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Sample records for acetyltransferase creb-binding protein

  1. An acetyltransferase assay for CREB-binding protein based on reverse phase-ultra-fast liquid chromatography of fluorescent histone H3 peptides.

    PubMed

    Duval, Romain; Fritsch, Lauriane; Bui, Linh-Chi; Berthelet, Jérémy; Guidez, Fabien; Mathieu, Cécile; Dupret, Jean-Marie; Chomienne, Christine; Ait-Si-Ali, Slimane; Rodrigues-Lima, Fernando

    2015-10-01

    CREB-binding protein (CBP) is a lysine acetyltransferase that regulates transcription by acetylating histone and non-histone substrates. Defects in CBP activity are associated with hematologic malignancies, neurodisorders, and congenital malformations. Sensitive and quantitative enzymatic assays are essential to better characterize the pathophysiological features of CBP. We describe a sensitive nonradioactive method to measure purified and immunopurified cellular CBP enzymatic activity through rapid reverse phase-ultra-fast liquid chromatography (RP-UFLC) analysis of fluorescent histone H3 peptide substrates. The applicability and biological relevance of the assay are supported by kinetic, inhibition, and immunoprecipitation studies. More broadly, this approach could be easily adapted to assay other lysine acetyltransferases or methyltransferases.

  2. Mechanical regulation of the proangiogenic factor CCN1/CYR61 gene requires the combined activities of MRTF-A and CREB-binding protein histone acetyltransferase.

    PubMed

    Hanna, Mary; Liu, Haibo; Amir, Jawaria; Sun, Yi; Morris, Stephan W; Siddiqui, M A Q; Lau, Lester F; Chaqour, Brahim

    2009-08-21

    Smooth muscle-rich tissues respond to mechanical overload by an adaptive hypertrophic growth combined with activation of angiogenesis, which potentiates their mechanical overload-bearing capabilities. Neovascularization is associated with mechanical strain-dependent induction of angiogenic factors such as CCN1, an immediate-early gene-encoded matricellular molecule critical for vascular development and repair. Here we have demonstrated that mechanical strain-dependent induction of the CCN1 gene involves signaling cascades through RhoA-mediated actin remodeling and the p38 stress-activated protein kinase (SAPK). Actin signaling controls serum response factor (SRF) activity via SRF interaction with the myocardin-related transcriptional activator (MRTF)-A and tethering to a single CArG box sequence within the CCN1 promoter. Such activity was abolished in mechanically stimulated mouse MRTF-A(-/-) cells or upon inhibition of CREB-binding protein (CBP) histone acetyltransferase (HAT) either pharmacologically or by siRNAs. Mechanical strain induced CBP-mediated acetylation of histones 3 and 4 at the SRF-binding site and within the CCN1 gene coding region. Inhibition of p38 SAPK reduced CBP HAT activity and its recruitment to the SRF.MRTF-A complex, whereas enforced induction of p38 by upstream activators (e.g. MKK3 and MKK6) enhanced both CBP HAT and CCN1 promoter activities. Similarly, mechanical overload-induced CCN1 gene expression in vivo was associated with nuclear localization of MRTF-A and enrichment of the CCN1 promoter with both MRTF-A and acetylated histone H3. Taken together, these data suggest that signal-controlled activation of SRF, MRTF-A, and CBP provides a novel connection between mechanical stimuli and angiogenic gene expression.

  3. Identification of a novel SNF2/SWI2 protein family member, SRCAP, which interacts with CREB-binding protein.

    PubMed

    Johnston, H; Kneer, J; Chackalaparampil, I; Yaciuk, P; Chrivia, J

    1999-06-04

    The ability of cAMP response-element binding protein (CREB)-binding protein (CBP) to function as a co-activator for a number of transcription factors appears to be mediated by its ability to act as a histone acetyltransferase and through its interaction with a number of other proteins (general transcription factors, histone acetyltransferases, and other co-activators). Here we report that CBP also interacts with a novel ATPase termed Snf2-Related CBP Activator Protein (SRCAP). Consistent with this activity, SRCAP contains the conserved ATPase domain found within members of the Snf2 family. Transfection experiments demonstrate that SRCAP is able to activate transcription when expressed as a Gal-SRCAP chimera and that SRCAP also enhances the ability of CBP to activate transcription. The adenoviral protein E1A was found to disrupt interaction between SRCAP and CBP possibly representing a mechanism for E1A-mediated transcriptional repression.

  4. p300/CREB Binding Protein-Related Protein p270 Is a Component of Mammalian SWI/SNF Complexes

    PubMed Central

    Dallas, Peter B.; Cheney, Ian Wayne; Liao, Da-Wei; Bowrin, Valerie; Byam, Whitney; Pacchione, Stephen; Kobayashi, Ryuji; Yaciuk, Peter; Moran, Elizabeth

    1998-01-01

    p300 and the closely related CREB binding protein (CBP) are transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and associated histone acetyltransferase activity, suggesting that chromatin modification is an essential part of their role in regulating transcription. Detailed characterization of a panel of antibodies raised against p300/CBP has revealed the existence of a 270-kDa cellular protein, p270, distinct from p300 and CBP but sharing at least two independent epitopes with p300. The subset of p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series a cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Purification and analysis of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of this complex. PMID:9584200

  5. SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells

    SciTech Connect

    Ryan, Colm M.; Kindle, Karin B.; Collins, Hilary M.; Heery, David M.

    2010-01-01

    The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBP{Delta}998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

  6. The CREB-binding protein affects the circadian regulation of behaviour.

    PubMed

    Maurer, Christian; Winter, Tobias; Chen, Siwei; Hung, Hsiu-Cheng; Weber, Frank

    2016-09-01

    Rhythmic changes in light and temperature conditions form the primary environmental cues that synchronize the molecular circadian clock of most species with the external cycles of day and night. Previous studies established a role for the CREB-binding protein (CBP) in molecular clock function by coactivation of circadian transcription. Here, we report that moderately increased levels of CBP strongly dampen circadian behavioural rhythms without affecting molecular oscillations of circadian transcription. Interestingly, light-dark cycles as well as high temperature facilitated a circadian control of behavioural activity. Based on these observations we propose that in addition to its coactivator function for circadian transcription, CBP is involved in the regulation of circadian behaviour down-stream of the circadian clock.

  7. Role of Hypothalamic Creb-Binding Protein in Obesity and Molecular Reprogramming of Metabolic Substrates

    PubMed Central

    Moreno, Cesar L.; Yang, Linda; Dacks, Penny A.; Isoda, Fumiko; van Deursen, Jan M. A.; Mobbs, Charles V.

    2016-01-01

    We have reported a correlation between hypothalamic expression of Creb-binding protein (Cbp) and lifespan, and that inhibition of Cbp prevents protective effects of dietary restriction during aging, suggesting that hypothalamic Cbp plays a role in responses to nutritional status and energy balance. Recent GWAS and network analyses have also implicated Cbp as the most connected gene in protein-protein interactions in human Type 2 diabetes. The present studies address mechanisms mediating the role of Cbp in diabetes by inhibiting hypothalamic Cbp using a Cre-lox strategy. Inhibition of hypothalamic Cbp results in profound obesity and impaired glucose homeostasis, increased food intake, and decreased body temperature. In addition, these changes are accompanied by molecular evidence in the hypothalamus for impaired leptin and insulin signaling, a shift from glucose to lipid metabolism, and decreased Pomc mRNA, with no effect on locomotion. Further assessment of the significance of the metabolic switch demonstrated that enhanced expression of hypothalamic Cpt1a, which promotes lipid metabolism, similarly resulted in increased body weight and reduced Pomc mRNA. PMID:27832201

  8. The CREB binding protein inhibitor ICG-001 suppresses pancreatic cancer growth

    PubMed Central

    Arensman, Michael D.; Telesca, Donatello; Lay, Anna R.; Kershaw, Kathleen M.; Wu, Nanping; Donahue, Timothy R.; Dawson, David W.

    2014-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer due in part to a lack of highly robust cytotoxic or molecular-based therapies. Recent studies investigating ligand-mediated Wnt/β-catenin signaling have highlighted its importance in pancreatic cancer initiation and progression, as well as its potential as a therapeutic target in PDAC. The small molecule ICG-001 binds CREB-binding protein (CBP) to disrupt its interaction with β-catenin and inhibit CBP function as a co-activator of Wnt/β-catenin-mediated transcription. Given its ability to inhibit Wnt/β-catenin-mediated transcription in vitro and in vivo, as well as its efficacy in preclinical models of colorectal cancer and other Wnt-driven diseases, we examined ICG-001 and its potential role as a therapeutic in PDAC. ICG-001 alone significantly inhibited anchorage-dependent and -independent growth of multiple PDAC lines, and augmented in vitro growth inhibition when used in combination with gemcitabine. ICG-001 had only variable modest effects on PDAC apoptosis and instead mediated PDAC growth inhibition primarily through robust induction of G1 cell cycle arrest. These effects, however, appeared decoupled from its inhibition of Wnt/β-catenin-mediated transcription. DNA microarrays performed on PDAC cells in the context of ICG-001 treatment revealed ICG-001 altered the expression of several genes with well-established roles in DNA replication and cell cycle progression, including direct actions on SKP2 and CDKN1A. ICG-001 also significantly prolonged survival in an in vivo orthotopic xenograft model of PDAC, indicating ICG-001 or derived compounds that disrupt CBP activity are potentially useful small molecule therapeutics for pancreatic cancer. PMID:25082960

  9. CREB-binding protein, p300, butyrate, and Wnt signaling in colorectal cancer

    PubMed Central

    Bordonaro, Michael; Lazarova, Darina L

    2015-01-01

    This paper reviews the distinctive roles played by the transcriptional coactivators CREB-binding protein (CBP) and p300 in Wnt/β-catenin signaling and cell physiology in colorectal cancer (CRC). Specifically, we focus on the effects of CBP- and p300-mediated Wnt activity on (1) neoplastic progression; (2) the activities of butyrate, a breakdown product of dietary fiber, on cell signaling and colonic cell physiology; (3) the development of resistance to histone deacetylase inhibitors (HDACis), including butyrate and synthetic HDACis, in colonic cells; and (4) the physiology and number of cancer stem cells. Mutations of the Wnt/β-catenin signaling pathway initiate the majority of CRC cases, and we have shown that hyperactivation of this pathway by butyrate and other HDACis promotes CRC cell apoptosis. This activity by butyrate may in part explain the preventive action of fiber against CRC. However, individuals with a high-fiber diet may still develop neoplasia; therefore, resistance to the chemopreventive action of butyrate likely contributes to CRC. CBP or p300 may modify the ability of butyrate to influence colonic cell physiology since the two transcriptional coactivators affect Wnt signaling, and likely, its hyperactivation by butyrate. Also, CBP and p300 likely affect colonic tumorigenesis, as well as stem cell pluripotency. Improvement of CRC prevention and therapy requires a better understanding of the alterations in Wnt signaling and gene expression that underlie neoplastic progression, stem cell fate, and the development of resistance to butyrate and clinically relevant HDACis. Detailed knowledge of how CBP- and p300 modulate colonic cell physiology may lead to new approaches for anti-CRC prevention and therapeutics, particularly with respect to combinatorial therapy of CBP/p300 inhibitors with HDACis. PMID:26217075

  10. Transgenic Mice Expressing a Truncated Form of CREB-Binding Protein (CBP) Exhibit Deficits in Hippocampal Synaptic Plasticity and Memory Storage

    ERIC Educational Resources Information Center

    Wood, Marcelo A.; Kaplan, Michael P.; Park, Alice; Blanchard, Edward J.; Oliveira, Ana M. M.; Lombardi, Thomas L.; Abel, Ted

    2005-01-01

    Deletions, translocations, or point mutations in the CREB-binding protein (CBP) gene have been associated with Rubinstein-Taybi Syndrome; a human developmental disorder characterized by retarded growth and reduced mental function. To examine the role of CBP in memory, transgenic mice were generated in which the CaMKII[alpha] promoter drives…

  11. Localization of human CREB-binding protein gene (CREBBP) to 16p13.2-p13.3 by fluorescence in situ hybridization

    SciTech Connect

    Wydner, K.L.; Lawrence, J.B.; Bhattacharya, S.; Eckner, R.; Livingston, D.M.

    1995-11-20

    The adenovirus E1a oncoprotein targets a number of negative regulators of cellular proliferation, including the retinoblastoma gene product RB, p107, p130, p300, and the CREB-binding protein (CBP). p300 and CBP are highly homologous proteins that are thought to function as transcriptional coactivators, with roles in the regulation of the cell cycle and in cell differentiation. Like the RB protein, these proteins also may function as tumor suppressors. We have mapped the chromosomal location of the human CBP gene 3 as a first step in defining its possible involvement in human neoplasia. 11 refs.

  12. Role of Intrinsic Protein Disorder in the Function and Interactions of the Transcriptional Coactivators CREB-binding Protein (CBP) and p300.

    PubMed

    Dyson, H Jane; Wright, Peter E

    2016-03-25

    The transcriptional coactivators CREB-binding protein (CBP) and p300 undergo a particularly rich set of interactions with disordered and partly ordered partners, as a part of their ubiquitous role in facilitating transcription of genes. CBP and p300 contain a number of small structured domains that provide scaffolds for the interaction of disordered transactivation domains from a wide variety of partners, including p53, hypoxia-inducible factor 1α (HIF-1α), NF-κB, and STAT proteins, and are the targets for the interactions of disordered viral proteins that compete with cellular factors to disrupt signaling and subvert the cell cycle. The functional diversity of the CBP/p300 interactome provides an excellent example of the power of intrinsic disorder to facilitate the complexity of living systems.

  13. Role of Intrinsic Protein Disorder in the Function and Interactions of the Transcriptional Coactivators CREB-binding Protein (CBP) and p300*

    PubMed Central

    2016-01-01

    The transcriptional coactivators CREB-binding protein (CBP) and p300 undergo a particularly rich set of interactions with disordered and partly ordered partners, as a part of their ubiquitous role in facilitating transcription of genes. CBP and p300 contain a number of small structured domains that provide scaffolds for the interaction of disordered transactivation domains from a wide variety of partners, including p53, hypoxia-inducible factor 1α (HIF-1α), NF-κB, and STAT proteins, and are the targets for the interactions of disordered viral proteins that compete with cellular factors to disrupt signaling and subvert the cell cycle. The functional diversity of the CBP/p300 interactome provides an excellent example of the power of intrinsic disorder to facilitate the complexity of living systems. PMID:26851278

  14. Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1

    SciTech Connect

    Zhang, Qingzhan; Shi, Kaichuang; Yoo, Dongwan

    2016-02-15

    Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression. - Highlights: • PEDV modulates the host innate immune system by suppressing the type I interferon production and ISGs expression. • Ten viral proteins were identified as IFN antagonists, and nsp1 was the most potent viral IFN antagonist. • PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP). • PEDV nsp1 caused the CBP degradation in the nucleus, which may be the key mechanism for PEDV-mediated IFN downregulation.

  15. A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor gene expression

    NASA Astrophysics Data System (ADS)

    Falvo, James V.; Brinkman, Brigitta M. N.; Tsytsykova, Alla V.; Tsai, Eunice Y.; Yao, Tso-Pang; Kung, Andrew L.; Goldfeld, Anne E.

    2000-04-01

    The cAMP response element binding protein (CREB)-binding protein (CBP)/p300 family of coactivator proteins regulates gene transcription through the integration of multiple signal transduction pathways. Here, we show that induction of tumor necrosis factor (TNF-) gene expression in T cells stimulated by engagement of the T cell receptor (TCR) or by virus infection requires CBP/p300. Strikingly, in mice lacking one copy of the CBP gene, TNF- gene induction by TCR activation is inhibited, whereas virus induction of the TNF- gene is not affected. Consistent with these findings, the transcriptional activity of CBP is strongly potentiated by TCR activation but not by virus infection of T cells. Thus, CBP gene dosage and transcriptional activity are critical in TCR-dependent TNF-α gene expression, demonstrating a stimulus-specific requirement for CBP in the regulation of a specific gene.

  16. Human T-Cell Leukemia Virus Type 1 Tax Requires Direct Access to DNA for Recruitment of CREB Binding Protein to the Viral Promoter

    PubMed Central

    Lenzmeier, Brian A.; Giebler, Holli A.; Nyborg, Jennifer K.

    1998-01-01

    Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5′-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter. PMID:9447968

  17. CREB-binding protein controls response to cocaine by acetylating histones at the fosB promoter in the mouse striatum

    PubMed Central

    Levine, Amir A.; Guan, Zhonghui; Barco, Angel; Xu, Shiqin; Kandel, Eric R.; Schwartz, James H.

    2005-01-01

    Remodeling chromatin is essential for cAMP-regulated gene expression, necessary not only for development but also for memory storage and other enduring mental states. Histone acetylation and deacetylation mediate long-lasting forms of synaptic plasticity in Aplysia as well as cognition in mice. Here, we show that histone acetylation by the cAMP-response element binding protein (CREB)-binding protein (CBP) mediates sensitivity to cocaine by regulating expression of the fosB gene and its splice variant, ΔfosB, a transcription factor previously implicated in addiction. Using the chromatin immunoprecipitation assay with antibodies against histone H4 or CBP, we find that CBP is recruited to the fosB promoter to acetylate histone H4 in response to acute exposure to cocaine. We show that mutant mice that lack one allele of the CBP gene and have normal levels of fosB expression are less sensitive to chronic (10-day) administration of cocaine than are wild-type mice. This decreased sensitivity is correlated with decreased histone acetylation and results in decreased fosB expression and diminished accumulation of ΔfosB. Thus, CBP, which forms part of the promoter complex with CREB, mediates sensitivity to cocaine by acetylating histones. PMID:16380431

  18. Upregulation of AKT1 protein expression in forskolin-stimulated macrophage: evidence from ChIP analysis that CREB binds to and activates the AKT1 promoter.

    PubMed

    Misra, Uma Kant; Pizzo, Salvatore Vincent

    2007-03-01

    Recently, we reported that silencing CREB gene expression by RNAi significantly attenuates forskolin-induced activation of Akt1. We now provide evidence that forskolin-treatment causes transcriptional and translational upregulation of Akt1 in macrophages. Akt synthesis was demonstrated by [(14)C]leucine or [(35)S] incorporation into newly synthesized Akt1 protein. Akt protein levels increased by approximately 1.5-fold after only a 5 min exposure of macrophages to forskolin. Akt1 levels thereafter rapidly returned to basal values (t(1/2) approximately 15 min). Maximal upregulation of Akt1 occurred in cells treated with 10 microM forskolin. Forskolin-dependent Akt1 synthesis was abolished by pretreating the cells with CREB-directed dsRNA as demonstrated at both the message and protein level, as well as by determining the synthesis of [(35)S]-labeled Akt1 protein. The PKA inhibitor H-89, greatly attenuated forskolin-induced Akt1 synthesis. Transcriptional and translational inhibitors also greatly reduced Akt1 synthesis in forskolin-stimulated [(14)C]leucine-labeled macrophages. Using a chromatin immunoprecipitation assay, we demonstrate that CREB binds to a CRE binding domain of the Akt1 gene promoter. In conclusion, we show here for the first time transcriptional upregulation of Akt1 by CREB, based upon Akt1 protein synthesis and its modulation by transitional and translational inhibitors in forskolin-stimulated cells, Akt1 protein. and mRNA levels upon silencing CREB gene expression, and binding of CREB to the Akt1 gene promoter.

  19. Modulation of type I interferon induction by porcine reproductive and respiratory syndrome virus and degradation of CREB-binding protein by non-structural protein 1 in MARC-145 and HeLa cells

    SciTech Connect

    Kim, Oekyung; Sun Yan; Lai, Frances W.; Song Cheng; Yoo, Dongwan

    2010-07-05

    Porcine reproductive and respiratory syndrome (PRRS) is an emerged disease of swine characterized by negligible response of type I IFNs and viral persistence. We show that the PRRSV non-structural protein 1 (Nsp1) is the viral component responsible for modulation of IFN response. Nsp1 blocked dsRNA-induced IRF3 and IFN promoter activities. Nsp1 did not block phosphorylation and nuclear translocation of IRF3 but inhibited IRF3 association with CREB-binding protein (CBP) in the nucleus. While IRF3 was stable, CBP was degraded, and CBP degradation was proteasome-dependent, suggesting that CBP degradation is not due to the protease activity of Nsp1 but an intermediary is involved. Our data suggest that the Nsp1-mediated CBP degradation inhibits the recruitment of CBP for enhanceosome assembly, leading to the block of IFN response. CBP degradation is a novel strategy for viral evasion from the host response, and Nsp1 may form a new class of viral antagonists for IFN modulation.

  20. The transcriptional integrator CREB-binding protein mediates positive cross talk between nuclear hormone receptors and the hematopoietic bZip protein p45/NF-E2.

    PubMed Central

    Cheng, X; Reginato, M J; Andrews, N C; Lazar, M A

    1997-01-01

    Thyroid hormone (T3) and retinoic acid (RA) play important roles in erythropoiesis. We found that the hematopoietic cell-specific bZip protein p45/NF-E2 interacts with T3 receptor (TR) and RA receptor (RAR) but not retinoid X receptor. The interaction is between the DNA-binding domain of the nuclear receptor and the leucine zipper region of p45/NF-E2 but is markedly enhanced by cognate ligand. Remarkably, ligand-dependent transactivation by TR and RAR is markedly potentiated by p45/NF-E2. This effect of p45/NF-E2 is prevented by maf-like protein p18, which functions positively as a heterodimer with p45/NF-E2 on DNA. Potentiation of hormone action by p45/NF-E2 requires its activation domain, which interacts strongly with the multifaceted coactivator cyclic AMP response element protein-binding protein (CBP). The region of CBP which interacts with p45/NF-E2 is the same interaction domain that mediates inhibition of hormone-stimulated transcription by AP1 transcription factors. Overexpression of the bZip interaction domain of CBP specifically abolishes the positive cross talk between TR and p45/NF-E2. Thus, positive cross talk between p45/NF-E2 and nuclear hormone receptors requires direct protein-protein interactions between these factors and with CBP, whose integration of positive signals from two transactivation domains provides a novel mechanism for potentiation of hormone action in hematopoietic cells. PMID:9032267

  1. Tomato heat stress transcription factor HsfB1 represents a novel type of general transcription coactivator with a histone-like motif interacting with the plant CREB binding protein ortholog HAC1.

    PubMed

    Bharti, Kapil; Von Koskull-Döring, Pascal; Bharti, Sanita; Kumar, Pravir; Tintschl-Körbitzer, Angelika; Treuter, Eckardt; Nover, Lutz

    2004-06-01

    In contrast with the class A heat stress transcription factors (HSFs) of plants, a considerable number of HSFs assigned to classes B and C have no evident function as transcription activators on their own. However, in the following article, we provide evidence that tomato (Lycopersicon peruvianum) HsfB1 represents a novel type of coactivator cooperating with class A HSFs (e.g., with tomato HsfA1). Provided the appropriate promoter architecture, the two HSFs assemble into an enhanceosome-like complex, resulting in strong synergistic activation of reporter gene expression. Moreover, HsfB1 also cooperates in a similar manner with other activators, for example, with the ASF1/2 enhancer binding proteins of the 35S promoter of Cauliflower mosaic virus or with yet unidentified activators controlling housekeeping gene expression. By these effects, HsfB1 may help to maintain and/or restore expression of certain viral or housekeeping genes during ongoing heat stress. The coactivator function of HsfB1 depends on a histone-like motif in its C-terminal domain with an indispensable Lys residue in the center (GRGKMMK). This motif is required for recruitment of the plant CREB binding protein (CBP) ortholog HAC1. HsfA1, HsfB1, and HAC1/CBP form ternary complexes in vitro and in vivo with markedly enhanced efficiency in promoter recognition and transcription activation in plant and mammalian (COS7) cells. Using small interfering RNA-mediated knock down of HAC1 expression in Arabidopsis thaliana mesophyll protoplasts, the crucial role for the coactivator function of HsfB1 was confirmed.

  2. The transcription factor GLI1 interacts with SMAD proteins to modulate transforming growth factor β-induced gene expression in a p300/CREB-binding protein-associated factor (PCAF)-dependent manner.

    PubMed

    Nye, Monica D; Almada, Luciana L; Fernandez-Barrena, Maite G; Marks, David L; Elsawa, Sherine F; Vrabel, Anne; Tolosa, Ezequiel J; Ellenrieder, Volker; Fernandez-Zapico, Martin E

    2014-05-30

    The biological role of the transcription factor GLI1 in the regulation of tumor growth is well established; however, the molecular events modulating this phenomenon remain elusive. Here, we demonstrate a novel mechanism underlying the role of GLI1 as an effector of TGFβ signaling in the regulation of gene expression in cancer cells. TGFβ stimulates GLI1 activity in cancer cells and requires its transcriptional activity to induce BCL2 expression. Analysis of the mechanism regulating this interplay identified a new transcriptional complex including GLI1 and the TGFβ-regulated transcription factor, SMAD4. We demonstrate that SMAD4 physically interacts with GLI1 for concerted regulation of gene expression and cellular survival. Activation of the TGFβ pathway induces GLI1-SMAD4 complex binding to the BCL2 promoter whereas disruption of the complex through SMAD4 RNAi depletion impairs GLI1-mediated transcription of BCL2 and cellular survival. Further characterization demonstrated that SMAD2 and the histone acetyltransferase, PCAF, participate in this regulatory mechanism. Both proteins bind to the BCL2 promoter and are required for TGFβ- and GLI1-stimulated gene expression. Moreover, SMAD2/4 RNAi experiments showed that these factors are required for the recruitment of GLI1 to the BCL2 promoter. Finally, we determined whether this novel GLI1 transcriptional pathway could regulate other TGFβ targets. We found that two additional TGFβ-stimulated genes, INTERLEUKIN-7 and CYCLIN D1, are dependent upon the intact GLI1-SMAD-PCAF complex for transcriptional activation. Collectively, these results define a novel epigenetic mechanism that uses the transcription factor GLI1 and its associated complex as a central effector to regulate gene expression in cancer cells.

  3. The Transcription Factor GLI1 Interacts with SMAD Proteins to Modulate Transforming Growth Factor β-Induced Gene Expression in a p300/CREB-binding Protein-associated Factor (PCAF)-dependent Manner*

    PubMed Central

    Nye, Monica D.; Almada, Luciana L.; Fernandez-Barrena, Maite G.; Marks, David L.; Elsawa, Sherine F.; Vrabel, Anne; Tolosa, Ezequiel J.; Ellenrieder, Volker; Fernandez-Zapico, Martin E.

    2014-01-01

    The biological role of the transcription factor GLI1 in the regulation of tumor growth is well established; however, the molecular events modulating this phenomenon remain elusive. Here, we demonstrate a novel mechanism underlying the role of GLI1 as an effector of TGFβ signaling in the regulation of gene expression in cancer cells. TGFβ stimulates GLI1 activity in cancer cells and requires its transcriptional activity to induce BCL2 expression. Analysis of the mechanism regulating this interplay identified a new transcriptional complex including GLI1 and the TGFβ-regulated transcription factor, SMAD4. We demonstrate that SMAD4 physically interacts with GLI1 for concerted regulation of gene expression and cellular survival. Activation of the TGFβ pathway induces GLI1-SMAD4 complex binding to the BCL2 promoter whereas disruption of the complex through SMAD4 RNAi depletion impairs GLI1-mediated transcription of BCL2 and cellular survival. Further characterization demonstrated that SMAD2 and the histone acetyltransferase, PCAF, participate in this regulatory mechanism. Both proteins bind to the BCL2 promoter and are required for TGFβ- and GLI1-stimulated gene expression. Moreover, SMAD2/4 RNAi experiments showed that these factors are required for the recruitment of GLI1 to the BCL2 promoter. Finally, we determined whether this novel GLI1 transcriptional pathway could regulate other TGFβ targets. We found that two additional TGFβ-stimulated genes, INTERLEUKIN-7 and CYCLIN D1, are dependent upon the intact GLI1-SMAD-PCAF complex for transcriptional activation. Collectively, these results define a novel epigenetic mechanism that uses the transcription factor GLI1 and its associated complex as a central effector to regulate gene expression in cancer cells. PMID:24739390

  4. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  5. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  6. 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.

  7. Structure and mechanism of non-histone protein acetyltransferase enzymes

    PubMed Central

    Friedmann, David R.

    2014-01-01

    Post translational modification (PTM) of proteins is ubiquitous and mediates many cellular processes including intracellular localization, protein-protein interactions, enzyme activity, transcriptional regulation and protein stability. While the role of phosphorylation as a key PTM has been well studied, the more evolutionarily conserved acetylation PTM has only recently attracted attention as a key regulator of cellular events. Protein acetylation has been largely studied in the context of its role in histone modification and gene regulation, where histones are modified by histone acetyltransferases (HATs) to promote transcription. However, more recent acetylomic and biochemical studies have revealed that acetylation is mediated by a broader family of protein acetyltransferases (PATs). The recent structure determination of several PATs has provided a wealth of molecular information regarding structural features of PATs, their enzymatic mechanisms, their mode of substrate-specific recognition and their regulatory elements. In this minireview, we will briefly describe what is known about non-histone protein substrates, but mainly focus on a few recent structures of PATs to compare and contrast them with HATs to better understand the molecular basis for protein recognition and modification by this burgeoning family of protein modification enzymes. PMID:23742047

  8. Assays for mechanistic investigations of protein/histone acetyltransferases.

    PubMed

    Berndsen, Christopher E; Denu, John M

    2005-08-01

    Protein/histone acetyltransferases (PATs/HATs) have been implicated in a number of cellular functions including gene regulation, DNA synthesis, and repair. This paper reviews methods that can be used to quantitatively determine the activity and ultimately the catalytic/kinetic mechanism of PAT/HATs in vitro. Two methods will be described in detail. The first method is a filter-binding assay that measures the transfer of radiolabeled acetate from acetyl-CoA to protein. The second method is a continuous, spectroscopic, enzyme-coupled assay that links the PAT/HAT reaction to the reduction of NAD+ by pyruvate or alpha-ketoglutarate dehydrogenase. Both methods are highly applicable in determining steady-state reaction rates, and obtaining the kinetic constants Vmax, Km, and V/K from substrate saturation curves. We describe a new application of the filter-binding assay to determine the kinetic parameters for HATs using low concentrations of nucleosomal substrates.

  9. Autoacetylation of the MYST lysine acetyltransferase MOF protein.

    PubMed

    Yang, Chao; Wu, Jiang; Sinha, Sarmistha H; Neveu, John M; Zheng, Yujun George

    2012-10-12

    The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation.

  10. Involvement of Arabidopsis histone acetyltransferase HAC family genes in the ethylene signaling pathway.

    PubMed

    Li, Chao; Xu, Jiang; Li, Jian; Li, Qingyun; Yang, Hongchun

    2014-02-01

    Epigenetic modifications play a fundamental role in regulating chromatin dynamics and gene expression. The level of histone acetylation is controlled by two functionally antagonistic enzymes, namely histone acetyltransferase (HAT) and histone deacetylase (HDAC). CREB-binding protein (CBP)/p300 proteins, a subfamily of highly conserved HATs, are involved in various physiological events including proliferation, differentiation and apoptosis. In this work, we study the poorly known function of their homologous genes, the HAC genes, in Arabidopsis. We found that hac1-involved mutants displayed pleiotropic phenotypes, in particular hypersensitivity to ethylene both in the dark and in the light. We also found that the transcriptional levels of ethylene-responsive genes are significantly higher in the hac1hac5 double mutant than in wild-type plants. Moreover, an ethylene synthesis inhibitor cannot release the triple responses of hac mutants. These results suggest that HACs are involved in the ethylene signaling pathway.

  11. 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.

  12. Comparison of protein acetyltransferase action of CRTAase with the prototypes of HAT.

    PubMed

    Ponnan, Prija; Kumar, Ajit; Singh, Prabhjot; Gupta, Prachi; Joshi, Rini; Gaspari, Marco; Saso, Luciano; Prasad, Ashok K; Rastogi, Ramesh C; Parmar, Virinder S; Raj, Hanumantharao G

    2014-01-01

    Our laboratory is credited for the discovery of enzymatic acetylation of protein, a phenomenon unknown till we identified an enzyme termed acetoxy drug: protein transacetylase (TAase), catalyzing the transfer of acetyl group from polyphenolic acetates to receptor proteins (RP). Later, TAase was identified as calreticulin (CR), an endoplasmic reticulum luminal protein. CR was termed calreticulin transacetylase (CRTAase). Our persistent study revealed that CR like other families of histone acetyltransferases (HATs) such as p300, Rtt109, PCAF, and ESA1, undergoes autoacetylation. The autoacetylated CR was characterized as a stable intermediate in CRTAase catalyzed protein acetylation, and similar was the case with ESA1. The autoacetylation of CR like that of HATs was found to enhance protein-protein interaction. CR like HAT-1, CBP, and p300 mediated the acylation of RP utilizing acetyl CoA and propionyl CoA as the substrates. The similarities between CRTAase and HATs in mediating protein acylation are highlighted in this review.

