobacter, Helicobacter, Neisseria, and Bacillus, including Bacillus anthracis. The process is a maturation event being catalyzed either by integral membrane O-acetylpeptidoglycan transferase (Oat) of gram-positive bacteria or by a two-component peptidoglycan O-acetyltransferase system (PatA/PatB) in gram-negative cells. Here, we describe the development of the first in vitro assay for any peptidoglycan O-acetyltransferase using PatB from Neisseria gonorrhoeae as the model enzyme. This assay is based on the use of chromogenic p-nitrophenyl acetate as the donor substrate and chitooligosaccharides as model acceptor substrates in place of peptidoglycan. The identity of the O-acetylated chitooligosaccharides was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rates of transacetylations were determined spectrophotometrically by monitoring p-nitrophenol release after accounting for both spontaneous and enzyme-catalyzed hydrolysis of the acetate donor. Conditions were established for use of the assay in microtiter plate format, and its applicability was demonstrated by determining the first Michaelis-Menten kinetic parameters for PatB. The assay is readily amenable for application in the high-throughput screening for potential inhibitors of peptidoglycan O-acetyltransferases that may prove to be leads for novel classes of antibiotics. PMID:23660013

  12. Comprehensive analysis of interacting proteins and genome-wide location studies of the Sas3-dependent NuA3 histone acetyltransferase complex

    PubMed Central

    Vicente-Muñoz, Sara; Romero, Paco; Magraner-Pardo, Lorena; Martinez-Jimenez, Celia P.; Tordera, Vicente; Pamblanco, Mercè

    2014-01-01

    Histone acetylation affects several aspects of gene regulation, from chromatin remodelling to gene expression, by modulating the interplay between chromatin and key transcriptional regulators. The exact molecular mechanism underlying acetylation patterns and crosstalk with other epigenetic modifications requires further investigation. In budding yeast, these epigenetic markers are produced partly by histone acetyltransferase enzymes, which act as multi-protein complexes. The Sas3-dependent NuA3 complex has received less attention than other histone acetyltransferases (HAT), such as Gcn5-dependent complexes. Here, we report our analysis of Sas3p-interacting proteins using tandem affinity purification (TAP), coupled with mass spectrometry. This analysis revealed Pdp3p, a recently described component of NuA3, to be one of the most abundant Sas3p-interacting proteins. The PDP3 gene, was TAP-tagged and protein complex purification confirmed that Pdp3p co-purified with the NuA3 protein complex, histones, and several transcription-related and chromatin remodelling proteins. Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa. We also provide evidence that Sas3p cannot substitute Gcn5p in acetylation of lysine 9 in histone H3 in vivo. Genome-wide occupancy of Sas3p using ChIP-on-chip tiled microarrays showed that Sas3p was located preferentially within the 5′-half of the coding regions of target genes, indicating its probable involvement in the transcriptional elongation process. Hence, this work further characterises the function and regulation of the NuA3 complex by identifying novel post-translational modifications in Pdp3p, additional Pdp3p-co-purifying chromatin regulatory proteins involved in chromatin-modifying complex dynamics and gene regulation, and a subset of genes whose transcriptional elongation is controlled by this complex. PMID:25473596

  13. New N-Acetyltransferase Fold in the Structure and Mechanism of the Phosphonate Biosynthetic Enzyme FrbF

    SciTech Connect

    Bae, Brian; Cobb, Ryan E.; DeSieno, Matthew A.; Zhao, Huimin; Nair, Satish K.

    2015-10-15

    The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 {angstrom} resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.

  14. New N-acetyltransferase fold in the structure and mechanism of the phosphonate biosynthetic enzyme FrbF.

    PubMed

    Bae, Brian; Cobb, Ryan E; DeSieno, Matthew A; Zhao, Huimin; Nair, Satish K

    2011-10-14

    The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5'-monophosphate-3-aminopropylphosphonate and cytidine 5'-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 Å resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098. PMID:21865168

  15. Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases

    PubMed Central

    Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A.; Zhang, Zhiguo

    2015-01-01

    During DNA replication, nucleosomes ahead of replication forks are disassembled to accommodate replication machinery. Following DNA replication, nucleosomes are then reassembled onto replicated DNA using both parental and newly synthesized histones. This process, termed DNA replication-coupled nucleosome assembly (RCNA), is critical for maintaining genome integrity and for the propagation of epigenetic information, dysfunctions of which have been implicated in cancers and aging. In recent years, it has been shown that RCNA is carefully orchestrated by a series of histone modifications, histone chaperones and histone-modifying enzymes. Interestingly, many features of RCNA are also found in processes involving DNA replication-independent nucleosome assembly like histone exchange and gene transcription. In yeast, histone H3 lysine K56 acetylation (H3K56ac) is found in newly synthesized histone H3 and is critical for proper nucleosome assembly and for maintaining genomic stability. The histone acetyltransferase (HAT) regulator of Ty1 transposition 109 (Rtt109) is the sole enzyme responsible for H3K56ac in yeast. Much research has centered on this particular histone modification and histone-modifying enzyme. This Critical Review summarizes much of our current understanding of nucleosome assembly and highlights many important insights learned from studying Rtt109 HATs in fungi. We highlight some seminal features in nucleosome assembly conserved in mammalian systems and describe some of the lingering questions in the field. Further studying fungal and mammalian chromatin assembly may have important public health implications, including deeper understandings of human cancers and aging as well as the pursuit of novel anti-fungal therapies. PMID:25365782

  16. New substrate analogues of human serotonin N-acetyltransferase produce in situ specific and potent inhibitors.

    PubMed

    Ferry, Gilles; Ubeaud, Caroline; Mozo, Julien; Péan, Christophe; Hennig, Philippe; Rodriguez, Marianne; Scoul, Catherine; Bonnaud, Anne; Nosjean, Olivier; Galizzi, Jean-Pierre; Delagrange, Philippe; Renard, Pierre; Volland, Jean-Paul; Yous, Said; Lesieur, Daniel; Boutin, Jean A

    2004-01-01

    Melatonin is synthesized by an enzymatic pathway, in which arylalkylamine (serotonin) N-acetyltransferase catalyzes the rate-limiting step. A previous study reported the discovery of bromoacetyltryptamine (BAT), a new type of inhibitor of this enzyme. This compound is the precursor of a potent bifunctional inhibitor (analogue of the transition state), capable of interfering with both the substrate and the cosubstrate binding sites. This inhibitor is biosynthesized by the enzyme itself in the presence of free coenzyme A. In the present report, we describe the potency of new N-halogenoacetyl derivatives leading to a strong in situ inhibition of serotonin N-acetyltransferase. The new concept behind the mechanism of action of these precursors was studied by following the biosynthesis of the inhibitor from tritiated-BAT in a living cell. The fate of tritiated-phenylethylamine (PEA), a natural substrate of the enzyme, in the presence or absence of [(3)H]BAT was also followed, leading to their incorporation into the reaction product or the inhibitor (N-acetyl[(3)H]PEA and coenzyme A-S[(3)H]acetyltryptamine, respectively). The biosynthesis of this bifunctional inhibitor derived from BAT was also followed by nuclear magnetic resonance during its catalytic production by the pure enzyme. In a similar manner we studied the production of another inhibitor generated from N-[2-(7-hydroxynaphth-1-yl)ethyl]bromoacetamide. New derivatives were also screened for their capacity to inhibit a purified enzyme, in addition to enzyme overexpressed in a cellular model. Some of these compounds proved to be extremely potent, with IC(50)s of approximately 30 nM. As these compounds, by definition, closely resemble the natural substrates of arylalkylamine N-acetyltransferase, we also show that they are potent ligands at the melatonin receptors. Nevertheless, these inhibitors form a series of pharmacological tools that could be used to understand more closely the inhibition of pineal melatonin

  17. The molecular structure of ornithine acetyltransferase from Mycobacterium tuberculosis bound to ornithine, a competitive inhibitor.

    PubMed

    Sankaranarayanan, Ramasamy; Cherney, Maia M; Garen, Craig; Garen, Grace; Niu, Chunying; Yuan, Marshall; James, Michael N G

    2010-04-01

    Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to L-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 A resolutions, respectively. ORN is a competitive inhibitor of this enzyme against L-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O(gamma 1) of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN. PMID:20184895

  18. Mutations in HISTONE ACETYLTRANSFERASE1 affect sugar response and gene expression in Arabidopsis

    PubMed Central

    Heisel, Timothy J.; Li, Chun Yao; Grey, Katia M.; Gibson, Susan I.

    2013-01-01

    Nutrient response networks are likely to have been among the first response networks to evolve, as the ability to sense and respond to the levels of available nutrients is critical for all organisms. Although several forward genetic screens have been successful in identifying components of plant sugar-response networks, many components remain to be identified. Toward this end, a reverse genetic screen was conducted in Arabidopsis thaliana to identify additional components of sugar-response networks. This screen was based on the rationale that some of the genes involved in sugar-response networks are likely to be themselves sugar regulated at the steady-state mRNA level and to encode proteins with activities commonly associated with response networks. This rationale was validated by the identification of hac1 mutants that are defective in sugar response. HAC1 encodes a histone acetyltransferase. Histone acetyltransferases increase transcription of specific genes by acetylating histones associated with those genes. Mutations in HAC1 also cause reduced fertility, a moderate degree of resistance to paclobutrazol and altered transcript levels of specific genes. Previous research has shown that hac1 mutants exhibit delayed flowering. The sugar-response and fertility defects of hac1 mutants may be partially explained by decreased expression of AtPV42a and AtPV42b, which are putative components of plant SnRK1 complexes. SnRK1 complexes have been shown to function as central regulators of plant nutrient and energy status. Involvement of a histone acetyltransferase in sugar response provides a possible mechanism whereby nutritional status could exert long-term effects on plant development and metabolism. PMID:23882272

  19. Activation Domain-Specific and General Transcription Stimulation by Native Histone Acetyltransferase Complexes

    PubMed Central

    Ikeda, Keiko; Steger, David J.; Eberharter, Anton; Workman, Jerry L.

    1999-01-01

    Recent progress in identifying the catalytic subunits of histone acetyltransferase (HAT) complexes has implicated histone acetylation in the regulation of transcription. Here, we have analyzed the function of two native yeast HAT complexes, SAGA (Spt-Ada-Gcn5 Acetyltransferase) and NuA4 (nucleosome acetyltransferase of H4), in activating transcription from preassembled nucleosomal array templates in vitro. Each complex was tested for the ability to enhance transcription driven by GAL4 derivatives containing either acidic, glutamine-rich, or proline-rich activation domains. On nucleosomal array templates, the SAGA complex selectively stimulates transcription driven by the VP16 acidic activation domain in an acetyl coenzyme A-dependent manner. In contrast, the NuA4 complex facilitates transcription mediated by any of the activation domains tested if allowed to preacetylate the nucleosomal template, indicating a general stimulatory effect of histone H4 acetylation. However, when the extent of acetylation by NuA4 is limited, the complex also preferentially stimulates VP16-driven transcription. SAGA and NuA4 interact directly with the VP16 activation domain but not with a glutamine-rich or proline-rich activation domain. These data suggest that recruitment of the SAGA and NuA4 HAT complexes by the VP16 activation domain contributes to HAT-dependent activation. In addition, extensive H4/H2B acetylation by NuA4 leads to a general activation of transcription, which is independent of activator-NuA4 interactions. PMID:9858608

  20. Cinnamoyl compounds as simple molecules that inhibit p300 histone acetyltransferase.

    PubMed

    Costi, Roberta; Di Santo, Roberto; Artico, Marino; Miele, Gaetano; Valentini, Paola; Novellino, Ettore; Cereseto, Anna

    2007-04-19

    Cinnamoly compounds 1a-c and 2a-d were designed, synthesized, and in vitro tested as p300 inhibitors. At different degrees, all tested compounds were proven to inactivate p300, particularly, derivative 2c was the most active inhibitor, also showing high specificity for p300 as compared to other histone acetyltransferases. Most notably, 2c showed anti-acetylase activity in mammalian cells. These compounds represent a new class of synthetic inhibitors of p300, characterized by simple chemical structures. PMID:17348637

  1. Choline Acetyltransferase Activity in Striatum of Neonatal Rats Increased by Nerve Growth Factor

    NASA Astrophysics Data System (ADS)

    Mobley, William C.; Rutkowski, J. Lynn; Tennekoon, Gihan I.; Buchanan, Karen; Johnston, Michael V.

    1985-07-01

    Some neurodegenerative disorders may be caused by abnormal synthesis or utilization of trophic molecules required to support neuronal survival. A test of this hypothesis requires that trophic agents specific for the affected neurons be identified. Cholinergic neurons in the corpus striatum of neonatal rats were found to respond to intracerebroventricular administration of nerve growth factor with prominent, dose-dependent, selective increases in choline acetyltransferase activity. Cholinergic neurons in the basal forebrain also respond to nerve growth factor in this way. These actions of nerve growth factor may indicate its involvement in the normal function of forebrain cholinergic neurons as well as in neurodegenerative disorders involving such cells.

  2. Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

    PubMed Central

    Maes, Dominique; Crabeel, Marjolaine; Van de Weerdt, Cécile; Martial, Joseph; Peeters, Eveline; Charlier, Daniël; Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid

    2006-01-01

    A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 Å, and a data set was collected to 2.76 Å. PMID:17142921

  3. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency. PMID:25749943

  4. Synthesis of 4'-aminopantetheine and derivatives to probe aminoglycoside N-6'-acetyltransferase.

    PubMed

    Yan, Xuxu; Akinnusi, T Olukayode; Larsen, Aaron T; Auclair, Karine

    2011-03-01

    A convenient synthesis of 4'-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4'-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6'-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA. PMID:21225062

  5. Synthesis of 4′-aminopantetheine and derivatives to probe aminoglycoside N-6′-acetyltransferase

    PubMed Central

    Yan, Xuxu; Akinnusi, T. Olukayode; Larsen, Aaron T.; Auclair, Karine

    2011-01-01

    Summary A convenient synthesis of 4′-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4′-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6′-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual flexibility and ability to adopt different folds, this enzyme is highly specific for AcCoA. PMID:21225062

  6. Biochemical analysis and structure determination of bacterial acetyltransferases responsible for the biosynthesis of UDP-N,N'-diacetylbacillosamine.