  13. 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.

  14. Lysine acetyltransferases CBP and p300 as therapeutic targets in cognitive and neurodegenerative disorders.

    PubMed

    Valor, Luis M; Viosca, Jose; Lopez-Atalaya, Jose P; Barco, Angel

    2013-01-01

    Neuropsychiatric pathologies, including neurodegenerative diseases and neurodevelopmental syndromes, are frequently associated with dysregulation of various essential cellular mechanisms, such as transcription, mitochondrial respiration and protein degradation. In these complex scenarios, it is difficult to pinpoint the specific molecular dysfunction that initiated the pathology or that led to the fatal cascade of events that ends with the death of the neuron. Among the possible original factors, epigenetic dysregulation has attracted special attention. This review focuses on two highly related epigenetic factors that are directly involved in a number of neurological disorders, the lysine acetyltransferases CREB-binding protein (CBP) and E1A-associated protein p300 (p300). We first comment on the role of chromatin acetylation and the enzymes that control it, particularly CBP and p300, in neuronal plasticity and cognition. Next, we describe the involvement of these proteins in intellectual disability and in different neurodegenerative diseases. Finally, we discuss the potential of ameliorative strategies targeting CBP/p300 for the treatment of these disorders.

  15. The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization*

    PubMed Central

    de Diego Puente, Teresa; Gallego-Jara, Julia; Castaño-Cerezo, Sara; Bernal Sánchez, Vicente; Fernández Espín, Vanesa; García de la Torre, José; Manjón Rubio, Arturo; Cánovas Díaz, Manuel

    2015-01-01

    Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism. PMID:26251518

  16. The Protein Acetyltransferase PatZ from Escherichia coli Is Regulated by Autoacetylation-induced Oligomerization.

    PubMed

    de Diego Puente, Teresa; Gallego-Jara, Julia; Castaño-Cerezo, Sara; Bernal Sánchez, Vicente; Fernández Espín, Vanesa; García de la Torre, José; Manjón Rubio, Arturo; Cánovas Díaz, Manuel

    2015-09-18

    Lysine acetylation is an important post-translational modification in the metabolic regulation of both prokaryotes and eukaryotes. In Escherichia coli, PatZ (formerly YfiQ) is the only known acetyltransferase protein and is responsible for acetyl-CoA synthetase acetylation. In this study, we demonstrated PatZ-positive cooperativity in response to acetyl-CoA and the regulation of acetyl-CoA synthetase activity by the acetylation level. Furthermore, functional analysis of an E809A mutant showed that the conserved glutamate residue is not relevant for the PatZ catalytic mechanism. Biophysical studies demonstrated that PatZ is a stable tetramer in solution and is transformed to its octameric form by autoacetylation. Moreover, this modification is reversed by the sirtuin CobB. Finally, an in silico PatZ tetramerization model based on hydrophobic and electrostatic interactions is proposed and validated by three-dimensional hydrodynamic analysis. These data reveal, for the first time, the structural regulation of an acetyltransferase by autoacetylation in a prokaryotic organism.

  17. GPS-PAIL: prediction of lysine acetyltransferase-specific modification sites from protein sequences

    PubMed Central

    Deng, Wankun; Wang, Chenwei; Zhang, Ying; Xu, Yang; Zhang, Shuang; Liu, Zexian; Xue, Yu

    2016-01-01

    Protein acetylation catalyzed by specific histone acetyltransferases (HATs) is an essential post-translational modification (PTM) and involved in the regulation a broad spectrum of biological processes in eukaryotes. Although several ten thousands of acetylation sites have been experimentally identified, the upstream HATs for most of the sites are unclear. Thus, the identification of HAT-specific acetylation sites is fundamental for understanding the regulatory mechanisms of protein acetylation. In this work, we first collected 702 known HAT-specific acetylation sites of 205 proteins from the literature and public data resources, and a motif-based analysis demonstrated that different types of HATs exhibit similar but considerably distinct sequence preferences for substrate recognition. Using 544 human HAT-specific sites for training, we constructed a highly useful tool of GPS-PAIL for the prediction of HAT-specific sites for up to seven HATs, including CREBBP, EP300, HAT1, KAT2A, KAT2B, KAT5 and KAT8. The prediction accuracy of GPS-PAIL was critically evaluated, with a satisfying performance. Using GPS-PAIL, we also performed a large-scale prediction of potential HATs for known acetylation sites identified from high-throughput experiments in nine eukaryotes. Both online service and local packages were implemented, and GPS-PAIL is freely available at: http://pail.biocuckoo.org. PMID:28004786

  18. GPS-PAIL: prediction of lysine acetyltransferase-specific modification sites from protein sequences.

    PubMed

    Deng, Wankun; Wang, Chenwei; Zhang, Ying; Xu, Yang; Zhang, Shuang; Liu, Zexian; Xue, Yu

    2016-12-22

    Protein acetylation catalyzed by specific histone acetyltransferases (HATs) is an essential post-translational modification (PTM) and involved in the regulation a broad spectrum of biological processes in eukaryotes. Although several ten thousands of acetylation sites have been experimentally identified, the upstream HATs for most of the sites are unclear. Thus, the identification of HAT-specific acetylation sites is fundamental for understanding the regulatory mechanisms of protein acetylation. In this work, we first collected 702 known HAT-specific acetylation sites of 205 proteins from the literature and public data resources, and a motif-based analysis demonstrated that different types of HATs exhibit similar but considerably distinct sequence preferences for substrate recognition. Using 544 human HAT-specific sites for training, we constructed a highly useful tool of GPS-PAIL for the prediction of HAT-specific sites for up to seven HATs, including CREBBP, EP300, HAT1, KAT2A, KAT2B, KAT5 and KAT8. The prediction accuracy of GPS-PAIL was critically evaluated, with a satisfying performance. Using GPS-PAIL, we also performed a large-scale prediction of potential HATs for known acetylation sites identified from high-throughput experiments in nine eukaryotes. Both online service and local packages were implemented, and GPS-PAIL is freely available at: http://pail.biocuckoo.org.

  19. A pair of transposon-derived proteins function in a histone acetyltransferase complex for active DNA demethylation

    PubMed Central

    Duan, Cheng-Guo; Wang, Xingang; Xie, Shaojun; Pan, Li; Miki, Daisuke; Tang, Kai; Hsu, Chuan-Chih; Lei, Mingguang; Zhong, Yingli; Hou, Yueh-Ju; Wang, Zhijuan; Zhang, Zhengjing; Mangrauthia, Satendra K; Xu, Huawei; Zhang, Heng; Dilkes, Brian; Tao, W Andy; Zhu, Jian-Kang

    2017-01-01

    Transposons are generally kept silent by epigenetic mechanisms including DNA methylation. Here, we identified a pair of Harbinger transposon-derived proteins (HDPs), HDP1 and HDP2, as anti-silencing factors in Arabidopsis. hdp1 and hdp2 mutants displayed an enhanced silencing of transgenes and some transposons. Phylogenetic analyses revealed that HDP1 and HDP2 were co-domesticated from the Harbinger transposon-encoded transposase and DNA-binding protein, respectively. HDP1 interacts with HDP2 in the nucleus, analogous to their transposon counterparts. Moreover, HDP1 and HDP2 are associated with IDM1, IDM2, IDM3 and MBD7 that constitute a histone acetyltransferase complex functioning in DNA demethylation. HDP2 and the methyl-DNA-binding protein MBD7 share a large set of common genomic binding sites, indicating that they jointly determine the target specificity of the histone acetyltransferase complex. Thus, our data revealed that HDP1 and HDP2 constitute a functional module that has been recruited to a histone acetyltransferase complex to prevent DNA hypermethylation and epigenetic silencing. PMID:27934869

  20. Mutation of the CH1 Domain in the Histone Acetyltransferase CREBBP Results in Autism-Relevant Behaviors in Mice

    PubMed Central

    Zheng, Fei; Kasper, Lawryn H.; Bedford, David C.; Lerach, Stephanie; Teubner, Brett J. W.; Brindle, Paul K.

    2016-01-01

    Autism spectrum disorders (ASDs) are a group of neurodevelopmental afflictions characterized by repetitive behaviors, deficits in social interaction, and impaired communication skills. For most ASD patients, the underlying causes are unknown. Genetic mutations have been identified in about 25 percent of ASD cases, including mutations in epigenetic regulators, suggesting that dysregulated chromatin or DNA function is a critical component of ASD. Mutations in the histone acetyltransferase CREB binding protein (CBP, CREBBP) cause Rubinstein-Taybi Syndrome (RTS), a developmental disorder that includes ASD-like symptoms. Recently, genomic studies involving large numbers of ASD patient families have theoretically modeled CBP and its paralog p300 (EP300) as critical hubs in ASD-associated protein and gene interaction networks, and have identified de novo missense mutations in highly conserved residues of the CBP acetyltransferase and CH1 domains. Here we provide animal model evidence that supports this notion that CBP and its CH1 domain are relevant to autism. We show that mice with a deletion mutation in the CBP CH1 (TAZ1) domain (CBPΔCH1/ΔCH1) have an RTS-like phenotype that includes ASD-relevant repetitive behaviors, hyperactivity, social interaction deficits, motor dysfunction, impaired recognition memory, and abnormal synaptic plasticity. Our results therefore indicate that loss of CBP CH1 domain function contributes to RTS, and possibly ASD, and that this domain plays an essential role in normal motor function, cognition and social behavior. Although the key physiological functions affected by ASD-associated mutation of epigenetic regulators have been enigmatic, our findings are consistent with theoretical models involving CBP and p300 in ASD, and with a causative role for recently described ASD-associated CBP mutations. PMID:26730956

  1. Spatiotemporal expression of histone acetyltransferases, p300 and CBP, in developing embryonic hearts

    PubMed Central

    Chen, Guozhen; Zhu, Jing; Lv, Tiewei; Wu, Gang; Sun, Huichao; Huang, Xupei; Tian, Jie

    2009-01-01

    Histone acetyltransferases (HATs), p300 and cAMP response element binding protein (CREB)-binding protein (CBP) are two structurally related transcriptional co-activators that activate expression of many eukaryotic genes involved in cellular growth and signaling, muscle differentiation and embryogenesis. However, whether these proteins play important and different roles in mouse cardiogenesis is not clear. Here, we investigate the protein distributions and mRNA expression of the two HATs in embryonic and adult mouse heart during normal heart development by using immunohistochemical and RT-PCR techniques. The data from immunohistochemical experiments revealed that p300 was extensively present in nearly every region of the hearts from embryonic stages to the adulthood. However, no CBP expression was detected in embryonic hearts at day E7.5. CBP expression appeared at the later stages, and the distribution of CBP was less than that of p300. In the developmental hearts after E10.5, both for p300 and CBP, the mRNA expression levels reached a peak on day E10.5, and then were gradually decreased afterwards. These results reveal that both p300 and CBP are related to embryonic heart development. The dynamic expression patterns of these two enzymes during mouse heart development indicate that they may play an important role on heart development. However, there is a difference in spatiotemporal expression patterns between these two enzymes during heart development. The expression of p300 is earlier and more predominate, suggesting that p300 may play a more important role in embryonic heart development especially during cardiac precursor cell induction and interventricular septum formation. PMID:19272189

  2. CBP/p300 acetyltransferases regulate the expression of NKG2D ligands on tumor cells

    PubMed Central

    Sauer, M; Schuldner, M; Hoffmann, N; Cetintas, A; Reiners, K S; Shatnyeva, O; Hallek, M; Hansen, H P; Gasser, S; von Strandmann, E P

    2017-01-01

    Tumor surveillance of natural killer (NK) cells is mediated by the cytotoxicity receptor natural-killer group 2 member D (NKG2D). Ligands for NKG2D are generally not expressed on healthy cells, but induced on the surface of malignant cells. To date, NKG2D ligand (NKG2D-L) induction was mainly described to depend on the activation of the DNA damage response, although the molecular mechanisms that regulate NKG2D-L expression remain largely unknown. Here, we show that the acetyltransferases CBP (CREB-binding protein) and p300 play a crucial role in the regulation of NKG2D-L on tumor cells. Loss of CBP/p300 decreased the basal cell surface expression of human ligands and reduced the upregulation of MICA/B and ULBP2 in response to histone deacetylase inhibitors or DNA damage. Furthermore, CBP/P300 deficiency abrogated the sensitivity of stressed cells to NK cell-mediated killing. CBP/p300 were also identified as major regulators of mouse NKG2D ligand RAE-1 in vitro and in vivo using the Eμ-Myc lymphoma model. Mechanistically, we observed an enhanced activation of the CBP/p300 binding transcription factor CREB (cAMP response element-binding protein) correlating to the NKG2D-L upregulation. Moreover, increased binding of CREB and CBP/p300 to NKG2D-L promoters and elevated histone acetylation were detectable. This study provides strong evidence for a major role of CBP and p300 in orchestrating NKG2D-L induction and consequently immunosurveillance of tumors in mice and humans. These findings might help to develop novel immunotherapeutic approaches against cancer. PMID:27477692

  3. Single neuron transcriptomics identify SRSF/SR protein B52 as a regulator of axon growth and Choline acetyltransferase splicing

    PubMed Central

    Liu, Boyin; Bossing, Torsten

    2016-01-01

    We removed single identified neurons from living Drosophila embryos to gain insight into the transcriptional control of developing neuronal networks. The microarray analysis of the transcriptome of two sibling neurons revealed seven differentially expressed transcripts between both neurons (threshold: log21.4). One transcript encodes the RNA splicing factor B52. Loss of B52 increases growth of axon branches. B52 function is also required for Choline acetyltransferase (ChAT ) splicing. At the end of embryogenesis, loss of B52 function impedes splicing of ChAT, reduces acetylcholine synthesis, and extends the period of uncoordinated muscle twitches during larval hatching. ChAT regulation by SRSF proteins may be a conserved feature since changes in SRSF5 expression and increased acetylcholine levels in brains of bipolar disease patients have been reported recently. PMID:27725692

  4. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-induced Lysine Acetylation of Mitochondrial Proteins

    PubMed Central

    Davies, Michael N.; Kjalarsdottir, Lilja; Thompson, J. Will; Dubois, Laura G.; Stevens, Robert D.; Ilkayeva, Olga R.; Brosnan, M. Julia; Rolph, Timothy P.; Grimsrud, Paul A.; Muoio, Deborah M.

    2016-01-01

    Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK. PMID:26748706

  5. Spatial memory consolidation is associated with induction of several lysine-acetyltransferase (histone acetyltransferase) expression levels and H2B/H4 acetylation-dependent transcriptional events in the rat hippocampus.

    PubMed

    Bousiges, Olivier; Vasconcelos, Anne Pereira de; Neidl, Romain; Cosquer, Brigitte; Herbeaux, Karine; Panteleeva, Irina; Loeffler, Jean-Philippe; Cassel, Jean-Christophe; Boutillier, Anne-Laurence

    2010-12-01

    Numerous genetic studies have shown that the CREB-binding protein (CBP) is an essential component of long-term memory formation, through its histone acetyltransferase (HAT) function. E1A-binding protein p300 and p300/CBP-associated factor (PCAF) have also recently been involved in memory formation. By contrast, only a few studies have reported on acetylation modifications during memory formation, and it remains unclear as to how the system is regulated during this dynamic phase. We investigated acetylation-dependent events and the expression profiles of these HATs during a hippocampus-dependent task taxing spatial reference memory in the Morris water maze. We found a specific increase in H2B and H4 acetylation in the rat dorsal hippocampus, while spatial memory was being consolidated. This increase correlated with the degree of specific acetylated histones enrichment on some memory/plasticity-related gene promoters. Overall, a global increase in HAT activity was measured during this memory consolidation phase, together with a global increase of CBP, p300, and PCAF expression. Interestingly, these regulations were altered in a model of hippocampal denervation disrupting spatial memory consolidation, making it impossible for the hippocampus to recruit the CBP pathway (CBP regulation and acetylated-H2B-dependent transcription). CBP has long been thought to be present in limited concentrations in the cells. These results show, for the first time, that CBP, p300, and PCAF are dynamically modulated during the establishment of a spatial memory and are likely to contribute to the induction of a specific epigenetic tagging of the genome for hippocampus-dependent (spatial) memory consolidation. These findings suggest the use of HAT-activating molecules in new therapeutic strategies of pathological aging, Alzheimer's disease, and other neurodegenerative disorders.

  6. Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

    PubMed Central

    2010-01-01

    Background In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator. Results MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes. Conclusions Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal

  7. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveal Substrate Specificity of Protein Acetyltransferases*

    PubMed Central

    Crosby, Heidi A.; Pelletier, Dale A.; Hurst, Gregory B.; Escalante-Semerena, Jorge C.

    2012-01-01

    N-Lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously. PMID:22416131

  8. Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

    PubMed

    Singh, Kuljit; Singh, Krishn Pratap; Equbal, Asif; Suman, Shashi S; Zaidi, Amir; Garg, Gaurav; Pandey, Krishna; Das, Pradeep; Ali, Vahab

    2016-12-01

    Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a Km of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania.

  9. The yeast SAS (something about silencing) protein complex contains a MYST-type putative acetyltransferase and functions with chromatin assembly factor ASF1

    PubMed Central

    Osada, Shigehiro; Sutton, Ann; Muster, Nemone; Brown, Christine E.; Yates, John R.; Sternglanz, Rolf; Workman, Jerry L.

    2001-01-01

    It is well established that acetylation of histone and nonhistone proteins is intimately linked to transcriptional activation. However, loss of acetyltransferase activity has also been shown to cause silencing defects, implicating acetylation in gene silencing. The something about silencing (Sas) 2 protein of Saccharomyces cerevisiae, a member of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, promotes silencing at HML and telomeres. Here we identify a ∼450-kD SAS complex containing Sas2p, Sas4p, and the tf2f-related Sas5 protein. Mutations in the conserved acetyl-CoA binding motif of Sas2p are shown to disrupt the ability of Sas2p to mediate the silencing at HML and telomeres, providing evidence for an important role for the acetyltransferase activity of the SAS complex in silencing. Furthermore, the SAS complex is found to interact with chromatin assembly factor Asf1p, and asf1 mutants show silencing defects similar to mutants in the SAS complex. Thus, ASF1-dependent chromatin assembly may mediate the role of the SAS complex in silencing. PMID:11731479

  10. Arachidonic acid increases choline acetyltransferase activity in spinal cord neurons through a protein kinase C-mediated mechanism.

    PubMed

    Chalimoniuk, Malgorzata; King-Pospisil, Kelley; Pedersen, Ward A; Malecki, Andrzej; Wylegala, Edward; Mattson, Mark P; Hennig, Bernhard; Toborek, Michal

    2004-08-01

    Arachidonic acid (AA) plays an important role as a signaling factor in the CNS. Therefore, exposure to AA may affect cholinergic neurons in the spinal cord. To test this hypothesis, mRNA expression and activity of choline acetyltransferase (ChAT) was measured in cultured spinal cord neurons treated with increasing concentrations (0.1-10 microm) of AA. Exposure to AA increased mRNA levels and activity of ChAT in dose- and time-dependent manners. The most marked effect of AA on ChAT expression was observed in spinal cord neurons treated with 10 microm AA for 1 h. To study the mechanisms associated with these effects, ChAT mRNA levels and activity were measured in cultured spinal cord neurons exposed to AA and inhibitors of protein kinase C (PKC), such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dichloride (H-7) and chelerythrine. Inhibition of PKC completely prevented an AA-induced increase in ChAT expression. In addition, exposure of spinal cord neurons to phorbol-12-myristate-13-acetate (PMA), an activator of PKC, mimicked AA-induced stimulation of ChAT activity. The AA-mediated increase in ChAT mRNA levels and activity was also prevented by treatments with EGTA, indicating the role of calcium metabolism in induction of this enzyme. In contrast, treatments with 7-nitroindazole (7-NI, a specific inhibitor of neuronal nitric oxide synthase), sodium vanadate (NaV, a non-specific inhibitor of phosphatases), and N-acetyl-cysteine (NAC, an antioxidant) had no effect on AA-induced changes in ChAT activity. The protein synthesis inhibitor cycloheximide completely blocked AA-mediated increase in ChAT activity. These results indicate that the AA-evoked increase in ChAT activity in spinal cord neurons is mediated by PKC, presumably at the transcriptional level.

  11. Histone acetyltransferase (HAT) activity of p300 modulates human T lymphotropic virus type 1 p30{sup II}-mediated repression of LTR transcriptional activity

    SciTech Connect

    Michael, Bindhu; Nair, Amrithraj M.; Datta, Antara; Hiraragi, Hajime; Ratner, Lee; Lairmore, Michael D. . E-mail: lairmore.1@osu.edu

    2006-10-25

    Human T-lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus that causes adult T cell leukemia/lymphoma, and is implicated in a variety of lymphocyte-mediated inflammatory disorders. HTLV-1 provirus has regulatory and accessory genes in four pX open reading frames. HTLV-1 pX ORF-II encodes two proteins, p13{sup II} and p30{sup II}, which are incompletely defined in virus replication or pathogenesis. We have demonstrated that pX ORF-II mutations block virus replication in vivo and that ORF-II encoded p30{sup II}, a nuclear-localizing protein that binds with CREB-binding protein (CBP)/p300, represses CREB and Tax responsive element (TRE)-mediated transcription. Herein, we have identified p30{sup II} motifs important for p300 binding and in regulating TRE-mediated transcription in the absence and presence of HTLV-1 provirus. Within amino acids 100-179 of p30{sup II}, a region important for repression of LTR-mediated transcription, we identified a single lysine residue at amino acid 106 (K3) that significantly modulates the ability of p30{sup II} to repress TRE-mediated transcription. Exogenous p300, in a dose-responsive manner, reverses p30{sup II}-dependent repression of TRE-mediated transcription, in the absence or presence of the provirus, In contrast to wild type p300, p300 HAT mutants (defective in histone acetyltransferase activity) only partially rescued p30{sup II}-mediated LTR repression. Deacetylation by histone deacetylase-1 (HDAC-1) enhanced p30{sup II}-mediated LTR repression, while inhibition of deacetylation by trichostatin A decreases p30{sup II}-mediated LTR repression. Collectively, our data indicate that HTLV-1 p30{sup II} modulates viral gene expression in a cooperative manner with p300-mediated acetylation.

  12. Acetylation of retinal histones in diabetes increases inflammatory proteins: effects of minocycline and manipulation of histone acetyltransferase (HAT) and histone deacetylase (HDAC).

    PubMed

    Kadiyala, Chandra Sekhar Rao; Zheng, Ling; Du, Yunpeng; Yohannes, Elizabeth; Kao, Hung-Ying; Miyagi, Masaru; Kern, Timothy S

    2012-07-27

    Histone acetylation was significantly increased in retinas from diabetic rats, and this acetylation was inhibited in diabetics treated with minocycline, a drug known to inhibit early diabetic retinopathy in animals. Histone acetylation and expression of inflammatory proteins that have been implicated in the pathogenesis of diabetic retinopathy were increased likewise in cultured retinal Müller glia grown in a diabetes-like concentration of glucose. Both the acetylation and induction of the inflammatory proteins in elevated glucose levels were significantly inhibited by inhibitors of histone acetyltransferase (garcinol and antisense against the histone acetylase, p300) or activators of histone deacetylase (theophylline and resveratrol) and were increased by the histone deacetylase inhibitor, suberolylanilide hydroxamic acid. We conclude that hyperglycemia causes acetylation of retinal histones (and probably other proteins) and that the acetylation contributes to the hyperglycemia-induced up-regulation of proinflammatory proteins and thereby to the development of diabetic retinopathy.

  13. The lac operon galactoside acetyltransferase.

    PubMed

    Roderick, Steven L

    2005-06-01

    Of the proteins encoded by the three structural genes of the lac operon, the galactoside acetyltransferase (thiogalactoside transacetylase, LacA, GAT) encoded by lacA is the only protein whose biological role remains in doubt. Here, we briefly note the classical literature that led to the identification and initial characterization of GAT, and focus on more recent results which have revealed its chemical mechanism of action and its membership in a large superfamily of structurally similar acyltransferases. The structural and sequence similarities of several members of this superfamily confirm the original claim for GAT as a CoA-dependent acetyltransferase specific for the 6-hydroxyl group of certain pyranosides, but do not yet point to the identity of the natural substrate(s) of the enzyme.

  14. 15-Deoxy-{Delta}{sup 12,14}-prostaglandin J{sub 2} impairs the functions of histone acetyltransferases through their insolubilization in cells

    SciTech Connect

    Hironaka, Asako; Morisugi, Toshiaki; Kawakami, Tetsuji; Miyagi, Ikuko; Tanaka, Yasuharu

    2009-12-11

    The cyclopentenonic prostaglandin 15-deoxy-{Delta}{sup 12,14}-PG J{sub 2} (15d-PGJ{sub 2}) is a metabolite derived from PGD{sub 2}. Although 15d-PGJ{sub 2} has been demonstrated to be a potent ligand for peroxisome proliferator activated receptor {gamma} (PPAR{gamma}), the functions are not fully understood. In order to examine the effect of 15d-PGJ{sub 2} on histone acetyltransferases (HATs), several lines of cell including mouse embryonic fibroblast (MEF) cells were exposed to 15d-PGJ{sub 2}. Three types of HAT, p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF), selectively disappeared from the soluble fraction in time- and dose-dependent manners. Inversely, HATs in the insoluble fraction increased, suggesting their conformational changes. The decrease in the soluble form of HATs resulted in the attenuation of NF-{kappa}B-, p53-, and heat shock factor-dependent reporter gene expressions, implying that the insoluble HATs are inactive. The resultant insoluble PCAF and p300 seemed to be digested by proteasome, because proteasome inhibitors caused the accumulation of insoluble HATs. Taken together, these results indicate that 15d-PGJ{sub 2} attenuates some gene expressions that require HATs. This inhibitory action of 15d-PGJ{sub 2} on the function of HATs was independent of PPAR{gamma}, because PPAR{gamma} agonists could not mimick 15d-PGJ{sub 2} and PPAR{gamma} antagonists did not inhibit 15d-PGJ{sub 2}.

  15. N-hydroxyarylamine O-acetyltransferase of Salmonella typhimurium: proposal for a common catalytic mechanism of arylamine acetyltransferase enzymes.

    PubMed Central

    Watanabe, M; Igarashi, T; Kaminuma, T; Sofuni, T; Nohmi, T

    1994-01-01

    Acetyl-CoA:N-hydroxyarylamine O-acetyltransferase is an enzyme involved in the metabolic activation of N-hydroxyarylamines derived from mutagenic and carcinogenic aromatic amines and nitroarenes. The O-acetyltransferase gene of Salmonella typhimurium has been cloned, and new Ames tester substrains highly sensitive to mutagenic aromatic amines and nitroarenes have been established in our laboratory. The nucleotide sequence of the O-acetyltransferase gene was determined. There was an open reading frame of 843 nucleotides coding for a protein with a calculated molecular weight of 32,177, which was close to the molecular weight of the O-acetyltransferase protein determined by using the maxicell technique. Only the residue of Cys69 in O-acetyltransferase of S. typhimurium and its corresponding residue (Cys68) in N-acetyltransferase of higher organisms were conserved in all acetyltransferase enzymes sequenced so far. The amino acid sequence Arg-Gly-Gly-X-Cys, including the Cys69, was highly conserved. A mutant O-acetyltransferase of S. typhimurium, which contained Ala69 instead of Cys69, no longer showed the activities of O- and N-acetyltransferase. These results suggest that the Cys69 of S. typhimurium and the corresponding cysteine residues of the higher organisms are essential for the enzyme activities as an acetyl-CoA binding site. We propose a new catalytic model of acetyltransferase for S. typhimurium and the higher organisms. PMID:7889864

  16. A Metazoan ATAC Acetyltransferase Subunit That Regulates Mitogen-activated Protein Kinase Signaling Is Related to an Ancient Molybdopterin Synthase Component*

    PubMed Central

    Suganuma, Tamaki; Mushegian, Arcady; Swanson, Selene K.; Florens, Laurence; Washburn, Michael P.; Workman, Jerry L.

    2012-01-01

    Molybdopterin (MPT) synthase is an essential enzyme involved in the synthesis of the molybdenum cofactor precursor molybdopterin. The molybdenum cofactor biosynthetic pathway is conserved from prokaryotes to Metazoa. CG10238 is the Drosophila homolog of the MoaE protein, a subunit of MPT synthase, and is found in a fusion with the mitogen-activated protein kinase (MAPK)-upstream protein kinase-binding inhibitory protein (MBIP). This fused protein inhibits the activation of c-Jun N-terminal kinase (JNK). dMoaE (CG10238) carries out this function as a subunit of the ATAC histone acetyltransferase complex. In this study, we demonstrate that Drosophila MoaE (CG10238) also interacts with Drosophila MoaD and with itself to form a complex with stoichiometry identical to the MPT synthase holoenzyme in addition to its function in ATAC. We also show that sequence determinants that regulate MAPK signaling are located within the MoaE region of dMoaE (CG10238). Analysis of other metazoan MBIPs reveals that MBIP protein sequences have an N-terminal region that appears to have been derived from the MoaE protein, although it has lost residues responsible for catalytic activity. Thus, intact and modified copies of the MoaE protein may have been conscripted to play a new, noncatalytic role in MAPK signaling in Metazoa as part of the ATAC complex. PMID:22345504

  17. A PWWP Domain-Containing Protein Targets the NuA3 Acetyltransferase Complex via Histone H3 Lysine 36 trimethylation to Coordinate Transcriptional Elongation at Coding Regions*

    PubMed Central

    Gilbert, Tonya M.; McDaniel, Stephen L.; Byrum, Stephanie D.; Cades, Jessica A.; Dancy, Blair C. R.; Wade, Herschel; Tackett, Alan J.; Strahl, Brian D.; Taverna, Sean D.

    2014-01-01

    Post-translational modifications of histones, such as acetylation and methylation, are differentially positioned in chromatin with respect to gene organization. For example, although histone H3 is often trimethylated on lysine 4 (H3K4me3) and acetylated on lysine 14 (H3K14ac) at active promoter regions, histone H3 lysine 36 trimethylation (H3K36me3) occurs throughout the open reading frames of transcriptionally active genes. The conserved yeast histone acetyltransferase complex, NuA3, specifically binds H3K4me3 through a plant homeodomain (PHD) finger in the Yng1 subunit, and subsequently catalyzes the acetylation of H3K14 through the histone acetyltransferase domain of Sas3, leading to transcription initiation at a subset of genes. We previously found that Ylr455w (Pdp3), an uncharacterized proline-tryptophan-tryptophan-proline (PWWP) domain-containing protein, copurifies with stable members of NuA3. Here, we employ mass-spectrometric analysis of affinity purified Pdp3, biophysical binding assays, and genetic analyses to classify NuA3 into two functionally distinct forms: NuA3a and NuA3b. Although NuA3a uses the PHD finger of Yng1 to interact with H3K4me3 at the 5′-end of open reading frames, NuA3b contains the unique member, Pdp3, which regulates an interaction between NuA3b and H3K36me3 at the transcribed regions of genes through its PWWP domain. We find that deletion of PDP3 decreases NuA3-directed transcription and results in growth defects when combined with transcription elongation mutants, suggesting NuA3b acts as a positive elongation factor. Finally, we determine that NuA3a, but not NuA3b, is synthetically lethal in combination with a deletion of the histone acetyltransferase GCN5, indicating NuA3b has a specialized role at coding regions that is independent of Gcn5 activity. Collectively, these studies define a new form of the NuA3 complex that associates with H3K36me3 to effect transcriptional elongation. MS data are available via ProteomeXchange with

  18. Bacillus anthracis acetyltransferases PatA1 and PatA2 modify the secondary cell wall polysaccharide and affect the assembly of S-layer proteins.