    PubMed

    Morrison, Michael J; Imperiali, Barbara

    2013-11-01

    UDP-N,N'-diacetylbacillosamine (UDP-diNAcBac) is a unique carbohydrate produced by a number of bacterial species and has been implicated in pathogenesis. The terminal step in the formation of this important bacterial sugar is catalyzed by an acetyl-CoA (AcCoA)-dependent acetyltransferase in both N- and O-linked protein glycosylation pathways. This bacterial acetyltransferase is a member of the left-handed β-helix family and forms a homotrimer as the functional unit. Whereas previous endeavors have focused on the Campylobacter jejuni acetyltransferase (PglD) from the N-linked glycosylation pathway, structural characterization of the homologous enzymes in the O-linked glycosylation pathways is lacking. Herein, we present the apo-crystal structures of the acetyltransferase domain (ATD) from the bifunctional enzyme PglB (Neisseria gonorrhoeae) and the full-length acetyltransferase WeeI (Acinetobacter baumannii). Additionally, a PglB-ATD structure was solved in complex with AcCoA. Surprisingly, this structure reveals a contrasting binding mechanism for this substrate when compared with the AcCoA-bound PglD structure. A comparison between these findings and the previously solved PglD crystal structures illustrates a dichotomy among N- and O-linked glycosylation pathway enzymes. Based upon these structures, key residues in the UDP-4-amino and AcCoA binding pockets were mutated to determine their effect on binding and catalysis in PglD, PglB-ATD, and WeeI. Last, a phylogenetic analysis of the aforementioned acetyltransferases was employed to illuminate the diversity among N- and O-linked glycosylation pathway enzymes. PMID:24064219

  7. Biochemical Analysis and Structure Determination of Bacterial Acetyltransferases Responsible for the Biosynthesis of UDP-N,N′-Diacetylbacillosamine*

    PubMed Central

    Morrison, Michael J.; Imperiali, Barbara

    2013-01-01

    UDP-N,N′-diacetylbacillosamine (UDP-diNAcBac) is a unique carbohydrate produced by a number of bacterial species and has been implicated in pathogenesis. The terminal step in the formation of this important bacterial sugar is catalyzed by an acetyl-CoA (AcCoA)-dependent acetyltransferase in both N- and O-linked protein glycosylation pathways. This bacterial acetyltransferase is a member of the left-handed β-helix family and forms a homotrimer as the functional unit. Whereas previous endeavors have focused on the Campylobacter jejuni acetyltransferase (PglD) from the N-linked glycosylation pathway, structural characterization of the homologous enzymes in the O-linked glycosylation pathways is lacking. Herein, we present the apo-crystal structures of the acetyltransferase domain (ATD) from the bifunctional enzyme PglB (Neisseria gonorrhoeae) and the full-length acetyltransferase WeeI (Acinetobacter baumannii). Additionally, a PglB-ATD structure was solved in complex with AcCoA. Surprisingly, this structure reveals a contrasting binding mechanism for this substrate when compared with the AcCoA-bound PglD structure. A comparison between these findings and the previously solved PglD crystal structures illustrates a dichotomy among N- and O-linked glycosylation pathway enzymes. Based upon these structures, key residues in the UDP-4-amino and AcCoA binding pockets were mutated to determine their effect on binding and catalysis in PglD, PglB-ATD, and WeeI. Last, a phylogenetic analysis of the aforementioned acetyltransferases was employed to illuminate the diversity among N- and O-linked glycosylation pathway enzymes. PMID:24064219

  8. Purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3].

    PubMed

    Kubiak, Xavier; Pluvinage, Benjamin; Li de la Sierra-Gallay, Inès; Weber, Patrick; Haouz, Ahmed; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2012-02-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3], a putative NAT isoenzyme that possesses a unique catalytic triad containing a glutamate residue, is reported. The crystal diffracted to 2.42 Å resolution and belonged to the monoclinic space group C121, with unit-cell parameters a = 90.44, b = 44.52, c = 132.98 Å, β = 103.8°. PMID:22297998

  9. Crystal Structure Analysis of the Polysialic Acid Specific O-Acetyltransferase NeuO

    PubMed Central

    Schulz, Eike C.; Bergfeld, Anne K.; Ficner, Ralf; Mühlenhoff, Martina

    2011-01-01

    The major virulence factor of the neuroinvasive pathogen Escherichia coli K1 is the K1 capsule composed of α2,8-linked polysialic acid (polySia). K1 strains harboring the CUS-3 prophage modify their capsular polysaccharide by phase-variable O-acetlyation, a step that is associated with increased virulence. Here we present the crystal structure of the prophage-encoded polysialate O-acetyltransferase NeuO. The homotrimeric enzyme belongs to the left-handed β-helix (LβH) family of acyltransferases and is characterized by an unusual funnel-shaped outline. Comparison with other members of the LβH family allowed the identification of active site residues and proposal of a catalytic mechanism and highlighted structural characteristics of polySia specific O-acetyltransferases. As a unique feature of NeuO, the enzymatic activity linearly increases with the length of the N-terminal poly-ψ-domain which is composed of a variable number of tandem copies of an RLKTQDS heptad. Since the poly-ψ-domain was not resolved in the crystal structure it is assumed to be unfolded in the apo-enyzme. PMID:21390252

  10. Mutations in KAT6B, Encoding a Histone Acetyltransferase, Cause Genitopatellar Syndrome

    PubMed Central

    Campeau, Philippe M.; Kim, Jaeseung C.; Lu, James T.; Schwartzentruber, Jeremy A.; Abdul-Rahman, Omar A.; Schlaubitz, Silke; Murdock, David M.; Jiang, Ming-Ming; Lammer, Edward J.; Enns, Gregory M.; Rhead, William J.; Rowland, Jon; Robertson, Stephen P.; Cormier-Daire, Valérie; Bainbridge, Matthew N.; Yang, Xiang-Jiao; Gingras, Marie-Claude; Gibbs, Richard A.; Rosenblatt, David S.; Majewski, Jacek; Lee, Brendan H.

    2012-01-01

    Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs. PMID:22265014

  11. The N-terminal acetyltransferase Naa10 is essential for zebrafish development

    PubMed Central

    Ree, Rasmus; Myklebust, Line M.; Thiel, Puja; Foyn, Håvard; Fladmark, Kari E.; Arnesen, Thomas

    2015-01-01

    N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish. PMID:26251455

  12. Intracellular localization of α-tubulin acetyltransferase ATAT1 in rat ciliated cells.

    PubMed

    Nakakura, Takashi; Suzuki, Takeshi; Nemoto, Takahiro; Tanaka, Hideyuki; Asano-Hoshino, Anshin; Arisawa, Kenjiro; Nishijima, Yoshimi; Kiuchi, Yoshiko; Hagiwara, Haruo

    2016-09-01

    Cilia are microtubule-based hair-like organelles on basal bodies located beneath the cell membrane in various tissues of multicellular animals, and are usually classified into motile cilia and primary cilia. Microtubules are assembled from the heterodimers of α- and β-tubulin. The lysine residue at position 40 (K40) of α-tubulin is an important site for acetylation, and this site is acetylated in the cilium. α-Tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to the K40 residue of α-tubulin; however, its intracellular distribution in mammalian tissues remains unclear. In this study, we analyzed ATAT1 localization in rat trachea, oviduct, kidney, retina, testis and the third ventricle of the brain by immunohistochemical techniques using a specific antibody against ATAT1. ATAT1 was distributed to the motile cilia of multiciliated cells of the trachea, third ventricle of the brain and oviduct, and in the primary cilia of the renal medullary collecting duct. ATAT1 also localized to the primary cilia, inner and outer segments of retinal photoreceptor cells, and at the Golgi apparatus of spermatocytes and spermatids of testis. These results indicated that α-tubulin acetylation by ATAT1 at distinct subcellular positions may influence the functional regulation of microtubules and cilia in a variety of ciliated cells. PMID:26700226

  13. K-Lysine acetyltransferase 2a regulates a hippocampal gene expression network linked to memory formation

    PubMed Central

    Stilling, Roman M; Rönicke, Raik; Benito, Eva; Urbanke, Hendrik; Capece, Vincenzo; Burkhardt, Susanne; Bahari-Javan, Sanaz; Barth, Jonas; Sananbenesi, Farahnaz; Schütz, Anna L; Dyczkowski, Jerzy; Martinez-Hernandez, Ana; Kerimoglu, Cemil; Dent, Sharon YR; Bonn, Stefan; Reymann, Klaus G; Fischer, Andre

    2014-01-01

    Neuronal histone acetylation has been linked to memory consolidation, and targeting histone acetylation has emerged as a promising therapeutic strategy for neuropsychiatric diseases. However, the role of histone-modifying enzymes in the adult brain is still far from being understood. Here we use RNA sequencing to screen the levels of all known histone acetyltransferases (HATs) in the hippocampal CA1 region and find that K-acetyltransferase 2a (Kat2a)—a HAT that has not been studied for its role in memory function so far—shows highest expression. Mice that lack Kat2a show impaired hippocampal synaptic plasticity and long-term memory consolidation. We furthermore show that Kat2a regulates a highly interconnected hippocampal gene expression network linked to neuroactive receptor signaling via a mechanism that involves nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). In conclusion, our data establish Kat2a as a novel and essential regulator of hippocampal memory consolidation. PMID:25024434

  14. Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT).

    PubMed

    Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

    2016-01-01

    General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. PMID:27367672

  15. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

    PubMed

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-02-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE. PMID:26790714

  16. Improvement of L-citrulline production in Corynebacterium glutamicum by ornithine acetyltransferase.

    PubMed

    Hao, N; Mu, J; Hu, N; Xu, S; Yan, M; Li, Y; Guo, K; Xu, L

    2015-02-01

    In this study, Corynebacterium glutamicum ATCC 13032 was engineered to produce L-citrulline through a metabolic engineering strategy. To prevent the flux away from L-citrulline and to increase the expression levels of genes involved in the citrulline biosynthesis pathway, the argininosuccinate synthase gene (argG) and the repressor gene (argR) were inactivated. The engineered C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2) produced higher amounts of L-citrulline (5.43 g/L) compared to the wildtype strain (0.15 g/L). To determine new strategies for further enhancement of L-citrulline production, the effect of L-citrulline on ornithine acetyltransferase (EC 2.3.1.35; OATase; ArgJ) was first investigated. Citrulline was determined to inhibit Ornithine acetyltransferase; for 50 % inhibition, citrulline concentration was 30 mM. The argJ gene from C. glutamicum ATCC 13032 was cloned, and the recombinant shuttle plasmid pXMJ19-argJ was constructed and expressed in C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2). Overexpression of the argJ gene exhibited increased OAT activity and resulted in a positive effect on citrulline production (8.51 g/L). These results indicate that OAT plays a vital role during L-citrulline production in C. glutamicum. PMID:25492493

  17. Purification and characterization of glutamate N-acetyltransferase involved in citrulline accumulation in wild watermelon.

    PubMed

    Takahara, Kentaro; Akashi, Kinya; Yokota, Akiho

    2005-10-01

    Citrulline is an efficient hydroxyl radical scavenger that can accumulate at concentrations of up to 30 mm in the leaves of wild watermelon during drought in the presence of strong light; however, the mechanism of this accumulation remains unclear. In this study, we characterized wild watermelon glutamate N-acetyltransferase (CLGAT) that catalyses the transacetylation reaction between acetylornithine and glutamate to form acetylglutamate and ornithine, thereby functioning in the first and fifth steps in citrulline biosynthesis. CLGAT enzyme purified 7000-fold from leaves was composed of two subunits with different N-terminal amino acid sequences. Analysis of the corresponding cDNA revealed that these two subunits have molecular masses of 21.3 and 23.5 kDa and are derived from a single precursor polypeptide, suggesting that the CLGAT precursor is cleaved autocatalytically at the conserved ATML motif, as in other glutamate N-acetyltransferases of microorganisms. A green fluorescence protein assay revealed that the first 26-amino acid sequence at the N-terminus of the precursor functions as a chloroplast transit peptide. The CLGAT exhibited thermostability up to 70 degrees C, suggesting an increase in enzyme activity under high leaf temperature conditions during drought/strong-light stresses. Moreover, CLGAT was not inhibited by citrulline or arginine at physiologically relevant high concentrations. These findings suggest that CLGAT can effectively participate in the biosynthesis of citrulline in wild watermelon leaves during drought/strong-light stress. PMID:16218965

  18. Histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and cancer cell proliferation.

    PubMed

    Malatesta, Martina; Steinhauer, Cornelia; Mohammad, Faizaan; Pandey, Deo P; Squatrito, Massimo; Helin, Kristian

    2013-10-15

    The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. In addition, we found that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally, we observed that PCAF silencing reduces the tumor-forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma. PMID:23943798

  19. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase

    PubMed Central

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-01-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE. PMID:26790714

  20. Chemoproteomic Profiling of Lysine Acetyltransferases Highlights an Expanded Landscape of Catalytic Acetylation

    PubMed Central

    2015-01-01

    Lysine acetyltransferases (KATs) play a critical role in the regulation of gene expression, metabolism, and other key cellular functions. One shortcoming of traditional KAT assays is their inability to study KAT activity in complex settings, a limitation that hinders efforts at KAT discovery, characterization, and inhibitor development. To address this challenge, here we describe a suite of cofactor-based affinity probes capable of profiling KAT activity in biological contexts. Conversion of KAT bisubstrate inhibitors to clickable photoaffinity probes enables the selective covalent labeling of three phylogenetically distinct families of KAT enzymes. Cofactor-based affinity probes report on KAT activity in cell lysates, where KATs exist as multiprotein complexes. Chemical affinity purification and unbiased LC–MS/MS profiling highlights an expanded landscape of orphan lysine acetyltransferases present in the human genome and provides insight into the global selectivity and sensitivity of CoA-based proteomic probes that will guide future applications. Chemoproteomic profiling provides a powerful method to study the molecular interactions of KATs in native contexts and will aid investigations into the role of KATs in cell state and disease. PMID:24836640

  1. Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity.

    PubMed

    Lalonde, Marie-Eve; Avvakumov, Nikita; Glass, Karen C; Joncas, France-Hélène; Saksouk, Nehmé; Holliday, Michael; Paquet, Eric; Yan, Kezhi; Tong, Qiong; Klein, Brianna J; Tan, Song; Yang, Xiang-Jiao; Kutateladze, Tatiana G; Côté, Jacques

    2013-09-15

    Histone acetyltransferases (HATs) assemble into multisubunit complexes in order to target distinct lysine residues on nucleosomal histones. Here, we characterize native HAT complexes assembled by the BRPF family of scaffold proteins. Their plant homeodomain (PHD)-Zn knuckle-PHD domain is essential for binding chromatin and is restricted to unmethylated H3K4, a specificity that is reversed by the associated ING subunit. Native BRPF1 complexes can contain either MOZ/MORF or HBO1 as catalytic acetyltransferase subunit. Interestingly, while the previously reported HBO1 complexes containing JADE scaffold proteins target histone H4, the HBO1-BRPF1 complex acetylates only H3 in chromatin. We mapped a small region to the N terminus of scaffold proteins responsible for histone tail selection on chromatin. Thus, alternate choice of subunits associated with HBO1 can switch its specificity between H4 and H3 tails. These results uncover a crucial new role for associated proteins within HAT complexes, previously thought to be intrinsic to the catalytic subunit. PMID:24065767

  2. Structural and functional characterization of the α-tubulin acetyltransferase MEC-17

    PubMed Central

    Davenport, Andrew M.; Collins, Leslie N.; Chiu, Hui; Minor, Paul J.; Sternberg, Paul W.; Hoelz, André

    2014-01-01

    Tubulin protomers undergo an extensive array of post-translational modifications to tailor microtubules to specific tasks. One such modification, the acetylation of lysine-40 of α-tubulin, located in the lumen of microtubules, is associated with stable, long-living microtubule structures. MEC-17 was recently identified as the acetyltransferase that mediates this event. We have determined the crystal structure of the catalytic core of human MEC-17 in complex with its cofactor acetyl-CoA at 1.7 Å resolution. The structure reveals that the MEC-17 core adopts a canonical Gcn5-related N-acetyltransferase (GNAT) fold that is decorated with extensive surface loops. An enzymatic analysis of 33 MEC-17 surface mutants identifies hot-spot residues for catalysis and substrate recognition. A large, evolutionarily conserved hydrophobic surface patch is identified that is critical for enzymatic activity, suggesting that specificity is achieved by interactions with the α-tubulin substrate that extend outside of the modified surface loop. An analysis of MEC-17 mutants in C. elegans shows that enzymatic activity is dispensable for touch sensitivity. PMID:24846647

  3. Crystal structure of bacillus subtilis YdaF protein : a putative ribosomal N-acetyltransferase.

    SciTech Connect

    Brunzelle, J. S.; Wu, R.; Korolev, S. V.; Collart, F. R.; Joachimiak, A.; Anderson, W. F.; Biosciences Division; Northwestern Univ.; Saint Louis Univ. School of Medicine

    2004-12-01

    Comparative sequence analysis suggests that the ydaF gene encodes a protein (YdaF) that functions as an N-acetyltransferase, more specifically, a ribosomal N-acetyltransferase. Sequence analysis using basic local alignment search tool (BLAST) suggests that YdaF belongs to a large family of proteins (199 proteins found in 88 unique species of bacteria, archaea, and eukaryotes). YdaF also belongs to the COG1670, which includes the Escherichia coli RimL protein that is known to acetylate ribosomal protein L12. N-acetylation (NAT) has been found in all kingdoms. NAT enzymes catalyze the transfer of an acetyl group from acetyl-CoA (AcCoA) to a primary amino group. For example, NATs can acetylate the N-terminal {alpha}-amino group, the {epsilon}-amino group of lysine residues, aminoglycoside antibiotics, spermine/speridine, or arylalkylamines such as serotonin. The crystal structure of the alleged ribosomal NAT protein, YdaF, from Bacillus subtilis presented here was determined as a part of the Midwest Center for Structural Genomics. The structure maintains the conserved tertiary structure of other known NATs and a high sequence similarity in the presumed AcCoA binding pocket in spite of a very low overall level of sequence identity to other NATs of known structure.

  4. Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

    PubMed Central

    Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

    2016-01-01

    General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. PMID:27367672

  5. Mutations in KAT6B, encoding a histone acetyltransferase, cause Genitopatellar syndrome.

    PubMed

    Campeau, Philippe M; Kim, Jaeseung C; Lu, James T; Schwartzentruber, Jeremy A; Abdul-Rahman, Omar A; Schlaubitz, Silke; Murdock, David M; Jiang, Ming-Ming; Lammer, Edward J; Enns, Gregory M; Rhead, William J; Rowland, Jon; Robertson, Stephen P; Cormier-Daire, Valérie; Bainbridge, Matthew N; Yang, Xiang-Jiao; Gingras, Marie-Claude; Gibbs, Richard A; Rosenblatt, David S; Majewski, Jacek; Lee, Brendan H

    2012-02-10

    Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with GPS. In addition, human pathological analyses and mouse expression studies point to systemic roles of KAT6B in controlling organismal growth and development. Myst4 (the mouse orthologous gene) is expressed in mouse tissues corresponding to those affected by GPS. Phenotypic differences and similarities between GPS, the Say-Barber-Biesecker variant of Ohdo syndrome (caused by different mutations of KAT6B), and Rubinstein-Taybi syndrome (caused by mutations in other histone acetyltransferases) are discussed. Together, the data support an epigenetic dysregulation of the limb, brain, and genital developmental programs. PMID:22265014

  6. Diurnal cycles in serotonin acetyltransferase activity and cyclic GMP content of cultured chick pineal glands.

    PubMed

    Wainwright, S D

    1980-06-12

    Levels of serotonin N-acetyltransferase (NAT: acetul CoA:arylamine N-acetyltransferase; EC 2.1.1.5.) activity in the chick pineal gland exhibit a marked diurnal variation in birds kept under a diurnal cycle of ilumination. Activity begins to rise rapidly at the start of the dark phase of the cycle and reaches maximum levels at mid-dark phase about 25-fold greater than the minimum basal level at mid-light phase. Thereafter, the level of activity declines to the basal level about the start of the light phase. This diurnal cycle in chick pineal NAT activity found in vivo has recently been reproduced in vitro with intact glands incubated in organ culture. The mechanism of the 'biological clock' which regulates these variations in level of chick pineal NAT activity is unknown. However, I now report that chick pineal glands cultured under a diurnal cycle of illumination exhibit a diurnal cycle in content of cyclic GMP which roughly parallels the cycles in NAT activity. In contrast, there was no correlation between variations in pineal content of cyclic AMP and in level of NAT activity. PMID:6250035

  7. Specific alkylation of a histidine residue in carnitine acetyltransferase by bromoacetyl-l-carnitine

    PubMed Central

    Chase, J. F. A.; Tubbs, P. K.

    1970-01-01

    Incubation of carnitine acetyltransferase with low concentrations of bromoacetyl-l-carnitine causes a rapid and irreversible loss of enzyme activity; one mol of inhibitor can inactivate one mol of enzyme. Bromoacetyl-d-carnitine, iodoacetate or iodoacetamide are ineffective. l-Carnitine protects the transferase from bromoacetyl-l-carnitine. Investigation shows that the enzyme first reversibly binds bromoacetyl-l-carnitine with an affinity similar to that shown for the normal substrate acetyl-l-carnitine; this binding is followed by an alkylation reaction, forming the carnitine ester of a monocarboxymethyl-protein, which is catalytically inactive. The carnitine is released at an appreciable rate by spontaneous hydrolysis, and the resulting carboxymethyl-enzyme is also inactive. Total acid hydrolysis of enzyme after treatment with 2-[14C]bromoacetyl-l-carnitine yields N-3-carboxy[14C]methylhistidine as the only labelled amino acid. These findings, taken in conjunction with previous work, suggest that the single active centre of carnitine acetyltransferase contains a histidine residue. PMID:5461620

  8. Characterization and transcriptional regulation of the 2'-N-acetyltransferase gene from Providencia stuartii.

    PubMed Central

    Rather, P N; Orosz, E; Shaw, K J; Hare, R; Miller, G

    1993-01-01

    We have cloned the chromosomally encoded 2'-N-acetyltransferase gene [aac(2')-Ia] from Providencia stuartii. DNA sequence analysis of the cloned insert identified a single open reading frame, which is capable of encoding a protein with a predicted molecular mass of 20,073 Da. The deduced AAC(2')-Ia protein showed no significant homology to other proteins, including all of the AAC(3) and AAC(6') proteins. Primer extension analysis was used to identify the aac(2')-Ia promoter, which contained an unusual sequence (CTTTTT) at the -35 region. Expression of the aac(2')-Ia gene occurs at low levels in wild-type P. stuartii strains; therefore, they are aminoglycoside susceptible. We have isolated mutants with high-level AAC(2')-Ia expression at a frequency of 4.8 x 10(-6). Detailed analysis of one mutant demonstrated a 12.2-fold increase in the accumulation of aac(2')-Ia mRNA. In addition, the levels of beta-galactosidase expression from a plasmid-encoded aac(2')-lacZ transcriptional fusion were increased 11.5-fold in this mutant relative to those in an isogenic wild-type strain. These results suggested that a trans-acting factor, designated aar (for aminoglycoside acetyltransferase regulator), controls AAC(2')-Ia expression in P. stuartii. Images PMID:8407825

  9. Studying aminoglycoside modification by the acetyltransferase class of resistance-causing enzymes via microarray

    PubMed Central

    Barrett, Olivia J.; Pushechnikov, Alexei; Wu, Meilan; Disney, Matthew D.

    2008-01-01

    Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AAC’s). Thus, the modification of aminoglycosides by AAC(2’) from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by resistance-causing enzymes have reduced affinities for binding their therapeutic target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC(3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2’). Interestingly, these effects mirror the biological functions of these enzymes: the AAC(3) used in this study is known to confer aminoglycoside resistance while the AAC(2’) is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes. PMID:18774127

  10. Identification and characterization of novel small molecule inhibitors of the acetyltransferase activity of Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU).

    PubMed

    Sharma, Rashmi; Rani, Chitra; Mehra, Rukmankesh; Nargotra, Amit; Chib, Reena; Rajput, Vikrant S; Kumar, Sunil; Singh, Samsher; Sharma, Parduman R; Khan, Inshad A

    2016-04-01

    This study aims at identifying novel chemical scaffolds as inhibitors specific to the acetyltransferase domain of a bifunctional enzyme, Escherichia coli GlmU, involved in the cell wall biosynthesis of Gram-negative organisms. A two-pronged approach was used to screen a 50,000 small-molecule library. Using the first approach, the library was in silico screened by docking the library against acetyltransferase domain of E. coli GlmU studies. In the second approach, complete library was screened against Escherichia coli ATCC 25922 to identify the whole cell active compounds. Active compounds from both the screens were screened in a colorimetric absorbance-based assay to identify inhibitors of acetyltransferase domain of E. coli GlmU which resulted in the identification of 1 inhibitor out of 56 hits identified by in silico screening and 4 inhibitors out of 35 whole cell active compounds on Gram-negative bacteria with the most potent inhibitor showing IC50 of 1.40 ± 0.69 μM. Mode of inhibition studies revealed these inhibitors to be competitive with AcCoA and uncompetitive with GlcN-1-P. These selected inhibitors were also tested for their antibacterial and cytotoxic activities. Compounds 5175178 and 5215319 exhibited antibacterial activity that co-related with GlmU inhibition. These compounds, therefore, represent novel chemical scaffolds targeting acetyltransferase activity of E. coli GlmU. PMID:26563552

  11. Investigating the Hydrolysis Reactions of a Chemical Warfare Agent Surrogate. A Systematic Study using 1H, 13C, 17O, 19F, 31P, and <sup>35Cl NMR Spectroscopy

    SciTech Connect

    Alam, Todd M.; Wilson, Brendan W.

    2015-07-24

    During the summer of 2015, I participated in the DHS HS-STEM fellowship at Sandia National Laboratories (SNL, NM) under the supervision of Dr. Todd M. Alam in his Nuclear Magnetic Resonance (NMR) Spectroscopy research group. While with the group, my main project involved pursing various hydrolysis reactions with Diethyl Chlorophosphate (DECP), a surrogate for the agent Sarin (GB). Specifically, I performed different hydrolysis reactions, monitored and tracked the different phosphorous containing species using phosphorous (31P) NMR spectroscopy. With the data collected, I performed kinetics studies mapping the rates of DECP hydrolysis. I also used the NMR of different nuclei such as 1H, 13C, 17O, and <sup>35Cl to help understand the complexity of the reactions that take place. Finally, my last task at SNL was to work with Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) NMR Spectroscopy optimizing conditions for 19F- 31P filtering NMR experiments.

  12. Comparative genomic, phylogenetic, and functional investigation of the xenobiotic metabolizing arylamine N-acetyltransferase enzyme family among fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated (Glenn and Bacon, 2009; Glenn et al., 2010). The NAT1 gene of Gibberella moniliformis was the...

  13. Construction and Use of a Replication-Competent Human Immunodeficiency Virus (HIV-1) that Expresses the Chloramphenicol Acetyltransferase Enzyme

    NASA Astrophysics Data System (ADS)

    Terwilliger, E. F.; Godin, B.; Sodroski, J. G.; Haseltine, W. A.

    1989-05-01

    The construction and properties of an infectious human immunodeficiency virus (HIV) that expresses the bacterial gene chloramphenicol acetyltransferase are described. This virus can be used in vitro to screen for drugs that inhibit HIV infection. The marked virus may also be used to trace the routes of infection from the site of inoculation in animal experiments.

  14. Homologues of xenobiotic metabolizing N-acetyltransferases in plant-associated fungi: Novel functions for an old enzyme family

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant-pathogenic fungi and their hosts engage in chemical warfare, attacking each other with toxic products of secondary metabolism and defending themselves via an arsenal of xenobiotic metabolizing enzymes. One such enzyme is homologous to arylamine N-acetyltransferase (NAT) and has been identified...

  15. Chloramphenicol acetyltransferase should not provide methanogens with resistance to chloramphenicol. [Methanococcus voltae; Methanococcus vannielii; Methanococcus deltae; Methanobrevibacter smithii

    SciTech Connect

    Beckler, G.S.; Hook, L.A.; Reeve, J.N.

    1984-04-01

    Growth of the four methanogens investigated was inhibitied by chloramphenicol-3-acetate; therefore, introduction of chloramphenicol acetyltransferase-encoding genes should not confer chloramphenicol resistance on these methanogens. Reduction of the aryl nitro group of chloramphenicol produced a compound which did not inhibit the growth of these methanogens. 9 references.

  16. Structural and Functional Evidence for Bacillus subtilis PaiA as a Novel N1-spermidine/spermine acetyltransferase (SSAT)

    SciTech Connect

    Forouhar,F.; Lee, I.; Vujcic, J.; Vujcic, S.; Shen, J.; Vorobiev, S.; Xiao, R.; Acton, T.; Montelione, G.; et al.

    2005-01-01

    Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates (SSATs). We have determined the crystal structure of PaiA in complex with CoA at 1.9 Angstrom resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is also consistent with this possibility since purified PaiA possesses N1-acetyltransferase activity towards polyamine substrates including spermidine and spermine. Further, conditional over-expression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation.

  17. Dissociable roles for histone acetyltransferases p300 and PCAF in hippocampus and perirhinal cortex-mediated object memory.

    PubMed

    Mitchnick, K A; Creighton, S D; Cloke, J M; Wolter, M; Zaika, O; Christen, B; Van Tiggelen, M; Kalisch, B E; Winters, B D

    2016-07-01

    The importance of histone acetylation for certain types of memory is now well established. However, the specific contributions of the various histone acetyltransferases to distinct memory functions remain to be determined; therefore, we employed selective histone acetyltransferase protein inhibitors and short-interference RNAs to evaluate the roles of CREB-binding protein (CBP), E1A-binding protein (p300) and p300/CBP-associated factor (PCAF) in hippocampus and perirhinal cortex (PRh)-mediated object memory. Rats were tested for short- (STM) and long-term memory (LTM) in the object-in-place task, which relies on the hippocampus and PRh for spatial memory and object identity processing, respectively. Selective inhibition of these histone acetyltransferases by small-interfering RNA and pharmacological inhibitors targeting the HAT domain produced dissociable effects. In the hippocampus, CBP or p300 inhibition impaired long-term but not short-term object memory, while inhibition of PCAF impaired memory at both delays. In PRh, HAT inhibition did not impair STM, and only CBP and PCAF inhibition disrupted LTM; p300 inhibition had no effects. Messenger RNA analyses revealed findings consistent with the pattern of behavioral effects, as all three enzymes were upregulated in the hippocampus (dentate gyrus) following learning, whereas only CBP and PCAF were upregulated in PRh. These results demonstrate, for the first time, the necessity of histone acetyltransferase activity for PRh-mediated object memory and indicate that the specific mnemonic roles of distinctive histone acetyltransferases can be dissociated according to specific brain regions and memory timeframe. PMID:27251651

  18. Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: Dihydrolipoamide acetyltransferase

    SciTech Connect

    Coppel, R.L.; McNeilage, L.J.; Surh, C.D.; Van De Water, J.; Spithill, T.W.; Whittingham, S.; Gershwin, M.E. )

    1988-10-01

    Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency of autoantibodies to a M{sub r} 70,000 mitochondrial antigen a component of the M2 antigen complex. The authors have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects.

  19. Role of histone acetyltransferases and histone deacetylases in adipocyte differentiation and adipogenesis.

    PubMed

    Zhou, Yuanfei; Peng, Jian; Jiang, Siwen

    2014-04-01

    Adipogenesis is a complex process strictly regulated by a well-established cascade that has been thoroughly studied in the last two decades. This process is governed by complex regulatory networks that involve the activation/inhibition of multiple functional genes, and is controlled by histone-modifying enzymes. Among such modification enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the transcriptional regulation and post-translational modification of protein acetylation. HATs and HDACs have been shown to respond to signals that regulate cell differentiation, participate in the regulation of protein acetylation, mediate transcription and post-translation modifications, and directly acetylate/deacetylate various transcription factors and regulatory proteins. In this paper, we review the role of HATs and HDACs in white and brown adipocyte differentiation and adipogenesis, to expand our knowledge on fat formation and adipose tissue biology. PMID:24810880

  20. Structure of homoserine O-acetyltransferase from Staphylococcus aureus: the first Gram-positive ortholog structure

    PubMed Central

    Thangavelu, Bharani; Pavlovsky, Alexander G.; Viola, Ronald

    2014-01-01

    Homoserine O-acetyltransferase (HTA) catalyzes the formation of l-O-acetyl-homoserine from l-homoserine through the transfer of an acetyl group from acetyl-CoA. This is the first committed step required for the biosynthesis of methionine in many fungi, Gram-positive bacteria and some Gram-negative bacteria. The structure of HTA from Staphylococcus aureus (SaHTA) has been determined to a resolution of 2.45 Å. The structure belongs to the α/β-hydrolase superfamily, consisting of two distinct domains: a core α/β-domain containing the catalytic site and a lid domain assembled into a helical bundle. The active site consists of a classical catalytic triad located at the end of a deep tunnel. Structure analysis revealed some important differences for SaHTA compared with the few known structures of HTA. PMID:25286936

  1. Structure of homoserine O-acetyltransferase from Staphylococcus aureus: the first Gram-positive ortholog structure.

    PubMed

    Thangavelu, Bharani; Pavlovsky, Alexander G; Viola, Ronald

    2014-10-01

    Homoserine O-acetyltransferase (HTA) catalyzes the formation of L-O-acetyl-homoserine from L-homoserine through the transfer of an acetyl group from acetyl-CoA. This is the first committed step required for the biosynthesis of methionine in many fungi, Gram-positive bacteria and some Gram-negative bacteria. The structure of HTA from Staphylococcus aureus (SaHTA) has been determined to a resolution of 2.45 Å. The structure belongs to the α/β-hydrolase superfamily, consisting of two distinct domains: a core α/β-domain containing the catalytic site and a lid domain assembled into a helical bundle. The active site consists of a classical catalytic triad located at the end of a deep tunnel. Structure analysis revealed some important differences for SaHTA compared with the few known structures of HTA. PMID:25286936

  2. Preliminary X-ray crystallographic analysis of ornithine acetyltransferase (Rv1653) from Mycobacterium tuberculosis.

    PubMed

    Sankaranarayanan, R; Garen, C R; Cherney, M M; Yuan, M; Lee, C; James, M N G

    2009-02-01

    The gene product of open reading frame Rv1653 from Mycobacterium tuberculosis is annotated as encoding a probable ornithine acetyltransferase (OATase; EC 2.3.1.35), an enzyme that catalyzes two steps in the arginine-biosynthesis pathway. It transfers an acetyl group from N-acetylornithine to L-glutamate to produce N-acetylglutamate and L-ornithine. Rv1653 was crystallized using the sitting-drop vapour-diffusion method. The native crystals diffracted to a resolution of 1.7 A and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 60.1, b = 99.7, c = 155.3 A. The preliminary X-ray study showed the presence of a dimer in the asymmetric unit of the crystals, which had a Matthews coefficient V(M) of 2.8 A(3) Da(-1). PMID:19194014

  3. A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

    SciTech Connect

    Narayanan, R.; Jastreboff, M.M.; Chiu, Chang Fang; Ito, Etsuro; Bertino, J.R. )

    1988-01-01

    A rapid procedure is described for assaying chloramphenicol acetyltransferase enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with ({sup 14}C)chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of {sup 14}C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated {sup 14}C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice.