    PubMed

    Lunderberg, J Mark; Nguyen-Mau, Sao-Mai; Richter, G Stefan; Wang, Ya-Ting; Dworkin, Jonathan; Missiakas, Dominique M; Schneewind, Olaf

    2013-03-01

    The envelope of Bacillus anthracis encompasses a proteinaceous S-layer with two S-layer proteins (Sap and EA1). Protein assembly in the envelope of B. anthracis requires S-layer homology domains (SLH) within S-layer proteins and S-layer-associated proteins (BSLs), which associate with the secondary cell wall polysaccharide (SCWP), an acetylated carbohydrate that is tethered to peptidoglycan. Here, we investigated the contributions of two putative acetyltransferases, PatA1 and PatA2, on SCWP acetylation and S-layer assembly. We show that mutations in patA1 and patA2 affect the chain lengths of B. anthracis vegetative forms and perturb the deposition of the BslO murein hydrolase at cell division septa. The patA1 and patA2 mutants are defective for the assembly of EA1 in the envelope but retain the ability of S-layer formation with Sap. SCWP isolated from the patA1 patA2 mutant lacked acetyl moieties identified in wild-type polysaccharide and failed to associate with the SLH domains of EA1. A model is discussed whereby patA1- and patA2-mediated acetylation of SCWP enables the deposition of EA1 as well as BslO near the septal region of the B. anthracis envelope.

  19. Repression of GCN5 Histone Acetyltransferase Activity via Bromodomain-Mediated Binding and Phosphorylation by the Ku–DNA-Dependent Protein Kinase Complex

    PubMed Central

    Barlev, Nickolai A.; Poltoratsky, Vladimir; Owen-Hughes, Tom; Ying, Carol; Liu, Lin; Workman, Jerry L.; Berger, Shelley L.

    1998-01-01

    GCN5, a putative transcriptional adapter in humans and yeast, possesses histone acetyltransferase (HAT) activity which has been linked to GCN5’s role in transcriptional activation in yeast. In this report, we demonstrate a functional interaction between human GCN5 (hGCN5) and the DNA-dependent protein kinase (DNA-PK) holoenzyme. Yeast two-hybrid screening detected an interaction between the bromodomain of hGCN5 and the p70 subunit of the human Ku heterodimer (p70-p80), which is the DNA-binding component of DNA-PK. Interaction between intact hGCN5 and Ku70 was shown biochemically using recombinant proteins and by coimmunoprecipitation of endogenous proteins following chromatography of HeLa nuclear extracts. We demonstrate that the catalytic subunit of DNA-PK phosphorylates hGCN5 both in vivo and in vitro and, moreover, that the phosphorylation inhibits the HAT activity of hGCN5. These findings suggest a possible regulatory mechanism of HAT activity. PMID:9488450

  20. Acetyltransferase p300 collaborates with chromodomain helicase DNA-binding protein 4 (CHD4) to facilitate DNA double-strand break repair.

    PubMed

    Qi, Wenjing; Chen, Hongyu; Xiao, Ting; Wang, Ruoxi; Li, Ting; Han, Liping; Zeng, Xianlu

    2016-03-01

    Chromatin remodelling is critical for repairing DNA damage and maintaining genomic integrity. Previous studies have reported that histone acetyltransferase p300 and ATP-dependent chromatin remodeler chromodomain helicase DNA-binding protein 4 (CHD4) functions, respectively, in DNA double-strand breaks (DSBs) repair. But the physiological significance of their interaction remains elusive. Here, we showed that p300 and CHD4 were both recruited to the sites of DSBs. Their ablation led to impaired DSBs repair and sensitised cells to laser and the anti-cancer drug, etoposide. Using DR-GFP and EJ5-GFP reporter systems, we found that knockdown of p300 or CHD4 impaired the homologous recombination (HR) repair but no the non-homologous end joining (NHEJ) repair. Furthermore, p300 or CHD4 knockdown respectively suppressed the recruitment of replication protein A (RPA), a key protein for HR, to the DSB sites. In addition, immunofluorescence results showed that knockdown of p300 reduced the recruitment of CHD4 at DSB sites. In turn, CHD4 knockdown also decreased p300 assembly. Moreover, immunoprecipitation and purified protein pull down assay revealed that p300 physically interacted with CHD4 at DNA damage sites, and this interaction was dependent on the chromodomain and ATPase/helicase domain of CHD4 and the CH2, Bd and HAT domains of p300. These results indicate that p300 and CHD4 could function cooperatively at DSB sites and provide a new insight into the detailed crosstalk among the chromatin remodelling proteins.

  1. Interactions of Histone Acetyltransferase p300 with the Nuclear Proteins Histone and HMGB1, As Revealed by Single Molecule Atomic Force Spectroscopy.

    PubMed

    Banerjee, S; Rakshit, T; Sett, S; Mukhopadhyay, R

    2015-10-22

    One of the important properties of the transcriptional coactivator p300 is histone acetyltransferase (HAT) activity that enables p300 to influence chromatin action via histone modulation. p300 can exert its HAT action upon the other nuclear proteins too--one notable example being the transcription-factor-like protein HMGB1, which functions also as a cytokine, and whose accumulation in the cytoplasm, as a response to tissue damage, is triggered by its acetylation. Hitherto, no information on the structure and stability of the complexes between full-length p300 (p300FL) (300 kDa) and the histone/HMGB1 proteins are available, probably due to the presence of unstructured regions within p300FL that makes it difficult to be crystallized. Herein, we have adopted the high-resolution atomic force microscopy (AFM) approach, which allows molecularly resolved three-dimensional contour mapping of a protein molecule of any size and structure. From the off-rate and activation barrier values, obtained using single molecule dynamic force spectroscopy, the biochemical proposition of preferential binding of p300FL to histone H3, compared to the octameric histone, can be validated. Importantly, from the energy landscape of the dissociation events, a model for the p300-histone and the p300-HMGB1 dynamic complexes that HAT forms, can be proposed. The lower unbinding forces of the complexes observed in acetylating conditions, compared to those observed in non-acetylating conditions, indicate that upon acetylation, p300 tends to weakly associate, probably as an outcome of charge alterations on the histone/HMGB1 surface and/or acetylation-induced conformational changes. To our knowledge, for the first time, a single molecule level treatment of the interactions of HAT, where the full-length protein is considered, is being reported.

  2. Coexpression of glutamate vesicular transporter (VGLUT1) and choline acetyltransferase (ChAT) proteins in fetal rat hippocampal neurons in culture.

    PubMed

    Bhargava, Neelima; Das, Mainak; Edwards, Darin; Stancescu, Maria; Kang, Jung-Fong; Hickman, James J

    2010-09-01

    A very small population of choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus. This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine. Using this method, a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons, and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in the culture. Interestingly, most of the glutamatergic neurons that expressed glutamate vesicular transporter (VGLUT1) also co-expressed ChAT proteins. On the contrary, when the cultures were double-stained with GABA and ChAT, colocalization was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate.

  3. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins

    PubMed Central

    Iyer, Lakshminarayan M.; Abhiman, Saraswathi; Burroughs, A. Maxwell; Aravind, L.

    2011-01-01

    Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family. PMID:20023723

  4. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Maxwell Burroughs, A; Aravind, L

    2009-12-01

    Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN-hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family.

  5. Regulation of cAMP-induced arylalkylamine N-acetyltransferase, Period1, and MKP-1 gene expression by mitogen-activated protein kinases in the rat pineal gland.

    PubMed

    Chansard, Mathieu; Iwahana, Eiko; Liang, Jian; Fukuhara, Chiaki

    2005-10-03

    In rodent pineal glands, sympathetic innervation, which leads to norepinephrine release, is a key process in the circadian regulation of physiology and certain gene expressions. It has been shown that gene expression of the rate-limiting enzyme in the melatonin synthesis arylalkylamine N-acetyltransferase (Aa-Nat), circadian clock gene Period1, and mitogen-activated protein kinase (MAPK) phosphtase-1 (MKP-1), is controlled mainly by a norepinephrine-beta-adrenergic receptor-cAMP signaling cascade in the rat pineal gland. To further dissect the signaling cascades that regulate those gene expressions, we examined whether MAPKs are involved in cAMP-induced gene expression. Western blot and immunohistochemical analyses showed that one of the three MAPKs, c-Jun N-terminal kinase (JNK), was expressed in the pineal, and was phosphorylated by cAMP analogue stimulation with a peak 20 min after start of the stimulation, in vitro. A specific JNK inhibitor SP600125 (Anthra[1,9-cd]pyrazol-6(2H)-one1,9-pyrazoloanthrone), but not its negative control (N1-Methyl-1,9-pyrazoloanthrone), significantly reduced cAMP-stimulated Aa-Nat, Period1, and MKP-1 mRNA levels. Although another MAPK, p38(MAPK), has also been shown to be activated by cAMP stimulation, a p38(MAPK) inhibitor, SB203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole, HCl), showed no effect on cAMP-induced Aa-Nat and Period1 mRNA levels; whereas SB203580, but not its negative analogue SB202474 (4-Ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole, DiHCl), significantly reduced cAMP-induced MKP-1 mRNA levels. Taken together, our data suggest that cAMP-induced Aa-Nat and Period1 are likely to be mediated by activation of JNK, whereas MKP-1 may be mediated by both p38(MAPK) and JNK activations.

  6. Host Cell Factor and an Uncharacterized SANT Domain Protein Are Stable Components of ATAC, a Novel dAda2A/dGcn5-Containing Histone Acetyltransferase Complex in Drosophila

    PubMed Central

    Guelman, Sebastián; Suganuma, Tamaki; Florens, Laurence; Swanson, Selene K.; Kiesecker, Cheri L.; Kusch, Thomas; Anderson, Scott; Yates, John R.; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2006-01-01

    Gcn5 is a conserved histone acetyltransferase (HAT) found in a number of multisubunit complexes from Saccharomyces cerevisiae, mammals, and flies. We previously identified Drosophila melanogaster homologues of the yeast proteins Ada2, Ada3, Spt3, and Tra1 and showed that they associate with dGcn5 to form at least two distinct HAT complexes. There are two different Ada2 homologues in Drosophila named dAda2A and dAda2B. dAda2B functions within the Drosophila version of the SAGA complex (dSAGA). To gain insight into dAda2A function, we sought to identify novel components of the complex containing this protein, ATAC (Ada two A containing) complex. Affinity purification and mass spectrometry revealed that, in addition to dAda3 and dGcn5, host cell factor (dHCF) and a novel SANT domain protein, named Atac1 (ATAC component 1), copurify with this complex. Coimmunoprecipitation experiments confirmed that these proteins associate with dGcn5 and dAda2A, but not with dSAGA-specific components such as dAda2B and dSpt3. Biochemical fractionation revealed that ATAC has an apparent molecular mass of 700 kDa and contains dAda2A, dGcn5, dAda3, dHCF, and Atac1 as stable subunits. Thus, ATAC represents a novel histone acetyltransferase complex that is distinct from previously purified Gcn5/Pcaf-containing complexes from yeast and mammalian cells. PMID:16428443

  7. The Purification of Choline Acetyltransferase of Squid-Head Ganglia

    PubMed Central

    Husain, S. S.; Mautner, Henry G.

    1973-01-01

    Choline acetyltransferase (EC 2.3.1.6) isolated from the head ganglia of squid could be purified by use of mercurial-Sepharose columns as well as Sepharose columns to which the enzyme inhibitor p-(m-bromophenyl)vinyl pyridinium had been attached. These columns, in conjunction with 30-55% ammonium sulfate precipitation, 40-30% ammonium sulfate extraction, chromatography on sulfopropyl-Sephadex and on cellulose phosphate and hydroxylapatite columns, led to the isolation of three factions of choline acetyltransferase ranging in activity from 1000 to 4000 μmole/mg of protein/per hr. Polyacrylamide gel electrophoresis suggests that two of these fractions are homogeneous. The squid choline acetyltransferase is different from the mammalian-brain enzymes in having a larger molecular weight under the conditions used and in being relatively poorly inhibited by styryl pyridinium compounds. Images PMID:4521199

  8. Neuregulin1 signaling targets SRF and CREB and activates the muscle spindle-specific gene Egr3 through a composite SRF-CREB-binding site.

    PubMed

    Herndon, Carter A; Ankenbruck, Nick; Lester, Bridget; Bailey, Julie; Fromm, Larry

    2013-03-10

    Muscle spindles are sensory receptors embedded within muscle that detect changes in muscle length. Each spindle is composed of specialized muscle fibers, known as intrafusal muscle fibers, along with the endings of axons from sensory neurons that innervate these muscle fibers. Formation of muscle spindles requires neuregulin1 (NRG1), which is released by sensory axons, activating ErbB receptors in muscle cells that are contacted. In muscle cells, the transcription factor Egr3 is transcriptionally induced by NRG1, which in turn activates various target genes involved in forming the intrafusal fibers of muscle spindles. The signaling relay within the NRG1-ErbB pathway that acts to induce Egr3 is presumably critical for muscle spindle formation but for the most part has not been determined. In the current studies, we examined, using cultured muscle cells, transcriptional regulatory mechanisms by which Egr3 responds to NRG1. We identified a composite regulatory element for the Egr3 gene, consisting adjacent sites that bind cAMP response element binding protein (CREB) and serum response factor (SRF), with a role in NRG1 responsiveness. The SRF element also influences Egr3 basal expression in unstimulated myotubes, and in the absence of the SRF element, the CREB element influences basal expression. We show that NRG1 signaling, to target SRF, acts on the SRF coactivators myocardian-related transcription factor (MRTF)-A and MRTF-B, which are known to activate SRF-mediated transcription, by stimulating their translocation from the cytoplasm to the nucleus. CREB is phosphorylated, which is known to contribute to its activation, in response to NRG1. These results suggest that NRG1 induces expression of the muscle spindle-specific gene Egr3 by stimulating the transcriptional activity of CREB and SRF.

  9. Prefrontal Consolidation Supports the Attainment of Fear Memory Accuracy

    ERIC Educational Resources Information Center

    Vieira, Philip A.; Lovelace, Jonathan W.; Corches, Alex; Rashid, Asim J.; Josselyn, Sheena A.; Korzus, Edward

    2014-01-01

    The neural mechanisms underlying the attainment of fear memory accuracy for appropriate discriminative responses to aversive and nonaversive stimuli are unclear. Considerable evidence indicates that coactivator of transcription and histone acetyltransferase cAMP response element binding protein (CREB) binding protein (CBP) is critically required…

  10. A Transcription Factor-Binding Domain of the Coactivator CBP Is Essential for Long-Term Memory and the Expression of Specific Target Genes

    ERIC Educational Resources Information Center

    Oliveira, Ana M. M.; Brindle, Paul K.; Abel, Ted; Wood, Marcelo A.; Attner, Michelle A.

    2006-01-01

    Transcriptional activation is a key process required for long-term memory formation. Recently, the transcriptional coactivator CREB-binding protein (CBP) was shown to be critical for hippocampus-dependent long-term memory and hippocampal synaptic plasticity. As a coactivator with intrinsic histone acetyltransferase activity, CBP interacts with…

  11. Arylamine N-Acetyltransferases in Mycobacteria

    PubMed Central

    Sim, Edith; Sandy, James; Evangelopoulos, Dimitrios; Fullam, Elizabeth; Bhakta, Sanjib; Westwood, Isaac; Krylova, Anna; Lack, Nathan; Noble, Martin

    2008-01-01

    Polymorphic Human arylamine N-acetyltransferase (NAT2) inactivates the anti-tubercular drug isoniazid by acetyltransfer from acetylCoA. There are active NAT proteins encoded by homologous genes in mycobacteria including M. tuberculosis, M. bovis BCG, M. smegmatis and M. marinum. Crystallographic structures of NATs from M. smegmatis and M. marinum, as native enzymes and with isoniazid bound share a similar fold with the first NAT structure, Salmonella typhimurium NAT. There are three approximately equal domains and an active site essential catalytic triad of cysteine, histidine and aspartate in the first two domains. An acetyl group from acetylCoA is transferred to cysteine and then to the acetyl acceptor e.g. isoniazid. M. marinum NAT binds CoA in a more open mode compared with CoA binding to human NAT2. The structure of mycobacterial NAT may promote its role in synthesis of cell wall lipids, identified through gene deletion studies. NAT protein is essential for survival of M. bovis BCG in macrophage as are the proteins encoded by other genes in the same gene cluster (hsaA-D). HsaA-D degrade cholesterol, essential for mycobacterial survival inside macrophage. Nat expression remains to be fully understood but is co-ordinated with hsaA-D and other stress response genes in mycobacteria. Amide synthase genes in the streptomyces are also nat homologues. The amide synthases are predicted to catalyse intramolecular amide bond formation and creation of cyclic molecules, e.g. geldanamycin. Lack of conservation of the CoA binding cleft residues of M. marinum NAT suggests the amide synthase reaction mechanism does not involve a soluble CoA intermediate during amide formation and ring closure. PMID:18680471

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

    DOEpatents

    Stephens, David S [Stone Mountain, GA; Gudlavalleti, Seshu K [Kensington, MD; Tzeng, Yih-Ling [Atlanta, GA; Datta, Anup K [San Diego, CA; Carlson, Russell W [Athens, GA

    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.

  13. Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity.

    PubMed Central

    Dobransky, T; Davis, W L; Xiao, G H; Rylett, R J

    2000-01-01

    Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity. PMID:10861222

  14. 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.

  15. 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

  16. Melatonin production: proteasomal proteolysis in serotonin N-acetyltransferase regulation.

    PubMed

    Gastel, J A; Roseboom, P H; Rinaldi, P A; Weller, J L; Klein, D C

    1998-02-27

    The nocturnal increase in circulating melatonin in vertebrates is regulated by 10- to 100-fold increases in pineal serotonin N-acetyltransferase (AA-NAT) activity. Changes in the amount of AA-NAT protein were shown to parallel changes in AA-NAT activity. When neural stimulation was switched off by either light exposure or L-propranolol-induced beta-adrenergic blockade, both AA-NAT activity and protein decreased rapidly. Effects of L-propranolol were blocked in vitro by dibutyryl adenosine 3',5'-monophosphate (cAMP) or inhibitors of proteasomal proteolysis. This result indicates that adrenergic-cAMP regulation of AA-NAT is mediated by rapid reversible control of selective proteasomal proteolysis. Similar proteasome-based mechanisms may function widely as selective molecular switches in vertebrate neural systems.

  17. Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex

    PubMed Central

    Wang, Haoyong; Chong, Shaorong

    2003-01-01

    During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors α and β, and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor β or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein–protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins. PMID:12515863

  18. 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

  19. The Novel SLIK Histone Acetyltransferase Complex Functions in the Yeast Retrograde Response Pathway

    PubMed Central

    Pray-Grant, Marilyn G.; Schieltz, David; McMahon, Stacey J.; Wood, Jennifer M.; Kennedy, Erin L.; Cook, Richard G.; Workman, Jerry L.; Yates III, John R.; Grant, Patrick A.

    2002-01-01

    The SAGA complex is a conserved histone acetyltransferase-coactivator that regulates gene expression in Saccharomyces cerevisiae. SAGA contains a number of subunits known to function in transcription including Spt and Ada proteins, the Gcn5 acetyltransferase, a subset of TATA-binding-protein-associated factors (TAFIIs), and Tra1. Here we report the identification of SLIK (SAGA-like), a complex related in composition to SAGA. Notably SLIK uniquely contains the protein Rtg2, linking the function of SLIK to the retrograde response pathway. Yeast harboring mutations in both SAGA and SLIK complexes displays synthetic phenotypes more severe than those of yeast with mutation of either complex alone. We present data indicating that distinct forms of the SAGA complex may regulate specific subsets of genes and that SAGA and SLIK have multiple partly overlapping activities, which play a critical role in transcription by RNA polymerase II. PMID:12446794

  20. 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.

  1. 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.

  2. 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.

  3. To learn better, keep the HAT on.

    PubMed

    Martin, Kelsey C; Sun, Yi E

    2004-06-24

    Long-lasting memories are known to require new transcription. Recent studies have highlighted a role for epigenetic alterations, including histone acetylation, in regulating gene expression. In this issue of Neuron, Alarcón et al. and Korzus et al. use two different mouse models of Rubinstein-Taybi syndrome to elucidate a role for the histone acetyltransferase activity of CREB binding protein (CBP) in long-term memory and plasticity.

  4. Spermidine/spermine-N(1)-acetyltransferase: a key metabolic regulator.

    PubMed

    Pegg, Anthony E

    2008-06-01

    Spermidine/spermine-N(1)-acetyltransferase (SSAT) regulates cellular polyamine content. Its acetylated products are either excreted from the cell or oxidized by acetylpolyamine oxidase. Since polyamines play critical roles in normal and neoplastic growth and in ion channel regulation, SSAT is a key enzyme in these processes. SSAT is very highly regulated. Its content is adjusted in response to alterations in polyamine content to maintain polyamine homeostasis. Certain polyamine analogs can mimic the induction of SSAT and cause a loss of normal polyamines. This may have utility in cancer chemotherapy. SSAT activity is also induced via a variety of other stimuli, including toxins, hormones, cytokines, nonsteroidal anti-inflammatory agents, natural products, and stress pathways, and by ischemia-reperfusion injury. These increases are initiated by alterations in Sat1 gene transcription reinforced by alterations at the other regulatory steps, including protein turnover, mRNA processing, and translation. Transgenic manipulation of SSAT activity has revealed that SSAT activity links polyamine metabolism to lipid and carbohydrate metabolism by means of alterations in the content of acetyl-CoA and ATP. A high level of SSAT stimulates flux through the polyamine biosynthetic pathway, since biosynthetic enzymes are induced in response to the fall in polyamines. This sets up a futile cycle in which ATP is used to generate S-adenosylmethionine for polyamine biosynthesis and acetyl-CoA is consumed in the acetylation reaction. A variety of other effects of increased SSAT activity include death of pancreatic cells, blockage of regenerative tissue growth, behavioral changes, keratosis follicularis spinulosa decalvans, and hair loss. These are very likely due to changes in polyamine and putrescine levels, although increased oxidative stress via the oxidation of acetylated polyamines may also contribute. Recently, it was found that the SSAT protein and/or a related protein, thialysine

  5. 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.

  6. Histone acetyltransferases: challenges in targeting bi-substrate enzymes.

    PubMed

    Wapenaar, Hannah; Dekker, Frank J

    2016-01-01

    Histone acetyltransferases (HATs) are epigenetic enzymes that install acetyl groups onto lysine residues of cellular proteins such as histones, transcription factors, nuclear receptors, and enzymes. HATs have been shown to play a role in diseases ranging from cancer and inflammatory diseases to neurological disorders, both through acetylations of histone proteins and non-histone proteins. Several HAT inhibitors, like bi-substrate inhibitors, natural product derivatives, small molecules, and protein-protein interaction inhibitors, have been developed. Despite their potential, a large gap remains between the biological activity of inhibitors in in vitro studies and their potential use as therapeutic agents. To bridge this gap, new potent HAT inhibitors with improved properties need to be developed. However, several challenges have been encountered in the investigation of HATs and HAT inhibitors that hinder the development of new HAT inhibitors. HATs have been shown to function in complexes consisting of many proteins. These complexes play a role in the activity and target specificity of HATs, which limits the translation of in vitro to in vivo experiments. The current HAT inhibitors suffer from undesired properties like anti-oxidant activity, reactivity, instability, low potency, or lack of selectivity between HAT subtypes and other enzymes. A characteristic feature of HATs is that they are bi-substrate enzymes that catalyze reactions between two substrates: the cofactor acetyl coenzyme A (Ac-CoA) and a lysine-containing substrate. This has important-but frequently overlooked-consequences for the determination of the inhibitory potency of small molecule HAT inhibitors and the reproducibility of enzyme inhibition experiments. We envision that a careful characterization of molecular aspects of HATs and HAT inhibitors, such as the HAT catalytic mechanism and the enzyme kinetics of small molecule HAT inhibitors, will greatly improve the development of potent and

  7. The histone acetyltransferase p300 promotes intrinsic axonal regeneration.

    PubMed

    Gaub, Perrine; Joshi, Yashashree; Wuttke, Anja; Naumann, Ulrike; Schnichels, Sven; Heiduschka, Peter; Di Giovanni, Simone

    2011-07-01

    Axonal regeneration and related functional recovery following axonal injury in the adult central nervous system are extremely limited, due to a lack of neuronal intrinsic competence and the presence of extrinsic inhibitory signals. As opposed to what occurs during nervous system development, a weak proregenerative gene expression programme contributes to the limited intrinsic capacity of adult injured central nervous system axons to regenerate. Here we show, in an optic nerve crush model of axonal injury, that adenoviral (cytomegalovirus promoter) overexpression of the acetyltransferase p300, which is regulated during retinal ganglion cell maturation and repressed in the adult, can promote axonal regeneration of the optic nerve beyond 0.5 mm. p300 acetylates histone H3 and the proregenerative transcription factors p53 and CCAAT-enhancer binding proteins in retinal ganglia cells. In addition, it directly occupies and acetylates the promoters of the growth-associated protein-43, coronin 1 b and Sprr1a and drives the gene expression programme of several regeneration-associated genes. On the contrary, overall increase in cellular acetylation using the histone deacetylase inhibitor trichostatin A, enhances retinal ganglion cell survival but not axonal regeneration after optic nerve crush. Therefore, p300 targets both the epigenome and transcription to unlock a post-injury silent gene expression programme that would support axonal regeneration.

  8. 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.

  9. 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.

  10. N-Acetyltransferase 1 Polymorphism and Breast Cancer Risk

    DTIC Science & Technology

    2011-10-01

    analysis of the N-acetyltransferase 1 gene (NAT1*) using polymerase chain reaction-restriction fragment- single strand conformation polymorphism assay...risk of smoking-induced lung cancer (Bouchardy et al., 1998). NAT1*14B is characterized by a single nucleotide polymorphism (SNP) G560A (rs4986782...Structure-function analyses of single nucleotide polymorphisms in human N-acetyltransferase 1. Drug Metab Rev 40, 169-184. Zheng, W., Deitz, A.C., Campbell

  11. Obesity and lipid stress inhibit carnitine acetyltransferase activity[S

    PubMed Central

    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.

    2014-01-01

    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

  12. Structure of the lac operon galactoside acetyltransferase.

    PubMed

    Wang, Xing-Guo; Olsen, Laurence R; Roderick, Steven L

    2002-04-01

    The galactoside acetyltransferase (thiogalactoside transacetylase) of Escherichia coli (GAT, LacA, EC 2.3.1.18) is a gene product of the classical lac operon. GAT may assist cellular detoxification by acetylating nonmetabolizable pyranosides, thereby preventing their reentry into the cell. The structure of GAT has been solved in binary complexes with acetyl-CoA or CoA and in ternary complexes with CoA and the nonphysiological acceptor substrates isopropyl beta-D-thiogalactoside (IPTG) or p-nitrophenyl beta-D-galactopyranoside (PNPbetaGal). A hydrophobic cleft that binds the thioisopropyl and p-nitrophenyl aglycones of IPTG and PNPbetaGal may discriminate against substrates with hydrophilic substituents at this position, such as lactose, or inducers of the lac operon. An extended loop projecting from the left-handed parallel beta helix domain contributes His115, which is in position to facilitate attack of the C6-hydroxyl group of the substrate on the thioester.

  13. Evidence for arylamine N-acetyltransferase in Hymenolepis nana.

    PubMed

    Chung, J G; Kuo, H M; Wu, L T; Lai, J M; Lee, J H; Hung, C F

    1997-02-01

    N-acetyltransferase activities with p-aminobenzoic acid and 2-aminofluorene were determined in Hymenolepis nana, a cestode found in the intestine of the Sprague-Dawley rats. The N-acetyltransferase activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. The N-acetyltransferase activities from a number of Hymenolepis nana whole tissue homogenizations were found to be 2.83 +/- 0.31 nmole/min/mg for 2-aminofluorene and 2.07 +/- 0.24 nmole/min/mg for p-aminobenzoic acid. The apparent Km and Vmax were 1.06 +/- 0.38 mM and 8.92 +/- 1.46 nmol/min/mg for 2-aminofluorene, and 2.16 +/- 0.19 mM and 12.68 +/- 2.26 nmol/min/mg for p-aminobenzoic acid. The optimal pH value for the enzyme activity was pH 8.0 for both substrates tested. The optimal temperature for enzyme activity was 37 degrees C for both substrates. The N-acetyltransferase activity was inhibited by iodacetamide. At 0.25 mM iodacetamide the activity was reduced 50% and 1.0 mM iodacetamide inhibited activity more than 90%. Among a series of divalent cations and salts, Fe2+, Ca2+ and Zn2+ were demonstrated to be the most potent inhibi-tors. Among the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected N-acetyltransferase. Iodoacetate, in contrast to other agents, markedly inhibited N-acetyltransferase activity. This is the first demonstration of acetyl CoA:arylamine N-acetyltransferase activity in a cestode and extends the number of phyla in which this activity has been found.

  14. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase

    SciTech Connect

    He, Yuan; Roth, Christian; Turkenburg, Johan P.; Davies, Gideon J.

    2014-01-01

    The crystal structure of a bacterial acetyltransferase with 27% sequence identity to the C-terminal domain of human O-GlcNAcase has been solved at 1.5 Å resolution. This S. sviceus protein is compared with known GCN5-related acetyltransferases, adding to the diversity observed in this superfamily. The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP-05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  15. Overexpression and characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.

    PubMed

    Franklin, K; Clarke, A J

    2001-08-01

    The gene coding for aminoglycoside 2'-N-acetyltransferase Ia [AAC(2')-Ia] from Providencia stuartii was amplified by PCR and cloned. The resulting construct, pACKF2, was transferred into Escherichia coli for overexpression of AAC(2')-Ia as a fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75. Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of both forms of the 2'-N-acetyltransferase were 20 degrees C and pH 6.0, respectively, while the enzymes were most stable at 15 degrees C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2')-Ia at 20 degrees C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2'-amino group and a concentration of acetyl coenzyme A fixed at 10 times its K(m) value of 8.75 microM. Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of both K(m) and k(cat)/K(m) values, whereas neomycin was the poorest. Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2')-Ia activity, while the enzyme exhibits greater tolerance further from these sites. No correlation was found between these kinetic parameters and MICs determined for P. stuartii PR50 expressing the 2'-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.

  16. Analysis of p300/CBP histone acetyltransferase regulation using circular permutation and semisynthesis.

    PubMed

    Karukurichi, Kannan R; Wang, Ling; Uzasci, Lerna; Manlandro, Cara Marie; Wang, Qing; Cole, Philip A

    2010-02-03

    The histone acetyltransferase (HAT) p300/CBP has been shown to undergo autoacetylation on lysines in an apparent regulatory loop that stimulates HAT activity. Here we have developed a strategy to introduce acetyl-Lys at up to six known modification sites in p300/CBP HAT using a combination of circular permutation and expressed protein ligation. We show that these semisynthetic, circularly permuted acetylated proteins retain high affinity for an acetyl-CoA substrate analogue and that HAT activity correlates positively with degree of acetylation. This study provides novel evidence for control of p300/CBP HAT activity by site-specific autoacetylation and outlines a potentially general strategy for using expressed protein ligation and circular permutation to chemically interrogate internal regions of proteins.

  17. Biochemical and structural analysis of an Eis family aminoglycoside acetyltransferase from bacillus anthracis.

    PubMed

    Green, Keith D; Biswas, Tapan; Chang, Changsoo; Wu, Ruiying; Chen, Wenjing; Janes, Brian K; Chalupska, Dominika; Gornicki, Piotr; Hanna, Philip C; Tsodikov, Oleg V; Joachimiak, Andrzej; Garneau-Tsodikova, Sylvie

    2015-05-26

    Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.

  18. 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

  19. Nucleotide sequence and phylogeny of a chloramphenicol acetyltransferase encoded by the plasmid pSCS7 from Staphylococcus aureus.

    PubMed

    Schwarz, S; Cardoso, M

    1991-08-01

    The nucleotide sequence of the chloramphenicol acetyltransferase gene (cat) and its regulatory region, encoded by the plasmid pSCS7 from Staphylococcus aureus, was determined. The structural cat gene encoded a protein of 209 amino acids, which represented one monomer of the enzyme chloramphenicol acetyltransferase (CAT). Comparisons between the amino acid sequences of the pSCS7-encoded CAT from S. aureus and the previously sequenced CAT variants from S. aureus, Staphylococcus intermedius, Staphylococcus haemolyticus, Bacillus pumilis, Clostridium difficile, Clostridium perfringens, Escherichia coli, Shigella flexneri, and Proteus mirabilis were performed. An alignment of CAT amino acid sequences demonstrated the presence of 34 conserved amino acids among all CAT variants. These conserved residues were considered for their possible roles in the structure and function of CAT. On the basis of the alignment, a phylogenetic tree was constructed. It demonstrated relatively large evolutionary distances between the CAT variants of enteric bacteria, Clostridium, Bacillus, and Staphylococcus species.

  20. Three-dimensional structure of a Streptomyces sviceus GNAT acetyltransferase with similarity to the C-terminal domain of the human GH84 O-GlcNAcase.

    PubMed

    He, Yuan; Roth, Christian; Turkenburg, Johan P; Davies, Gideon J

    2014-01-01

    The mammalian O-GlcNAc hydrolysing enzyme O-GlcNAcase (OGA) is a multi-domain protein with glycoside hydrolase activity in the N-terminus and with a C-terminal domain that has low sequence similarity to known acetyltransferases, prompting speculation, albeit controversial, that the C-terminal domain may function as a histone acetyltransferase (HAT). There are currently scarce data available regarding the structure and function of this C-terminal region. Here, a bacterial homologue of the human OGA C-terminal domain, an acetyltransferase protein (accession No. ZP_05014886) from Streptomyces sviceus (SsAT), was cloned and its crystal structure was solved to high resolution. The structure reveals a conserved protein core that has considerable structural homology to the acetyl-CoA (AcCoA) binding site of GCN5-related acetyltransferases (GNATs). Calorimetric data further confirm that SsAT is indeed able to bind AcCoA in solution with micromolar affinity. Detailed structural analysis provided insight into the binding of AcCoA. An acceptor-binding cavity was identified, indicating that the physiological substrate of SsAT may be a small molecule. Consistent with recently published work, the SsAT structure further questions a HAT function for the human OGA domain.

  1. 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.

  2. 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

  3. 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

  4. Molecular Determinants of the N-Terminal Acetyltransferase Naa60 Anchoring to the Golgi Membrane.

    PubMed

    Aksnes, Henriette; Goris, Marianne; Strømland, Øyvind; Drazic, Adrian; Waheed, Qaiser; Reuter, Nathalie; Arnesen, Thomas

    2017-02-14

    Nα-acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) since it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N-termini of transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling, in vitro assays, and cellular localization studies to study secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C-terminus bind the membrane directly in a parallel position relative to the lipid bilayer via hydrophobic and electrostatic interactions. A peptide corresponding to the C-terminus is unstructured in solution and only folds into an α-helical conformation in the presence of liposomes. Computational modeling and cellular mutational analysis revealed the hydrophobic face of two α-helices to be critical for membranous localization. Furthermore, we found a strong and specific binding preference of Naa60 towards membranes containing the phosphatidylinositol PI4P, thus possibly explaining the primary residency of Naa60 at the PI4P-rich Golgi. In conclusion, we have defined the mode of cytosolic Naa60 anchoring to the Golgi apparatus, most likely occurring post-translationally and specifically facilitating post-translational N-terminal acetylation of many transmembrane proteins.

  5. 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.

  6. New perspectives for the regulation of acetyltransferase MOF.

    PubMed

    Li, Xiangzhi; Dou, Yali

    2010-04-01

    In higher eukaryotes, histone acetyltransferase MOF (male absent on the first) is the major enzyme that acetylates histone H4 lysine 16, a prevalent mark associated with chromatin decondensation. Recent studies show that MOF resides in two different but evolutionarily conserved complexes, MSL and MOF-MSL1v1. Although these two MOF complexes have indistinguishable activity on histone H4 K16, they differ dramatically in acetylating non-histone substrate p53. The regulation of MOF activity in these complexes remains elusive. Given the evolution conservation of MOF and the importance of H4 K16 acetylation in maintaining higher order chromatin structures, understanding the function and regulation of MOF bears great significance. Here, we discussed the key differences in two MOF complexes that may shed light on the regulation of their distinct acetyltransferase activities. We also discussed coordinated functions of two MOF complexes with different histone methyltransferase complexes in transcription regulation.

  7. The ADA Complex Is a Distinct Histone Acetyltransferase Complex in Saccharomyces cerevisiae

    PubMed Central

    Eberharter, Anton; Sterner, David E.; Schieltz, David; Hassan, Ahmed; Yates, John R.; Berger, Shelley L.; Workman, Jerry L.

    1999-01-01

    We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex. In this study, we report the purification of the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023C have not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A (J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999–2009, 1995). Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada− phenotypes. These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA. PMID:10490601

  8. 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.

  9. 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.

  10. 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

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

    PubMed

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

    2011-06-14

    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.

  12. Polyamine-Regulated Translation of Spermidine/Spermine-N1-Acetyltransferase

    PubMed Central

    Perez-Leal, Oscar; Barrero, Carlos A.; Clarkson, Allen B.; Casero, Robert A.

    2012-01-01

    Rapid synthesis of the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT) in response to increased polyamines is an important polyamine homeostatic mechanism. Indirect evidence has suggested that there is an important control mechanism involving the release of a translational repressor protein that allows the immediate initiation of SSAT protein synthesis without RNA transcription, maturation, or translocation. To identify a repressor protein, we used a mass spectroscopy-based RNA-protein interaction system and found six proteins that bind to the coding region of SSAT mRNA. Individual small interfering RNA (siRNA) experiments showed that nucleolin knockdown enhances SSAT translation. Nucleolin exists in several isoforms, and we report that the isoform that binds to SSAT mRNA undergoes autocatalysis in the presence of polyamines, a result suggesting that there is a negative feedback system that helps control the cellular content of polyamines. Preliminary molecular interaction data show that a nucleolin isoform binds to a 5′ stem-loop of the coding region of SSAT mRNA. The glycine/arginine-rich C terminus of nucleolin is required for binding, and the four RNA recognition motif domains are included in the isoform that blocks SSAT translation. Understanding SSAT translational control mechanisms has the potential for the development of therapeutic strategies against cancer and obesity. PMID:22354986

  13. Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse.

    PubMed

    Sadananda, Aparna; Hamid, Runa; Doodhi, Harinath; Ghosal, Debnath; Girotra, Mukul; Jana, Swadhin Chandra; Ray, Krishanu

    2012-07-01

    Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin-based movement of large protein-aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution.

  14. Chemical biology of histone acetyltransferase natural compounds modulators.

    PubMed

    Piaz, Fabrizio Dal; Vassallo, Antonio; Rubio, Osmany Cuesta; Castellano, Sabrina; Sbardella, Gianluca; De Tommasi, Nunziatina

    2011-05-01

    Histone acetyltransferases (HATs) are a class of epigenetic enzymes crucial for chromatin restructuring and transcriptional regulation in eukaryotic cells, thus being a promising target for therapeutic development. Nonetheless, differently from histone deacetylases (HDACs) inhibitors, there is still paucity of small-molecule modulators of HAT activity. After a decline during past decade, natural products and their derivatives could be once again a valuable tool in the lead discovery process and meet such need of Novel Chemical Entities (NCEs). In this review, we will provide a comprehensive summary on the discovery of small-molecule HAT modulators from naturally occurring molecular scaffolds.

  15. Choline acetyltransferase expression does not identify early pathogenic events in fetal SMA spinal cord.

    PubMed

    Soler-Botija, Carolina; Cuscó, Ivón; López, Eva; Clua, Agustín; Gich, Ignasi; Baiget, Montserrat; Ferrer, Isidre; Tizzano, Eduardo F

    2005-03-01

    We investigated the expression of choline acetyltransferase, a specific marker for cholinergic neurons, in control and spinal muscular atrophy fetuses and newborns. By immunoblot we observed at 12 and 15 weeks a similar pattern of choline acetyltransferase expression in spinal muscular atrophy with respect to controls, although at 22 weeks this expression was reduced, probably due to a smaller number of motor neurons in the spinal muscular atrophy spinal cord. By immunohistochemistry, the counting of positive and negative motor neurons for choline acetyltransferase immunostaining in control and spinal muscular atrophy fetuses showed a similar proportion at all stages analyzed. The choline acetyltransferase-negative motor neurons were of similar appearance in both groups. After birth, chromatolytic motor neurons were detected in spinal muscular atrophy, all of which were choline acetyltransferase-negative. Our results in spinal muscular atrophy fetuses indicate that choline acetyltransferase immunostaining does not identify early events in neuronal pathogenesis and suggest that the spinal muscular atrophy surviving motor neurons may not be dysfunctional during this period. Furthermore, spinal muscular atrophy choline acetyltransferase-negative motor neurons showed detectable pathological changes only after birth, indicating that choline acetyltransferase is a late marker for motor neuron degeneration and not a primary contributing factor in this process.

  16. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

    PubMed Central

    Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; Li, Mei-Jun; Tan, Kemin; Yang, Xiaohan; Yun, Cai-Hong

    2016-01-01

    N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε-acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity. PMID:27550639

  17. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

    SciTech Connect

    Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; Li, Mei-Jun; Tan, Kemin; Yang, Xiaohan; Yun, Caihong

    2016-08-23

    N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε -acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that may contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.

  18. Structure and function of human Naa60 (NatF), a Golgi-localized bi-functional acetyltransferase

    DOE PAGES

    Chen, Ji-Yun; Liu, Liang; Cao, Chun-Ling; ...

    2016-08-23

    N-terminal acetylation (Nt-acetylation), carried out by N-terminal acetyltransferases (NATs), is a conserved and primary modification of nascent peptide chains. Naa60 (also named NatF) is a recently identified NAT found only in multicellular eukaryotes. This protein was shown to locate on the Golgi apparatus and mainly catalyze the Nt-acetylation of transmembrane proteins, and it also harbors lysine Nε -acetyltransferase (KAT) activity to catalyze the acetylation of lysine ε-amine. Here, we report the crystal structures of human Naa60 (hNaa60) in complex with Acetyl-Coenzyme A (Ac-CoA) or Coenzyme A (CoA). The hNaa60 protein contains an amphipathic helix following its GNAT domain that maymore » contribute to Golgi localization of hNaa60, and the β7-β8 hairpin adopted different conformations in the hNaa60(1-242) and hNaa60(1-199) crystal structures. Remarkably, we found that the side-chain of Phe 34 can influence the position of the coenzyme, indicating a new regulatory mechanism involving enzyme, co-factor and substrates interactions. Moreover, structural comparison and biochemical studies indicated that Tyr 97 and His 138 are key residues for catalytic reaction and that a non-conserved β3-β4 long loop participates in the regulation of hNaa60 activity.« less

  19. 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.

  20. Ligand promiscuity through the eyes of the aminoglycoside N3 acetyltransferase IIa

    PubMed Central

    Norris, Adrianne L; Serpersu, Engin H

    2013-01-01

    Aminoglycoside-modifying enzymes (AGMEs) are expressed in many pathogenic bacteria and cause resistance to aminoglycoside (AG) antibiotics. Remarkably, the substrate promiscuity of AGMEs is quite variable. The molecular basis for such ligand promiscuity is largely unknown as there is not an obvious link between amino acid sequence or structure and the antibiotic profiles of AGMEs. To address this issue, this article presents the first kinetic and thermodynamic characterization of one of the least promiscuous AGMEs, the AG N3 acetyltransferase-IIa (AAC-IIa) and its comparison to two highly promiscuous AGMEs, the AG N3-acetyltransferase-IIIb (AAC-IIIb) and the AG phosphotransferase(3′)-IIIa (APH). Despite having similar antibiotic selectivities, AAC-IIIb and APH catalyze different reactions and share no homology to one another. AAC-IIa and AAC-IIIb catalyze the same reaction and are very similar in both amino acid sequence and structure. However, they demonstrate strong differences in their substrate profiles and kinetic and thermodynamic properties. AAC-IIa and APH are also polar opposites in terms of ligand promiscuity but share no sequence or apparent structural homology. However, they both are highly dynamic and may even contain disordered segments and both adopt well-defined conformations when AGs are bound. Contrary to this AAC-IIIb maintains a well-defined structure even in apo form. Data presented herein suggest that the antibiotic promiscuity of AGMEs may be determined neither by the flexibility of the protein nor the size of the active site cavity alone but strongly modulated or controlled by the effects of the cosubstrate on the dynamic and thermodynamic properties of the enzyme. PMID:23640799

  1. Structural and Functional Role of Acetyltransferase hMOF K274 Autoacetylation

    SciTech Connect

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

    2016-07-05

    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.

  2. 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

  3. Homology modeling and identification of amino acids involved in the catalytic process of Mycobacterium tuberculosis serine acetyltransferase.

    PubMed

    Qiu, Juanjuan; Zang, Shizhu; Ma, Yufang; Owusu, Lawrence; Zhou, Lei; Jiang, Tao; Xin, Yi

    2017-03-01

    Serine acetyltransferase (CysE) belongs to the hexapeptide acetyltransferase family and is involved in the biosynthesis of L‑cysteine in microorganisms. Mycobacterium tuberculosis CysE is regarded as a potential target for anti‑tuberculosis (TB) drugs; however, the structure and active sites of M. tuberculosis CysE remain unknown. The present study aimed to predict the secondary structure and to construct a 3D model for M. tuberculosis CysE using bioinformatics analysis. To determine the essential amino acids that are associated with CysE enzymatic activity, amino acid sequences from several microorganisms were compared, and a consensus sequence was identified. Subsequently, site‑directed mutagenesis was used to generate mutant M. tuberculosis CysE proteins. Enzyme assays demonstrated that D67A, H82A and H117A mutants abolished ~75% activity of M. tuberculosis CysE. Prediction of the protein structure and identification of the active amino acids for M. tuberculosis CysE is essential for designing inhibitors, which may aid the discovery of effective anti‑TB drugs.

  4. Molecular and Biochemical Analysis of a Madagascar Periwinkle Root-Specific Minovincinine-19-Hydroxy-O-Acetyltransferase1

    PubMed Central

    Laflamme, Pierre; St-Pierre, Benoit; De Luca, Vincenzo

    2001-01-01

    The terminal steps in the biosynthesis of the monoterpenoid indole alkaloids vindoline and minovincinine are catalyzed by separate acetyl coenzyme A-dependent O-acetyltransferases in Madagascar periwinkle (Catharanthus roseus G. Don). Two genes were isolated that had 63% nucleic acid identity and whose deduced amino acid sequences were 78% identical. Active enzymes that were expressed as recombinant His-tagged proteins in Escherichia coli were named minovincinine-19-O-acetyltransferase (MAT) and deacetylvindoline-4-O-acetyltransferase (DAT) because they catalyzed the 19-O-acetylation of indole alkaloids such as minovincinine and hörhammericine and the 4-O-acetylation of deacetylvindoline, respectively. Kinetic studies showed that the catalytic efficiency of recombinant MAT (rMAT) was very poor compared with that of recombinant DAT (rDAT), whose turnover rates for Acetyl-coenzyme A and deacetylvindoline were approximately 240- and 10,000-fold greater than those of rMAT. Northern-blot analyses showed that MAT is expressed in cortical cells of the root tip, whereas DAT is only expressed in specialized idioblast and laticifer cells within light exposed tissues like leaves and stems. The coincident expression of trytophan decarboxylase, strictosidine synthase, and MAT within root cortical cells suggests that the entire pathway for the biosynthesis of tabersonine and its substituted analogs occurs within these cells. The ability of MAT to catalyze the 4-O-acetylation of deacetylvindoline with low efficiency suggests that this enzyme, rather than DAT, is involved in vindoline biosynthesis within transformed cell and root cultures, which accumulate low levels of this alkaloid under certain circumstances. PMID:11154328

  5. A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds

    PubMed Central

    Durrett, Timothy P.; McClosky, Daniel D.; Tumaney, Ajay W.; Elzinga, Dezi A.; Ohlrogge, John; Pollard, Mike

    2010-01-01

    Endosperm and embryo tissues from the seeds of Euonymus alatus (Burning Bush) accumulate high levels of 3-acetyl-1,2-diacyl-sn-glycerols (acTAGs) as their major storage lipids. In contrast, the aril tissue surrounding the seed produces long-chain triacylglycerols (lcTAGs) typical of most other organisms. The presence of the sn-3 acetyl group imparts acTAGs with different physical and chemical properties, such as a 30% reduction in viscosity, compared to lcTAGs. Comparative transcriptome analysis of developing endosperm and aril tissues using pyrosequencing technology was performed to isolate the enzyme necessary for the synthesis of acTAGs. An uncharacterized membrane-bound O-acyltransferase (MBOAT) family member was the most abundant acyltransferase in the endosperm but was absent from the aril. Expression of this MBOAT in yeast resulted in the accumulation of acTAGs but not lcTAG; hence, the enzyme was named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Yeast microsomes expressing EaDAcT possessed acetyl-CoA diacylglycerol acetyltransferase activity but lacked long-chain acyl-CoA diacylglycerol acyltransferase activity. Expression of EaDAcT under the control of a strong, seed-specific promoter in Arabidopsis resulted in the accumulation of acTAGs, up to 40 mol % of total TAG in the seed oil. These results demonstrate the utility of deep transcriptional profiling with multiple tissues as a gene discovery strategy for low-abundance proteins. They also show that EaDAcT is the acetyltransferase necessary and sufficient for the production of acTAGs in Euonymus seeds, and that this activity can be introduced into the seeds of other plants, allowing the evaluation of these unusual TAGs for biofuel and other applications. PMID:20439724

  6. Structural Analysis of a Putative Aminoglycoside N-Acetyltransferase from Bacillus anthracis

    SciTech Connect

    Klimecka, Maria M.; Chruszcz, Maksymilian; Font, Jose; Skarina, Tatiana; Shumilin, Igor; Onopryienko, Olena; Porebski, Przemyslaw J.; Cymborowski, Marcin; Zimmerman, Matthew D.; Hasseman, Jeremy; Glomski, Ian J.; Lebioda, Lukasz; Savchenko, Alexei; Edwards, Aled; Minor, Wladek

    2012-02-15

    For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic-NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic-NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.

  7. DNA damage induces N-acetyltransferase NAT10 gene expression through transcriptional activation.

    PubMed

    Liu, Haijing; Ling, Yun; Gong, Yilei; Sun, Ying; Hou, Lin; Zhang, Bo

    2007-06-01

    NAT10 (N-acetyltransferase 10) is a protein with histone acetylation activity and primarily identified to be involved in regulation of telomerase activity. The presented research shows its transcriptional activation by genotoxic agents and possible role in DNA damage. NAT10 mRNA could be markedly increased by using hydrogen peroxide (H2O2) or cisplatin in a dose- and time-dependent way, and the immunofluorescent staining revealed that the treatment of H2O2 or cisplatin induced focal accumulation of NAT10 protein in cellular nuclei. Both H2O2 and cisplatin could stimulate the transcriptional activity of the NAT10 promoter through the upstream sequences from -615 bp to +110 bp, with which some nuclear proteins interacted. Ectopic expression of NAT10 could enhance the number of survival cells in the presence of H2O2 or cisplatin. The above results suggested that NAT10 could be involved in DNA damage response and increased cellular resistance to genotoxicity.

  8. Cloning and characterization of a serotonin N-acetyltransferase from a gymnosperm, loblolly pine (Pinus taeda).

    PubMed

    Park, Sangkyu; Byeon, Yeong; Lee, Hyoung Yool; Kim, Young-Soon; Ahn, Taeho; Back, Kyoungwhan

    2014-10-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K(m) (428 μM) and Vmax (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis.

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

    PubMed Central

    2015-01-01

    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

  10. Identification of arylamine N-acetyltransferase inhibitors as an approach towards novel anti-tuberculars.

    PubMed

    Westwood, Isaac M; Bhakta, Sanjib; Russell, Angela J; Fullam, Elizabeth; Anderton, Matthew C; Kawamura, Akane; Mulvaney, Andrew W; Vickers, Richard J; Bhowruth, Veemal; Besra, Gurdyal S; Lalvani, Ajit; Davies, Stephen G; Sim, Edith

    2010-01-01

    New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules-triazoles-in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.

  11. Molecular cloning of cDNAs encoding human carnitine acetyltransferase and mapping of the corresponding gene to chromosome 9q34.1

    SciTech Connect

    Corti, O.; Finocchiaro, G.; DiDonato, S.

    1994-09-01

    Using a combination of PCR screening of cDNA libraries and reverse transcription PCR, we have cloned three overlapping DNA fragments that encode human carnitine acetyltransferase (CAT), a key enzyme for metabolic pathways involved with the control of the acyl-Co/CoA ratio in mitochondria, peroxisomes, and endoplasmic reticulum. The resulting cDNA (2436 bp) hybridizes to a mRNA species of {approximately}2.9 kb that is particularly abundant in skeletal muscle and encodes a 68-kDa protein containing a peroxisomal targeting signal. The sequence matches those of several tryptic peptides obtained from purified human liver CAT and shows striking similarities with other members of the carnitine/choline acetyltransferase family very distant throughout evolution. CAT cDNA has also been used for fluorescence in situ hybridization on metaphase spreads of human chromosomes, and the corresponding gene, CAT1, has been mapped to chromosome 9q34.1. 29 refs., 4 figs.

  12. The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase

    PubMed Central

    1988-01-01

    Autoantibodies to mitochondrial antigens are characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC), but the precise antigenic determinants recognized by these antibodies have not been defined. Recently, our laboratory identified a 1,370-bp rat liver cDNA clone that coded for a polypeptide recognized specifically by sera from patients with PBC but not by sera from patients with other forms of liver disease. This recombinant protein was identified as the 74-kD M2 mitochondrial inner membrane autoantigen, now known to be dihydrolipoamide acetyltransferase. In the present study, we have identified a 603-bp fragment that codes for a polypeptide containing all of the autoreactivity of the original clone. In addition, based on hydrophobicity/hydrophilicity plots of the amino acid sequence of this polypeptide segment, several peptides were synthesized and tested for reactivity by an inhibition assay using sera from patients with PBC. One peptide, defined by the amino acids AEIETDKATIGFEVQEEGYL, absorbed serum reactivity to the protein product of the original clone. Of particular interest was the finding that this peptide contains the lipoic acid binding site KATIGF of the dihydrolipoamide acetyltransferase found in the inner mitochondrial membrane. Thus, it appears that for this autoantigen, the target of the autoantibodies corresponds to a functional site of the dihydrolipoamide acetyltransferase. PMID:2455013

  13. 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

  14. A chromosomal chloramphenicol acetyltransferase determinant from a probiotic strain of Bacillus clausii.

    PubMed

    Galopin, Sébastien; Cattoir, Vincent; Leclercq, Roland

    2009-06-01

    The mechanism of resistance to chloramphenicol was studied in four strains of Bacillus clausii included in a probiotic mixture, which is administered to humans for prevention of gastrointestinal side effects due to oral antibiotic therapy. By cloning experiments, a chloramphenicol acetyltransferase (CAT) gene, cat(Bcl), coding for a putative 228-amino acid CAT protein was identified in B. clausii SIN. The deduced amino acid sequence displayed from 31% to 85% identity with 56 CAT proteins from other Gram-positive bacterial strains. The cat(Bcl) gene was also detected by PCR in the three other B. clausii strains resistant to chloramphenicol, whereas it was absent in the three control strains susceptible to chloramphenicol. Pulse-field gel electrophoresis of total DNA digested by I-CeuI followed by hybridization with a cat-specific probe as well as unsuccessful repeated attempts of in vitro transfer of chloramphenicol resistance to various recipient cells indicated that cat(Bcl) was chromosomally located in all four resistant B. clausii strains.

  15. Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii

    PubMed Central

    Jeffers, Victoria; Gao, Hongyu; Checkley, Lisa A.; Liu, Yunlong; Ferdig, Michael T.

    2016-01-01

    Lysine acetylation is a critical posttranslational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasite Toxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product garcinol was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5 family KAT known to be required for Toxoplasma tachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed transcriptome sequencing (RNA-seq), which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previously reported chromatin immunoprecipitation assay with microarray technology (ChIP-chip) analysis. The dysregulated gene expression induced by garcinol significantly inhibits Toxoplasma tachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibits Plasmodium falciparum asexual replication with a 50% inhibitory concentration (IC50) similar to that for Toxoplasma. Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication. PMID:26810649

  16. The Role of Sas2, an Acetyltransferase Homologue of Saccharomyces Cerevisiae, in Silencing and Orc Function

    PubMed Central

    Ehrenhofer-Murray, A. E.; Rivier, D. H.; Rine, J.

    1997-01-01

    Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers. Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMRa-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR. Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMRa-e**. Silencing conferred by the removal of SAS2 (sas2Δ) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC). Restoration of silencing by sas2Δ required ORC2 and ORC5, but not SIR1 or RAP1. Furthermore, sas2Δ suppressed the temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1. Moreover, sas2Δ had opposing effects on silencing of HML and HMR. The putative Sas2 protein bears similarities to known protein acetyltransferases. Several models for the role of Sas2 in silencing are discussed. PMID:9093847

  17. Inference of Functionally-Relevant N-acetyltransferase Residues Based on Statistical Correlations.

    PubMed

    Neuwald, Andrew F; Altschul, Stephen F

    2016-12-01

    Over evolutionary time, members of a superfamily of homologous proteins sharing a common structural core diverge into subgroups filling various functional niches. At the sequence level, such divergence appears as correlations that arise from residue patterns distinct to each subgroup. Such a superfamily may be viewed as a population of sequences corresponding to a complex, high-dimensional probability distribution. Here we model this distribution as hierarchical interrelated hidden Markov models (hiHMMs), which describe these sequence correlations implicitly. By characterizing such correlations one may hope to obtain information regarding functionally-relevant properties that have thus far evaded detection. To do so, we infer a hiHMM distribution from sequence data using Bayes' theorem and Markov chain Monte Carlo (MCMC) sampling, which is widely recognized as the most effective approach for characterizing a complex, high dimensional distribution. Other routines then map correlated residue patterns to available structures with a view to hypothesis generation. When applied to N-acetyltransferases, this reveals sequence and structural features indicative of functionally important, yet generally unknown biochemical properties. Even for sets of proteins for which nothing is known beyond unannotated sequences and structures, this can lead to helpful insights. We describe, for example, a putative coenzyme-A-induced-fit substrate binding mechanism mediated by arginine residue switching between salt bridge and π-π stacking interactions. A suite of programs implementing this approach is available (psed.igs.umaryland.edu).

  18. Schizosaccharomyces pombe mst2+ Encodes a MYST Family Histone Acetyltransferase That Negatively Regulates Telomere Silencing†

    PubMed Central

    Gómez, Eliana B.; Espinosa, Joaquín M.; Forsburg, Susan L.

    2005-01-01

    Histone acetylation and deacetylation are associated with transcriptional activity and the formation of constitutively silent heterochromatin. Increasingly, histone acetylation is also implicated in other chromosome transactions, including replication and segregation. We have cloned the only Schizosaccharomyces pombe MYST family histone acetyltransferase genes, mst1+ and mst2+. Mst1p, but not Mst2p, is essential for viability. Both proteins are localized to the nucleus and bound to chromatin throughout the cell cycle. Δmst2 genetically interacts with mutants that affect heterochromatin, cohesion, and telomere structure. Mst2p is a negative regulator of silencing at the telomere but does not affect silencing in the centromere or mating type region. We generated a census of proteins and histone modifications at wild-type telomeres. A histone acetylation gradient at the telomeres is lost in Δmst2 cells without affecting the distribution of Taz1p, Swi6p, Rad21p, or Sir2p. We propose that the increased telomeric silencing is caused by histone hypoacetylation and/or an increase in the ratio of methylated to acetylated histones. Although telomere length is normal, meiosis is aberrant in Δmst2 diploid homozygote mutants, suggesting that telomeric histone acetylation contributes to normal meiotic progression. PMID:16199868

  19. In silico identification and characterization of N-Terminal acetyltransferase genes of poplar (Populus trichocarpa).

    PubMed

    Zhu, Hang-Yong; Li, Chun-Ming; Wang, Li-Feng; Bai, Hui; Li, Yan-Ping; Yu, Wen-Xi; Xia, De-An; Liu, Chang-Cai

    2014-01-27

    N-terminal acetyltransferase (Nats) complex is responsible for protein N-terminal acetylation (Nα-acetylation), which is one of the most common covalent modifications of eukaryotic proteins. Although genome-wide investigation and characterization of Nat catalytic subunits (CS) and auxiliary subunits (AS) have been conducted in yeast and humans they remain unexplored in plants. Here we report on the identification of eleven genes encoding eleven putative Nat CS polypeptides, and five genes encoding five putative Nat AS polypeptides in Populus. We document that the expansion of Nat CS genes occurs as duplicated blocks distributed across 10 of the 19 poplar chromosomes, likely only as a result of segmental duplication events. Based on phylogenetic analysis, poplar Nat CS were assigned to six subgroups, which corresponded well to the Nat CS types (CS of Nat A-F), being consistent with previous reports in humans and yeast. In silico analysis of microarray data showed that in the process of normal development of the poplar, their Nat CS and AS genes are commonly expressed at one relatively low level but share distinct tissue-specific expression patterns. This exhaustive survey of Nat genes in poplar provides important information to assist future studies on their functional role in poplar.

  20. 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.

  1. Moco biosynthesis and the ATAC acetyltransferase engage translation initiation by inhibiting latent PKR activity.

    PubMed

    Suganuma, Tamaki; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Workman, Jerry L

    2016-02-01

    Molybdenum cofactor (Moco) biosynthesis is linked to c-Jun N-terminal kinase (JNK) signaling in Drosophila through MoaE, a molybdopterin (MPT) synthase subunit that is also a component of the Ada Two A containing (ATAC) acetyltransferase complex. Here, we show that human MPT synthase and ATAC inhibited PKR, a double-stranded RNA-dependent protein kinase, to facilitate translation initiation of iron-responsive mRNA. MPT synthase and ATAC directly interacted with PKR and suppressed latent autophosphorylation of PKR and its downstream phosphorylation of JNK and eukaryotic initiation factor 2α (eIF2α). The suppression of eIF2α phosphorylation via MPT synthase and ATAC prevented sequestration of the guanine nucleotide exchange factor eIF2B, which recycles eIF2-GDP to eIF2-GTP, resulting in the promotion of translation initiation. Indeed, translation of the iron storage protein, ferritin, was reduced in the absence of MPT synthase or ATAC subunits. Thus, MPT synthase and ATAC regulate latent PKR signaling and link transcription and translation initiation.

  2. Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

    PubMed Central

    Howes, Stuart C.; Alushin, Gregory M.; Shida, Toshinobu; Nachury, Maxence V.; Nogales, Eva

    2014-01-01

    Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments. PMID:24227885

  3. Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60.

    PubMed

    Gao, Yuan; Koppen, Arjen; Rakhshandehroo, Maryam; Tasdelen, Ismayil; van de Graaf, Stan F; van Loosdregt, Jorg; van Beekum, Olivier; Hamers, Nicole; van Leenen, Dik; Berkers, Celia R; Berger, Ruud; Holstege, Frank C P; Coffer, Paul J; Brenkman, Arjan B; Ovaa, Huib; Kalkhoven, Eric

    2013-01-01

    Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.

  4. Inference of Functionally-Relevant N-acetyltransferase Residues Based on Statistical Correlations

    PubMed Central

    Neuwald, Andrew F.