  4. CBP histone acetyltransferase activity is a critical component of memory consolidation.

    PubMed

    Korzus, Edward; Rosenfeld, Michael G; Mayford, Mark

    2004-06-24

    The stabilization of learned information into long-term memories requires new gene expression. CREB binding protein (CBP) is a coactivator of transcription that can be independently regulated in neurons. CBP functions both as a platform for recruiting other required components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. To dissect the chromatin remodeling versus platform function of CBP or the developmental versus adult role of this gene, we generated transgenic mice that express CBP in which HAT activity is eliminated. Acquisition of new information and short-term memory is spared in these mice, while the stabilization of short-term memory into long-term memory is impaired. The behavioral phenotype is due to an acute requirement for CBP HAT activity in the adult as it is rescued by both suppression of transgene expression or by administration of the histone deacetylase inhibitor Trichostatin A (TSA) in adult animals. PMID:15207240

  5. Isolation of bacteria producing chloramphenicol acetyltransferase from soil and their characterization.

    PubMed

    Datta, K; Mukherjee, S K; Majumdar, M K; Roy, S K

    1982-07-01

    After screening 107 soil samples collected from different spots around Calcutta, 579 chloramphenicol resistant colonies were isolated. Out of these only 58 colonies could inactivate chloramphenicol in detectable amounts. By noting the production of inactivating factor, 5 high yielding strains were further characterized to species level. Three of them were Escherichia coli strains, the two others were Alcaligenes faecalis and Klebsiella pneumoniae strains. All strains inactivated chloramphenicol by acetylation, with the production of chloramphenicol acetyltransferase. Production of this latter enzyme was not inducible. Minimum inhibitory concentrations for these 5 strains were studied against 14 antimicrobial agents. All strains were found to be resistant to most antimicrobial agents, but sensitive to polymyxin B. The strain A. faecalis was also sensitive to carbenicillin but other four strains were resistant to this antibiotic. PMID:6956790

  6. Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

    SciTech Connect

    Chen, Wenjing; Biswas, Tapan; Porter, Vanessa R.; Tsodikov, Oleg V.; Garneau-Tsodikova, Sylvie

    2011-09-06

    The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

  7. Purification and characterization of aspartate N-acetyltransferase: A critical enzyme in brain metabolism.

    PubMed

    Wang, Qinzhe; Zhao, Mojun; Parungao, Gwenn G; Viola, Ronald E

    2016-03-01

    Canavan disease (CD) is a neurological disorder caused by an interruption in the metabolism of N-acetylaspartate (NAA). Numerous mutations have been found in the enzyme that hydrolyzes NAA, and the catalytic activity of aspartoacylase is significantly impaired in CD patients. Recent studies have also supported an important role in CD for the enzyme that catalyzes the synthesis of NAA in the brain. However, previous attempts to study this enzyme had not succeeded in obtaining a soluble, stable and active form of this membrane-associated protein. We have now utilized fusion constructs with solubilizing protein partners to obtain an active and soluble form of aspartate N-acetyltransferase. Characterization of the properties of this enzyme has set the stage for the development of selective inhibitors that can lower the elevated levels of NAA that are observed in CD patients and potentially serve as a new treatment therapy. PMID:26550943

  8. Inhibition of lyso-PAF: acetyl-CoA acetyltransferase by salicylates and other compounds.

    PubMed

    White, H L; Faison, L D

    1988-06-01

    Diflunisal and benoxaprofen (20-100 microM) produced dose-dependent inhibitions of lyso-platelet activating factor: acetyl-CoA acetyltransferase in a lysate of rat pleural neutrophils. Salicylate and aspirin were inhibitory at concentrations of 1 mM and above. Nordihydroguaiaretic acid was a relatively potent inhibitor (I50 = 6 microM). Other compounds, including anti-inflammatory steroids, cyclooxygenase and 5-lipoxygenase inhibitors, appeared ineffective at relevant concentrations. Inhibitions by diflunisal and salicylate occurred at concentrations similar to expected plasma levels in humans at therapeutic doses. An inhibition of platelet-activating factor synthesis may contribute to the antiinflammatory, analgesic, or antipyretic actions of these compounds. PMID:2903520

  9. Integration of Bioorthogonal Probes and Q-FRET for the Detection of Histone Acetyltransferase Activity.

    PubMed

    Han, Zhen; Luan, Yepeng; Zheng, Yujun George

    2015-12-01

    Histone acetyltransferases (HATs) are key players in the epigenetic regulation of gene function. The recent discovery of diverse HAT substrates implies a broad spectrum of cellular functions of HATs. Many pathological processes are also intimately associated with the dysregulation of HAT levels and activities. However, detecting the enzymatic activity of HATs has been challenging, and this has significantly impeded drug discovery. To advance the field, we developed a convenient one-pot, mix-and-read strategy that is capable of directly detecting the acylated histone product through a fluorescent readout. The strategy integrates three technological platforms-bioorthogonal HAT substrate labeling, alkyne-azide click chemistry, and quenching FRET-into one system for effective probing of HAT enzyme activity. PMID:26455821

  10. Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis.

    PubMed

    Willby, Melisa J; Green, Keith D; Gajadeera, Chathurada S; Hou, Caixia; Tsodikov, Oleg V; Posey, James E; Garneau-Tsodikova, Sylvie

    2016-06-17

    A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB. PMID:27010218

  11. Interferon-Induced Spermidine-Spermine Acetyltransferase and Polyamine Depletion Restrict Zika and Chikungunya Viruses.

    PubMed

    Mounce, Bryan C; Poirier, Enzo Z; Passoni, Gabriella; Simon-Loriere, Etienne; Cesaro, Teresa; Prot, Matthieu; Stapleford, Kenneth A; Moratorio, Gonzalo; Sakuntabhai, Anavaj; Levraud, Jean-Pierre; Vignuzzi, Marco

    2016-08-10

    Polyamines are small, positively charged molecules derived from ornithine and synthesized through an intricately regulated enzymatic pathway. Within cells, they are abundant and play several roles in diverse processes. We find that polyamines are required for the life cycle of the RNA viruses chikungunya virus (CHIKV) and Zika virus (ZIKV). Depletion of spermidine and spermine via type I interferon signaling-mediated induction of spermidine/spermine N1-acetyltransferase (SAT1), a key catabolic enzyme in the polyamine pathway, restricts CHIKV and ZIKV replication. Polyamine depletion restricts these viruses in vitro and in vivo, due to impairment of viral translation and RNA replication. The restriction is released by exogenous replenishment of polyamines, further supporting a role for these molecules in virus replication. Thus, SAT1 and, more broadly, polyamine depletion restrict viral replication and suggest promising avenues for antiviral therapies. PMID:27427208

  12. Structural Basis of Substrate-Binding Specificity of Human Arylamine N-acetyltransferases

    SciTech Connect

    Wu,H.; Dombrovsky, L.; Tempel, W.; Martin, F.; Loppnau, P.; Goodfellow, G.; Grant, D.; Plotnikov, A.

    2007-01-01

    The human arylamine N-acetyltransferases NAT1 and NAT2 play an important role in the biotransformation of a plethora of aromatic amine and hydrazine drugs. They are also able to participate in the bioactivation of several known carcinogens. Each of these enzymes is genetically variable in human populations, and polymorphisms in NAT genes have been associated with various cancers. Here we have solved the high resolution crystal structures of human NAT1 and NAT2, including NAT1 in complex with the irreversible inhibitor 2-bromoacetanilide, a NAT1 active site mutant, and NAT2 in complex with CoA, and have refined them to 1.7-, 1.8-, and 1.9- Angstroms resolution, respectively. The crystal structures reveal novel structural features unique to human NATs and provide insights into the structural basis of the substrate specificity and genetic polymorphism of these enzymes.

  13. Effects of acute ethanol administration on nocturnal pineal serotonin N-acetyltransferase activity

    SciTech Connect

    Creighton, J.A.; Rudeen, P.K.

    1988-01-01

    The effect of acute ethanol administration on pineal serotonin N-acetyltransferase (NAT) activity, norepinephrine and indoleamine content was examined in male rats. When ethanol was administered in two equal doses (2 g/kg body weight) over a 4 hour period during the light phase, the nocturnal rise in NAT activity was delayed by seven hours. The nocturnal pineal norepinephrine content was not altered by ethanol except for a delay in the reduction of NE with the onset of the following light phase. Although ethanol treatment led to a significant reduction in nocturnal levels of pineal serotonin content, there was no significant effect upon pineal content of 5-hydroxyindoleacetic acid (5-HIAA). The data indicate that ethanol delays the onset of the rise of nocturnal pineal NAT activity.

  14. Absence of N-terminal acetyltransferase diversification during evolution of eukaryotic organisms

    PubMed Central

    Rathore, Om Singh; Faustino, Alexandra; Prudêncio, Pedro; Van Damme, Petra; Cox, Cymon J.; Martinho, Rui Gonçalo

    2016-01-01

    Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes. PMID:26861501

  15. Biochemical and structural analysis of aminoglycoside acetyltransferase Eis from Anabaena variabilis.

    PubMed

    Pricer, Rachel E; Houghton, Jacob L; Green, Keith D; Mayhoub, Abdelrahman S; Garneau-Tsodikova, Sylvie

    2012-10-30

    The Mycobacterium tuberculosis enhanced intracellular survival (Eis_Mtb) protein is a clinically important aminoglycoside (AG) multi-acetylating enzyme. Eis homologues are found in a variety of mycobacterial and non-mycobacterial species. Variation of the residues lining the AG-binding pocket and positions of the loops bearing these residues in the Eis homologues dictates the substrate specificity and, thus, Eis homologues are Nature-made tools for elucidating principles of AG recognition by Eis. Here, we demonstrate that the Eis from Anabaena variabilis (Eis_Ava), the first non-mycobacterial Eis homologue reported, is a multi-acetylating AG-acetyltransferase. Eis_Ava, Eis from Mycobacterium tuberculosis (Eis_Mtb), and Eis from Mycobacterium smegmatis (Eis_Msm) have different structures of their AG-binding pockets. We perform comparative analysis of these differences and investigate how they dictate the substrate and cosubstrate recognition and acetylation of AGs by Eis. PMID:23090428

  16. First Things First: Vital Protein Marks by N-Terminal Acetyltransferases.

    PubMed

    Aksnes, Henriette; Drazic, Adrian; Marie, Michaël; Arnesen, Thomas

    2016-09-01

    N-terminal (Nt) acetylation is known to be a highly abundant co-translational protein modification, but the recent discovery of Golgi- and chloroplast-resident N-terminal acetyltransferases (NATs) revealed that it can also be added post-translationally. Nt-acetylation may act as a degradation signal in a novel branch of the N-end rule pathway, whose functions include the regulation of human blood pressure. Nt-acetylation also modulates protein interactions, targeting, and folding. In plants, Nt-acetylation plays a role in the control of resistance to drought and in regulation of immune responses. Mutations of specific human NATs that decrease their activity can cause either the lethal Ogden syndrome or severe intellectual disability and cardiovascular defects. In sum, recent advances highlight Nt-acetylation as a key factor in many biological pathways. PMID:27498224

  17. A new arylalkylamine N-acetyltransferase in silkworm (Bombyx mori) affects integument pigmentation.

    PubMed

    Long, Yaohang; Li, Jiaorong; Zhao, Tianfu; Li, Guannan; Zhu, Yong

    2015-04-01

    Dopamine is a precursor for melanin synthesis. Arylalkylamine N-acetyltransferase (AANAT) is involved in the melatonin formation in insects because it could catalyze the transformation from dopamine to dopamine-N-acetyldopamine. In this study, we identified a new AANAT gene in the silkworm (Bombyx mori) and assessed its role in the silkworm. The cDNA of this gene encodes 233 amino acids that shares 57 % amino acid identity with the Bm-iAANAT protein. We thus refer to this gene as Bm-iAANAT2. To investigate the role of Bm-iAANAT2, we constructed a transgenic interference system using a 3xp3 promoter to suppress the expression of Bm-iAANAT2 in the silkworm. We observed that melanin deposition occurs in the head and integument in transgenic lines. To verify the melanism pattern, dopamine content and the enzyme activity of AANAT were determined by high-performance liquid chromatography (HPLC). We found that an increase in dopamine levels affects melanism patterns on the heads of transgenic B. mori. A reduction in the enzyme activity of AANAT leads to changes in dopamine levels. We analyzed the expression of the Bm-iAANAT2 genes by qPCR and found that the expression of Bm-iAANAT2 gene is significantly lower in transgenic lines. Our results lead us to conclude that Bm-iAANAT2 is a new arylalkylamine N-acetyltransferase gene in the silkworm and is involved in the metabolism of the dopamine to avoid the generation of melanin. PMID:25712907

  18. Suppression of exogenous gene expression by spermidine/spermine N1-acetyltransferase 1 (SSAT1) cotransfection.

    PubMed

    Lee, Seung Bum; Park, Jong Hwan; Woster, Patrick M; Casero, Robert A; Park, Myung Hee

    2010-05-14

    Spermidine/spermine N(1)-acetyltransferase 1 (SSAT1), which catalyzes the N(1)-acetylation of spermidine and spermine to form acetyl derivatives, is a rate-limiting enzyme in polyamine catabolism. We now report a novel activity of transiently transfected SSAT1 in suppressing the exogenous expression of other proteins, i.e. green fluorescent protein (GFP) or GFP-eIF5A. Spermidine/spermine N(1)-acetyltransferase 2 (SSAT2) or inactive SSAT1 mutant enzymes (R101A or R101K) were without effect. The loss of exogenous gene expression is not due to accelerated protein degradation, because various inhibitors of proteases, lysosome, or autophagy did not mitigate the effects. This SSAT1 effect cannot be attributed to the depletion of overall cellular polyamines or accumulation of N(1)-acetylspermidine (N(1)-AcSpd) because of the following: (i) addition of putrescine, spermidine, spermine, or N(1)-AcSpd did not restore the expression of GFP or GFP-eIF5A; (ii) depletion of cellular polyamines with alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, did not inhibit exogenous gene expression; and (iii) N(1),N(11)-bis(ethyl)norspermine caused a drastic depletion of cellular polyamines through induction of endogenous SSAT1 but did not block exogenous gene expression. SSAT1 transient transfection did not affect stable expression of GFP, and stably expressed SSAT1 did not affect exogenous expression of GFP, suggesting that only transiently (episomally) expressed SSAT1 blocks exogenous (episomal) expression of other proteins. SSAT1 may regulate exogenous gene expression by blocking steps involved in transcription/translation from an episomal vector by targeting non-polyamine substrate(s) critical for this pathway. PMID:20212040

  19. Structural and functional characterization of TRI3 trichothecene 15-O-acetyltransferase from Fusarium sporotrichioides

    SciTech Connect

    Garvey, Graeme S.; McCormick, Susan P.; Alexander, Nancy J.; Rayment, Ivan

    2009-08-14

    Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13-epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15-O-trichothecene acetyltransferase isolated from F. sporotrichioides and the 'in vivo' characterization of Deltatri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15-decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15-decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3-O-trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.

  20. Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

    PubMed Central

    Lönnerberg, P; Lendahl, U; Funakoshi, H; Arhlund-Richter, L; Persson, H; Ibáñez, C F

    1995-01-01

    Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic alpha-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons. Images Fig. 1 Fig. 2 PMID:7732028

  1. Sulphoacetaldehyde acetyltransferase yields acetyl phosphate: purification from Alcaligenes defragrans and gene clusters in taurine degradation.

    PubMed

    Ruff, Jürgen; Denger, Karin; Cook, Alasdair M

    2003-01-15

    The facultatively anaerobic bacterium Alcaligenes defragrans NKNTAU was found to oxidize taurine (2-aminoethanesulphonate) with nitrate as the terminal electron acceptor. Taurine was transaminated to 2-sulphoacetaldehyde. This was not converted into sulphite and acetate by a "sulphoacetaldehyde sulpho-lyase" (EC 4.4.1.12), but into sulphite and acetyl phosphate, which was identified by three methods. The enzyme, which required the addition of phosphate, thiamin diphosphate and Mg(2+) ions for activity, was renamed sulphoacetaldehyde acetyltransferase (Xsc; EC 2.3.1.-). Inducible Xsc was expressed at high levels, and a three-step 11-fold purification yielded an essentially homogeneous soluble protein, which was a homotetramer in its native form; the molecular mass of the subunit was found to be between about 63 kDa (SDS/PAGE) and 65.3 kDa (matrix-assisted laser-desorption ionization-time-of-flight MS). The N-terminal and two internal amino acid sequences were determined, and PCR primers were generated. The xsc gene was amplified and sequenced; the derived molecular mass of the processed protein was 65.0 kDa. The downstream gene presumably encoded the inducible phosphate acetyltransferase (Pta) found in crude extracts. The desulphonative enzymes ("EC 4.4.1.12") from Achromobacter xylosoxidans NCIMB 10751 and Desulfonispora thiosulfatigenes GKNTAU were shown to be Xscs. We detected at least three subclasses of xsc in Proteobacteria and in Gram-positive bacteria, and they comprised a distinct group within the acetohydroxyacid synthase supergene family. Genome sequencing data revealed xsc genes in Burkholderia fungorum (80% sequence identity) and Sinorhizobium meliloti (61%) with closely linked pta genes. Different patterns of regulation for the transport and dissimilation of taurine were hypothesized for S. meliloti and B. fungorum. PMID:12358600

  2. Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans.

    PubMed

    Chang, Peng; Fan, Xueyi; Chen, Jiangye

    2015-08-01

    Candida albicans is an opportunistic fungal pathogen commonly found in humans. It has the ability to switch reversibly between three growth forms: budding yeast, pseudohypha, and hypha. The transition between yeast and hyphal growth forms is critical for the pathogenesis of C. albicans. During the yeast-to-hypha morphologic transition, gene expression is regulated by transcriptional regulators including histone modifying complexes and chromatin remodeling complexes. We previously reported that Esa1, a catalytic subunit in the histone acetyltransferase complex NuA4, is essential for the hyphal development of C. albicans. In this study, we analyzed the functional roles of Gcn5, a catalytic subunit in the histone acetyltransferase complex SAGA, in C. albicans. Gcn5 is required for the invasive and filamentous growth of C. albicans. Deletion of GCN5 impaired hyphal elongation in sensing serum and attenuated the virulence of C. albicans in a mouse systemic infection model. The C. albicans gcn5/gcn5 mutant cells also exhibited sensitivity to cell wall stress. Functional analysis showed that the HAT domain and Bromodomain in Gcn5 play distinct roles in morphogenesis and cell wall stress response of C. albicans. Our results show that the conserved residue Glu188 is crucial for the Gcn5 HAT activity and for Gcn5 function during filamentous growth. In addition, the subcellular distribution of ectopically expressed GFP-Gcn5 correlates with the different growth states of C. albicans. In stationary phase, Gcn5 accumulated in the nucleus, while during vegetative growth it localized in the cytoplasm in a morpha-independent manner. Our results suggest that the nuclear localization of Gcn5 depends on the existence of its N-terminal NLS and HAT domains. PMID:25656079

  3. Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells

    PubMed Central

    Di Martile, Marta; Desideri, Marianna; De Luca, Teresa; Gabellini, Chiara; Buglioni, Simonetta; Eramo, Adriana; Sette, Giovanni; Milella, Michele; Rotili, Dante; Mai, Antonello; Carradori, Simone; Secci, Daniela; De Maria, Ruggero; Del Bufalo, Donatella; Trisciuoglio, Daniela

    2016-01-01

    Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4′-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC. PMID:26870991

  4. Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells.

    PubMed

    Di Martile, Marta; Desideri, Marianna; De Luca, Teresa; Gabellini, Chiara; Buglioni, Simonetta; Eramo, Adriana; Sette, Giovanni; Milella, Michele; Rotili, Dante; Mai, Antonello; Carradori, Simone; Secci, Daniela; De Maria, Ruggero; Del Bufalo, Donatella; Trisciuoglio, Daniela

    2016-03-01

    Cancer stem cells (CSCs) play an important role in tumor initiation, progression, therapeutic failure and tumor relapse. In this study, we evaluated the efficacy of the thiazole derivative 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6), a novel pCAF and Gcn5 histone acetyltransferase inhibitor, as a small molecule that preferentially targets lung cancer stem-like cells (LCSCs) derived from non-small cell lung cancer (NSCLC) patients. Notably, although CPTH6 inhibits the growth of both LCSC and NSCLC cell lines, LCSCs exhibit greater growth inhibition than established NSCLC cells. Growth inhibitory effect of CPTH6 in LCSC lines is primarily due to apoptosis induction. Of note, differentiated progeny of LCSC lines is more resistant to CPTH6 in terms of loss of cell viability and reduction of protein acetylation, when compared to their undifferentiated counterparts. Interestingly, in LCSC lines CPTH6 treatment is also associated with a reduction of stemness markers. By using different HAT inhibitors we provide clear evidence that inhibition of HAT confers a strong preferential inhibitory effect on cell viability of undifferentiated LCSC lines when compared to their differentiated progeny. In vivo, CPTH6 is able to inhibit the growth of LCSC-derived xenografts and to reduce cancer stem cell content in treated tumors, as evidenced by marked reduction of tumor-initiating capacity in limiting dilution assays. Strikingly, the ability of CPTH6 to inhibit tubulin acetylation is also confirmed in vivo. Overall, our studies propose histone acetyltransferase inhibition as an attractive target for cancer therapy of NSCLC. PMID:26870991

  5. The transcriptional coactivator and acetyltransferase p300 in fibroblast biology and fibrosis.

    PubMed

    Ghosh, Asish K; Varga, John

    2007-12-01

    The transcriptional coactivator p300 is a ubiquitous nuclear phosphoprotein and transcriptional cofactor with intrinsic acetyltransferase activity. p300 controls the expression of numerous genes in cell-type and signal-specific manner, and plays a pivotal role in cellular proliferation, apoptosis, and embryogenesis. By catalyzing acetylation of histones and transcription factors, p300 plays a significant role in epigenetic regulation. Recent evidence suggests that abnormal p300 function is associated with deregulated target gene expression, and is implicated in inflammation, cancer, cardiac hypertrophy, and genetic disorders such as the Rubinstein-Taybi syndrome. The activity of p300 is regulated at multiple levels, including developmental stage-specific expression, post-translational modifications, subcellular localization, and cell-type and gene-specific interactions with transcription factors. Although p300 has been investigated extensively in epithelial and hematopoietic cells, its role in fibroblast biology and tissue repair has received little attention to date. Recent studies implicate p300 in the regulation of collagen synthesis by transforming growth factor-beta (TGF-beta). Both the acetyltransferase activity of p300 and its inducible interaction with Smad3 are essential for mediating TGF-beta-induced stimulation of collagen synthesis. As a signal integrator whose availability for intracellular interactions with transcription factors is strictly limiting, p300 mediates the antagonistic regulation of TGF-beta-induced collagen synthesis by IFN-gamma and TNF-alpha via intracellular competition for limiting amount of p300. Significantly, p300 is itself a direct transcriptional target of TGF-beta in normal fibroblasts, and its levels are significantly elevated in fibrotic lesions as well as in experimental models of fibrosis. The emerging appreciation of the importance of p300 in extracellular matrix (ECM) remodeling and fibrosis and novel insights concerning

  6. Structural and Functional Role of Acetyltransferase hMOF K274 Autoacetylation.

    PubMed

    McCullough, Cheryl E; Song, Shufei; Shin, Michael H; Johnson, F Brad; Marmorstein, Ronen

    2016-08-26

    Many histone acetyltransferases undergo autoacetylation, either through chemical or enzymatic means, to potentiate enzymatic cognate substrate lysine acetylation, although the mode and molecular role of such autoacetylation is poorly understood. The MYST family of histone acetyltransferases is autoacetylated at an active site lysine residue to facilitate cognate substrate lysine binding and acetylation. Here, we report on a detailed molecular investigation of Lys-274 autoacetylation of the human MYST protein Males Absent on the First (hMOF). A mutational scan of hMOF Lys-274 reveals that all amino acid substitutions of this residue are able to bind cofactor but are significantly destabilized, both in vitro and in cells, and are catalytically inactive for cognate histone H4 peptide lysine acetylation. The x-ray crystal structure of a hMOF K274P mutant suggests that the reduced stability and catalytic activity stems from a disordering of the residue 274-harboring a α2-β7 loop. We also provide structural evidence that a C316S/E350Q mutant, which is defective for cognate substrate lysine acetylation; and biochemical evidence that a K268M mutant, which is defective for Lys-274 chemical acetylation in the context of a K274-peptide, can still undergo quantitative K274 autoacetylation. Together, these studies point to the critical and specific role of hMOF Lys-274 autoacetylation in hMOF stability and cognate substrate acetylation and argues that binding of Ac-CoA to hMOF likely drives Lys-274 autoacetylation for subsequent cognate substrate acetylation. PMID:27382063

  7. Sulphoacetaldehyde acetyltransferase yields acetyl phosphate: purification from Alcaligenes defragrans and gene clusters in taurine degradation.

    PubMed Central

    Ruff, Jürgen; Denger, Karin; Cook, Alasdair M

    2003-01-01

    The facultatively anaerobic bacterium Alcaligenes defragrans NKNTAU was found to oxidize taurine (2-aminoethanesulphonate) with nitrate as the terminal electron acceptor. Taurine was transaminated to 2-sulphoacetaldehyde. This was not converted into sulphite and acetate by a "sulphoacetaldehyde sulpho-lyase" (EC 4.4.1.12), but into sulphite and acetyl phosphate, which was identified by three methods. The enzyme, which required the addition of phosphate, thiamin diphosphate and Mg(2+) ions for activity, was renamed sulphoacetaldehyde acetyltransferase (Xsc; EC 2.3.1.-). Inducible Xsc was expressed at high levels, and a three-step 11-fold purification yielded an essentially homogeneous soluble protein, which was a homotetramer in its native form; the molecular mass of the subunit was found to be between about 63 kDa (SDS/PAGE) and 65.3 kDa (matrix-assisted laser-desorption ionization-time-of-flight MS). The N-terminal and two internal amino acid sequences were determined, and PCR primers were generated. The xsc gene was amplified and sequenced; the derived molecular mass of the processed protein was 65.0 kDa. The downstream gene presumably encoded the inducible phosphate acetyltransferase (Pta) found in crude extracts. The desulphonative enzymes ("EC 4.4.1.12") from Achromobacter xylosoxidans NCIMB 10751 and Desulfonispora thiosulfatigenes GKNTAU were shown to be Xscs. We detected at least three subclasses of xsc in Proteobacteria and in Gram-positive bacteria, and they comprised a distinct group within the acetohydroxyacid synthase supergene family. Genome sequencing data revealed xsc genes in Burkholderia fungorum (80% sequence identity) and Sinorhizobium meliloti (61%) with closely linked pta genes. Different patterns of regulation for the transport and dissimilation of taurine were hypothesized for S. meliloti and B. fungorum. PMID:12358600

  8. Epigenetic change in kidney tumor: downregulation of histone acetyltransferase MYST1 in human renal cell carcinoma

    PubMed Central

    2013-01-01

    Background MYST1 (also known as hMOF), a member of the MYST family of histone acetyltransferases (HATs) as an epigenetic mark of active genes, is mainly responsible for histone H4K16 acetylation in the cells. Recent studies have shown that the abnormal gene expression of hMOF is involved in certain primary cancers. Here we examined the involvement of hMOF expression and histone H4K16 acetylation in primary renal cell carcinoma (RCC). Simultaneously, we investigated the correlation between the expression of hMOF and clear cell RCC (ccRCC) biomarker carbohydrase IX (CA9) in RCC. Materials and methods The frozen RCC tissues and RCC cell lines as materials, the reverse transcription polymerase chain reaction (RT-PCR), western blotting and immunohistochemical staining approaches were used. Results RT-PCR results indicate that hMOF gene expression levels frequently downregulated in 90.5% of patients (19/21) with RCC. The reduction of hMOF protein in both RCC tissues and RCC cell lines is tightly correlated with acetylation of histone H4K16. In addition, overexpression of CA9 was detected in 100% of ccRCC patients (21/21). However, transient transfection of hMOF in ccRCC 786–0 cells did not affect both the gene and protein expression of CA9. Conclusion hMOF as an acetyltransferase of H4K16 might be involved in the pathogenesis of kidney cancer, and this epigenetic changes might be a new CA9-independent RCC diagnostic maker. PMID:23394073

  9. Structures and functions of insect arylalkylamine N-acetyltransferase (iaaNAT); a key enzyme for physiological and behavioral switch in arthropods

    PubMed Central

    Hiragaki, Susumu; Suzuki, Takeshi; Mohamed, Ahmed A. M.; Takeda, Makio

    2015-01-01

    The evolution of N-acetyltransfeases (NATs) seems complex. Vertebrate arylalkylamine N-acetyltransferase (aaNAT) has been extensively studied since it leads to the synthesis of melatonin, a multifunctional neurohormone prevalent in photoreceptor cells, and is known as a chemical token of the night. Melatonin also serves as a scavenger for reactive oxygen species. This is also true with invertebrates. NAT therefore has distinct functional implications in circadian function, as timezymes (aaNAT), and also xenobiotic reactions (arylamine NAT or simply NAT). NATs belong to a broader enzyme group, the GCN5-related N-acetyltransferase superfamily. Due to low sequence homology and a seemingly fast rate of structural differentiation, the nomenclature for NATs can be confusing. The advent of bioinformatics, however, has helped to classify this group of enzymes; vertebrates have two distinct subgroups, the timezyme type and the xenobiotic type, which has a wider substrate range including imidazolamine, pharmacological drugs, environmental toxicants and even histone. Insect aaNAT (iaaNAT) form their own clade in the phylogeny, distinct from vertebrate aaNATs. Arthropods are unique, since the phylum has exoskeleton in which quinones derived from N-acetylated monoamines function in coupling chitin and arthropodins. Monoamine oxidase (MAO) activity is limited in insects, but NAT-mediated degradation prevails. However, unexpectedly iaaNAT occurs not only among arthropods but also among basal deuterostomia, and is therefore more apomorphic. Our analyses illustrate that iaaNATs has unique physiological roles but at the same time it plays a role in a timezyme function, at least in photoperiodism. Photoperiodism has been considered as a function of circadian system but the detailed molecular mechanism is not well understood. We propose a molecular hypothesis for photoperiodism in Antheraea pernyi based on the transcription regulation of NAT interlocked by the circadian system

  10. Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus.

    PubMed

    Sharma, Rashmi; Lambu, Mallikharjuna Rao; Jamwal, Urmila; Rani, Chitra; Chib, Reena; Wazir, Priya; Mukherjee, Debaraj; Chaubey, Asha; Khan, Inshad Ali

    2016-04-01

    Secondary metabolite of Aspergillus terreus, terreic acid, is a reported potent antibacterial that was identified more than 60 years ago, but its cellular target(s) are still unknown. Here we screen its activity against the acetyltransferase domain of a bifunctional enzyme, Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). An absorbance-based assay was used to screen terreic acid against the acetyltransferase activity of E. coli GlmU. Terreic acid was found to inhibit the acetyltransferase domain of E. coli GlmU with an IC50 of 44.24 ± 1.85 µM. Mode of inhibition studies revealed that terreic acid was competitive with AcCoA and uncompetitive with GlcN-1-P. It also exhibited concentration-dependent killing of E. coli ATCC 25922 up to 4× minimum inhibitory concentration and inhibited the growth of biofilms generated by E. coli. Characterization of resistant mutants established mutation in the acetyltransferase domain of GlmU. Terreic acid was also found to be metabolically stable in the in vitro incubations with rat liver microsome in the presence of a NADPH regenerating system. The studies reported here suggest that terreic acid is a potent antimicrobial agent and support that E. coli GlmU acetyltransferase is a molecular target of terreic acid, resulting in its antibacterial activity. PMID:26762501

  11. Regulation of KAT6 Acetyltransferases and Their Roles in Cell Cycle Progression, Stem Cell Maintenance, and Human Disease.

    PubMed

    Huang, Fu; Abmayr, Susan M; Workman, Jerry L

    2016-07-15

    The lysine acetyltransferase 6 (KAT6) histone acetyltransferase (HAT) complexes are highly conserved from yeast to higher organisms. They acetylate histone H3 and other nonhistone substrates and are involved in cell cycle regulation and stem cell maintenance. In addition, the human KAT6 HATs are recurrently mutated in leukemia and solid tumors. Therefore, it is important to understand the mechanisms underlying the regulation of KAT6 HATs and their roles in cell cycle progression. In this minireview, we summarize the identification and analysis of the KAT6 complexes and discuss the regulatory mechanisms governing their enzymatic activities and substrate specificities. We further focus on the roles of KAT6 HATs in regulating cell proliferation and stem cell maintenance and review recent insights that aid in understanding their involvement in human diseases. PMID:27185879

  12. The histone acetyltransferase p300 inhibitor C646 reduces pro-inflammatory gene expression and inhibits histone deacetylases

    PubMed Central

    van den Bosch, Thea; Boichenko, Alexander; Leus, Niek G. J.; Eleni Ourailidou, Maria; Wapenaar, Hannah; Rotili, Dante; Mai, Antonello; Imhof, Axel; Bischoff, Rainer; Haisma, Hidde J.; Dekker, Frank J.