    2016-01-01

    Over evolutionary time, members of a superfamily of homologous proteins sharing a common structural core diverge into subgroups filling various functional niches. At the sequence level, such divergence appears as correlations that arise from residue patterns distinct to each subgroup. Such a superfamily may be viewed as a population of sequences corresponding to a complex, high-dimensional probability distribution. Here we model this distribution as hierarchical interrelated hidden Markov models (hiHMMs), which describe these sequence correlations implicitly. By characterizing such correlations one may hope to obtain information regarding functionally-relevant properties that have thus far evaded detection. To do so, we infer a hiHMM distribution from sequence data using Bayes’ theorem and Markov chain Monte Carlo (MCMC) sampling, which is widely recognized as the most effective approach for characterizing a complex, high dimensional distribution. Other routines then map correlated residue patterns to available structures with a view to hypothesis generation. When applied to N-acetyltransferases, this reveals sequence and structural features indicative of functionally important, yet generally unknown biochemical properties. Even for sets of proteins for which nothing is known beyond unannotated sequences and structures, this can lead to helpful insights. We describe, for example, a putative coenzyme-A-induced-fit substrate binding mechanism mediated by arginine residue switching between salt bridge and π-π stacking interactions. A suite of programs implementing this approach is available (psed.igs.umaryland.edu). PMID:28002465

  5. Polymorphisms in the Human Cytochrome P450 and Arylamine N-Acetyltransferase: Susceptibility to Head and Neck Cancers

    PubMed Central

    Khlifi, Rim; Messaoud, Olfa; Rebai, Ahmed; Hamza-Chaffai, Amel

    2013-01-01

    The occurrence of head and neck cancer (HNC) is associated with smoking and alcohol drinking. Tobacco smoking exposes smokers to a series of carcinogenic chemicals. Cytochrome P450 enzymes (CYP450s), such as CYP1A1, CYP1B1, and CYP2D6, usually metabolize carcinogens to their inactive derivatives, but they occasionally convert the chemicals to more potent carcinogens. In addition, via CYP450 (CYP2E1) oxidase, alcohol is metabolized to acetaldehyde, a highly toxic compound, which plays an important role in carcinogenesis. Furthermore, two N-acetyltransferase isozymes (NATs), NAT1 and NAT2, are polymorphic and catalyze both N-acetylation and O-acetylation of aromatic and heterocyclic amine carcinogens. Genetic polymorphisms are associated with a number of enzymes involved in the metabolism of carcinogens important in the induction of HNC. It has been suggested that such polymorphisms may be linked to cancer susceptibility. In this paper, we select four cytochrome P450 enzymes (CYP1A1, CYP1BA1, CYP2D6, and CYP2E1), and two N-acetyltransferase isozymes (NAT1 and NAT2) in order to summarize and analyze findings from the literature related to HNC risk by focusing on (i) the interaction between these genes and the environment, (ii) the impact of genetic defect on protein activity and/or expression, and (iii) the eventual involvement of race in such associations. PMID:24151610

  6. The Polyamine N-Acetyltransferase-Like Enzyme PmvE Plays a Role in the Virulence of Enterococcus faecalis

    PubMed Central

    Martini, Cecilia; Michaux, Charlotte; Bugli, Francesca; Arcovito, Alessandro; Iavarone, Federica; Cacaci, Margherita; Sterbini, Francesco Paroni; Hartke, Axel; Sauvageot, Nicolas; Sanguinetti, Maurizio; Posteraro, Brunella

    2014-01-01

    We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N1-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism. PMID:25385793

  7. The human serotonin N-acetyltransferase (EC 2.3.1.87) gene (AANAT): Structure, chromosomal localization, and tissue expression

    SciTech Connect

    Coon, S.L.; Bernard, M.; Roseboom, P.H.

    1996-05-15

    Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT, HGMW-approved symbol AANAT;EC 2.3.1.87) is the penultimate enzyme in melatonin synthesis and controls the night/day rhythm in melatonin production in the vertebrate pineal gland. We have found that the human AA-NAT gene spans {approx}2.5 kb, contains four exons, and is located at chromosome 17q25. The open reading frame encodes a 23.2-kDa protein that is {approx}80% identical to sheep and rat AA-NAT. The AA-NAT transcript ({approx}1 kb) is highly abundant in the pineal gland and is expressed at lower levels in the retina and in the Y79 retinoblastoma cell line. AA-NAT mRNA is also detectable at low levels in several brain regions and the pituitary gland, but not in several peripheral tissues examined. Brain and pituitary AA-NAT could modulate serotonin-dependent aspects of human behavior and pituitary function. 31 refs., 5 figs.

  8. 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 ...

  9. Diversity among the gram-positive acetyltransferases inactivating streptogramin A and structurally related compounds and characterization of a new staphylococcal determinant, vatB.

    PubMed

    Allignet, J; el Solh, N

    1995-09-01

    A gene encoding an acetyltransferase inactivating streptogramin A (SgA) and structurally similar compounds was isolated from a staphylococcal plasmid and sequenced. This gene, designated vatB, potentially encodes a 212-amino-acid protein, VatB, of 23,320 Da with 47.4 and 58.4% amino acid identities with two other enzymes with the same activity, Vat and SatA, respectively, which are encoded by a staphylococcal plasmid and an enterococcal plasmid, respectively. The C-terminal parts of these three enzymes share significant homology with the C-terminal parts of 10 other acetyltransferases modifying various substrates. A pair of degenerate primers representing the conserved motifs shared by VatB, Vat, and SatA was designed to detect the three genes encoding these SgA acetyltransferases. Five of 12 clinical SgAr Staphylococcus aureus isolates tested carried neither these genes nor the gene vga, which confers resistance to SgA by a different mechanism, suggesting that another gene(s) and possibly another mechanism of resistance to SgA in staphylococci remains to be characterized.

  10. Diversity among the gram-positive acetyltransferases inactivating streptogramin A and structurally related compounds and characterization of a new staphylococcal determinant, vatB.

    PubMed Central

    Allignet, J; el Solh, N

    1995-01-01

    A gene encoding an acetyltransferase inactivating streptogramin A (SgA) and structurally similar compounds was isolated from a staphylococcal plasmid and sequenced. This gene, designated vatB, potentially encodes a 212-amino-acid protein, VatB, of 23,320 Da with 47.4 and 58.4% amino acid identities with two other enzymes with the same activity, Vat and SatA, respectively, which are encoded by a staphylococcal plasmid and an enterococcal plasmid, respectively. The C-terminal parts of these three enzymes share significant homology with the C-terminal parts of 10 other acetyltransferases modifying various substrates. A pair of degenerate primers representing the conserved motifs shared by VatB, Vat, and SatA was designed to detect the three genes encoding these SgA acetyltransferases. Five of 12 clinical SgAr Staphylococcus aureus isolates tested carried neither these genes nor the gene vga, which confers resistance to SgA by a different mechanism, suggesting that another gene(s) and possibly another mechanism of resistance to SgA in staphylococci remains to be characterized. PMID:8540711

  11. Angiotensin II regulates brain (pro)renin receptor expression through activation of cAMP response element-binding protein.

    PubMed

    Li, Wencheng; Liu, Jiao; Hammond, Sean L; Tjalkens, Ronald B; Saifudeen, Zubaida; Feng, Yumei

    2015-07-15

    We reported that brain (pro)renin receptor (PRR) expression levels are elevated in DOCA-salt-induced hypertension; however, the underlying mechanism remained unknown. To address whether ANG II type 1 receptor (AT1R) signaling is involved in this regulation, we implanted a DOCA pellet and supplied 0.9% saline as the drinking solution to C57BL/6J mice. Sham pellet-implanted mice that were provided regular drinking water served as controls. Concurrently, mice were intracerebroventricularly infused with the AT1R blocker losartan, angiotensin-converting-enzyme inhibitor captopril, or artificial cerebrospinal fluid for 3 wk. Intracerebroventricular infusion of losartan or captopril attenuated DOCA-salt-induced PRR mRNA elevation in the paraventricular nucleus of the hypothalamus, suggesting a role for ANG II/AT1R signaling in regulating PRR expression during DOCA-salt hypertension. To test which ANG II/AT1R downstream transcription factors were involved in PRR regulation, we treated Neuro-2A cells with ANG II with or without CREB (cAMP response element-binding protein) or AP-1 (activator protein-1) inhibitors, or CREB siRNA. CREB and AP-1 inhibitors, as well as CREB knockdown abolished ANG II-induced increases in PRR levels. ANG II also induced PRR upregulation in primary cultured neurons. Chromatin immunoprecipitation assays revealed that ANG II treatment increased CREB binding to the endogenous PRR promoter in both cultured neurons and hypothalamic tissues of DOCA-salt hypertensive mice. This increase in CREB activity was reversed by AT1R blockade. Collectively, these findings indicate that ANG II acts via AT1R to upregulate PRR expression both in cultured cells and in DOCA-salt hypertensive mice by increasing CREB binding to the PRR promoter.

  12. An Intrinsically Disordered Region of the Acetyltransferase p300 with Similarity to Prion-Like Domains Plays a Role in Aggregation

    PubMed Central

    Kirilyuk, Alexander; Shimoji, Mika; Catania, Jason; Sahu, Geetaram; Pattabiraman, Nagarajan; Giordano, Antonio; Albanese, Christopher; Mocchetti, Italo; Toretsky, Jeffrey A.; Uversky, Vladimir N.; Avantaggiati, Maria Laura

    2012-01-01

    Several human diseases including neurodegenerative disorders and cancer are associated with abnormal accumulation and aggregation of misfolded proteins. Proteins with high tendency to aggregate include the p53 gene product, TAU and alpha synuclein. The potential toxicity of aberrantly folded proteins is limited via their transport into intracellular sub-compartments, the aggresomes, where misfolded proteins are stored or cleared via autophagy. We have identified a region of the acetyltransferase p300 that is highly disordered and displays similarities with prion-like domains. We show that this region is encoded as an alternative spliced variant independently of the acetyltransferase domain, and provides an interaction interface for various misfolded proteins, promoting their aggregation. p300 enhances aggregation of TAU and of p53 and is a component of cellular aggregates in both tissue culture cells and in alpha-synuclein positive Lewy bodies of patients affected by Parkinson disease. Down-regulation of p300 impairs aggresome formation and enhances cytotoxicity induced by misfolded protein stress. These data unravel a novel activity of p300, offer new insights into the function of disordered domains and implicate p300 in pathological aggregation that occurs in neurodegeneration and cancer. PMID:23133622

  13. Identification of novel CBP interacting proteins in embryonic orofacial tissue

    SciTech Connect

    Yin Xiaolong; Warner, Dennis R.; Roberts, Emily A.; Pisano, M. Michele; Greene, Robert M. . E-mail: greene@louisville.edu

    2005-04-15

    cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a general co-integrator of multiple signaling pathways and interacts with a large number of transcription factors and co-factors, through its numerous protein-binding domains. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from orofacial tissue from gestational day 11 to 13 mouse embryos was conducted. Using the carboxy terminus (amino acid residues 1676-2441) of CBP as bait, several novel proteins that bind CBP were identified, including an Msx-interacting-zinc finger protein, CDC42 interaction protein 4/thyroid hormone receptor interactor 10, SH3-domain GRB2-like 1, CCR4-NOT transcription complex subunit 3, adaptor protein complex AP-1 {beta}1 subunit, eukaryotic translation initiation factor 2B subunit 1 ({alpha}), and cyclin G-associated kinase. Results of the yeast two-hybrid screen were confirmed by glutathione S-transferase pull-down assays. The identification of these proteins as novel CBP-binding partners allows exploration of new mechanisms by which CBP regulates and integrates diverse cell signaling pathways.

  14. 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.

  15. 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

  16. Polyamine regulation of heat-shock-induced spermidine N1-acetyltransferase activity.

    PubMed Central

    Fuller, D J; Carper, S W; Clay, L; Chen, J R; Gerner, E W

    1990-01-01

    The enzyme spermidine/spermine N1-acetyltransferase (N1-SAT) is rapidly induced by heat shock in CHO and A549 cells, with activity declining by 24 h. Depletion of intracellular polyamines by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, blocks this induction. Re-addition of putrescine to these cultures restores the response to heat shock, with a concomitant increase in intracellular N1-acetylspermidine. Diaminopropane is more than twice as effective as the naturally occurring diamine putrescine, suggesting that the propylamine moiety of spermidine is involved in the regulation of N1-SAT induction. Inhibitor studies indicate transcriptional activation and that the enzyme has an apparent half-life of 30-60 min. A second heat shock rapidly inhibits induced N1-SAT activity, which decays with a half-life of 2-3 min. Despite its induction by heat, N1-SAT is not a stable enzyme, suggesting that the activity observed is not due to a modification of an existing peptide, but is due to a transcriptional event, which may justify the inclusion of this enzyme in the family of heat-shock proteins. Images Fig. 2. PMID:2111132

  17. The Lysine Acetyltransferase Activator Brpf1 Governs Dentate Gyrus Development through Neural Stem Cells and Progenitors

    PubMed Central

    You, Linya; Yan, Kezhi; Zhou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

    2015-01-01

    Lysine acetylation has recently emerged as an important post-translational modification in diverse organisms, but relatively little is known about its roles in mammalian development and stem cells. Bromodomain- and PHD finger-containing protein 1 (BRPF1) is a multidomain histone binder and a master activator of three lysine acetyltransferases, MOZ, MORF and HBO1, which are also known as KAT6A, KAT6B and KAT7, respectively. While the MOZ and MORF genes are rearranged in leukemia, the MORF gene is also mutated in prostate and other cancers and in four genetic disorders with intellectual disability. Here we show that forebrain-specific inactivation of the mouse Brpf1 gene causes hypoplasia in the dentate gyrus, including underdevelopment of the suprapyramidal blade and complete loss of the infrapyramidal blade. We trace the developmental origin to compromised Sox2+ neural stem cells and Tbr2+ intermediate neuronal progenitors. We further demonstrate that Brpf1 loss deregulates neuronal migration, cell cycle progression and transcriptional control, thereby causing abnormal morphogenesis of the hippocampus. These results link histone binding and acetylation control to hippocampus development and identify an important epigenetic regulator for patterning the dentate gyrus, a brain structure critical for learning, memory and adult neurogenesis. PMID:25757017

  18. Choline acetyltransferase in the hippocampus is associated with learning strategy preference in adult male rats.

    PubMed

    Hawley, Wayne R; Witty, Christine F; Daniel, Jill M; Dohanich, Gary P

    2015-08-01

    One principle of the multiple memory systems hypothesis posits that the hippocampus-based and striatum-based memory systems compete for control over learning. Consistent with this notion, previous research indicates that the cholinergic system of the hippocampus plays a role in modulating the preference for a hippocampus-based place learning strategy over a striatum-based stimulus--response learning strategy. Interestingly, in the hippocampus, greater activity and higher protein levels of choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, are associated with better performance on hippocampus-based learning and memory tasks. With this in mind, the primary aim of the current study was to determine if higher levels of ChAT and the high-affinity choline uptake transporter (CHT) in the hippocampus were associated with a preference for a hippocampus-based place learning strategy on a task that also could be solved by relying on a striatum-based stimulus--response learning strategy. Results confirmed that levels of ChAT in the dorsal region of the hippocampus were associated with a preference for a place learning strategy on a water maze task that could also be solved by adopting a stimulus-response learning strategy. Consistent with previous studies, the current results support the hypothesis that the cholinergic system of the hippocampus plays a role in balancing competition between memory systems that modulate learning strategy preference.

  19. Molecular Basis of Substrate Specific Acetylation by N-Terminal Acetyltransferase NatB.

    PubMed

    Hong, Haiyan; Cai, Yongfei; Zhang, Shijun; Ding, Hongyan; Wang, Haitao; Han, Aidong

    2017-04-04

    The NatB N-terminal acetyltransferase specifically acetylates the N-terminal group of substrate protein peptides starting with Met-Asp/Glu/Asn/Gln. How NatB recognizes and acetylates these substrates remains unknown. Here, we report crystal structures of a NatB holoenzyme from Candida albicans in the presence of its co-factor CoA and substrate peptides. The auxiliary subunit Naa25 of NatB forms a horseshoe-like deck to hold specifically its catalytic subunit Naa20. The first two amino acids Met and Asp of a substrate peptide mediate the major interactions with the active site in the Naa20 subunit. The hydrogen bonds between the substrate Asp and pocket residues of Naa20 are essential to determine the NatB substrate specificity. Moreover, a hydrogen bond between the amino group of the substrate Met and a carbonyl group in the Naa20 active site directly anchors the substrate toward acetyl-CoA. Together, these structures define a unique molecular mechanism of specific N-terminal acetylation acted by NatB.

  20. Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis.

    PubMed

    Zaunbrecher, M Analise; Sikes, R David; Metchock, Beverly; Shinnick, Thomas M; Posey, James E

    2009-11-24

    The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the -10 and -35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20-180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 microg/mL < minimum inhibitory concentration (MIC)

  1. 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

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

    PubMed Central

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

    2015-01-01

    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

  3. Effect of undernutrition on the regional development of transmitter enzymes: glutamate decarboxylase and choline acetyltransferase.

    PubMed

    Patel, A J; del Vecchio, M; Atkinson, D J

    1978-01-01

    The effect of undernutrition on the activity of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAc) (markers for the GABA-ergic and the cholinergic transmitter system, respectively) was studied in various parts of the rat brain at the age of 10, 15 and 21 days, and at day 54 following 33 days of rehabilitation. The brain regions investigated were the olfactory bulbs, cerebellum, pons-medulla, hypothalamus, colliculi, cerebral cortex hippocampus and the residual brain. Undernutrition resulted in a marked retardation of the developmental rise of the activities of both enzymes, expressed in terms of either total brain part or unit weight or protein. The effect diminished with age even during the period of nutritional deprivation. In most brain regions the enzyme activities were restored to normal after rehabilitation. In the cerebral cortex the total activity of both enzymes was persistently reduced, although the concentration of GAD exceeded the control levels. A negative correlation was manifested between the activities of GAD and ChAc in the different brain parts (except the cerebellum) during development. The correlation became significant by day 21 in the controls, but only after postweaning rehabilitation of the undernourished rats. The results showed therefore that undernutrition caused a reversible retardation in the development of these two transmitter enzymes, and they suggested that even the balance of the GABA-ergic and cholinergic systems throughout the brain can be restored to normal by rehabilitation.

  4. The Histone Acetyltransferase MOF Promotes Induces Generation of Pluripotent Stem Cells.

    PubMed

    Mu, Xupeng; Yan, Shaohua; Fu, Changhao; Wei, Anhui

    2015-08-01

    Histone modification plays an important role in maintaining pluripotency and self-renewal of embryonic stem cells (ESCs). The histone acetyltransferase MOF is a key regulator of ESCs; however, the role of MOF in the process of reprogramming back to induced pluripotent stem cells (iPSCs) remains unclear. In this study, we investigated the function of MOF on the generation of iPSCs. We show that iPSCs contain high levels of MOF mRNA, and the expression level of MOF protein is dramatically upregulated following reprogramming. Most importantly, overexpression of MOF improves reprogramming efficiency and facilitates the formation of iPSCs, whereas small hairpin RNA (shRNA)-mediated knockdown of MOF impairs iPSCs generation during reprogramming. Further investigation reveals that MOF interacts with the H3K4 methyltransferase Wdr5 to promote endogenous Oct4 expression during the reprogramming process. Knockdown of MOF reduces H4K16ac and H3K4me3 modification at the Oct4 promoter. In conclusion, our data indicate that MOF is an important epigenetic regulator that is critical for efficient reprogramming.

  5. 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

    2015-12-31

    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.

  6. Histone Acetyltransferase Complexes Can Mediate Transcriptional Activation by the Major Glucocorticoid Receptor Activation Domain

    PubMed Central

    Wallberg, Annika E.; Neely, Kristen E.; Gustafsson, Jan-Åke; Workman, Jerry L.; Wright, Anthony P. H.; Grant, Patrick A.

    1999-01-01

    Previous studies have shown that the Ada adapter proteins are important for glucocorticoid receptor (GR)-mediated gene activation in yeast. The N-terminal transactivation domain of GR, τ1, is dependent upon Ada2, Ada3, and Gcn5 for transactivation in vitro and in vivo. Using in vitro techniques, we demonstrate that the GR-τ1 interacts directly with the native Ada containing histone acetyltransferase (HAT) complex SAGA but not the related Ada complex. Mutations in τ1 that reduce τ1 transactivation activity in vivo lead to a reduced binding of τ1 to the SAGA complex and conversely, mutations increasing the transactivation activity of τ1 lead to an increased binding of τ1 to SAGA. In addition, the Ada-independent NuA4 HAT complex also interacts with τ1. GAL4-τ1-driven transcription from chromatin templates is stimulated by SAGA and NuA4 in an acetyl coenzyme A-dependent manner. Low-activity τ1 mutants reduce SAGA- and NuA4-stimulated transcription while high-activity τ1 mutants increase transcriptional activation, specifically from chromatin templates. Our results demonstrate that the targeting of native HAT complexes by the GR-τ1 activation domain mediates transcriptional stimulation from chromatin templates. PMID:10454542

  7. Crystallization and preliminary X-ray analysis of maltose O-acetyltransferase.

    PubMed

    Lo Leggio, L; Dal Degan, F; Poulsen, P; Sørensen, S O; Harlow, K; Harris, P; Larsen, S

    2001-12-01

    Maltose O-acetyltransferase (Mac) is a member of the hexapeptide-repeat family of enzymes, which contains proteins with left-handed parallel beta-helix architecture forming homotrimers. Diffraction data for four well diffracting crystal forms were collected. Crystal form I diffracted beyond 1.53 A resolution but was perfectly merohedrally twinned with an apparent space group P622. Crystal forms II and III (space groups R3 and C2, respectively) could be obtained under very similar conditions by adjusting the buffer pH differently. Crystal forms II and III had several monomers in the asymmetric unit and were difficult to derivatize. However, during soaking with trimethyl lead acetate, the form III crystals dissolved and crystals with a different habit and space group grew in their place (form IV). In three of the crystal forms, a ladder of peaks was visible in the native Patterson maps along the c axis. These peaks were interpreted as corresponding to the vectors between the beta-strands in the turns of the beta-helix. Crystal form IV is suitable for structure determination of Mac exploiting the anomalous scattering of lead.

  8. 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.

  9. Carnitine Acetyltransferase Mitigates Metabolic Inertia and Muscle Fatigue during Exercise.

    PubMed

    Seiler, Sarah E; Koves, Timothy R; Gooding, Jessica R; Wong, Kari E; Stevens, Robert D; Ilkayeva, Olga R; Wittmann, April H; DeBalsi, Karen L; Davies, Michael N; Lindeboom, Lucas; Schrauwen, Patrick; Schrauwen-Hinderling, Vera B; Muoio, Deborah M

    2015-07-07

    Acylcarnitine metabolites have gained attention as biomarkers of nutrient stress, but their physiological relevance and metabolic purpose remain poorly understood. Short-chain carnitine conjugates, including acetylcarnitine, derive from their corresponding acyl-CoA precursors via the action of carnitine acetyltransferase (CrAT), a bidirectional mitochondrial matrix enzyme. We show here that contractile activity reverses acetylcarnitine flux in muscle, from net production and efflux at rest to net uptake and consumption during exercise. Disruption of this switch in mice with muscle-specific CrAT deficiency resulted in acetyl-CoA deficit, perturbed energy charge, and diminished exercise tolerance, whereas acetylcarnitine supplementation produced opposite outcomes in a CrAT-dependent manner. Likewise, in exercise-trained compared to untrained humans, post-exercise phosphocreatine recovery rates were positively associated with CrAT activity and coincided with dramatic shifts in muscle acetylcarnitine dynamics. These findings show acetylcarnitine serves as a critical acetyl buffer for working muscles and provide insight into potential therapeutic strategies for combatting exercise intolerance.

  10. 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

  11. Epigenetic Modulation using Small Molecules - Targeting Histone Acetyltransferases in Disease.

    PubMed

    Richters, André; Koehler, Angela N

    2017-02-23

    Histone acetyltransferases (HATs) are epigenetic drivers that catalyze the acetyl transfer from acetyl-CoA to lysines of both histone and non-histone substrates and thereby induce transcription either by chromatin remodeling or direct transcription factor activation. Histone deacetylases (HDACs) conduct the reverse reaction to counter HAT activity. Physiological processes such as cell cycle progression or apoptosis require a thoroughly balanced equilibrium of the interplay between acetylation and deacetylation processes to maintain or, if required, alter the global acetylome status. Aberrant HAT activity has recently been demonstrated to play a crucial role in the progression of various diseases such as prostate, lung, and colon cancers as well as glioblastomas and neurodegenerative diseases. Recent investigations have aimed for the identification of HAT modulators to further decipher the complexity of acetyl transferase related signaling cascades and discover potential leads for drug design approaches. HDACs have been extensively characterized and targeted by small molecules, including four FDA-approved HDAC inhibitors; in contrast, HATs have not been active targets for therapeutic development. This review will summarize the status of HAT associated diseases and the arsenal of currently known and available HAT inhibitors with respect to their discovery, further improvements, and current applications.

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

    PubMed

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

    2016-01-14

    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.

  13. 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%).

  14. Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

    PubMed

    Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

    2013-12-06

    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

  15. Camello, a novel family of Histone Acetyltransferases that acetylate histone H4 and is essential for zebrafish development

    PubMed Central

    Karmodiya, Krishanpal; Anamika, Krishanpal; Muley, Vijaykumar; Pradhan, Saurabh J.; Bhide, Yoshita; Galande, Sanjeev

    2014-01-01

    In this study, we have investigated genome-wide occurrence of Histone Acetyltransferases (HATs) in genomes of Mus musculus and Danio rerio on the basis of presence of HAT domain. Our study identified a group of proteins that lacks characteristic features of known HAT families, relatively smaller in size and has no other associated domains. Most of the proteins in this unclassified group are Camello proteins, which are not yet known and classified as functional HATs. Our in vitro and in vivo analysis revealed that Camello family proteins are active HATs and exhibit specificity towards histone H4. Interestingly, Camello proteins are among the first identified HATs showing perinuclear localization. Moreover, Camello proteins are evolutionarily conserved in all chordates and are observed for the first time in cnidarians in phylogeny. Furthermore, knockdown of Camello protein (CMLO3) in zebrafish embryos exhibited defects in axis elongation and head formation. Thus, our study identified a novel family of active HATs that is specific for histone H4 acetylation, exhibits perinuclear localization and is essential for zebrafish development. PMID:25123547

  16. Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity.

    PubMed

    Stimson, Lindsay; Rowlands, Martin G; Newbatt, Yvette M; Smith, Nicola F; Raynaud, Florence I; Rogers, Paul; Bavetsias, Vassilios; Gorsuch, Stephen; Jarman, Michael; Bannister, Andrew; Kouzarides, Tony; McDonald, Edward; Workman, Paul; Aherne, G Wynne

    2005-10-01

    Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF) and p300 (1 to >50 micromol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 micromol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2-48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of alpha-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition.

  17. Stimulation of chloramphenicol acetyltransferase mRNA translation by reovirus capsid polypeptide sigma 3 in cotransfected COS cells.

    PubMed Central

    Giantini, M; Shatkin, A J

    1989-01-01

    The mammalian reovirus S4 gene has been implicated in the serotype-dependent inhibition of host cell protein synthesis during viral replication in mouse L cells. To examine the effect(s) of this gene on transcription or translation or both, a DNA copy of the serotype 3 S4 gene was inserted into a eucaryotic expression vector. Cotransfection of COS cells with plasmids containing S4 and the reporter gene, chloramphenicol acetyltransferase (CAT), resulted in a marked stimulation of CAT expression, predominantly at the level of translation. The significance of these findings is discussed in relation to the double-stranded-RNA-binding activity of the S4 gene product, polypeptide sigma 3. Images PMID:2724407

  18. Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

    PubMed Central

    Gomez-Cambronero, J; Mato, J M; Vivanco, F; Sanchez-Crespo, M

    1987-01-01

    A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP. Images Fig. 2. Fig. 3. Fig. 4. PMID:3663199

  19. The facC Gene of Aspergillus nidulans Encodes an Acetate-Inducible Carnitine Acetyltransferase

    PubMed Central

    Stemple, Christopher J.; Davis, Meryl A.; Hynes, Michael J.

    1998-01-01

    Mutations in the facC gene of Aspergillus nidulans result in an inability to use acetate as a sole carbon source. This gene has been cloned by complementation. The proposed translation product of the facC gene has significant similarity to carnitine acetyltransferases (CAT) from other organisms. Total CAT activity was found to be inducible by acetate and fatty acids and repressed by glucose. Acetate-inducible activity was found to be absent in facC mutants, while fatty acid-inducible activity was absent in an acuJ mutant. Acetate induction of facC expression was dependent on the facB regulatory gene, and an expressed FacB fusion protein was demonstrated to bind to 5′ facC sequences. Carbon catabolite repression of facC expression was affected by mutations in the creA gene and a CreA fusion protein bound to 5′ facC sequences. Mutations in the acuJ gene led to increased acetate induction of facC expression and also of an amdS-lacZ reporter gene, and it is proposed that this results from accumulation of acetate, as well as increased expression of facB. A model is presented in which facC encodes a cytosolic CAT enzyme, while a different CAT enzyme, which is acuJ dependent, is present in peroxisomes and mitochondria, and these activities are required for the movement of acetyl groups between intracellular compartments. PMID:9829933

  20. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    SciTech Connect

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

  1. Neural restrictive silencer factor and choline acetyltransferase expression in cerebral tissue of Alzheimer’s Disease patients: A pilot study

    PubMed Central

    González-Castañeda, Rocío E.; Sánchez-González, Víctor J.; Flores-Soto, Mario; Vázquez-Camacho, Gonzalo; Macías-Islas, Miguel A.; Ortiz, Genaro G.

    2013-01-01

    Decreased Choline Acetyltransferase (ChAT) brain level is one of the main biochemical disorders in Alzheimer’s Disease (AD). In rodents, recent data show that the CHAT gene can be regulated by a neural restrictive silencer factor (NRSF). The aim of the present work was to evaluate the gene and protein expression of CHAT and NRSF in frontal, temporal, entorhinal and parietal cortices of AD patient brains. Four brains from patients with AD and four brains from subjects without dementia were studied. Cerebral tissues were obtained and processed by the guanidine isothiocyanate method for RNA extraction. CHAT and NRSF gene and protein expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CHAT gene expression levels were 39% lower in AD patients as compared to the control group (p < 0.05, U test). ChAT protein levels were reduced by 17% (p = 0.02, U test). NRSF gene expression levels were 86% higher in the AD group (p = 0.001, U test) as compared to the control group. In the AD subjects, the NRSF protein levels were 57% higher (p > 0.05, U test) than in the control subjects. These findings suggest for the first time that in the brain of AD patients high NRSF protein levels are related to low CHAT gene expression levels. PMID:23569405

  2. Neural restrictive silencer factor and choline acetyltransferase expression in cerebral tissue of Alzheimer's Disease patients: A pilot study.

    PubMed

    González-Castañeda, Rocío E; Sánchez-González, Víctor J; Flores-Soto, Mario; Vázquez-Camacho, Gonzalo; Macías-Islas, Miguel A; Ortiz, Genaro G

    2013-03-01

    Decreased Choline Acetyltransferase (ChAT) brain level is one of the main biochemical disorders in Alzheimer's Disease (AD). In rodents, recent data show that the CHAT gene can be regulated by a neural restrictive silencer factor (NRSF). The aim of the present work was to evaluate the gene and protein expression of CHAT and NRSF in frontal, temporal, entorhinal and parietal cortices of AD patient brains. Four brains from patients with AD and four brains from subjects without dementia were studied. Cerebral tissues were obtained and processed by the guanidine isothiocyanate method for RNA extraction. CHAT and NRSF gene and protein expression were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CHAT gene expression levels were 39% lower in AD patients as compared to the control group (p < 0.05, U test). ChAT protein levels were reduced by 17% (p = 0.02, U test). NRSF gene expression levels were 86% higher in the AD group (p = 0.001, U test) as compared to the control group. In the AD subjects, the NRSF protein levels were 57% higher (p > 0.05, U test) than in the control subjects. These findings suggest for the first time that in the brain of AD patients high NRSF protein levels are related to low CHAT gene expression levels.

  3. Acetyl-CoA:benzylalcohol acetyltransferase--an enzyme involved in floral scent production in Clarkia breweri.

    PubMed

    Dudareva, N; D'Auria, J C; Nam, K H; Raguso, R A; Pichersky, E

    1998-05-01

    Volatile esters impart distinct characteristics to the floral scent of many plants, and are important in attracting insect pollinators. They are also important flavor compounds in fruits. The ester benzylacetate is a major constituent of the floral scent of Clarkia breweri, an annual plant native to California. The enzyme acetyl-CoA:benzylalcohol acetyltransferase (BEAT), which catalyzes the formation of benzylacetate, has been purified from C. breweri petals, and a cDNA encoding this enzyme has been isolated and characterized. The sequence of the 433-residue BEAT protein does not show high similarity to any previously characterized protein, but a 35-residue region from position 135-163 has significant similarity (42-56% identity) to several proteins known or suspected to use an acyl-CoA substrate. E. coli cells expressing C. breweri BEAT produced enzymatically active protein, and also synthesized benzylacetate and secreted it into the medium. Of the different parts of the C. breweri flower, petals contained the majority of BEAT transcripts, and no BEAT mRNA was detected in leaves. The levels of BEAT mRNA in the petals increased as the bud matured, and peaked at anthesis, paralleling changes in BEAT activity. However, three days after anthesis, mRNA levels began a steep decline, whereas BEAT activity remained high for the next two days, suggesting that the BEAT protein is relatively stable.

  4. Structures of Wild-Type and Mutant Human Spermidine/Spermine N1-acetyltransferase, a Potential Therapeutic Drug Target

    SciTech Connect

    Bewley,M.; Graziano, V.; Jiang, J.; Matz, E.; Studier, F.; Pegg, A.; Coleman, C.; Flanagan, J.