    2016-01-01

    Lysine acetylations are reversible posttranslational modifications of histone and non-histone proteins that play important regulatory roles in signal transduction cascades and gene expression. Lysine acetylations are regulated by histone acetyltransferases as writers and histone deacetylases as erasers. Because of their role in signal transduction cascades, these enzymes are important players in inflammation. Therefore, applications of histone acetyltransferase inhibitors to reduce inflammatory responses are interesting. Among the few histone acetyltransferase inhibitors described, C646 is one of the most potent (Ki of 0.4 μM for histone acetyltransferase p300). C646 was described to regulate the NF-κB pathway; an important pathway in inflammatory responses, which is regulated by acetylation. Interestingly, this pathway has been implicated in asthma and COPD. Therefore we hypothesized that via regulation of the NF-κB signaling pathway, C646 can inhibit pro-inflammatory gene expression, and have potential for the treatment of inflammatory lung diseases. In line with this, here we demonstrate that C646 reduces pro-inflammatory gene expression in RAW264.7 murine macrophages and murine precision-cut lung slices. To unravel its effects on cellular substrates we applied mass spectrometry and found, counterintuitively, a slight increase in acetylation of histone H3. Based on this finding, and structural features of C646, we presumed inhibitory activity of C646 on histone deacetylases, and indeed found inhibition of histone deacetylases from 7 μM and higher concentrations. This indicates that C646 has potential for further development towards applications in the treatment of inflammation, however, its newly discovered lack of selectivity at higher concentrations needs to be taken into account. PMID:26718586

  13. The histone acetyltransferase p300 inhibitor C646 reduces pro-inflammatory gene expression and inhibits histone deacetylases.

    PubMed

    van den Bosch, Thea; Boichenko, Alexander; Leus, Niek G J; Ourailidou, Maria E; Wapenaar, Hannah; Rotili, Dante; Mai, Antonello; Imhof, Axel; Bischoff, Rainer; Haisma, Hidde J; Dekker, Frank J

    2016-02-15

    Lysine acetylations are reversible posttranslational modifications of histone and non-histone proteins that play important regulatory roles in signal transduction cascades and gene expression. Lysine acetylations are regulated by histone acetyltransferases as writers and histone deacetylases as erasers. Because of their role in signal transduction cascades, these enzymes are important players in inflammation. Therefore, histone acetyltransferase inhibitors could reduce inflammatory responses. Among the few histone acetyltransferase inhibitors described, C646 is one of the most potent (Ki of 0.4μM for histone acetyltransferase p300). C646 was described to affect the NF-κB pathway; an important pathway in inflammatory responses, which is regulated by acetylation. This pathway has been implicated in asthma and COPD. Therefore, we hypothesized that via regulation of the NF-κB signaling pathway, C646 can inhibit pro-inflammatory gene expression, and have potential for the treatment of inflammatory lung diseases. In line with this, we demonstrate here that C646 reduces pro-inflammatory gene expression in RAW264.7 murine macrophages and murine precision-cut lung slices. To unravel its effects on cellular substrates we applied mass spectrometry and found, counterintuitively, a slight increase in acetylation of histone H3. Based on this finding, and structural features of C646, we presumed inhibitory activity of C646 on histone deacetylases, and indeed found inhibition of histone deacetylases from 7μM and higher concentrations. This indicates that C646 has potential for further development towards applications in the treatment of inflammation, however, its newly discovered lack of selectivity at higher concentrations needs to be taken into account. PMID:26718586

  14. Structure of Arabidopsis thaliana At1g77540 Protein, a Minimal Acetyltransferase from the COG2388 Family †,‡

    PubMed Central

    Tyler, Robert C.; Bitto, Eduard; Berndsen, Christopher E.; Bingman, Craig A.; Singh, Shanteri; Lee, Min S.; Wesenberg, Gary E.; Denu, John M.; Phillips, George N.; Markley, John L.

    2008-01-01

    We describe X-ray crystal and NMR solution structures of the protein coded for by Arabidopsis thaliana gene At1g77540.1 (At1g77540). The crystal structure was determined to 1.15 Å with an R factor of 14.9% (Rfree = 17.0%) by multiple-wavelength anomalous diffraction using sodium bromide derivatized crystals. The ensemble of NMR conformers was determined with protein samples labeled with 15N and 13C+15N. The X-ray structure and NMR ensemble were closely similar with r.m.s.d 1.4 Å for residues 8–93. At1g77540 was found to adopt a fold similar to that of GCN5-related N-acetyltransferases. Enzymatic activity assays established that At1g77540 possesses weak acetyltransferase activity against histones H3 and H4. Chemical shift perturbations observed in 15N-HSQC spectra upon the addition of CoA indicated that the cofactor binds and identified its binding site. The molecular details of this interaction were further elucidated by solving the X-ray structure of the At1g77540–CoA complex. This work establishes that the domain family COG2388 represents a novel class of acetyltransferase and provides insight into possible mechanistic roles of the conserved Cys76 and His41 residues of this family. PMID:17128971

  15. Two N-Terminal Acetyltransferases Antagonistically Regulate the Stability of a Nod-Like Receptor in Arabidopsis

    PubMed Central

    Li, Lin; Gannon, Patrick; Linster, Eric; Huber, Monika; Kapos, Paul; Bienvenut, Willy; Giglione, Carmela; Zhang, Yuelin; Chen, She

    2015-01-01

    Nod-like receptors (NLRs) serve as immune receptors in plants and animals. The stability of NLRs is tightly regulated, though its mechanism is not well understood. Here, we show the crucial impact of N-terminal acetylation on the turnover of one plant NLR, Suppressor of NPR1, Constitutive 1 (SNC1), in Arabidopsis thaliana. Genetic and biochemical analyses of SNC1 uncovered its multilayered regulation by different N-terminal acetyltransferase (Nat) complexes. SNC1 exhibits a few distinct N-terminal isoforms generated through alternative initiation and N-terminal acetylation. Its first Met is acetylated by N-terminal acetyltransferase complex A (NatA), while the second Met is acetylated by N-terminal acetyltransferase complex B (NatB). Unexpectedly, the NatA-mediated acetylation serves as a degradation signal, while NatB-mediated acetylation stabilizes the NLR protein, thus revealing antagonistic N-terminal acetylation of a single protein substrate. Moreover, NatA also contributes to the turnover of another NLR, RESISTANCE TO P. syringae pv maculicola 1. The intricate regulation of protein stability by Nats is speculated to provide flexibility for the target protein in maintaining its homeostasis. PMID:25966763

  16. SIAH-mediated ubiquitination and degradation of acetyl-transferases regulate the p53 response and protein acetylation.

    PubMed

    Grishina, Inna; Debus, Katherina; García-Limones, Carmen; Schneider, Constanze; Shresta, Amit; García, Carlos; Calzado, Marco A; Schmitz, M Lienhard

    2012-12-01

    Posttranslational modification of proteins by lysine acetylation regulates many biological processes ranging from signal transduction to chromatin compaction. Here we identify the acetyl-transferases CBP/p300, Tip60 and PCAF as new substrates for the ubiquitin E3 ligases SIAH1 and SIAH2. While CBP/p300 can undergo ubiquitin/proteasome-dependent degradation by SIAH1 and SIAH2, the two other acetyl-transferases are exclusively degraded by SIAH2. Accordingly, SIAH-deficient cells show enhanced protein acetylation, thus revealing SIAH proteins as indirect regulators of the cellular acetylation status. Functional experiments show that Tip60/PCAF-mediated acetylation of the tumor suppressor p53 is antagonized by the p53 target gene SIAH2 which mediates ubiquitin/proteasome-mediated degradation of both acetyl-transferases and consequently diminishes p53 acetylation and transcriptional activity. The p53 kinase HIPK2 mediates hierarchical phosphorylation of SIAH2 at 5 sites, which further boosts its activity as a ubiquitin E3 ligase for several substrates and therefore dampens the late p53 response. PMID:23044042

  17. Identification and Functional Characterization of Arylamine N-Acetyltransferases in Eubacteria: Evidence for Highly Selective Acetylation of 5-Aminosalicylic Acid

    PubMed Central

    Deloménie, Claudine; Fouix, Sylvaine; Longuemaux, Sandrine; Brahimi, Naïma; Bizet, Chantal; Picard, Bertrand; Denamur, Erick; Dupret, Jean-Marie

    2001-01-01

    Arylamine N-acetyltransferase activity has been described in various bacterial species. Bacterial N-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time N-acetyltransferase activity in 11 species of Proteobacteriaceae from seven genera: Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter freundii, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Morganella morganii, Serratia marcescens, Shigella flexneri, Plesiomonas shigelloides, and Vibrio cholerae. We estimated apparent kinetic parameters and found that 5-aminosalicylic acid, a compound efficient in the treatment of inflammatory bowel diseases, was acetylated with a catalytic efficiency 27 to 645 times higher than that for its isomer, 4-aminosalicylic acid. In contrast, para-aminobenzoic acid, a folate precursor in bacteria, was poorly acetylated. Of the wild-type strains studied, Pseudomonas aeruginosa was the best acetylator in terms of both substrate spectrum and catalytic efficiency. DNA hybridization with a Salmonella enterica serovar Typhimurium-derived probe suggested the presence of this enzyme in eight proteobacterial and four gram-positive species. Molecular aspects together with the kinetic data suggest distinct functional features for this class of microbial enzymes. PMID:11344150

  18. Cloning, sequencing, characterisation and implications for vaccine design of the novel dihydrolipoyl acetyltransferase of Neisseria meningitidis.

    PubMed

    Ala' Aldeen, D A; Westphal, A H; De Kok, A; Weston, V; Atta, M S; Baldwin, T J; Bartley, J; Borriello, S P

    1996-12-01

    A lambdaZap-II expression library of Neisseria meningitidis was screened with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from preparative SDS-polyacrylamide gels. Selected clones were isolated, further purified, and their recombinant pBluescript SKII plasmids were excised. The cloned DNA insert was sequenced from positive clones and analysed. Four open reading frames (ORFs) were identified, three of which showed a high degree of homology with the pyruvate dehydrogenase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase complex (PDHC) of a number of prokaryotic and eukaryotic species. Sequence analysis indicated that the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl domains, an E1/E3 binding domain and a catalytic domain. The domains are separated by hinge regions rich in alanine, proline and charged residues. Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A further ORF, coding for a 16.5-kDa protein, was found between the ORFs encoding the E2p and E3 components. The identity and functional characteristics of the expressed and purified heterologous Men-E2p were confirmed as dihydrolipoyl acetyltransferase by immunological and biochemical assays. N-terminal amino-acid analysis confirmed the sequence of the DNA-derived mature protein. Purified Men-E2p reacted with monospecific antisera raised against the whole E2p molecule and against the lipoyl domain of the Azotobacter vinelandii E2p. Conversely, rabbit antiserum raised against Men-E2p reacted with protein extracts of A. vinelandii, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalytic domains of E2p obtained by limited proteolysis. In contrast, the original R-70 antiserum reacted almost exclusively with the lipoyl domain, indicating the strong immunogenicity

  19. Catalytic mechanism of bleomycin N-acetyltransferase proposed on the basis of its crystal structure.

    PubMed

    Oda, Kosuke; Matoba, Yasuyuki; Noda, Masafumi; Kumagai, Takanori; Sugiyama, Masanori

    2010-01-01

    Bleomycin (Bm) N-acetyltransferase, BAT, is a self-resistance determinant in Bm-producing Streptomyces verticillus ATCC15003. In our present study, we crystallized BAT under both a terrestrial and a microgravity environment in the International Space Station. In addition to substrate-free BAT, the crystal structures of BAT in a binary complex with CoA and in a ternary complex with Bm and CoA were determined. BAT forms a dimer structure via interaction of its C-terminal domains in the monomers. However, each N-terminal domain in the dimer is positioned without mutual interaction. The tunnel observed in the N-terminal domain of BAT has two entrances: one that adopts a wide funnel-like structure necessary to accommodate the metal-binding domain of Bm, and another narrow entrance that accommodates acetyl-CoA (AcCoA). A groove formed on the dimer interface of two BAT C-terminal domains accommodates the DNA-binding domain of Bm. In a ternary complex of BAT, BmA(2), and CoA, a thiol group of CoA is positioned near the primary amine of Bm at the midpoint of the tunnel. This proximity ensures efficient transfer of an acetyl group from AcCoA to the primary amine of Bm. Based on the BAT crystal structure and the enzymatic kinetic study, we propose that the catalytic mode of BAT takes an ordered-like mechanism. PMID:19889644

  20. Characterizing the Covalent Targets of a Small Molecule Inhibitor of the Lysine Acetyltransferase P300.

    PubMed

    Shrimp, Jonathan H; Sorum, Alexander W; Garlick, Julie M; Guasch, Laura; Nicklaus, Marc C; Meier, Jordan L

    2016-02-11

    C646 inhibits the lysine acetyltransferases (KATs) p300 and CBP and represents the most potent and selective small molecule KAT inhibitor identified to date. To gain insights into the cellular activity of this epigenetic probe, we applied chemoproteomics to identify covalent targets of the C646 chemotype. Modeling and synthetic derivatization was used to develop a clickable analogue (C646-yne) that inhibits p300 similarly to the parent compound and enables enrichment of bound proteins. LC-MS/MS identified the major covalent targets of C646-yne as highly abundant cysteine-containing proteins, and follow-up studies found that C646 can inhibit tubulin polymerization in vitro. Finally, we provide evidence that thiol reactivity of C646 may limit its ability to antagonize acetylation in cells. These findings should enable a more precise interpretation of studies utilizing C646 as a chemical probe of KAT activity and suggest that an underappreciated liability of electrophile-containing inhibitors is a reduction in their cellular potency due to consumption by abundant protein and metabolite thiol sinks. PMID:26985290

  1. Microfluidic Mobility Shift Profiling of Lysine Acetyltransferases Enables Screening and Mechanistic Analysis of Cellular Acetylation Inhibitors.

    PubMed

    Sorum, Alexander W; Shrimp, Jonathan H; Roberts, Allison M; Montgomery, David C; Tiwari, Neil K; Lal-Nag, Madhu; Simeonov, Anton; Jadhav, Ajit; Meier, Jordan L

    2016-03-18

    Lysine acetyltransferases (KATs) are critical regulators of signaling in many diseases, including cancer. A major challenge in establishing the targetable functions of KATs in disease is a lack of well-characterized, cell-active KAT inhibitors. To confront this challenge, here we report a microfluidic mobility shift platform for the discovery and characterization of small molecule KAT inhibitors. Novel fluorescent peptide substrates were developed for four well-known KAT enzymes (p300, Crebbp, Morf, and Gcn5). Enzyme-catalyzed acetylation alters the electrophoretic mobility of these peptides in a microfluidic chip, allowing facile and direct monitoring of KAT activity. A pilot screen was used to demonstrate the utility of microfluidic mobility shift profiling to identify known and novel modulators of KAT activity. Real-time kinetic monitoring of KAT activity revealed that garcinol, a natural product KAT inhibitor used in cellular studies, exhibits time-dependent and detergent-sensitive inhibition, consistent with an aggregation-based mechanism. In contrast, the cell-permeable bisubstrate inhibitor Tat-CoA exhibited potent and time-independent KAT inhibition, highlighting its potential utility as a cellular inhibitor of KAT activity. These studies define microfluidic mobility shift profiling as a powerful platform for the discovery and characterization of small molecule inhibitors of KAT activity, and provide mechanistic insights potentially important for the application of KAT inhibitors in cellular contexts. PMID:26428393

  2. X-ray crystal structure of ornithine acetyltransferase from the clavulanic acid biosynthesis gene cluster.

    PubMed

    Elkins, Jonathan M; Kershaw, Nadia J; Schofield, Christopher J

    2005-01-15

    The orf6 gene from the clavulanic acid biosynthesis gene cluster encodes an OAT (ornithine acetyltransferase). Similar to other OATs the enzyme has been shown to catalyse the reversible transfer of an acetyl group from N-acetylornithine to glutamate. OATs are Ntn (N-terminal nucleophile) enzymes, but are distinct from the better-characterized Ntn hydrolase enzymes as they catalyse acetyl transfer rather than a hydrolysis reaction. In the present study, we describe the X-ray crystal structure of the OAT, corresponding to the orf6 gene product, to 2.8 A (1 A=0.1 nm) resolution. The larger domain of the structure consists of an alphabetabetaalpha sandwich as in the structures of Ntn hydrolase enzymes. However, differences in the connectivity reveal that OATs belong to a structural family different from that of other structurally characterized Ntn enzymes, with one exception: unexpectedly, the alphabetabetaalpha sandwich of ORF6 (where ORF stands for open reading frame) displays the same fold as an DmpA (L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi), and so the OATs and DmpA form a new structural subfamily of Ntn enzymes. The structure reveals an alpha2beta2-heterotetrameric oligomerization state in which the intermolecular interface partly defines the active site. Models of the enzyme-substrate complexes suggest a probable oxyanion stabilization mechanism as well as providing insight into how the enzyme binds its two differently charged substrates. PMID:15352873

  3. P300 acetyltransferase regulates fatty acid synthase expression, lipid metabolism and prostate cancer growth

    PubMed Central

    Zhong, Jian; Jiang, Kui; Pan, Yunqian; Karnes, R. Jeffrey; Zhang, Jun; Xu, Wanhai; Wang, Guixia; Huang, Haojie

    2016-01-01

    De novo fatty acid (FA) synthesis is required for prostate cancer (PCa) survival and progression. As a key enzyme for FA synthesis fatty acid synthase (FASN) is often overexpressed in human prostate cancers and its expression correlates with worse prognosis and poor survival. P300 is an acetyltransferase that acts as a transcription co-activator. Increasing evidence suggests that P300 is a major PCa promoter, although the underlying mechanism remains poorly understood. Here, we demonstrated that P300 binds to and increases histone H3 lysine 27 acetylation (H3K27Ac) in the FASN gene promoter. We provided evidence that P300 transcriptionally upregulates FASN expression and promotes lipid accumulation in human PCa cells in culture and Pten knockout prostate tumors in mice. Pharmacological inhibition of P300 decreased FASN expression and lipid droplet accumulation in PCa cells. Immunohistochemistry analysis revealed that expression of P300 protein positively correlates with FASN protein levels in a cohort of human PCa specimens. We further showed that FASN is a key mediator of P300-induced growth of PCa cells in culture and in mice. Together, our findings demonstrate P300 as a key factor that regulates FASN expression, lipid accumulation and cell growth in PCa. They also suggest that this regulatory pathway can serve as a new therapeutic target for PCa treatment. PMID:26934656

  4. Disubstituted naphthyl β-D-xylopyranosides: Synthesis, GAG priming, and histone acetyltransferase (HAT) inhibition.

    PubMed

    Thorsheim, Karin; Persson, Andrea; Siegbahn, Anna; Tykesson, Emil; Westergren-Thorsson, Gunilla; Mani, Katrin; Ellervik, Ulf

    2016-04-01

    Xylosides are a group of compounds that can induce glycosaminoglycan (GAG) chain synthesis independently of a proteoglycan core protein. We have previously shown that the xyloside 2-(6-hydroxynaphthyl)β-D-xylopyranoside has a tumor-selective growth inhibitory effect both in vitro and in vivo, and that the effect in vitro was correlated to a reduction in histone H3 acetylation. In addition, GAG chains have previously been reported to inhibit histone acetyltransferases (HAT). To investigate if xylosides, or the corresponding xyloside-primed GAG chains, can be used as HAT inhibitors, we have synthesized a series of naphthoxylosides carrying structural motifs similar to the aromatic moieties of the known HAT inhibitors garcinol and curcumin, and studied their biological activities. Here, we show that the disubstituted naphthoxylosides induced GAG chain synthesis, and that the ones with at least one free phenolic group exhibited moderate HAT inhibition in vitro, without affecting histone H3 acetylation in cell culture. The xyloside-primed GAG chains, on the other hand, had no effect on HAT activity, possibly explaining why the effect of the xylosides on histone H3 acetylation was absent in cell culture as the xylosides were recruited for GAG chain synthesis. Further investigations are required to find xylosides that are effective HAT inhibitors or xylosides producing GAG chains with HAT inhibitory effects. PMID:27023911

  5. Interaction of human arylamine N-acetyltransferase 1 with different nanomaterials.

    PubMed

    Deng, Zhou J; Butcher, Neville J; Mortimer, Gysell M; Jia, Zhongfan; Monteiro, Michael J; Martin, Darren J; Minchin, Rodney F

    2014-03-01

    Humans are exposed to nanoparticles in the environment as well as those in nanomaterials developed for biomedical applications. However, the safety and biologic effects of many nanoparticles remain to be elucidated. Over the past decade, our understanding of the interaction of proteins with various nanomaterials has grown. The protein corona can determine not only how nanoparticles interact with cells but also their biologic effects and toxicity. In this study, we describe the effects that several different classes of nanoparticles exert on the enzymatic activity of the cytosolic protein human arylamine N-acetyltransferase 1 (NAT1), a drug-metabolizing enzyme widely distributed in the body that is also responsible for the activation and detoxification of known carcinogens. We investigated three metal oxides (zinc oxide, titanium dioxide, and silicon dioxide), two synthetic clay nanoparticles (layered double hydroxide and layered silicate nanoparticles), and a self-assembling thermo-responsive polymeric nanoparticle that differ in size and surface characteristics. We found that the different nanoparticles induced very different responses, ranging from inhibition to marked enhancement of enzyme activity. The layered silicates did not directly inactivate NAT1, but was found to enhance substrate-dependent inhibition. These differing effects demonstrate the multiplicity of nanoparticle-protein interactions and suggest that enzyme activity may be compromised in organs exposed to nanoparticles, such as the lungs or reticulo-endothelial system. PMID:24346836

  6. Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization

    PubMed Central

    Kumar, Rajnish; Långström, Bengt; Darreh-Shori, Taher

    2016-01-01

    Recent reports have brought back the acetylcholine synthesizing enzyme, choline acetyltransferase in the mainstream research in dementia and the cholinergic anti-inflammatory pathway. Here we report, a specific strategy for the design of novel ChAT ligands based on molecular docking, Hologram Quantitative Structure Activity Relationship (HQSAR) and lead optimization. Molecular docking was performed on a series of ChAT inhibitors to decipher the molecular fingerprint of their interaction with the active site of ChAT. Then robust statistical fragment HQSAR models were developed. A library of novel ligands was generated based on the pharmacophoric and shape similarity scoring function, and evaluated in silico for their molecular interactions with ChAT. Ten of the top scoring invented compounds are reported here. We confirmed the activity of α-NETA, the only commercially available ChAT inhibitor, and one of the seed compounds in our model, using a new simple colorimetric ChAT assay (IC50 ~ 88 nM). In contrast, α-NETA exhibited an IC50 of ~30 μM for the ACh-degrading cholinesterases. In conclusion, the overall results may provide useful insight for discovering novel ChAT ligands and potential positron emission tomography tracers as in vivo functional biomarkers of the health of central cholinergic system in neurodegenerative disorders, such as Alzheimer’s disease. PMID:27507101

  7. Structural model of carnitine palmitoyltransferase I based on the carnitine acetyltransferase crystal.

    PubMed Central

    Morillas, Montserrat; López-VViñas, Eduardo; Valencia, Alfonso; Serra, Dolors; Gómez-Puertas, Paulino; Hegardt, Fausto G; Asins, Guillermina

    2004-01-01

    CPT I (carnitine palmitoyltransferase I) catalyses the conversion of palmitoyl-CoA into palmitoylcarnitine in the presence of L-carnitine, facilitating the entry of fatty acids into mitochondria. We propose a 3-D (three-dimensional) structural model for L-CPT I (liver CPT I), based on the similarity of this enzyme to the recently crystallized mouse carnitine acetyltransferase. The model includes 607 of the 773 amino acids of L-CPT I, and the positions of carnitine, CoA and the palmitoyl group were assigned by superposition and docking analysis. Functional analysis of this 3-D model included the mutagenesis of several amino acids in order to identify putative catalytic residues. Mutants D477A, D567A and E590D showed reduced L-CPT I activity. In addition, individual mutation of amino acids forming the conserved Ser685-Thr686-Ser687 motif abolished enzyme activity in mutants T686A and S687A and altered K(m) and the catalytic efficiency for carnitine in mutant S685A. We conclude that the catalytic residues are His473 and Asp477, while Ser687 probably stabilizes the transition state. Several conserved lysines, i.e. Lys455, Lys505, Lys560 and Lys561, were also mutated. Only mutants K455A and K560A showed decreases in activity of 50%. The model rationalizes the finding of nine natural mutations in patients with hereditary L-CPT I deficiencies. PMID:14711372

  8. Mechanistic and Structural Analysis of Drosophila melanogaster Arylalkylamine N-Acetyltransferases

    PubMed Central

    2015-01-01

    Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH–activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure–function relationships, pH–rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA. PMID:25406072

  9. Fungal Rtt109 Histone Acetyltransferase is an Unexpected Structural Homolog of Metazoan p300/CBP

    SciTech Connect

    Tang,Y.; Holbert, M.; Wurtele, H.; Meeth, K.; Rocha, W.; Gharib, M.; Jiang, E.; Thibault, P.; Verreault, A.; et al

    2008-01-01

    Rtt109, also known as KAT11, is a recently characterized fungal-specific histone acetyltransferase (HAT) that modifies histone H3 lysine 56 (H3K56) to promote genome stability. Rtt109 does not show sequence conservation with other known HATs and depends on association with either of two histone chaperones, Asf1 or Vps75, for HAT activity. Here we report the X-ray crystal structure of an Rtt109-acetyl coenzyme A complex and carry out structure-based mutagenesis, combined with in vitro biochemical studies of the Rtt109-Vps75 complex and studies of Rtt109 function in vivo. The Rtt109 structure reveals noteworthy homology to the metazoan p300/CBP HAT domain but exhibits functional divergence, including atypical catalytic properties and mode of cofactor regulation. The structure reveals a buried autoacetylated lysine residue that we show is also acetylated in the Rtt109 protein purified from yeast cells. Implications for understanding histone substrate and chaperone binding by Rtt109 are discussed.

  10. MYST2 acetyltransferase expression and Histone H4 Lysine acetylation are suppressed in AML.

    PubMed

    Sauer, Tim; Arteaga, Maria Francisca; Isken, Fabienne; Rohde, Christian; Hebestreit, Katja; Mikesch, Jan-Henrik; Stelljes, Matthias; Cui, Chunhong; Zhou, Fengbiao; Göllner, Stefanie; Bäumer, Nicole; Köhler, Gabriele; Krug, Utz; Thiede, Christian; Ehninger, Gerhard; Edemir, Bayram; Schlenke, Peter; Berdel, Wolfgang E; Dugas, Martin; Müller-Tidow, Carsten

    2015-09-01

    Chromatin-modifying enzymes are frequently altered in acute myeloid leukemia (AML). In the current study, we identified MYST2, a core histone acetyltransferase, to be suppressed in blast cells from AML patients compared with nonmalignant hematopoietic progenitor cells. Functionally, loss of MYST2 accelerated leukemic growth and colony formation, while forced expression of MYST2 induced H4K5 acetylation (H4K5Ac) and suppressed hematopoietic progenitor cell growth. Consistently, global H4K5Ac levels were frequently decreased in AML blasts. Low levels of H4K5Ac were most prominent in patients with complex karyotype AML and were associated with inferior overall survival in univariate but not multivariate analysis. ChIP-seq experiments in primary AML patients' blasts revealed widespread H4K5Ac deregulation, most prominent at gene promoters. Taken together, MYST2 is a repressed growth suppressor in AML mediating reduced acetylation of histone 4 at residue 5 and is associated with inferior AML patient survival. PMID:26072331

  11. Implication of ornithine acetyltransferase activity on l-ornithine production in Corynebacterium glutamicum.

    PubMed

    Hao, Ning; Mu, Jingrui; Hu, Nan; Xu, Sheng; Shen, Peng; Yan, Ming; Li, Yan; Xu, Lin

    2016-01-01

    l-Ornithine is an intermediate of the l-arginine biosynthetic pathway in Corynebacterium glutamicum. The effect of ornithine acetyltransferase (OATase; ArgJ) on l-ornithine production was investigated, and C. glutamicum 1006 was engineered to overproduce l-ornithine as a major product by inactivating regulatory repressor argR gene and overexpressing argJ gene. A genome sequence analysis indicated that the argF gene encoding ornithine carbamoyltransferase in C. glutamicum 1006 was mutated, resulting in the accumulation of a certain amount of l-ornithine (20.5 g/L). The assays using a crude extract of C. glutamicum 1006 indicated that the l-ornithine concentration for 50% inhibition of OAT was 5 mM. To enhance l-ornithine production, the argJ gene from C. glutamicum ATCC 13032 was overexpressed. In flask cultures, the resulting strain, C. glutamicum 1006∆argR-argJ, produced 31.6 g/L l-ornithine, which is 54.15% more than that produced by C. glutamicum 1006. The OAT activity of C. glutamicum 1006∆argR-argJ was significantly greater than that of C. glutamicum 1006, and this study achieved the highest conversion ratio of sugar to acid (0.396 g/g) compared with those of previous reports. ArgJ strongly influences the production of l-ornithine in C. glutamicum. PMID:25630515

  12. The acetyltransferase Tip60 contributes to mammary tumorigenesis by modulating DNA repair.

    PubMed

    Bassi, C; Li, Y-T; Khu, K; Mateo, F; Baniasadi, P S; Elia, A; Mason, J; Stambolic, V; Pujana, M A; Mak, T W; Gorrini, C

    2016-07-01

    The acetyltransferase Tip60/Kat5 acetylates both histone and non-histone proteins, and is involved in a variety of biological processes. By acetylating p53, Tip60 controls p53-dependent transcriptional activity and so is implicated as a tumor suppressor. However, many breast cancers with low Tip60 also show p53 mutation, implying that Tip60 has a tumor suppressor function independent of its acetylation of p53. Here, we show in a p53-null mouse model of sporadic invasive breast adenocarcinoma that heterozygosity for Tip60 deletion promotes mammary tumorigenesis. Low Tip60 reduces DNA repair in normal and tumor mammary epithelial cells, both under resting conditions and following genotoxic stress. We demonstrate that Tip60 controls homologous recombination (HR)-directed DNA repair, and that Tip60 levels correlate inversely with a gene expression signature associated with defective HR-directed DNA repair. In human breast cancer data sets, Tip60 mRNA is downregulated, with low Tip60 levels correlating with p53 mutations in basal-like breast cancers. Our findings indicate that Tip60 is a novel breast tumor suppressor gene whose loss results in genomic instability leading to cancer formation. PMID:26915295

  13. Acceptor substrate binding revealed by crystal structure of human glucosamine-6-phosphate N-acetyltransferase 1.

    PubMed

    Wang, Juan; Liu, Xiang; Liang, Yu-He; Li, Lan-Fen; Su, Xiao-Dong

    2008-09-01

    Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP-N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. PMID:18675810

  14. Mechanistic and structural analysis of Drosophila melanogaster arylalkylamine N-acetyltransferases.

    PubMed

    Dempsey, Daniel R; Jeffries, Kristen A; Bond, Jason D; Carpenter, Anne-Marie; Rodriguez-Ospina, Santiago; Breydo, Leonid; Caswell, K Kenneth; Merkler, David J

    2014-12-16

    Arylalkylamine N-acetyltransferase (AANAT) catalyzes the penultimate step in the biosynthesis of melatonin and other N-acetylarylalkylamides from the corresponding arylalkylamine and acetyl-CoA. The N-acetylation of arylalkylamines is a critical step in Drosophila melanogaster for the inactivation of the bioactive amines and the sclerotization of the cuticle. Two AANAT variants (AANATA and AANATB) have been identified in D. melanogaster, in which AANATA differs from AANATB by the truncation of 35 amino acids from the N-terminus. We have expressed and purified both D. melanogaster AANAT variants (AANATA and AANATB) in Escherichia coli and used the purified enzymes to demonstrate that this N-terminal truncation does not affect the activity of the enzyme. Subsequent characterization of the kinetic and chemical mechanism of AANATA identified an ordered sequential mechanism, with acetyl-CoA binding first, followed by tyramine. We used a combination of pH-activity profiling and site-directed mutagenesis to study prospective residues believed to function in AANATA catalysis. These data led to an assignment of Glu-47 as the general base in catalysis with an apparent pKa of 7.0. Using the data generated for the kinetic mechanism, structure-function relationships, pH-rate profiles, and site-directed mutagenesis, we propose a chemical mechanism for AANATA. PMID:25406072

  15. Genome-Wide Relationships between TAF1 and Histone Acetyltransferases in Saccharomyces cerevisiae†

    PubMed Central

    Durant, Melissa; Pugh, B. Franklin

    2006-01-01

    Histone acetylation regulates gene expression, yet the functional contributions of the numerous histone acetyltransferases (HATs) to gene expression and their relationships with each other remain largely unexplored. The central role of the putative HAT-containing TAF1 subunit of TFIID in gene expression raises the fundamental question as to what extent, if any, TAF1 contributes to acetylation in vivo and to what extent it is redundant with other HATs. Our findings herein do not support the basic tenet that TAF1 is a major HAT in Saccharomyces cerevisiae, nor do we find that TAF1 is functionally redundant with other HATs, including Gcn5, Elp3, Hat1, Hpa2, Sas3, and Esa1, which is in contrast to previous conclusions regarding Gcn5. Our findings do reveal that of these HATs, only Gcn5 and Esa1 contribute substantially to gene expression genome wide. Interestingly, histone acetylation at promoter regions throughout the genome does not require TAF1 or RNA polymerase II, indicating that most acetylation is likely to precede transcription and not depend upon it. TAF1 function has been linked to Bdf1, which binds TFIID and acetylated histone H4 tails, but no linkage between TAF1 and the H4 HAT Esa1 has been established. Here, we present evidence for such a linkage through Bdf1. PMID:16537921

  16. Structural Basis for Microcin C7 Inactivation by the MccE Acetyltransferase

    SciTech Connect

    Agarwal, Vinayak; Metlitskaya, Anastasiya; Severinov, Konstantin; Nair, Satish K.