    2006-01-01

    Spermidine/spermine N{sup 1}-acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, and compounds that affect the expression, activity, or stability of SSAT are being explored as potential therapeutic drugs. We have expressed human SSAT from the cloned cDNA in Escherichia coli and have determined high-resolution structures of wild-type and mutant SSAT, as the free dimer and in binary and ternary complexes with CoA, acetyl-CoA (AcCoA), spermine, and the inhibitor N{sup 1},N{sup 11}-bis-(ethyl)-norspermine (BE-3-3-3). These structures show details of binding sites for cofactor, substrates, and inhibitor and provide a framework to understand enzymatic activity, mutations, and the action of potential drugs. Two dimer conformations were observed: a symmetric form with two open surface channels capable of binding substrate or cofactor, and an asymmetric form in which only one of the surface channels appears capable of binding and acetylating polyamines. SSAT was found to self-acetylate lysine-26 in the presence of AcCoA and absence of substrate, a reaction apparently catalyzed by AcCoA bound in the second channel of the asymmetric dimer. These unexpected and intriguing complexities seem likely to have some as yet undefined role in regulating SSAT activity or stability as a part of polyamine homeostasis. Sequence signatures group SSAT with proteins that appear to have thialysine N{sup {var_epsilon}}-acetyltransferase activity.

  5. Choline acetyltransferase and organic cation transporters are responsible for synthesis and propionate-induced release of acetylcholine in colon epithelium.

    PubMed

    Bader, Sandra; Klein, Jochen; Diener, Martin

    2014-06-15

    Acetylcholine is not only a neurotransmitter, but is found in a variety of non-neuronal cells. For example, the enzyme choline acetyltransferase (ChAT), catalyzing acetylcholine synthesis, is expressed by the colonic epithelium of different species. These cells release acetylcholine across the basolateral membrane after luminal exposure to propionate, a short-chain fatty acid. The functional consequence is the induction of chloride secretion, measurable as increase in short-circuit current (Isc) in Ussing chamber experiments. It is unclear how acetylcholine is produced and released by colonic epithelium. Therefore, the aim of the present study was the identification (on mRNA and protein level) and functional characterization (in Ussing chamber experiments combined with HPLC detection of acetylcholine) of transporters/enzymes in the cholinergic system of rat colonic epithelium. Immunohistochemical staining as well as RT-PCR revealed the expression of high-affinity choline transporter, ChAT, carnitine acetyltransferase (CarAT), vesicular acetylcholine transporter (VAChT), and organic cation transporters (OCT 1, 2, 3) in colonic epithelium. In contrast to blockade of ChAT with bromoacetylcholine, inhibition of CarAT with mildronate did not inhibit the propionate-induced increase in Isc, suggesting a predominant synthesis of epithelial acetylcholine by ChAT. Although being expressed, blockade of VAChT with vesamicol was ineffective, whereas inhibition of OCTs with omeprazole and corticosterone inhibited propionate-induced Isc and the release of acetylcholine into the basolateral compartment. In summary, OCTs seem to be involved in regulated acetylcholine release by colonic epithelium, which is assumed to be involved in chemosensing of luminal short-chain fatty acids by the intestinal epithelium.

  6. Nucleotide sequence analysis of the gene specifying the bifunctional 6'-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

    PubMed

    Ferretti, J J; Gilmore, K S; Courvalin, P

    1986-08-01

    The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.

  7. Method to produce acetyldiacylglycerols (ac-TAGs) by expression of an acetyltransferase gene isolated from Euonymus alatus (burning bush)

    DOEpatents

    Durrett, Timothy; Ohlrogge, John; Pollard, Michael

    2016-05-03

    The present invention relates to novel diacylglycerol acyltransferase genes and proteins, and methods of their use. In particular, the invention describes genes encoding proteins having diacylglycerol acetyltransferase activity, specifically for transferring an acetyl group to a diacylglycerol substrate to form acetyl-Triacylglycerols (ac-TAGS), for example, a 3-acetyl-1,2-diacyl-sn-glycerol. The present invention encompasses both native and recombinant wild-type forms of the transferase, as well as mutants and variant forms. The present invention also relates to methods of using novel diacylglycerol acyltransferase genes and proteins, including their expression in transgenic organisms at commercially viable levels, for increasing production of 3-acetyl-1,2-diacyl-sn-glycerols in plant oils and altering the composition of oils produced by microorganisms, such as yeast, by increasing ac-TAG production. Additionally, oils produced by methods of the present inventions comprising genes and proteins are contemplated for use as biodiesel fuel, in polymer production and as naturally produced food oils with reduced calories.

  8. Garcinol, a Histone Acetyltransferase Inhibitor, Radiosensitizes Cancer Cells by Inhibiting Non-Homologous End Joining

    SciTech Connect

    Oike, Takahiro; Ogiwara, Hideaki; Torikai, Kohta; Nakano, Takashi; Yokota, Jun; Kohno, Takashi

    2012-11-01

    Purpose: Non-homologous end joining (NHEJ), a major pathway used to repair DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), requires chromatin remodeling at DSB sites through the acetylation of histones by histone acetyltransferases (HATs). However, the effect of compounds with HAT inhibitory activities on the DNA damage response (DDR), including the NHEJ and cell cycle checkpoint, as well as on the radiosensitivity of cancer cells, remains largely unclear. Here, we investigated whether garcinol, a HAT inhibitor found in the rinds of Garcinia indica fruit (called mangosteens), has effects on DDR, and whether it can be used for radiosensitization. Methods and Materials: The following assays were used to examine the effect of garcinol on the inhibition of DSB repair, including the following: a conventional neutral comet assay; a cell-based assay recently developed by us, in which NHEJ repair of DSBs on chromosomal DNA was evaluated; the micrococcal nuclease sensitivity assay; and immunoblotting for autophosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs). We assessed the effect of garcinol on the cell cycle checkpoint after IR treatment by analyzing the phosphorylation levels of checkpoint kinases CHK1 and CHK2 and histone H3, and by cell cycle profile analysis using flow cytometry. The radiosensitizing effect of garcinol was assessed by a clonogenic survival assay, whereas its effects on apoptosis and senescence were examined by annexin V and senescence-associated {beta}-galactosidase (SA-{beta}-Gal) staining, respectively. Results: We found that garcinol inhibits DSB repair, including NHEJ, without affecting cell cycle checkpoint. Garcinol radiosensitized A549 lung and HeLa cervical carcinoma cells with dose enhancement ratios (at 10% surviving fraction) of 1.6 and 1.5, respectively. Cellular senescence induced by IR was enhanced by garcinol. Conclusion: These results suggest that garcinol is a radiosensitizer that

  9. Arylamine N-acetyltransferases: from drug metabolism and pharmacogenetics to drug discovery

    PubMed Central

    Sim, E; Abuhammad, A; Ryan, A

    2014-01-01

    Arylamine N-acetyltransferases (NATs) are polymorphic drug-metabolizing enzymes, acetylating arylamine carcinogens and drugs including hydralazine and sulphonamides. The slow NAT phenotype increases susceptibility to hydralazine and isoniazid toxicity and to occupational bladder cancer. The two polymorphic human NAT loci show linkage disequilibrium. All mammalian Nat genes have an intronless open reading frame and non-coding exons. The human gene products NAT1 and NAT2 have distinct substrate specificities: NAT2 acetylates hydralazine and human NAT1 acetylates p-aminosalicylate (p-AS) and the folate catabolite para-aminobenzoylglutamate (p-abaglu). Human NAT2 is mainly in liver and gut. Human NAT1 and its murine homologue are in many adult tissues and in early embryos. Human NAT1 is strongly expressed in oestrogen receptor-positive breast cancer and may contribute to folate and acetyl CoA homeostasis. NAT enzymes act through a catalytic triad of Cys, His and Asp with the architecture of the active site-modulating specificity. Polymorphisms may cause unfolded protein. The C-terminus helps bind acetyl CoA and differs among NATs including prokaryotic homologues. NAT in Salmonella typhimurium supports carcinogen activation and NAT in mycobacteria metabolizes isoniazid with polymorphism a minor factor in isoniazid resistance. Importantly, nat is in a gene cluster essential for Mycobacterium tuberculosis survival inside macrophages. NAT inhibitors are a starting point for novel anti-tuberculosis drugs. Human NAT1-specific inhibitors may act in biomarker detection in breast cancer and in cancer therapy. NAT inhibitors for co-administration with 5-aminosalicylate (5-AS) in inflammatory bowel disease has prompted ongoing investigations of azoreductases in gut bacteria which release 5-AS from prodrugs including balsalazide. PMID:24467436

  10. Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures.

    PubMed

    Kobayashi, Jyumpei; Furukawa, Megumi; Ohshiro, Takashi; Suzuki, Hirokazu

    2015-07-01

    Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CAT(A138T) may be useful as a genetic marker in Geobacillus spp. Notably, CAT(A138T) generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.

  11. Choline Acetyltransferase Mutations Causing Congenital Myasthenic Syndrome: Molecular Findings and Genotype-Phenotype Correlations.

    PubMed

    Arredondo, Juan; Lara, Marian; Gospe, Sídney M; Mazia, Claudio G; Vaccarezza, Maria; Garcia-Erro, Marcela; Bowe, Constance M; Chang, Celia H; Mezei, Michelle M; Maselli, Ricardo A

    2015-09-01

    Choline acetyltransferase catalyzes the synthesis of acetylcholine at cholinergic nerves. Mutations in human CHAT cause a congenital myasthenic syndrome due to impaired synthesis of ACh; this severe variant of the disease is frequently associated with unexpected episodes of potentially fatal apnea. The severity of this condition varies remarkably, and the molecular factors determining this variability are poorly understood. Furthermore, genotype-phenotype correlations have been difficult to establish in patients with biallelic mutations. We analyzed the protein expression of phosphorylated ChAT of seven CHAT mutations, p.Val136Met, p.Arg207His, p.Arg186Trp, p.Val194Leu, p.Pro211Ala, p.Arg566Cys, and p.Ser694Cys, in HEK-293 cells to phosphorylated ChAT, determined their enzyme kinetics and thermal stability, and examined their structural changes. Three mutations, p.Arg207His, p.Arg186Trp, and p.Arg566Cys, are novel, and p.Val136Met and p.Arg207His are homozygous in three families and associated with severe disease. The characterization of mutants showed a decrease in the overall catalytic efficiency of ChAT; in particular, those located near the active-site tunnel produced the most seriously disruptive phenotypic effects. On the other hand, p.Val136Met, which is located far from both active and substrate-binding sites, produced the most drastic reduction of ChAT expression. Overall, CHAT mutations producing low enzyme expression and severe kinetic effects are associated with the most severe phenotypes.

  12. Choline acetyltransferase mutations causing congenital myasthenic syndrome: molecular findings and genotype-phenotype correlations

    PubMed Central

    Arredondo, Juan; Lara, Marian; Gospe, Sídney M.; Mazia, Claudio G.; Vaccarezza, Maria; Garcia-Erro, Marcela; Bowe, Constance; Chang, Celia; Mezei, Michelle; Maselli, Ricardo A.

    2015-01-01

    Choline acetyltransferase catalyzes the synthesis of acetylcholine at cholinergic nerves. Mutations in human CHAT cause a congenital myasthenic syndrome (CMS) due to impaired synthesis of ACh; this severe variant of the disease is frequently associated with unexpected episodes of potentially fatal apnea. The severity of this condition varies remarkably, and the molecular factors determining this variability are poorly understood. Furthermore, genotype–phenotype correlations have been difficult to establish in patients with biallelic mutations. We analyzed the protein expression of seven ChAT mutations, p.Val136Met, p.Arg207His, p.Arg186Trp, p.Val194Leu, p.Pro211Ala, p.Arg566Cys and p.Ser694Cys, in HEK-293 cells to phosphorylated ChAT, determined their enzyme kinetics and thermal instability, and examined their structural changes. Three mutations, p.Arg207His, p.Arg186Trp and p.Arg566Cys, are novel, and p.Val136Met and p.Arg207His are homozygous in three families and associated with severe disease. The characterization of mutants showed a decrease in the overall catalytic efficiency of ChAT; in particular, those located near the active-site tunnel produced the most seriously disruptive phenotypic effects. On the other hand, p.Val136Met is located far from both active and substrate-binding sites produced the most drastic reduction of ChAT expression. Overall, CHAT mutations producing low enzyme expression and severe kinetic effects are associated with the most severe phenotypes. PMID:26080897

  13. Role of Jade-1 in the histone acetyltransferase (HAT) HBO1 complex.

    PubMed

    Foy, Rebecca L; Song, Ihn Young; Chitalia, Vipul C; Cohen, Herbert T; Saksouk, Nehme; Cayrou, Christelle; Vaziri, Cyrus; Côté, Jacques; Panchenko, Maria V

    2008-10-24

    Regulation of global chromatin acetylation is important for chromatin remodeling. A small family of Jade proteins includes Jade-1L, Jade-2, and Jade-3, each bearing two mid-molecule tandem plant homology domain (PHD) zinc fingers. We previously demonstrated that the short isoform of Jade-1L protein, Jade-1, is associated with endogenous histone acetyltransferase (HAT) activity. It has been found that Jade-1L/2/3 proteins co-purify with a novel HAT complex, consisting of HBO1, ING4/5, and Eaf6. We investigated a role for Jade-1/1L in the HBO1 complex. When overexpressed individually, neither Jade-1/1L nor HBO1 affected histone acetylation. However, co-expression of Jade-1/1L and HBO1 increased acetylation of the bulk of endogenous histone H4 in epithelial cells in a synergistic manner, suggesting that Jade1/1L positively regulates HBO1 HAT activity. Conversely, small interfering RNA-mediated depletion of endogenous Jade resulted in reduced levels of H4 acetylation. Moreover, HBO1-mediated H4 acetylation activity was enhanced severalfold by the presence of Jade-1/1L in vitro. The removal of PHD fingers affected neither binding nor mutual Jade-1-HBO1 stabilization but completely abrogated the synergistic Jade-1/1L- and HBO1-mediated histone H4 acetylation in live cells and in vitro with reconstituted oligonucleosome substrates. Therefore, PHDs are necessary for Jade-1/1L-induced acetylation of nucleosomal histones by HBO1. In contrast to Jade-1/1L, the PHD zinc finger protein ING4/5 failed to synergize with HBO1 to promote histone acetylation. The physical interaction of ING4/5 with HBO1 occurred in the presence of Jade-1L or Jade-3 but not with the Jade-1 short isoform. In summary, this study demonstrates that Jade-1/1L are crucial co-factors for HBO1-mediated histone H4 acetylation.

  14. Crystal Structure of the Golgi-Associated Human Nα-Acetyltransferase 60 Reveals the Molecular Determinants for Substrate-Specific Acetylation.

    PubMed

    Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas

    2016-07-06

    N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions.

  15. 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...

  16. Conditions for the self-catalysed inactivation of carnitine acetyltransferase. A novel form of enzyme inhibition

    PubMed Central

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

    1969-01-01

    1. Carnitine acetyltransferase is very rapidly inhibited in the presence of bromoacetyl-(−)-carnitine plus CoA or of bromoacetyl-CoA plus (−)-carnitine. 2. Under appropriate conditions, the enzyme may be titrated with either bromoacetyl substrate analogue; in each case about 1mole of inhibitor is required to inactivate completely 1mole of enzyme of molecular weight 58000±3000. 3. Inhibition by bromoacetyl-CoA plus (−)-carnitine results in the formation of an inactive enzyme species, containing stoicheiometric amounts of bound adenine nucleotide and (−)-carnitine in a form that is not removed by gel filtration. This is shown to be S-carboxymethyl-CoA (−)-carnitine ester. 4. The inhibited enzyme recovers activity slowly on prolonged standing at 4°. 5. Incubation with S-carboxymethyl-CoA (−)-carnitine ester causes a slow inhibition of carnitine acetyltransferase. 6. The formation of bound S-carboxymethyl-CoA (−)-carnitine ester by the enzyme is discussed. Presumably the resulting inhibition reflects binding of the ester to both the CoA- and carnitine-binding sites on the enzyme and its consequent very slow dissociation. These observations confirm that carnitine acetyltransferase can form ternary enzyme–substrate complexes; this also appears to be the case with carnitine palmitoyltransferase and choline acetyltransferase. PMID:5763788

  17. 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...

  18. 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...

  19. Histone acetyltransferase activity of MOF is required for adult but not early fetal hematopoiesis in mice.

    PubMed

    Valerio, Daria G; Xu, Haiming; Eisold, Meghan E; Woolthuis, Carolien M; Pandita, Tej K; Armstrong, Scott A

    2017-01-05

    K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre-induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage-specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis.

  20. 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...

  1. CBP and p300 histone acetyltransferases contribute to homologous recombination by transcriptionally activating the BRCA1 and RAD51 genes.

    PubMed

    Ogiwara, Hideaki; Kohno, Takashi

    2012-01-01

    Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the BRCA1 and RAD51 genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.

  2. Thogoto virus ML protein suppresses IRF3 function

    SciTech Connect

    Jennings, Stephanie . E-mail: stephanie.jennings@uniklinik-freiburg.de; Martinez-Sobrido, Luis . E-mail: Luis.Martinez@mssm.edu; Garcia-Sastre, Adolfo . E-mail: adolfo.garcia-sastre@mssm.edu; Weber, Friedemann . E-mail: friedemann.weber@uniklinik-freiburg.de; Kochs, Georg . E-mail: georg.kochs@uniklinik-freiburg.de

    2005-01-05

    The Thogoto virus (THOV) is a member of the family Orthomyxoviridae. It prevents induction of alpha/beta interferons (IFN) in cell culture and in vivo via the action of the viral ML protein. Phenotypically, the effect of THOV ML resembles that of the NS1 protein of influenza A virus (FLUAV) in that it blocks the expression of IFN genes. IFN expression depends on IFN regulatory factor 3 (IRF3). Upon activation, IRF3 forms homodimers and accumulates in the nucleus where it binds the transcriptional coactivator CREB-binding protein (CBP). Here, we show that expression of ML blocked the transcriptional activity of IRF3 after stimulation by virus infection. Further biochemical analysis revealed that ML acts by blocking IRF3 dimerization and association with CBP. Surprisingly, however, ML did not interfere with the nuclear transport of IRF3. Thus, the action of ML differs strikingly from that of FLUAV NS1 that prevents IFN induction by retaining IRF3 in the cytoplasm.

  3. Structural basis for concerted recruitment and activation of IRF-3 by innate immune adaptor proteins

    PubMed Central

    Zhao, Baoyu; Shu, Chang; Gao, Xinsheng; Sankaran, Banumathi; Du, Fenglei; Shelton, Catherine L.; Herr, Andrew B.; Ji, Jun-Yuan; Li, Pingwei

    2016-01-01

    Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses. PMID:27302953

  4. Nuclear Arc Interacts with the Histone Acetyltransferase Tip60 to Modify H4K12 Acetylation1,2,3

    PubMed Central

    Wee, Caroline L.; Teo, Shaun; Oey, Nicodemus E.; Wright, Graham D.; VanDongen, Hendrika M.A.

    2014-01-01

    Abstract Arc is an immediate-early gene whose genetic ablation selectively abrogates long-term memory, indicating a critical role in memory consolidation. Although Arc protein is found at synapses, it also localizes to the neuronal nucleus, where its function is less understood. Nuclear Arc forms a complex with the β-spectrin isoform βSpIVΣ5 and associates with PML bodies, sites of epigenetic regulation of gene expression. We report here a novel interaction between Arc and Tip60, a histone-acetyltransferase and subunit of a chromatin-remodelling complex, using biochemistry and super-resolution microscopy in primary rat hippocampal neurons. Arc and βSpIVΣ5 are recruited to nuclear Tip60 speckles, and the three proteins form a tight complex that localizes to nuclear perichromatin regions, sites of transcriptional activity. Neuronal activity-induced expression of Arc (1) increases endogenous nuclear Tip60 puncta, (2) recruits Tip60 to PML bodies, and (3) increases histone acetylation of Tip60 substrate H4K12, a learning-induced chromatin modification. These mechanisms point to an epigenetic role for Arc in regulating memory consolidation. PMID:26464963

  5. Insights into the O-Acetylation Reaction of Hydroxylated Heterocyclic Amines by Human Arylamine N-Acetyltransferases: A Computational Study

    SciTech Connect

    Lau, E Y; Felton, J S; Lightstone, F C

    2006-06-06

    A computational study was performed to better understand the differences between human arylamine N-acetyltransferase (NAT) 1 and 2. Homology models were constructed from available crystal structures and comparisons of the active site residues 125, 127, and 129 for these two enzymes provide insight into observed substrate differences. The NAT2 model provided a basis for understanding how some of the common mutations may affect the structure of the protein. Molecular dynamics simulations of the human NAT models and the template structure (NAT from Mycobacterium smegmatis) were performed and showed the models to be stable and reasonable. Docking studies of hydroxylated heterocyclic amines in the models of NAT1 and NAT2 probed the differences exhibited by these two proteins with mutagenic agents. The hydroxylated heterocyclic amines were only able to fit into the NAT2 active site, and an alternative binding site by the P-loop was found using our models and will be discussed. Additionally, quantum mechanical calculations were performed to study the O-acetylation reaction of the hydroxylated heterocyclic amines N-OH MeIQx and N-OH PhIP. This study has given us insight into why there are substrate differences among isoenzymes and explains some of the polymorphic activity differences.

  6. Different functions of the histone acetyltransferase HAC1 gene traced in the model species Medicago truncatula, Lotus japonicus and Arabidopsis thaliana.

    PubMed

    Boycheva, Irina; Vassileva, Valya; Revalska, Miglena; Zehirov, Grigor; Iantcheva, Anelia

    2017-03-01

    In eukaryotes, histone acetyltransferases regulate the acetylation of histones and transcription factors, affecting chromatin structural organization, transcriptional regulation, and gene activation. To assess the role of HAC1, a gene encoding for a histone acetyltransferase in Medicago truncatula, stable transgenic lines with modified HAC1 expression in the model plants M. truncatula, Lotus japonicus, and Arabidopsis thaliana were generated by Agrobacterium-mediated transformation and used for functional analyses. Histochemical, transcriptional, flow cytometric, and morphological analyses demonstrated the involvement of HAC1 in plant growth and development, responses to internal stimuli, and cell cycle progression. Expression patterns of a reporter gene encoding beta-glucuronidase (GUS) fused to the HAC1 promoter sequence were associated with young tissues comprised of actively dividing cells in different plant organs. The green fluorescent protein (GFP) signal, driven by the HAC1 promoter, was detected in the nuclei and cytoplasm of root cells. Transgenic lines with HAC1 overexpression and knockdown showed a wide range of phenotypic deviations and developmental abnormalities, which provided lines of evidence for the role of HAC1 in plant development. Synchronization of A. thaliana root tips in a line with HAC1 knockdown showed the involvement of this gene in the acetylation of two core histones during S phase of the plant cell cycle.

  7. Depletion of histone N-terminal-acetyltransferase Naa40 induces p53-independent apoptosis in colorectal cancer cells via the mitochondrial pathway.

    PubMed

    Pavlou, Demetria; Kirmizis, Antonis

    2016-03-01

    Protein N-terminal acetylation is an abundant post-translational modification in eukaryotes implicated in various fundamental cellular and biochemical processes. This modification is catalysed by evolutionarily conserved N-terminal acetyltransferases (NATs) whose deregulation has been linked to cancer development and thus, are emerging as useful diagnostic and therapeutic targets. Naa40 is a highly selective NAT that acetylates the amino-termini of histones H4 and H2A and acts as a sensor of cell growth in yeast. In the present study, we examine the role of Naa40 in cancer cell survival. We demonstrate that depletion of Naa40 in HCT116 and HT-29 colorectal cancer cells decreases cell survival by enhancing apoptosis, whereas Naa40 reduction in non-cancerous mouse embryonic fibroblasts has no effect on cell viability. Specifically, Naa40 knockdown in colon cancer cells activates the mitochondrial caspase-9-mediated apoptotic cascade. Consistent with this, we show that caspase-9 activation is required for the induced apoptosis because treatment of cells with an irreversible caspase-9 inhibitor impedes apoptosis when Naa40 is depleted. Furthermore, the effect of Naa40-depletion on cell-death is mediated through a p53-independent mechanism since p53-null HCT116 cells still undergo apoptosis upon reduction of the acetyltransferase. Altogether, these findings reveal an anti-apoptotic role for Naa40 and exhibit its potential as a therapeutic target in colorectal cancers.

  8. Structural and functional characterization of an arylamine N-acetyltransferase from the pathogen Mycobacterium abscessus: differences from other mycobacterial isoforms and implications for selective inhibition.

    PubMed

    Cocaign, Angélique; Kubiak, Xavier; Xu, Ximing; Garnier, Guillaume; Li de la Sierra-Gallay, Inès; Chi-Bui, Linh; Dairou, Julien; Busi, Florent; Abuhammad, Areej; Haouz, Ahmed; Dupret, Jean Marie; Herrmann, Jean Louis; Rodrigues-Lima, Fernando

    2014-11-01

    Mycobacterium abscessus is the most pathogenic rapid-growing mycobacterium and is one of the most resistant organisms to chemotherapeutic agents. However, structural and functional studies of M. abscessus proteins that could modify/inactivate antibiotics remain nonexistent. Here, the structural and functional characterization of an arylamine N-acetyltransferase (NAT) from M. abscessus [(MYCAB)NAT1] are reported. This novel prokaryotic NAT displays significant N-acetyltransferase activity towards aromatic substrates, including antibiotics such as isoniazid and p-aminosalicylate. The enzyme is endogenously expressed and functional in both the rough and smooth M. abscessus morphotypes. The crystal structure of (MYCAB)NAT1 at 1.8 Å resolution reveals that it is more closely related to Nocardia farcinica NAT than to mycobacterial isoforms. In particular, structural and physicochemical differences from other mycobacterial NATs were found in the active site. Peculiarities of (MYCAB)NAT1 were further supported by kinetic and docking studies showing that the enzyme was poorly inhibited by the piperidinol inhibitor of mycobacterial NATs. This study describes the first structure of an antibiotic-modifying enzyme from M. abscessus and provides bases to better understand the substrate/inhibitor-binding specificities among mycobacterial NATs and to identify/optimize specific inhibitors. These data should also contribute to the understanding of the mechanisms that are responsible for the pathogenicity and extensive chemotherapeutic resistance of M. abscessus.

  9. Cloning and characterization of the serotonin N-acetyltransferase-2 gene (SNAT2) in rice (Oryza sativa).

    PubMed

    Byeon, Yeong; Lee, Hyoung Yool; Back, Kyoungwhan

    2016-09-01

    The penultimate enzyme in melatonin synthesis is serotonin N-acetyltransferase (SNAT), which exists as a single copy in mammals and plants. Our recent studies of the Arabidopsis snat-knockout mutant and SNAT RNAi rice (Oryza sativa) plants predicted the presence of at least one other SNAT isogene in plants; that is, the snat-knockout mutant of Arabidopsis and the SNAT RNAi rice plants still produced melatonin, even in the absence or the suppression of SNAT expression. Here, we report a molecular cloning of an SNAT isogene (OsSNAT2) from rice. The mature amino acid sequences of SNAT proteins indicated that OsSNAT2 and OsSNAT1 proteins had 39% identity values and 60% similarity. The Km and Vmax values of the purified recombinant OsSNAT2 were 371 μm and 4700 pmol/min/mg protein, respectively; the enzyme's optimal activity temperature was 45°C. Confocal microscopy showed that the OsSNAT2 protein was localized to both the cytoplasm and chloroplasts. The in vitro enzyme activity of OsSNAT2 was severely inhibited by melatonin, but the activities of sheep SNAT (OaSNAT) and rice OsSNAT1 proteins were not. The enzyme activity of OsSNAT2 was threefold higher than that of OsSNAT1, but 232-fold lower than that of OaSNAT. The OsSNAT1 and OsSNAT2 transcripts were similarly suppressed in rice leaves during the melatonin induction after cadmium treatment. Phylogenetic analyses indicated that OsSNAT1 and OsSNAT2 are distantly related, suggesting that they evolved independently from Cyanobacteria prior to the endosymbiosis event.

  10. Sulfur assimilation in soybean ( Glycine max [L.] Merr.): molecular cloning and characterization of a cytosolic isoform of serine acetyltransferase.

    PubMed

    Chronis, Demosthenis; Krishnan, Hari B

    2004-01-01

    A full-length cDNA clone encoding a cytosolic isoform of serine acetyltransferase (SATase; EC 2.3.1.30) was isolated by screening a soybean seedling cDNA library with a (32)P-labeled expressed sequence tag. Nucleotide sequence analysis of the isolated cDNA revealed a single open-reading frame of 858 base pairs encoding a 30-kDa polypeptide. The deduced amino acid sequence of soybean SATase revealed significant homology with other plant SATases. Analysis of genomic DNA by Southern blotting indicated that SATase is encoded by a small gene family. The authenticity of the isolated SATase cDNA was confirmed by the expression of the cDNA in an Escherichia coli cysteine-auxotrophic mutant resulting in the growth of the mutant in minimal medium without cysteine. Expression of soybean SATase in E. coli resulted in the production of a 34-kDa protein that was subsequently purified by nickel-affinity column chromatography. The purified protein exhibited SATase activity, indicating that the E. coli-expressed protein is a functionally active SATase. The recombinant soybean SATase was inhibited by l-cysteine, the end product of the cysteine biosynthetic pathway. Antibodies raised against the recombinant soybean SATase cross-reacted with a 34-kDa protein from Arabidopsis leaves, but failed to detect any proteins from soybean leaves and seeds. Reverse transcriptase-polymerase chain reaction analysis indicated that SATase mRNA was expressed at low levels during soybean seed development. In comparison to Arabidopsis leaves, the SATase activity was several-fold lower in soybean leaves and seeds, suggesting that SATase is a low-abundance enzyme.

  11. The chromosomal 2'-N-acetyltransferase of Providencia stuartii: physiological functions and genetic regulation.

    PubMed

    Macinga, D R; Rather, P N

    1999-02-01

    Intrinsic chromosomal acetyltransferases involved in aminoglycoside resistance have been identified in a number of bacteria. In Providencia stuartii, a chromosomal acetyltransferase (AAC(2')-Ia) has been characterized in detail. In addition to the ability to acetylate aminoglycosides, the AAC(2')-Ia enzyme has at least one physiological function, which is the acetylation of peptidoglycan. This modification is likely to influence the autolytic system in P. stuartii. The regulation of aac(2')-Ia expression is extremely complex involving at least seven regulatory genes acting in at least two pathways. This complexity in regulation indicates that aac(2')-Ia expression must be tightly controlled in response to different environmental conditions. This presumably reflects the importance of maintaining correct levels of peptidoglycan acetylation. In this review, a summary of data will be presented involving both the physiological and genetic aspects of aac(2')-Ia in P. stuartii.

  12. Resistance to apramycin in two enterobacterial clinical isolates: detection of a 3-N-acetyltransferase IV.

    PubMed

    Gómez-Lus, R; Rivera, M J; Gómez-Lus, M L; Gil, J; Gómez-Lus, S; Castillo, J; Goñi, P; Madero, P; Rubio, M C

    1990-08-01

    Considering the possible role of farm animals in the contamination of human consumers by plasmid-mediated apramycin-resistant enterobacteria strains, this type of resistance should be tested more systematically in human isolates. Very recently we isolated in Zaragoza one apramycin-resistant Escheria coli strain obtained from the blood of a hospitalized patient; this clinical isolate produced a plasmid-mediated 3-N-aminoglycoside acetyltransferase IV. We describe also the isolation in Madrid of one multiresistant Klebsiella pneumoniae clinical strain. This isolate harbored a single plasmid and carried determinants for apramycin, gentamicin, tobramycin, hygromycin B, streptomycin, and ampicillin, which could be transferred en bloc to E. coli K-12 J62. Extracts from donor and transconjugant strains carrying pUZ6776 plasmid produce acetyltransferase activity AAC(3)-IV and double phosphotransferase activity (HPH and APH(3'')).

  13. Expression profiling of S. pombe acetyltransferase mutants identifies redundant pathways of gene regulation

    PubMed Central

    2010-01-01

    Background Histone acetyltransferase enzymes (HATs) are implicated in regulation of transcription. HATs from different families may overlap in target and substrate specificity. Results We isolated the elp3+ gene encoding the histone acetyltransferase subunit of the Elongator complex in fission yeast and characterized the phenotype of an Δelp3 mutant. We examined genetic interactions between Δelp3 and two other HAT mutants, Δmst2 and Δgcn5 and used whole genome microarray analysis to analyze their effects on gene expression. Conclusions Comparison of phenotypes and expression profiles in single, double and triple mutants indicate that these HAT enzymes have overlapping functions. Consistent with this, overlapping specificity in histone H3 acetylation is observed. However, there is no evidence for overlap with another HAT enzyme, encoded by the essential mst1+ gene. PMID:20096118

  14. 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.

  15. von Hippel-Lindau partner Jade-1 is a transcriptional co-activator associated with histone acetyltransferase activity.

    PubMed

    Panchenko, Maria V; Zhou, Mina I; Cohen, Herbert T

    2004-12-31

    Jade-1 was identified as a protein partner of the von Hippel-Lindau tumor suppressor pVHL. The interaction of Jade-1 and pVHL correlates with renal cancer risk. We have investigated the molecular function of Jade-1. Jade-1 has two zinc finger motifs called plant homeodomains (PHD). A line of evidence suggests that the PHD finger functions in chromatin remodeling and protein-protein interactions. We determined the cellular localization of Jade-1 and examined whether Jade-1 might have transcriptional and histone acetyltransferase (HAT) functions. Biochemical cell fractionation studies as well as confocal images of cells immunostained with a specific Jade-1 antibody revealed that endogenous Jade-1 is localized predominantly in the cell nucleus. Tethering of Gal4-Jade-1 fusion protein to Gal4-responsive promoters in co-transfection experiments activated transcription 5-6-fold, indicating that Jade-1 is a possible transcriptional activator. It was remarkable that overexpression of Jade-1 in cultured cells specifically increased levels of endogenous acetylated histone H4, but not histone H3, strongly suggesting that Jade-1 associates with HAT activity specific for histone H4. Deletion of the two PHD fingers completely abolished Jade-1 transcriptional and HAT activities, indicating that these domains are indispensable for Jade-1 nuclear functions. In addition, we demonstrated that TIP60, a known HAT with histone H4/H2A specificity, physically associates with Jade-1 and is able to augment Jade-1 HAT function in live cells, strongly suggesting that TIP60 might mediate Jade-1 HAT activity. Thus, Jade-1 is a novel candidate transcriptional co-activator associated with HAT activity and may play a key role in the pathogenesis of renal cancer and von Hippel-Lindau disease.

  16. Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses

    SciTech Connect

    Thoden, James B.; Reinhardt, Laurie A.; Cook, Paul D.; Menden, Patrick; Cleland, W.W.; Holden, Hazel M.

    2012-09-17

    N-Acetylperosamine is an unusual dideoxysugar found in the O-antigens of some Gram-negative bacteria, including the pathogenic Escherichia coli strain O157:H7. The last step in its biosynthesis is catalyzed by PerB, an N-acetyltransferase belonging to the left-handed {beta}-helix superfamily of proteins. Here we describe a combined structural and functional investigation of PerB from Caulobacter crescentus. For this study, three structures were determined to 1.0 {angstrom} resolution or better: the enzyme in complex with CoA and GDP-perosamine, the protein with bound CoA and GDP-N-acetylperosamine, and the enzyme containing a tetrahedral transition state mimic bound in the active site. Each subunit of the trimeric enzyme folds into two distinct regions. The N-terminal domain is globular and dominated by a six-stranded mainly parallel {beta}-sheet. It provides most of the interactions between the protein and GDP-perosamine. The C-terminal domain consists of a left-handed {beta}-helix, which has nearly seven turns. This region provides the scaffold for CoA binding. On the basis of these high-resolution structures, site-directed mutant proteins were constructed to test the roles of His 141 and Asp 142 in the catalytic mechanism. Kinetic data and pH-rate profiles are indicative of His 141 serving as a general base. In addition, the backbone amide group of Gly 159 provides an oxyanion hole for stabilization of the tetrahedral transition state. The pH-rate profiles are also consistent with the GDP-linked amino sugar substrate entering the active site in its unprotonated form. Finally, for this investigation, we show that PerB can accept GDP-3-deoxyperosamine as an alternative substrate, thus representing the production of a novel trideoxysugar.

  17. N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

    PubMed Central

    Van Damme, Petra; Lasa, Marta; Polevoda, Bogdan; Gazquez, Cristina; Elosegui-Artola, Alberto; Kim, Duk Soo; De Juan-Pardo, Elena; Demeyer, Kimberly; Hole, Kristine; Larrea, Esther; Timmerman, Evy; Prieto, Jesus; Arnesen, Thomas; Sherman, Fred; Gevaert, Kris; Aldabe, Rafael

    2012-01-01

    Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human. PMID:22814378

  18. 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.

  19. Histone H3 specific acetyltransferases are essential for cell cycle progression

    PubMed Central

    Howe, LeAnn; Auston, Darryl; Grant, Patrick; John, Sam; Cook, Richard G.; Workman, Jerry L.; Pillus, Lorraine

    2001-01-01

    Longstanding observations suggest that acetylation and/or amino-terminal tail structure of histones H3 and H4 are critical for eukaryotic cells. For Saccharomyces cerevisiae, loss of a single H4-specific histone acetyltransferase (HAT), Esa1p, results in cell cycle defects and death. In contrast, although several yeast HAT complexes preferentially acetylate histone H3, the catalytic subunits of these complexes are not essential for viability. To resolve the apparent paradox between the significance of H3 versus H4 acetylation, we tested the hypothesis that H3 modification is essential, but is accomplished through combined activities of two enzymes. We observed that Sas3p and Gcn5p HAT complexes have overlapping patterns of acetylation. Simultaneous disruption of SAS3, the homolog of the MOZ leukemia gene, and GCN5, the hGCN5/PCAF homolog, is synthetically lethal due to loss of acetyltransferase activity. This key combination of activities is specific for these two HATs because neither is synthetically lethal with mutations of other MYST family or H3-specific acetyltransferases. Further, the combined loss of GCN5 and SAS3 functions results in an extensive, global loss of H3 acetylation and arrest in the G2/M phase of the cell cycle. The strikingly similar effect of loss of combined essential H3 HAT activities and the loss of a single essential H4 HAT underscores the fundamental biological significance of each of these chromatin-modifying activities. PMID:11731478

  20. Diencephalic Size Is Restricted by a Novel Interplay Between GCN5 Acetyltransferase Activity and Retinoic Acid Signaling.

    PubMed

    Wilde, Jonathan J; Siegenthaler, Julie A; Dent, Sharon Y R; Niswander, Lee A

    2017-03-08

    Diencephalic defects underlie an array of neurological diseases. Previous studies have suggested that retinoic acid (RA) signaling is involved in diencephalic development at late stages of embryonic development, but its roles and mechanisms of action during early neural development are still unclear. Here we demonstrate that mice lacking enzymatic activity of the acetyltransferase GCN5 ((Gcn5(hat/hat) )), which were previously characterized with respect to their exencephalic phenotype, exhibit significant diencephalic expansion, decreased diencephalic RA signaling, and increased diencephalic WNT and SHH signaling. Using a variety of molecular biology techniques in both cultured neuroepithelial cells treated with a GCN5 inhibitor and forebrain tissue from (Gcn5(hat/hat) ) embryos, we demonstrate that GCN5, RARα/γ, and the poorly characterized protein TACC1 form a complex in the nucleus that binds specific retinoic acid response elements in the absence of RA. Furthermore, RA triggers GCN5-mediated acetylation of TACC1, which results in dissociation of TACC1 from retinoic acid response elements and leads to transcriptional activation of RA target genes. Intriguingly, RA signaling defects caused by in vitro inhibition of GCN5 can be rescued through RA-dependent mechanisms that require RARβ. Last, we demonstrate that the diencephalic expansion and transcriptional defects seen in (Gcn5(hat/hat) ) mutants can be rescued with gestational RA supplementation, supporting a direct link between GCN5, TACC1, and RA signaling in the developing diencephalon. Together, our studies identify a novel, nonhistone substrate for GCN5 whose modification regulates a previously undescribed, tissue-specific mechanism of RA signaling that is required to restrict diencephalic size during early forebrain development.SIGNIFICANCE STATEMENT Changes in diencephalic size and shape, as well as SNPs associated with retinoic acid (RA) signaling-associated genes, have been linked to neuropsychiatric

  1. Structural analysis of PseH, the Campylobacter jejuni N-acetyltransferase involved in bacterial O-linked glycosylation

    SciTech Connect

    Song, Wan Seok; Nam, Mi Sun; Namgung, Byeol; Yoon, Sung-il

    2015-03-20

    Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central β-sheet decorated by four α-helices with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis. - Highlights: • cjPseH adopts a single-domain structure of a central β-sheet decorated by α-helices. • cjPseH features two continuously connected grooves on the protein surface. • Acetyl coenzyme A (AcCoA) binds into a deep groove of cjPseH in an ‘L’ shape. • The acetyl end of AcCoA points to a wide groove, a potential substrate-binding site.

  2. The actin family protein ARP6 contributes to the structure and the function of the nucleolus

    SciTech Connect

    Kitamura, Hiroshi; Matsumori, Haruka; Kalendova, Alzbeta; Hozak, Pavel; Goldberg, Ilya G.; Nakao, Mitsuyoshi; Saitoh, Noriko; Harata, Masahiko

    2015-08-21

    The actin family members, consisting of actin and actin-related proteins (ARPs), are essential components of chromatin remodeling complexes. ARP6, one of the nuclear ARPs, is part of the Snf-2-related CREB-binding protein activator protein (SRCAP) chromatin remodeling complex, which promotes the deposition of the histone variant H2A.Z into the chromatin. In this study, we showed that ARP6 influences the structure and the function of the nucleolus. ARP6 is localized in the central region of the nucleolus, and its knockdown induced a morphological change in the nucleolus. We also found that in the presence of high concentrations of glucose ARP6 contributed to the maintenance of active ribosomal DNA (rDNA) transcription by placing H2A.Z into the chromatin. In contrast, under starvation, ARP6 was required for cell survival through the repression of rDNA transcription independently of H2A.Z. These findings reveal novel pleiotropic roles for the actin family in nuclear organization and metabolic homeostasis. - Highlights: • ARP6, an actin related protein, is important for nucleolar function and structure. • A population of ARP6 is localized in the center of nucleolus. • Depletion of ARP6 resulted in aberrant shape of the nucleolus. • ARP6 maintains the active rDNA transcription under high glucose. • ARP6 is required for the repression of rDNA transcription under starvation.

  3. Substrate-induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG

    SciTech Connect

    Filippova, Ekaterina V.; Weigand, Steven J.; Osipiuk, Jerzy; Kiryukhina, Olga; Joachimiak, Andrzej; Anderson, Wayne F.

    2015-09-26

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. As a result, our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.

  4. Induction of spermidine/spermine N1-acetyltransferase (SSAT) by aspirin in Caco-2 colon cancer cells.

    PubMed

    Babbar, Naveen; Gerner, Eugene W; Casero, Robert A

    2006-02-15

    Epidemiological, experimental and clinical results suggest that aspirin and other NSAIDs (non-steroidal anti-inflammatory drugs) inhibit the development of colon cancer. It has been shown that the NSAID sulindac induces apoptosis and suppresses carcinogenesis, in part, by a mechanism leading to the transcriptional activation of the gene encoding SSAT (spermidine/spermine N1-acetyltransferase), a rate-limiting enzyme in polyamine catabolism. In the present study, we show that a variety of NSAIDs, including aspirin, sulindac, ibuprofen and indomethacin, can induce SSAT gene expression in Caco-2 cells. Aspirin, at physiological concentrations, can induce SSAT mRNA via transcriptional initiation mechanisms. This induction leads to increased SSAT protein levels and enzyme activity. Promoter deletion analysis of the 5' SSAT promoter-flanking region led to the identification of two NF-kappaB (nuclear factor kappaB) response elements. Electrophoretic mobility-shift assays showed binding of NF-kappaB complexes at these sequences after aspirin treatment. Aspirin treatment led to the activation of NF-kappaB signalling and increased binding at these NF-kappaB sites in the SSAT promoter, hence providing a potential mechanism for the induction of SSAT by aspirin in these cells. Aspirin-induced SSAT ultimately leads to a decrease in cellular polyamine content, which has been associated with decreased carcinogenesis. These results suggest that activation of SSAT by aspirin and different NSAIDs may be a common property of NSAIDs that plays an important role in their chemopreventive actions in colorectal cancer.

  5. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean

    PubMed Central

    Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan

    2015-01-01

    Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR, and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at fourfold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops. PMID:26528311

  6. Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield, and plant biomass in rice.

    PubMed

    Song, Xian Jun; Kuroha, Takeshi; Ayano, Madoka; Furuta, Tomoyuki; Nagai, Keisuke; Komeda, Norio; Segami, Shuhei; Miura, Kotaro; Ogawa, Daisuke; Kamura, Takumi; Suzuki, Takamasa; Higashiyama, Tetsuya; Yamasaki, Masanori; Mori, Hitoshi; Inukai, Yoshiaki; Wu, Jianzhong; Kitano, Hidemi; Sakakibara, Hitoshi; Jacobsen, Steven E; Ashikari, Motoyuki

    2015-01-06

    Grain weight is an important crop yield component; however, its underlying regulatory mechanisms are largely unknown. Here, we identify a grain-weight quantitative trait locus (QTL) encoding a new-type GNAT-like protein that harbors intrinsic histone acetyltransferase activity (OsglHAT1). Our genetic and molecular evidences pinpointed the QTL-OsglHAT1's allelic variations to a 1.2-kb region upstream of the gene body, which is consistent with its function as a positive regulator of the traits. Elevated OsglHAT1 expression enhances grain weight and yield by enlarging spikelet hulls via increasing cell number and accelerating grain filling, and increases global acetylation levels of histone H4. OsglHAT1 localizes to the nucleus, where it likely functions through the regulation of transcription. Despite its positive agronomical effects on grain weight, yield, and plant biomass, the rare allele elevating OsglHAT1 expression has so far escaped human selection. Our findings reveal the first example, to our knowledge, of a QTL for a yield component trait being due to a chromatin modifier that has the potential to improve crop high-yield breeding.

  7. Co-expression of G2-EPSPS and glyphosate acetyltransferase GAT genes conferring high tolerance to glyphosate in soybean.

    PubMed

    Guo, Bingfu; Guo, Yong; Hong, Huilong; Jin, Longguo; Zhang, Lijuan; Chang, Ru-Zhen; Lu, Wei; Lin, Min; Qiu, Li-Juan

    2015-01-01

    Glyphosate is a widely used non-selective herbicide with broad spectrum of weed control around the world. At present, most of the commercial glyphosate tolerant soybeans utilize glyphosate tolerant gene CP4-EPSPS or glyphosate acetyltransferase gene GAT separately. In this study, both glyphosate tolerant gene G2-EPSPS and glyphosate degraded gene GAT were co-transferred into soybean and transgenic plants showed high tolerance to glyphosate. Molecular analysis including PCR, Sothern blot, qRT-PCR, and Western blot revealed that target genes have been integrated into genome and expressed effectively at both mRNA and protein levels. Furthermore, the glyphosate tolerance analysis showed that no typical symptom was observed when compared with a glyphosate tolerant line HJ06-698 derived from GR1 transgenic soybean even at fourfold labeled rate of Roundup. Chlorophyll and shikimic acid content analysis of transgenic plant also revealed that these two indexes were not significantly altered after glyphosate application. These results indicated that co-expression of G2-EPSPS and GAT conferred high tolerance to the herbicide glyphosate in soybean. Therefore, combination of tolerant and degraded genes provides a new strategy for developing glyphosate tolerant transgenic crops.

  8. Substrate-induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG

    DOE PAGES

    Filippova, Ekaterina V.; Weigand, Steven J.; Osipiuk, Jerzy; ...

    2015-09-26

    The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Twomore » hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. As a result, our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.« less

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

    PubMed Central

    Karagianni, Eleni P.; Kontomina, Evanthia; Davis, Britton; Kotseli, Barbara; Tsirka, Theodora; Garefalaki, Vasiliki; Sim, Edith; Glenn, Anthony E.; Boukouvala, Sotiria

    2015-01-01

    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 in Fusarium infecting cereal plants as responsible for detoxification of host defence compound 2-benzoxazolinone. Here we investigate functional diversification of NAT enzymes in crop-compromising species of Fusarium and Aspergillus, identifying three groups of homologues: Isoenzymes of the first group are found in all species and catalyse reactions with acetyl-CoA or propionyl-CoA. The second group is restricted to the plant pathogens and is active with malonyl-CoA in Fusarium species infecting cereals. The third group generates minimal activity with acyl-CoA compounds that bind non-selectively to the proteins. We propose that fungal NAT isoenzymes may have evolved to perform diverse functions, potentially relevant to pathogen fitness, acetyl-CoA/propionyl-CoA intracellular balance and secondary metabolism. PMID:26245863

  10. Opposing Functions of the N-terminal Acetyltransferases Naa50 and NatA in Sister-chromatid Cohesion.

    PubMed

    Rong, Ziye; Ouyang, Zhuqing; Magin, Robert S; Marmorstein, Ronen; Yu, Hongtao

    2016-09-02

    During the cell cycle, sister-chromatid cohesion tethers sister chromatids together from S phase to the metaphase-anaphase transition and ensures accurate segregation of chromatids into daughter cells. N-terminal acetylation is one of the most prevalent protein covalent modifications in eukaryotes and is mediated by a family of N-terminal acetyltransferases (NAT). Naa50 (also called San) has previously been shown to play a role in sister-chromatid cohesion in metazoans. The mechanism by which Naa50 contributes to cohesion is not understood however. Here, we show that depletion of Naa50 in HeLa cells weakens the interaction between cohesin and its positive regulator sororin and causes cohesion defects in S phase, consistent with a role of Naa50 in cohesion establishment. Strikingly, co-depletion of NatA, a heterodimeric NAT complex that physically interacts with Naa50, rescues the sister-chromatid cohesion defects and the resulting mitotic arrest caused by Naa50 depletion, indicating that NatA and Naa50 play antagonistic roles in cohesion. Purified recombinant NatA and Naa50 do not affect each other's NAT activity in vitro Because NatA and Naa50 exhibit distinct substrate specificity, we propose that they modify different effectors and regulate sister-chromatid cohesion in opposing ways.

  11. Treatment of Rats with Apocynin Has Considerable Inhibitory Effects on Arylamine N-Acetyltransferase Activity in the Liver

    PubMed Central

    Francis, Sheena; Laurieri, Nicola; Nwokocha, Chukwuemeka; Delgoda, Rupika

    2016-01-01

    The effect of apocynin on the activity of arylamine N-acetyltransferases (NATs) in excised liver samples was examined using eighteen Sprague-Dawley rats. Three groups of six animals each were fed a normal diet alone or a treatment of 50 or 100 mg/kg/day of apocynin via gavages for eight (8) weeks. Chronic in vivo administration of apocynin led to significant (p < 0.001) reduction of in vitro liver NAT activity up to 93% as compared with untreated rats (18.80 ± 2.10 μmols p-anisidine/min/μg liver protein). In vitro exposure of untreated liver homogenates to apocynin led to a dose-dependent inhibition of NAT activity with IC50 = 0.69 ± 0.02 mM. In silico modelling of apocynin tautomers and radical species into human NAT crystal structures supported the hypothesis that thiol functionalities in NAT enzymes may be crucial in apocynin binding. The involvement of human NAT enzymes in different pathological conditions, such as cancer, has encouraged the research for selective NAT inhibitors in both humans and animal models with possible chemopreventive properties. PMID:27242013

  12. Coordination of a transcriptional switch by HMGI(Y) acetylation.

    PubMed

    Munshi, N; Agalioti, T; Lomvardas, S; Merika, M; Chen, G; Thanos, D

    2001-08-10

    Dynamic control of interferon-beta (IFN-beta) gene expression requires the regulated assembly and disassembly of the enhanceosome, a higher-order nucleoprotein complex formed in response to virus infection. The enhanceosome activates transcription by recruiting the histone acetyltransferase proteins CREB binding protein (CBP) and p300/CBP-associated factors (PCAF)/GCN5, which, in addition to modifying histones, acetylate HMGI(Y), the architectural component required for enhanceosome assembly. We show that the accurate execution of the IFN-beta transcriptional switch depends on the ordered acetylation of the high-mobility group I protein HMGI(Y) by PCAF/GCN5 and CBP, which acetylate HMGI(Y) at distinct lysine residues on endogenous promoters. Whereas acetylation of HMGI(Y) by CBP at lysine-65 destabilizes the enhanceosome, acetylation of HMGI(Y) by PCAF/GCN5 at lysine-71 potentiates transcription by stabilizing the enhanceosome and preventing acetylation by CBP.

  13. Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates.

    PubMed

    Lilly, M; Lambrechts, M G; Pretorius, I S

    2000-02-01

    The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid

  14. Single dose exposure of sarin and physostigmine differentially regulates expression of choline acetyltransferase and vesicular acetylcholine transporter in rat brain.

    PubMed

    Bhardwaj, Sonika; Musalgaonkar, Nidhi; Waghmare, Chandrakant; Bhattacharya, Bijoy K

    2012-06-25

    Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are the key components of cholinergic system apart from acetylcholinesterase. Effects of subcutaneous exposures of 0.25 and 0.5 LD(50) sarin and 0.75 mg/kg physostigmine on immunoreactivity levels of these two proteins (ChAT and VAChT) were studied. Immunoreactivity levels of ChAT decreased significantly after 1 and 3 days in cortex and 3 days of 0.25 LD(50) sarin administration in cerebellum. While 0.5 LD(50) sarin exposure caused significant down regulation after 2.5 h to 7 days in cortex and 1 and 3 days in cerebellum with respect to controls. Physostigmine at 0.75 mg/kg dose showed enhanced levels of ChAT after 1 day which decreased significantly after 3 and 7 days both in cortex and cerebellum compared to controls. VAChT level decreased significantly after 1 day in cortex and 3 and 7 days in cerebellum after 0.25 LD(50) sarin administration, while 0.5 LD(50) sarin significantly lowered VAChT immunoreactivity level after 2.5 h and 7 days in cortex and 2.5 h and 1 day in cerebellum. Physostigmine at 0.75 mg/kg dose showed significant enhanced immunoreactivity levels of VAChT after 1, 3, and 7 days in cortex and 3 days in cerebellum. Results show that acetylcholinesterase inhibition by sarin caused reduction in cholinergic neurotransmission at cholinergic proteins expression levels, while physostigmine caused differential expression of key cholinergic proteins. Moreover, cortex, which receives greater cholinergic innervations, is more susceptible to anticholinesterase effect on cholinergic gene expression. These changes can explain delayed neurocognitive changes during anticholinesterases induced chronic neurotoxicity.

  15. 82-kDa choline acetyltransferase and SATB1 localize to β-amyloid induced matrix attachment regions

    PubMed Central

    Winick-Ng, Warren; Caetano, Fabiana A.; Winick-Ng, Jennifer; Morey, Trevor M.; Heit, Bryan; Rylett, R. Jane

    2016-01-01

    The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-β1–42 (Aβ1–42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aβ1–42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aβ1–42 -exposure increases 82-kDa ChAT association with gene promoters and introns. The Aβ1–42 -induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aβ1–42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aβ -exposure. PMID:27052102

  16. Choline acetyltransferase: further studies on the reverse reaction

    SciTech Connect

    Hsu, L.L.; Chao, L.P.

    1982-01-01

    In order to further characterize the reaction mechanism of brain ChAc in its purified form, we have investigated the reverse reaction of ChAc in terms of pH optimum, salt effects, and substrate kinetics using a radiochemical assay. We directly measured the reaction product acetylcoenzyme A which was separated from the substrate ACh by a cation exchange column. Dowex 50W-X8 (Na+ form). The reverse reaction of ChAc was linear with incubation time up to 40 minutes, and with enzyme protein concentration up to 5 micrograms. It had a pH optimum at 7.0. At 0.22 M the monovalent chloride and bromide salts activated the reverse ChAc activity by 23-47% but the fluoride and iodide salts inhibited the reverse enzyme activity by 10-30%. Kinetic studies in the absence of salt showed that KACh was 0.62 +/- 0.06 mM, KCoA . SH was 12.68 +/- 1.21 microM, and Vmax was 11.6 +/- 1.0 nmol AcCoA/mg protein/min. These data are in disagreement with the values reported on partially purified ChAc from bovine brain by Glover and Potter (1971) and Hersh (1980). This indicates that further investigations are necessary to clarify or resolve these differences.

  17. 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.

  18. The Chromatin Regulator BRPF3 Preferentially Activates the HBO1 Acetyltransferase but Is Dispensable for Mouse Development and Survival*

    PubMed Central

    Yan, Kezhi; You, Linya; Degerny, Cindy; Ghorbani, Mohammad; Liu, Xin; Chen, Lulu; Li, Lin; Miao, Dengshun; Yang, Xiang-Jiao

    2016-01-01

    To interpret epigenetic information, chromatin readers utilize various protein domains for recognition of DNA and histone modifications. Some readers possess multidomains for modification recognition and are thus multivalent. Bromodomain- and plant homeodomain-linked finger-containing protein 3 (BRPF3) is such a chromatin reader, containing two plant homeodomain-linked fingers, one bromodomain and a PWWP domain. However, its molecular and biological functions remain to be investigated. Here, we report that endogenous BRPF3 preferentially forms a tetrameric complex with HBO1 (also known as KAT7) and two other subunits but not with related acetyltransferases such as MOZ, MORF, TIP60, and MOF (also known as KAT6A, KAT6B, KAT5, and KAT8, respectively). We have also characterized a mutant mouse strain with a lacZ reporter inserted at the Brpf3 locus. Systematic analysis of β-galactosidase activity revealed dynamic spatiotemporal expression of Brpf3 during mouse embryogenesis and high expression in the adult brain and testis. Brpf3 disruption, however, resulted in no obvious gross phenotypes. This is in stark contrast to Brpf1 and Brpf2, whose loss causes lethality at E9.5 and E15.5, respectively. In Brpf3-null mice and embryonic fibroblasts, RT-quantitative PCR uncovered no changes in levels of Brpf1 and Brpf2 transcripts, confirming no compensation from them. These results indicate that BRPF3 forms a functional tetrameric complex with HBO1 but is not required for mouse development and survival, thereby distinguishing BRPF3 from its paralogs, BRPF1 and BRPF2. PMID:26677226

  19. Novel interaction between nuclear co-activator CBP and the CDK5 activator binding protein - C53.

    PubMed

    Yin, Xiaolong; Warner, Dennis R; Roberts, Emily A; Pisano, M Michele; Greene, Robert M

    2005-08-01

    cAMP response element-binding protein (CREB)-binding protein (CBP) is a multifunctional transcriptional co-activator that plays important roles in cell proliferation and differentiation. CBP is expressed in murine embryonic orofacial tissue and is developmentally regulated. To identify nuclear factors associating with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from embryonic orofacial tissue from gestational days 11-13 mouse embryos was conducted. The carboxy terminal region of CBP (including the C/H3 region) was utilized as a bait. C53, a 57 kDa protein known to bind to the p25 activator of cyclin-dependent kinase 5, was identified as a novel binding partner of CBP. The association of C53 with CBP was confirmed in vitro by glutathione S-transferase pull-down assays, and in vivo by co-immunoprecipitation. Reporter assays demonstrated that C53 had little effect on CBP mediated transcriptional activation. These results identify C53 as a novel binding partner for CBP. Recent research on presenilin-loss induced neurodegeneration demonstrated decreased expression of CBP and increased levels of the Cdk5 activator p25, both C53 binding proteins, suggesting that C53 might play a role in regulating neuronal proliferation, migration and/or differentiation in embryonic development.

  20. 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

  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. No association between apolipoprotein E or N‐Acetyltransferase 2 gene polymorphisms and age‐related hearing loss

    PubMed Central

    Dawes, Piers; Platt, Hazel; Horan, Michael; Ollier, William; Munro, Kevin; Pendleton, Neil

    2014-01-01

    Objectives/Hypothesis Age‐related hearing loss has a genetic component, but there have been limited genetic studies in this field. Both N‐acetyltransferase 2 and apolipoprotein E genes have previously been associated. However, these studies have either used small sample sizes, examined a limited number of polymorphisms, or have produced conflicting results. Here we use a haplotype tagging approach to determine association with age‐related hearing loss and investigate epistasis between these two genes. Study Design Candidate gene association study of a continuous phenotype. Methods We investigated haplotype tagging single nucleotide polymorphisms in the N‐acetyltransferase 2 gene and the presence/absence of the apolipoprotein E ε4 allele for association with age‐related hearing loss in a cohort of 265 Caucasian elderly volunteers from Greater Manchester, United Kingdom. Hearing phenotypes were generated using principal component analysis of the hearing threshold levels for the better ear (severity, slope, and concavity). Genotype data for the N‐acetyltransferase 2 gene was obtained from existing genome‐wide association study data from the Illumina 610‐Quadv1 chip. Apolipoprotein E genotyping was performed using Sequenom technology. Linear regression analysis was performed using Plink and Stata software. Results No significant associations (P value, > 0.05) were observed between the N‐acetyltransferase 2 or apolipoprotein E gene polymorphisms and any hearing factor. No significant association was observed for epistasis analysis of apolipoprotein E ε4 and the N‐acetyltransferase 2 single nucleotide polymorphism rs1799930 (NAT2*6A). Conclusion We found no evidence to support that either N‐acetyltransferase 2 or apolipoprotein E gene polymorphisms are associated with age‐related hearing loss in a cohort of 265 elderly volunteers. Level of Evidence N/A. Laryngoscope, 125:E33–E38, 2015 PMID:25155015

  3. Combined Action of Histone Reader Modules Regulates NuA4 Local Acetyltransferase Function but Not Its Recruitment on the Genome.

    PubMed

    Steunou, Anne-Lise; Cramet, Myriam; Rossetto, Dorine; Aristizabal, Maria J; Lacoste, Nicolas; Drouin, Simon; Côté, Valérie; Paquet, Eric; Utley, Rhea T; Krogan, Nevan; Robert, François; Kobor, Michael S; Côté, Jacques

    2016-11-15

    Recognition of histone marks by reader modules is thought to be at the heart of epigenetic mechanisms. These protein domains are considered to function by targeting regulators to chromosomal loci carrying specific histone modifications. This is important for proper gene regulation as well as propagation of epigenetic information. The NuA4 acetyltransferase complex contains two of these reader modules, an H3K4me3-specific plant homeodomain (PHD) within the Yng2 subunit and an H3K36me2/3-specific chromodomain in the Eaf3 subunit. While each domain showed a close functional interaction with the respective histone mark that it recognizes, at the biochemical level, genetic level (as assessed with epistatic miniarray profile screens), and phenotypic level, cells with the combined loss of both readers showed greatly enhanced phenotypes. Chromatin immunoprecipitation coupled with next-generation sequencing experiments demonstrated that the Yng2 PHD specifically directs H4 acetylation near the transcription start site of highly expressed genes, while Eaf3 is important downstream on the body of the genes. Strikingly, the recruitment of the NuA4 complex to these loci was not significantly affected. Furthermore, RNA polymerase II occupancy was decreased only under conditions where both PHD and chromodomains were lost, generally in the second half of the gene coding regions. Altogether, these results argue that methylated histone reader modules in NuA4 are not responsible for its recruitment to the promoter or coding regions but, rather, are required to orient its acetyltransferase catalytic site to the methylated histone 3-bearing nucleosomes in the surrounding chromatin, cooperating to allow proper transition from transcription initiation to elongation.