    2015-10-15

    The antibiotic microcin C7 (McC) acts as a bacteriocide by inhibiting aspartyl-tRNA synthetase and stalling the protein translation machinery. McC is synthesized as a heptapeptide-nucleotide conjugate, which is processed by cellular peptidases within target strains to yield the biologically active compound. As unwanted processing of intact McC can result in self-toxicity, producing strains utilize multiple mechanisms for autoimmunity against processed McC. We have shown previously that the mccE gene within the biosynthetic cluster can inactivate processed McC by acetylating the antibiotic. Here, we present the characterization of this acetylation mechanism through biochemical and structural biological studies of the MccE acetyltransferase domain (MccE{sup AcTase}). We have also determined five crystal structures of the MccE-acetyl-CoA complex with bound substrates, inhibitor, and reaction product. The structural data reveal an unexpected mode of substrate recognition through p-stacking interactions similar to those found in cap-binding proteins and nucleotidyltransferases. These studies provide a rationale for the observation that MccE{sup AcTase} can detoxify a range of aminoacylnucleotides, including those that are structurally distinct from microcin C7.

  17. Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey.

    PubMed

    Li, Jia; You, Xinxin; Bian, Chao; Yu, Hui; Coon, Steven L; Shi, Qiong

    2016-01-01

    All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2. PMID:26729109

  18. Arylamine N-acetyltransferase activity in bronchial epithelial cells and its inhibition by cellular oxidants

    SciTech Connect

    Dairou, Julien; Petit, Emile; Ragunathan, Nilusha; Baeza-Squiban, Armelle; Marano, Francelyne; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2009-05-01

    Bronchial epithelial cells express xenobiotic-metabolizing enzymes (XMEs) that are involved in the biotransformation of inhaled toxic compounds. The activities of these XMEs in the lung may modulate respiratory toxicity and have been linked to several diseases of the airways. Arylamine N-acetyltransferases (NAT) are conjugating XMEs that play a key role in the biotransformation of aromatic amine pollutants such as the tobacco-smoke carcinogens 4-aminobiphenyl (4-ABP) and {beta}-naphthylamine ({beta}-NA). We show here that functional human NAT1 or its murine counterpart Nat2 are present in different lung epithelial cells i.e. Clara cells, type II alveolar cells and bronchial epithelial cells, thus indicating that inhaled aromatic amines may undergo NAT-dependent biotransformation in lung epithelium. Exposure of these cells to pathophysiologically relevant amounts of oxidants known to contribute to lung dysfunction, such as H{sub 2}O{sub 2} or peroxynitrite, was found to impair the NAT1/Nat2-dependent cellular biotransformation of aromatic amines. Genetic and non genetic impairment of intracellular NAT enzyme activities has been suggested to compromise the important detoxification pathway of aromatic amine N-acetylation and subsequently to contribute to an exacerbation of untoward effects of these pollutants on health. Our study suggests that oxidative/nitroxidative stress in lung epithelial cells, due to air pollution and/or inflammation, could contribute to local and/or systemic dysfunctions through the alteration of the functions of pulmonary NAT enzymes.

  19. N-Acetyltransferase 2 genotype, exfoliated urothelial cells and benzidine exposure.

    PubMed

    Ma, Qing-wen; Lin, Guo-fang; Chen, Ji-gang; Guo, Wei-Chao; Qin, Yi-qiu; Golka, Klaus; Shen, Jian-hua

    2012-01-01

    Most studies report an association of the slow N-acetyltransferase 2 (NAT2) status with elevated bladder cancer risk. In this study, NAT2 genotypes and the decades-long records of Papanicolaou's grading of exfoliated urothelial cells in a former benzidine-exposed cohort of the Shanghai dyestuff industry (29 bladder cancer patients; 307 non-cancer cohort members, some of them presenting different grades of pre-malignant alterations of exfoliated urothelial cells) were investigated. The cohort members had been enrolled in regular medical surveillance since mid-1980s. No overall increase of slow NAT2 genotypes in the former benzidine-exposed bladder cancer patients was found, compared with non-diseased members of the same cohort. A lower presentation of the homozygous wild genotype NAT2 4/4 was observed in bladder cancer patients, compared with non-diseased members with averaged Papanicolaou's grading (APG)3 II (OR=0.31, 95 percent CI 0.10-0.96, p=0.034) or with APG less than II (OR=0.36,95 percent CI 0.12-1.10, p=0.063). Nevertheless, neither a protective influence of rapid NAT2 genotypes on bladder cancer risk nor on pre-malignant cytological alterations could be confirmed by the present data. PMID:22202012

  20. Histone acetyltransferase Hbo1: catalytic activity, cellular abundance, and links to primary cancers.

    PubMed

    Iizuka, Masayoshi; Takahashi, Yoshihisa; Mizzen, Craig A; Cook, Richard G; Fujita, Masatoshi; Allis, C David; Frierson, Henry F; Fukusato, Toshio; Smith, M Mitchell

    2009-05-01

    In addition to the well-characterized proteins that comprise the pre-replicative complex, recent studies suggest that chromatin structure plays an important role in DNA replication initiation. One of these chromatin factors is the histone acetyltransferase (HAT) Hbo1 which is unique among HAT enzymes in that it serves as a positive regulator of DNA replication. However, several of the basic properties of Hbo1 have not been previously examined, including its intrinsic catalytic activity, its molecular abundance in cells, and its pattern of expression in primary cancer cells. Here we show that recombinant Hbo1 can acetylate nucleosomal histone H4 in vitro, with a preference for lysines 5 and 12. Using semi-quantitative western blot analysis, we find that Hbo1 is approximately equimolar with the number of active replication origins in normal human fibroblasts but is an order of magnitude more abundant in both MCF7 and Saos-2 established cancer cell lines. Immunohistochemistry for Hbo1 in 11 primary human tumor types revealed strong Hbo1 protein expression in carcinomas of the testis, ovary, breast, stomach/esophagus, and bladder. PMID:19393168

  1. Catalytic Mechanism of Bleomycin N-Acetyltransferase Proposed on the Basis of Its Crystal Structure*

    PubMed Central

    Oda, Kosuke; Matoba, Yasuyuki; Noda, Masafumi; Kumagai, Takanori; Sugiyama, Masanori

    2010-01-01

    Bleomycin (Bm) N-acetyltransferase, BAT, is a self-resistance determinant in Bm-producing Streptomyces verticillus ATCC15003. In our present study, we crystallized BAT under both a terrestrial and a microgravity environment in the International Space Station. In addition to substrate-free BAT, the crystal structures of BAT in a binary complex with CoA and in a ternary complex with Bm and CoA were determined. BAT forms a dimer structure via interaction of its C-terminal domains in the monomers. However, each N-terminal domain in the dimer is positioned without mutual interaction. The tunnel observed in the N-terminal domain of BAT has two entrances: one that adopts a wide funnel-like structure necessary to accommodate the metal-binding domain of Bm, and another narrow entrance that accommodates acetyl-CoA (AcCoA). A groove formed on the dimer interface of two BAT C-terminal domains accommodates the DNA-binding domain of Bm. In a ternary complex of BAT, BmA2, and CoA, a thiol group of CoA is positioned near the primary amine of Bm at the midpoint of the tunnel. This proximity ensures efficient transfer of an acetyl group from AcCoA to the primary amine of Bm. Based on the BAT crystal structure and the enzymatic kinetic study, we propose that the catalytic mode of BAT takes an ordered-like mechanism. PMID:19889644

  2. Regulation of Antisense Transcription by NuA4 Histone Acetyltransferase and Other Chromatin Regulatory Factors.

    PubMed

    Uprety, Bhawana; Kaja, Amala; Ferdoush, Jannatul; Sen, Rwik; Bhaumik, Sukesh R

    2016-01-01

    NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3' end of the GAL10 coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription of GAL10 also requires NuA4 KAT. Here, we show that NuA4 KAT associates with the GAL10 antisense transcription initiation site at the 3' end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to the GAL10 antisense transcription initiation site. Targeted recruitment of NuA4 KAT to the GAL10 antisense transcription initiation site promotes GAL10 antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitate GAL10 antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription of GAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription. PMID:26755557

  3. Structure of the E. Coli Bifunctional GlmU Acetyltransferase Active Site with Substrates and Products

    SciTech Connect

    Olsen,L.; Vetting, M.; Roderick, S.

    2007-01-01

    The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO{sub 4} to form GlcNAc-1-PO{sub 4} and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO{sub 4}, and desulpho-CoA/GlcNAc-1-PO{sub 4}. These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO{sub 4} is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.

  4. Salt-induced changes in the subunit structure of the Bacillus stearothermophilus lipoate acetyltransferase.

    PubMed

    Shigeoka, Yuichi; Fujisawa, Tetsuro; Teshiba, Satoshi; Fukumori, Hisayoshi; Yamamoto, Kohji; Banno, Yutaka; Aso, Yoichi

    2013-01-01

    The Bacillus stearothermophilus lipoate acetyltransferase (E2), composed of sixty identical, subunits is the core component of the pyruvate dehydrogenase complex (PDC). E2 polypeptide is composed of LD, PSBD, and CD domains. Most studies had focused on a truncated E2 that is deficient in LD and PSBD, because CD mainly contributes to maintaining the multimeric structure. We examined salt-induced changes in E2 without truncation and constructed reaction models. We speculate that in the presence of KCl, E2 is dissociated into a monomer and then assembled into an aggregative complex (C(A)) and a quasi-stable complex (C(Q)). C(A) was larger than C(Q), but smaller than intact E2. C(A) and C(Q), were dominant complexes at about neutral pH and at basic pH respectively. PDC, in which PSBD is occupied by other components, and a truncated E2 undergo dissociation only. LD-PSBD region besides CD might then contribute to the partial association of dissociated E2. PMID:23924725

  5. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance*

    PubMed Central

    Guo, Wei-dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

    2009-01-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance. PMID:19353742

  6. Response of ATP sulfurylase and serine acetyltransferase towards cadmium in hyperaccumulator Sedum alfredii Hance.

    PubMed

    Guo, Wei-dong; Liang, Jun; Yang, Xiao-e; Chao, Yue-en; Feng, Ying

    2009-04-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much higher in HE than in NHE along with increasing treatment time and Cd supply levels. ATPS activity was higher in HE than in NHE in all Cd treatments, and increased with increasing Cd supply levels in both HE and NHE when exposed to Cd treatment within 8 h. However, a marked difference of ATPS activity between HE and NHE was found with Cd treatment for 168 h, where ATPS activity increased in HE but decreased in NHE. Similarly, SAT activity was higher in HE than in NHE at all Cd treatments, but was more sensitive in NHE than in HE. Both ATPS and SAT activities in NHE leaves tended to decrease with increasing treatment time after 8 h at all Cd levels. The results reveal the different responses in sulfur assimilation enzymes and Cd accumulation between HE and NHE. With increasing Cd stress, the activities of sulfur assimilation enzymes (ATPS and SAT) were induced in HE, which may contribute to Cd accumulation in the hyperaccumulator Sedum alfredii Hance. PMID:19353742

  7. Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75

    SciTech Connect

    Berndsen, Christopher E; Tsubota, Toshiaki; Lindner, Scott E; Lee, Susan; Holton, James M; Kaufman, Paul D; Keck, James L; Denu, John M

    2010-01-12

    Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by {approx}100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rtt109. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.

  8. Human Neural Stem Cells Overexpressing Choline Acetyltransferase Restore Unconditioned Fear in Rats with Amygdala Injury

    PubMed Central

    Shin, Kyungha; Cha, Yeseul; Kim, Kwang Sei; Choi, Ehn-Kyoung; Choi, Youngjin; Guo, Haiyu; Ban, Young-Hwan; Kim, Jong-Choon; Park, Dongsun; Kim, Yun-Bae

    2016-01-01

    Amygdala is involved in the fear memory that recognizes certain environmental cues predicting threatening events. Manipulation of neurotransmission within the amygdala affects the expression of conditioned and unconditioned emotional memories such as fear freezing behaviour. We previously demonstrated that F3.ChAT human neural stem cells (NSCs) overexpressing choline acetyltransferase (ChAT) improve cognitive function of Alzheimer's disease model rats with hippocampal or cholinergic nerve injuries by increasing acetylcholine (ACh) level. In the present study, we examined the effect of F3.ChAT cells on the deficit of unconditioned fear freezing. Rats given N-methyl-d-aspartate (NMDA) in their amygdala 2 weeks prior to cat odor exposure displayed very short resting (freezing) time compared to normal animals. NMDA induced neuronal degeneration in the amygdala, leading to a decreased ACh concentration in cerebrospinal fluid. However, intracerebroventricular transplantation of F3.ChAT cells attenuated amygdala lesions 4 weeks after transplantation. The transplanted cells were found in the NMDA-injury sites and produced ChAT protein. In addition, F3.ChAT-receiving rats recuperated freezing time staying remote from the cat odor source, according to the recovery of brain ACh concentration. The results indicate that human NSCs overexpressing ChAT may facilitate retrieval of unconditioned fear memory by increasing ACh level. PMID:27087745

  9. Rational design and validation of a Tip60 histone acetyltransferase inhibitor

    NASA Astrophysics Data System (ADS)

    Gao, Chunxia; Bourke, Emer; Scobie, Martin; Famme, Melina Arcos; Koolmeister, Tobias; Helleday, Thomas; Eriksson, Leif A.; Lowndes, Noel F.; Brown, James A. L.

    2014-06-01

    Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.

  10. The acetyltransferase Tip60 contributes to mammary tumorigenesis by modulating DNA repair

    PubMed Central

    Bassi, C; Li, Y-T; Khu, K; Mateo, F; Baniasadi, P S; Elia, A; Mason, J; Stambolic, V; Pujana, M A; Mak, T W; Gorrini, C

    2016-01-01

    The acetyltransferase Tip60/Kat5 acetylates both histone and non-histone proteins, and is involved in a variety of biological processes. By acetylating p53, Tip60 controls p53-dependent transcriptional activity and so is implicated as a tumor suppressor. However, many breast cancers with low Tip60 also show p53 mutation, implying that Tip60 has a tumor suppressor function independent of its acetylation of p53. Here, we show in a p53-null mouse model of sporadic invasive breast adenocarcinoma that heterozygosity for Tip60 deletion promotes mammary tumorigenesis. Low Tip60 reduces DNA repair in normal and tumor mammary epithelial cells, both under resting conditions and following genotoxic stress. We demonstrate that Tip60 controls homologous recombination (HR)-directed DNA repair, and that Tip60 levels correlate inversely with a gene expression signature associated with defective HR-directed DNA repair. In human breast cancer data sets, Tip60 mRNA is downregulated, with low Tip60 levels correlating with p53 mutations in basal-like breast cancers. Our findings indicate that Tip60 is a novel breast tumor suppressor gene whose loss results in genomic instability leading to cancer formation. PMID:26915295