  4. The Human SWI-SNF Complex Protein p270 Is an ARID Family Member with Non-Sequence-Specific DNA Binding Activity

    PubMed Central

    Dallas, Peter B.; Pacchione, Stephen; Wilsker, Deborah; Bowrin, Valerie; Kobayashi, Ryuji; Moran, Elizabeth

    2000-01-01

    p270 is an integral member of human SWI-SNF complexes, first identified through its shared antigenic specificity with p300 and CREB binding protein. The deduced amino acid sequence of p270 reported here indicates that it is a member of an evolutionarily conserved family of proteins distinguished by the presence of a DNA binding motif termed ARID (AT-rich interactive domain). The ARID consensus and other structural features are common to both p270 and yeast SWI1, suggesting that p270 is a human counterpart of SWI1. The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a Drosophila melanogaster gene product required for normal development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) have been analyzed directly in regard to their DNA binding properties. Each binds preferentially to AT-rich sites. In contrast, p270 shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions. PMID:10757798

  5. Acetylation of EGF Receptor Contributes to Tumor Cell Resistance to Histone Deacetylase Inhibitors

    PubMed Central

    Song, Hui; Li, Chia-Wei; Labaff, Adam M.; Lim, Seung-Oe; Li, Long-Yuan; Kan, Shu-Fen; Chen, Yue; Zhang, Kai; Lang, Jingyu; Xie, Xiaoming; Wang, Yan; Huo, Long-Fei; Hsu, Sheng-Chieh; Chen, Xiaomin; Zhao, Yingming; Hung, Mien-Chie

    2011-01-01

    Alteration of epidermal growth factor receptor (EGFR) is involved in various human cancers and has been intensively investigated. A plethora of evidence demonstrates that posttranslational modifications of EGFR play a pivotal role in controlling its function and metabolism. Here, we show that EGFR can be acetylated by CREB binding protein (CBP) acetyltransferase. Interestingly, EGFR acetylation affects its tyrosine phosphorylation, which may contribute to cancer cell resistance to histone deacetylase inhibitors (HDACIs). Since there is an increasing interest in using HDACIs to treat various cancers in the clinic, our current study provides insights and rationale for selecting effective therapeutic regimen. Consistent with the previous reports, we also show that HDACI combined with EGFR inhibitors achieves better therapeutic outcomes and provides a molecular rationale for the enhanced effect of combination therapy. Our results unveil a critical role of EGFR acetylation that regulates EGFR function, which may have an important clinical implication. PMID:21094134

  6. Learning and memory deficits consequent to reduction of the fragile X mental retardation protein result from metabotropic glutamate receptor-mediated inhibition of cAMP signaling in Drosophila.

    PubMed

    Kanellopoulos, Alexandros K; Semelidou, Ourania; Kotini, Andriana G; Anezaki, Maria; Skoulakis, Efthimios M C

    2012-09-19

    Loss of the RNA-binding fragile X protein [fragile X mental retardation protein (FMRP)] results in a spectrum of cognitive deficits, the fragile X syndrome (FXS), while aging individuals with decreased protein levels present with a subset of these symptoms and tremor. The broad range of behavioral deficits likely reflects the ubiquitous distribution and multiple functions of the protein. FMRP loss is expected to affect multiple neuronal proteins and intracellular signaling pathways, whose identity and interactions are essential in understanding and ameliorating FXS symptoms. We used heterozygous mutants and targeted RNA interference-mediated abrogation in Drosophila to uncover molecular pathways affected by FMRP reduction. We present evidence that FMRP loss results in excess metabotropic glutamate receptor (mGluR) activity, attributable at least in part to elevation of the protein in affected neurons. Using high-resolution behavioral, genetic, and biochemical analyses, we present evidence that excess mGluR upon FMRP attenuation is linked to the cAMP decrement reported in patients and models, and underlies olfactory associative learning and memory deficits. Furthermore, our data indicate positive transcriptional regulation of the fly fmr1 gene by cAMP, via protein kinase A, likely through the transcription factor CREB. Because the human Fmr1 gene also contains CREB binding sites, the interaction of mGluR excess and cAMP signaling defects we present suggests novel combinatorial pharmaceutical approaches to symptom amelioration upon FMRP attenuation.

  7. 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

  8. Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris

    PubMed Central

    Casini, A.; Vaccaro, R.; D'Este, L.; Sakaue, Y.; Bellier, J.P.; Kimura, H.; Renda, T.G.

    2012-01-01

    Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes. PMID:23027350

  9. An extracellular factor regulating expression of the chromosomal aminoglycoside 2'-N-acetyltransferase of Providencia stuartii.

    PubMed

    Rather, P N; Parojcic, M M; Paradise, M R

    1997-08-01

    The chromosomal aac(2')-Ia gene in Providencia stuartii encodes a housekeeping 2'-N-acetyltransferase [AAC(2')-Ia] involved in the acetylation of peptidoglycan. In addition, the AAC(2')-Ia enzyme also acetylates and confers resistance to the clinically important aminoglycoside antibiotics gentamicin, tobramycin, and netilmicin. Expression of the aac(2')-Ia gene was found to be strongly influenced by cell density, with a sharp decrease in aac(2')-Ia mRNA accumulation as cells approached stationary phase. This decrease was mediated by the accumulation of an extracellular factor, designated AR (for acetyltransferase repressing)-factor. AR-factor was produced in both minimal and rich media and acted in a manner that was strongly dose dependent. The activity of AR-factor was also pH dependent, with optimal activity at pH 8.0 and above. Biochemical characterization of conditioned media from P. stuartii has shown that AR-factor is between 500 and 1,000 Da in molecular size and is heat stable. In addition, AR-factor was inactivated by a variety of proteases, suggesting that it may be a small peptide.

  10. An acetyltransferase-independent function of Eso1 regulates centromere cohesion

    PubMed Central

    Lin, Su-Jiun; Tapia-Alveal, Claudia; Jabado, Omar J.; Germain, Doris; O’Connell, Matthew J.

    2016-01-01

    Eukaryotes contain three essential Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin, and Smc5/6. Cohesin forms a ring-shaped structure that embraces sister chromatids to promote their cohesion. The cohesiveness of cohesin is promoted by acetylation of N-terminal lysines of the Smc3 subunit by the acetyltransferases Eco1 in Saccharomyces cerevisiae and the homologue, Eso1, in Schizosaccharomyces pombe. In both yeasts, these acetyltransferases are essential for cell viability. However, whereas nonacetylatable Smc3 mutants are lethal in S. cerevisiae, they are not in S. pombe. We show that the lethality of a temperature-sensitive allele of eso1 (eso1-H17) is due to activation of the spindle assembly checkpoint (SAC) and is associated with premature centromere separation. The lack of cohesion at the centromeres does not correlate with Psm3 acetylation or cohesin levels at the centromeres, but is associated ith significantly reduced recruitment of the cohesin regulator Pds5. The SAC activation in this context is dependent on Smc5/6 function, which is required to remove cohesin from chromosome arms but not centromeres. The mitotic defects caused by Smc5/6 and Eso1 dysfunction are cosuppressed in double mutants. This identifies a novel function (or functions) for Eso1 and Smc5/6 at centromeres and extends the functional relationships between these SMC complexes. PMID:27798241

  11. Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris.

    PubMed

    Casini, A; Vaccaro, R; D'Este, L; Sakaue, Y; Bellier, J P; Kimura, H; Renda, T G

    2012-07-19

    Acetylcholine, the first neurotransmitter to be identified in the vertebrate frog, is widely distributed among the animal kingdom. The presence of a large amount of acetylcholine in the nervous system of cephalopods is well known from several biochemical and physiological studies. However, little is known about the precise distribution of cholinergic structures due to a lack of a suitable histochemical technique for detecting acetylcholine. The most reliable method to visualize the cholinergic neurons is the immunohistochemical localization of the enzyme choline acetyltransferase, the synthetic enzyme of acetylcholine. Following our previous study on the distribution patterns of cholinergic neurons in the Octopus vulgaris visual system, using a novel antibody that recognizes choline acetyltransferase of the common type (cChAT), now we extend our investigation on the octopus central brain mass. When applied on sections of octopus central ganglia, immunoreactivity for cChAT was detected in cell bodies of all central brain mass lobes with the notable exception of the subfrontal and subvertical lobes. Positive varicosed nerves fibers where observed in the neuropil of all central brain mass lobes.

  12. The Drosophila putative histone acetyltransferase Enok maintains female germline stem cells through regulating Bruno and the niche.

    PubMed

    Xin, Tianchi; Xuan, Tao; Tan, Jieqiong; Li, Mengjie; Zhao, Gengchun; Li, Mingfa

    2013-12-01

    Maintenance of adult stem cells is largely dependent on the balance between their self-renewal and differentiation. The Drosophila ovarian germline stem cells (GSCs) provide a powerful in vivo system for studying stem cell fate regulation. It has been shown that maintaining the GSC population involves both genetic and epigenetic mechanisms. Although the role of epigenetic regulation in this process is evident, the underlying mechanisms remain to be further explored. In this study, we find that Enoki mushroom (Enok), a Drosophila putative MYST family histone acetyltransferase controls GSC maintenance in the ovary at multiple levels. Removal or knockdown of Enok in the germline causes a GSC maintenance defect. Further studies show that the cell-autonomous role of Enok in maintaining GSCs is not dependent on the BMP/Bam pathway. Interestingly, molecular studies reveal an ectopic expression of Bruno, an RNA binding protein, in the GSCs and their differentiating daughter cells elicited by the germline Enok deficiency. Misexpression of Bruno in GSCs and their immediate descendants results in a GSC loss that can be exacerbated by incorporating one copy of enok mutant allele. These data suggest a role for Bruno in Enok-controlled GSC maintenance. In addition, we observe that Enok is required for maintaining GSCs non-autonomously. Compromised expression of enok in the niche cells impairs the niche maintenance and BMP signal output, thereby causing defective GSC maintenance. This is the first demonstration that the niche size control requires an epigenetic mechanism. Taken together, studies in this paper provide new insights into the GSC fate regulation.

  13. Spt-Ada-Gcn5-Acetyltransferase (SAGA) Complex in Plants: Genome Wide Identification, Evolutionary Conservation and Functional Determination.

    PubMed

    Srivastava, Rakesh; Rai, Krishan Mohan; Pandey, Bindu; Singh, Sudhir P; Sawant, Samir V

    2015-01-01

    The recruitment of RNA polymerase II on a promoter is assisted by the assembly of basal transcriptional machinery in eukaryotes. The Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex plays an important role in transcription regulation in eukaryotes. However, even in the advent of genome sequencing of various plants, SAGA complex has been poorly defined for their components and roles in plant development and physiological functions. Computational analysis of Arabidopsis thaliana and Oryza sativa genomes for SAGA complex resulted in the identification of 17 to 18 potential candidates for SAGA subunits. We have further classified the SAGA complex based on the conserved domains. Phylogenetic analysis revealed that the SAGA complex proteins are evolutionary conserved between plants, yeast and mammals. Functional annotation showed that they participate not only in chromatin remodeling and gene regulation, but also in different biological processes, which could be indirect and possibly mediated via the regulation of gene expression. The in silico expression analysis of the SAGA components in Arabidopsis and O. sativa clearly indicates that its components have a distinct expression profile at different developmental stages. The co-expression analysis of the SAGA components suggests that many of these subunits co-express at different developmental stages, during hormonal interaction and in response to stress conditions. Quantitative real-time PCR analysis of SAGA component genes further confirmed their expression in different plant tissues and stresses. The expression of representative salt, heat and light inducible genes were affected in mutant lines of SAGA subunits in Arabidopsis. Altogether, the present study reveals expedient evidences of involvement of the SAGA complex in plant gene regulation and stress responses.

  14. Spt-Ada-Gcn5-Acetyltransferase (SAGA) Complex in Plants: Genome Wide Identification, Evolutionary Conservation and Functional Determination

    PubMed Central

    Srivastava, Rakesh; Rai, Krishan Mohan; Pandey, Bindu; Singh, Sudhir P.; Sawant, Samir V.

    2015-01-01

    The recruitment of RNA polymerase II on a promoter is assisted by the assembly of basal transcriptional machinery in eukaryotes. The Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex plays an important role in transcription regulation in eukaryotes. However, even in the advent of genome sequencing of various plants, SAGA complex has been poorly defined for their components and roles in plant development and physiological functions. Computational analysis of Arabidopsis thaliana and Oryza sativa genomes for SAGA complex resulted in the identification of 17 to 18 potential candidates for SAGA subunits. We have further classified the SAGA complex based on the conserved domains. Phylogenetic analysis revealed that the SAGA complex proteins are evolutionary conserved between plants, yeast and mammals. Functional annotation showed that they participate not only in chromatin remodeling and gene regulation, but also in different biological processes, which could be indirect and possibly mediated via the regulation of gene expression. The in silico expression analysis of the SAGA components in Arabidopsis and O. sativa clearly indicates that its components have a distinct expression profile at different developmental stages. The co-expression analysis of the SAGA components suggests that many of these subunits co-express at different developmental stages, during hormonal interaction and in response to stress conditions. Quantitative real-time PCR analysis of SAGA component genes further confirmed their expression in different plant tissues and stresses. The expression of representative salt, heat and light inducible genes were affected in mutant lines of SAGA subunits in Arabidopsis. Altogether, the present study reveals expedient evidences of involvement of the SAGA complex in plant gene regulation and stress responses. PMID:26263547

  15. Production of tetraacetyl phytosphingosine (TAPS) in Wickerhamomyces ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p.

    PubMed

    Ter Veld, Frank; Wolff, Daniel; Schorsch, Christoph; Köhler, Tim; Boles, Eckhard; Poetsch, Ansgar

    2013-10-01

    Wickerhamomyces ciferrii secretes tetraacetyl phytosphingosine (TAPS), and in this study, the catalyzing acetyltransferases were identified using mass spectrometry-based proteomics. The proteome of wild-type strain NRRL Y-1031 served as control and was compared to the tetraacetyl phytosphingosine defective mating type NRRL Y-1031-27. Acetylation of phytosphingosine in W. ciferrii is catalyzed by acetyltransferases Sli1p and Atf2p, encoded by genes similar to Saccharomyces cerevisiae YGR212W and YGR177C, respectively. Ablation of SLI1 resulted in an almost complete loss of tri- and tetraacetyl phytosphingosines, whereas the loss ATF2 resulted in an 15-fold increase in triacetyl phytosphingosine. Most likely, it is the concerted action of these two acetyltransferases that yields tetraacetyl phytosphingosine, in which Sli1p catalyzes initial O- and N-acetylation, producing triacetyl phytosphingosine. Finally, Atf2p catalyzes final O-acetylation to yield tetraacetyl phytosphingosine. The current study demonstrates that mass spectrometry-based proteomics can be employed to identify key steps in ill-explored metabolite biosynthesis pathways of nonconventional microorganisms. Furthermore, the identification of phytosphingosine as substrate for alcohol acetyltransferase Atf2p broadens the known substrate range of this enzyme. This interesting property of Atf2p may be exploited to enhance the secretion of heterologous compounds.

  16. 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...

  17. 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.

  18. Arylamine N-acetyltransferases: a structural perspective. Comments regarding the BJP paper by Zhou et al., 2013

    PubMed Central

    Xu, Ximing; Kubiak, Xavier; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2014-01-01

    This letter is a comment on Zhou et al. (2013). Arylamine N-acetyltransferases: a structural perspective. Br J Pharmacol 169: 748–760. To view this article visit http://dx.doi.org/10.1111/bph.12182 PMID:24328723

  19. 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...

  20. Small Molecule Inhibition of cAMP Response Element Binding Protein in Human Acute Myeloid Leukemia Cells

    PubMed Central

    Mitton, Bryan; Chae, Hee-Don; Hsu, Katie; Dutta, Ritika; Aldana-Masangkay, Grace; Ferrari, Roberto; Davis, Kara; Tiu, Bruce C.; Kaul, Arya; Lacayo, Norman; Dahl, Gary; Xie, Fuchun; Li, Bingbing X.; Breese, Marcus R.; Landaw, Elliot M.; Nolan, Garry; Pellegrini, Matteo; Romanov, Sergei; Xiao, Xiangshu; Sakamoto, Kathleen M.

    2016-01-01

    The transcription factor CREB (cAMP Response Element Binding Protein) is overexpressed in the majority of acute myeloid leukemia (AML) patients, and this is associated with a worse prognosis. Previous work revealed that CREB overexpression augmented AML cell growth, while CREB knockdown disrupted key AML cell functions in vitro. In contrast, CREB knockdown had no effect on long-term hematopoietic stem cell activity in mouse transduction/transplantation assays. Together, these studies position CREB as a promising drug target for AML. To test this concept, a small molecule inhibitor of CREB, XX-650-23, was developed. This molecule blocks a critical interaction between CREB and its required co-activator CBP (CREB Binding Protein), leading to disruption of CREB-driven gene expression. Inhibition of CBP-CREB interaction induced apoptosis and cell cycle arrest in AML cells, and prolonged survival in vivo in mice injected with human AML cells. XX-650-23 had little toxicity on normal human hematopoietic cells and tissues in mice. To understand the mechanism of XX-650-23, we performed RNA-seq, ChIP-seq and Cytometry Time of Flight with human AML cells. Our results demonstrate that small molecule inhibition of CBP-CREB interaction mostly affects apoptotic, cell cycle, and survival pathways, which may represent a novel approach for AML therapy. PMID:27211267

  1. Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N-Acetyltransferase from Trypanosoma brucei ▿ ‡ §

    PubMed Central

    Mariño, Karina; Güther, M. Lucia Sampaio; Wernimont, Amy K.; Qiu, Wei; Hui, Raymond; Ferguson, Michael A. J.

    2011-01-01

    A gene predicted to encode Trypanosoma brucei glucosamine 6-phosphate N-acetyltransferase (TbGNA1; EC 2.3.1.4) was cloned and expressed in Escherichia coli. The recombinant protein was enzymatically active, and its high-resolution crystal structure was obtained at 1.86 Å. Endogenous TbGNA1 protein was localized to the peroxisome-like microbody, the glycosome. A bloodstream-form T. brucei GNA1 conditional null mutant was constructed and shown to be unable to sustain growth in vitro under nonpermissive conditions, demonstrating that there are no metabolic or nutritional routes to UDP-GlcNAc other than via GlcNAc-6-phosphate. Analysis of the protein glycosylation phenotype of the TbGNA1 mutant under nonpermissive conditions revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite and that the glycosylation profile of the principal parasite surface coat component, the variant surface glycoprotein (VSG), was modified. The significance of results and the potential of TbGNA1 as a novel drug target for African sleeping sickness are discussed. PMID:21531872

  2. An approach to identify SNPs in the gene encoding acetyl-CoA acetyltransferase-2 (ACAT-2) and their proposed role in metabolic processes in pig.

    PubMed

    Sodhi, Simrinder Singh; Ghosh, Mrinmoy; Song, Ki Duk; Sharma, Neelesh; Kim, Jeong Hyun; Kim, Nam Eun; Lee, Sung Jin; Kang, Chul Woong; Oh, Sung Jong; Jeong, Dong Kee

    2014-01-01

    The novel liver protein acetyl-CoA acetyltransferase-2 (ACAT2) is involved in the beta-oxidation and lipid metabolism. Its comprehensive relative expression, in silico non-synonymous single nucleotide polymorphism (nsSNP) analysis, as well as its annotation in terms of metabolic process with another protein from the same family, namely, acetyl-CoA acyltransferase-2 (ACAA2) was performed in Sus scrofa. This investigation was conducted to understand the most important nsSNPs of ACAT2 in terms of their effects on metabolic activities and protein conformation. The two most deleterious mutations at residues 122 (I to V) and 281 (R to H) were found in ACAT2. Validation of expression of genes in the laboratory also supported the idea of differential expression of ACAT2 and ACAA2 conceived through the in silico analysis. Analysis of the relative expression of ACAT2 and ACAA2 in the liver tissue of Jeju native pig showed that the former expressed significantly higher (P<0.05). Overall, the computational prediction supported by wet laboratory analysis suggests that ACAT2 might contribute more to metabolic processes than ACAA2 in swine. Further associations of SNPs in ACAT2 with production traits might guide efforts to improve growth performance in Jeju native pigs.

  3. Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1).

    PubMed

    Zhao, Minzhi; Li, Haiyun; Ma, Yan; Gong, He; Yang, Shu; Fang, Qiaojun; Hu, Zhiyuan

    2017-01-01

    Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression (P<0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes.

  4. Cloning of Arabidopsis serotonin N-acetyltransferase and its role with caffeic acid O-methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations.

    PubMed

    Lee, Hyoung Yool; Byeon, Yeong; Lee, Kyungjin; Lee, Hye-Jung; Back, Kyoungwhan

    2014-11-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45 °C (Km , 309 μm; Vmax , 1400 pmol/min/mg protein). AtSNAT also catalyzed 5-methoxytryptamine (5-MT) into melatonin with high catalytic activity (Km , 51 μm; Vmax , 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O-methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5-MT with low catalytic activity (Km , 3.396 mm; Vmax , 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N-acetylserotonin by SNAT or into 5-MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N-acetylserotonin was predominantly produced at high temperatures (45 °C), while low temperatures (37 °C) favored the production of 5-MT. Our results provide biochemical evidence for the presence of a serotonin O-methylation pathway in plant melatonin biosynthesis.

  5. Nanoparticle abraxane possesses impaired proliferation in A549 cells due to the underexpression of glucosamine 6-phosphate N-acetyltransferase 1 (GNPNAT1/GNA1)

    PubMed Central

    Zhao, Minzhi; Li, Haiyun; Ma, Yan; Gong, He; Yang, Shu; Fang, Qiaojun; Hu, Zhiyuan

    2017-01-01

    Abraxane (Abr), a US Food and Drug Administration-approved albumin-bound nanoparticle applied for the treatment of non-small-cell lung cancer, has been reported to be more effective than paclitaxel (PTX). To further understand the molecular mechanisms that produce this superior drug efficacy of Abr, a quantitative proteomic approach has been applied to investigate the global protein expression profiles of lung cancer cell A549 treated with Abr and PTX. Only one protein, namely, glucosamine 6-phosphate N-acetyltransferase 1 (GNA1), showed significant differential expression (P<0.05) in the cutoff of 2.0 fold, suggesting that Abr can be used safely as a substitute for PTX. GNA1 is a key enzyme in the biosynthesis of uridine diphosphate-N-acetylglucosamine, which is an important donor substrate for N-linked glycosylation and has several important functions such as embryonic development and growth. Albumin plays a major role in the regulation of this protein. In summary, this study first shows that the superior drug effect of Abr is mainly due to the downregulation of GNA1, which causes proliferative delay and cell adhesion defect. It is also noteworthy that the deficiency of GNA1 might reduce insulin secretion which correlates with type 2 diabetes. PMID:28280335

  6. Heterogeneous ribonucleoprotein R regulates arylalkylamine N-acetyltransferase synthesis via internal ribosomal entry site-mediated translation in a circadian manner.

    PubMed

    Lee, Hwa-Rim; Kim, Tae-Don; Kim, Hyo-Jin; Jung, Youngseob; Lee, Dohyun; Lee, Kyung-Ha; Kim, Do-Yeon; Woo, Kyung-Chul; Kim, Kyong-Tai

    2015-11-01

    Rhythmic arylalkylamine N-acetyltransferase (AANAT) synthesis is a prominent circadian-controlled response that occurs in most mammals. AANAT is the core enzyme in melatonin production; because melatonin participates in many physiological processes, the regulation of AANAT is an important research topic. In this study, we focused on the role of heterogeneous ribonucleoprotein R (hnRNP R) in the translation of AANAT. A novel RNA-binding protein hnRNP R widely interacted with the 5' untranslated region (UTR) of AANAT mRNA and contributed to translation through an internal ribosomal entry site (IRES). Fine-tuning of AANAT protein synthesis occurred in response to knockdown and overexpression of hnRNP R. Nocturnal elevation of AANAT protein was dependent on the rhythmic changes of hnRNP R, whose levels are elevated in the pineal gland during nighttime. Increases in hnRNP R additionally improved AANAT production in rat pinealocytes under norepinephrine (NE) treatment. These results suggest that cap-independent translation of AANAT mRNA plays a role in the rhythmic synthesis of melatonin through the recruitment of translational machinery to hnRNP R-bound AANAT mRNA.

  7. The Yeast ATF1 Acetyltransferase Efficiently Acetylates Insect Pheromone Alcohols: Implications for the Biological Production of Moth Pheromones.

    PubMed

    Ding, Bao-Jian; Lager, Ida; Bansal, Sunil; Durrett, Timothy P; Stymne, Sten; Löfstedt, Christer

    2016-04-01

    Many moth pheromones are composed of mixtures of acetates of long-chain (≥10 carbon) fatty alcohols. Moth pheromone precursors such as fatty acids and fatty alcohols can be produced in yeast by the heterologous expression of genes involved in insect pheromone production. Acetyltransferases that subsequently catalyze the formation of acetates by transfer of the acetate unit from acetyl-CoA to a fatty alcohol have been postulated in pheromone biosynthesis. However, so far no fatty alcohol acetyltransferases responsible for the production of straight chain alkyl acetate pheromone components in insects have been identified. In search for a non-insect acetyltransferase alternative, we expressed a plant-derived diacylglycerol acetyltransferase (EaDAcT) (EC 2.3.1.20) cloned from the seed of the burning bush (Euonymus alatus) in a yeast system. EaDAcT transformed various fatty alcohol insect pheromone precursors into acetates but we also found high background acetylation activities. Only one enzyme in yeast was shown to be responsible for the majority of that background activity, the acetyltransferase ATF1 (EC 2.3.1.84). We further investigated the usefulness of ATF1 for the conversion of moth pheromone alcohols into acetates in comparison with Ea DAcT. Overexpression of ATF1 revealed that it was capable of acetylating these fatty alcohols with chain lengths from 10 to 18 carbons with up to 27- and 10-fold higher in vivo and in vitro efficiency, respectively, compared to Ea DAcT. The ATF1 enzyme thus has the potential to serve as the missing enzyme in the reconstruction of the biosynthetic pathway of insect acetate pheromones from precursor fatty acids in yeast.

  8. Dietary phenolic acids attenuate multiple stages of protein glycation and high-glucose-stimulated proinflammatory IL-1beta activation by interfering with chromatin remodeling and transcription in monocytes.

    PubMed

    Wu, Chi-Hao; Yeh, Chi-Tai; Shih, Ping-Hsiao; Yen, Gow-Chin

    2010-07-01

    This study examined the effects of dietary phenolic acids on individual stages of protein glycation and utilized monocyte cultures to assess whether these phytochemicals modulate the activation of proinflammatory cytokine under high glucose (HG, 15 mmol/L) conditions mimicking diabetes. In vitro glycation assays showed that a number of phenolic acids exerted inhibitory effects on the glycation reaction and its subsequent crosslinking. Phenolic acids, especially methoxyphenolic acids, prevented increase in both levels of the interleukin-1beta (IL-1beta) and oxidative stress caused by HG. The effect appeared to be mediated by modulation of the protein kinase C/nuclear factor-kappaB axis. Chromatin immunoprecipitation demonstrated for the first time that HG increased the recruitment of nuclear factor-kappaB p65 and CREB-binding protein to the IL-1beta promoter. Interestingly, HG also increased histone acetylation and methylation within the IL-1beta promoter and decreased histone deacetylase activities in monocytes, thus facilitating chromatin remodeling and transcription. Such inappropriate inflammatory responses were found to be controlled effectively by treatment with methoxyphenolic compounds. In conclusion, this study suggests that phenolic acids could exert their anti-inflammatory activities as antiglycation agents and as modifiers of signaling pathways. It provides evidence for a novel mechanism by which phenolics supplementation might have additional protective effects against diabetic complications.

  9. Effects of nefiracetam on the levels of brain-derived neurotrophic factor and synapsin I mRNA and protein in the hippocampus of microsphere-embolized rats.

    PubMed

    Ando, Tsuyoshi; Takagi, Norio; Takagi, Keiko; Kago, Tomoyuki; Takeo, Satoshi

    2005-01-10

    Our recent study demonstrated that nefiracetam, N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide, prevented impairment of the cyclic AMP (cAMP)/cAMP-responsive element binding (CREB) protein signaling pathway in sustained cerebral ischemia. The purpose of the present study was to determine whether nefiracetam has an effect on the expression of brain-derived neurotrophic factor (BDNF) and synapsin I mRNAs that are believed to be produced via CREB, and the alteration in their protein contents in the hippocampus after cerebral ischemia. Sustained cerebral ischemia was induced by injection of 700 microspheres into the right hemisphere of each rat. The rats were treated once daily with 10 mg/kg nefiracetam, p.o., from 15 h after the operation. Treatment with nefiracetam reduced the prolongation of the escape latency in the water maze test on days 7-9 after microsphere embolism-induced sustained cerebral ischemia, suggesting an improvement in the spatial learning function. Microsphere-embolized rats on day 5 showed decreases in BDNF and synapsin I mRNA levels and their protein contents in the ipsilateral hippocampus. Treatment with nefiracetam partially attenuated the decreases. These results suggest that enhancement of BDNF and synapsin I expression by nefiracetam treatment may be, at least in part, due to the improvement in the CREB binding activity, contributing to the prevention of learning and memory dysfunction after sustained cerebral ischemia.

  10. Circadian clock controlling arylalkylamine N-acetyltransferase-like activity in the cricket (Gryllus bimaculatus) egg.

    PubMed

    Itoh, M T; Sumi, Y

    1998-07-13

    When cricket (Gryllus bimaculatus) eggs were incubated under a 12-h light/12-h dark (LD) cycle for 6 days after oviposition at 24-26 degrees C and thereafter transferred to constant darkness (DD), arylalkylamine N-acetyltransferase (NAT)-like activity fluctuated in a circadian manner, peaking during the subjective dark period, and the rhythmic activity persisted during the 3rd day of incubation in DD. When the eggs were transferred from LD to a lighting regime in which the light and dark periods were reversed, the rhythm of NAT-like activity continued to oscillate in phase with the light/dark cycle. These data demonstrate that the cricket egg (probably the embryo) contains a circadian clock controlling NAT-like activity, and that the circadian clock entrains to environmental light/dark cycles.

  11. 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.

  12. Metabolic Regulation of Histone Acetyltransferases by Endogenous Acyl-CoA Cofactors

    PubMed Central

    Guasch, Laura; Nicklaus, Marc C.; Meier, Jordan L.

    2015-01-01

    SUMMARY The finding that chromatin modifications are sensitive to changes in cellular cofactor levels potentially links altered tumor cell metabolism and gene expression. However, the specific enzymes and metabolites that connect these two processes remain obscure. Characterizing these metabolic-epigenetic axes is critical to understanding how metabolism supports signaling in cancer, and developing therapeutic strategies to disrupt this process. Here, we describe a chemical approach to define the metabolic regulation of lysine acetyltransferase (KAT) enzymes. Using a novel chemoproteomic probe, we identify a previously unreported interaction between fatty acyl-CoAs and KAT enzymes. Further analysis reveals that palmitoyl-CoA is a potent inhibitor of KAT activity and that fatty acyl-CoA precursors reduce cellular acetylation levels. These studies implicate fatty acyl-CoAs as endogenous regulators of histone acetylation, and suggest novel strategies for the investigation and metabolic modulation of epigenetic signaling. PMID:26190825

  13. 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.

  14. 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.

  15. The histone acetyltransferase MOF activates hypothalamic polysialylation to prevent diet-induced obesity in mice

    PubMed Central

    Brenachot, Xavier; Rigault, Caroline; Nédélec, Emmanuelle; Laderrière, Amélie; Khanam, Tasneem; Gouazé, Alexandra; Chaudy, Sylvie; Lemoine, Aleth; Datiche, Frédérique; Gascuel, Jean; Pénicaud, Luc; Benani, Alexandre

    2014-01-01

    Overfeeding causes rapid synaptic remodeling in hypothalamus feeding circuits. Polysialylation of cell surface molecules is a key step in this neuronal rewiring and allows normalization of food intake. Here we examined the role of hypothalamic polysialylation in the long-term maintenance of body weight, and deciphered the molecular sequence underlying its nutritional regulation. We found that upon high fat diet (HFD), reduced hypothalamic polysialylation exacerbated the diet-induced obese phenotype in mice. Upon HFD, the histone acetyltransferase MOF was rapidly recruited on the St8sia4 polysialyltransferase-encoding gene. Mof silencing in the mediobasal hypothalamus of adult mice prevented activation of the St8sia4 gene transcription, reduced polysialylation, altered the acute homeostatic feeding response to HFD and increased the body weight gain. These findings indicate that impaired hypothalamic polysialylation contribute to the development of obesity, and establish a role for MOF in the brain control of energy balance. PMID:25161885

  16. Muscle-specific deletion of carnitine acetyltransferase compromises glucose tolerance and metabolic flexibility.

    PubMed

    Muoio, Deborah M; Noland, Robert C; Kovalik, Jean-Paul; Seiler, Sarah E; Davies, Michael N; DeBalsi, Karen L; Ilkayeva, Olga R; Stevens, Robert D; Kheterpal, Indu; Zhang, Jingying; Covington, Jeffrey D; Bajpeyi, Sudip; Ravussin, Eric; Kraus, William; Koves, Timothy R; Mynatt, Randall L

    2012-05-02

    The concept of "metabolic inflexibility" was first introduced to describe the failure of insulin-resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes, and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility, and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility.

  17. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis.

    PubMed

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

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28-37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  18. 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.

  19. The place of choline acetyltransferase activity measurement in the "cholinergic hypothesis" of neurodegenerative diseases.

    PubMed

    Contestabile, Antonio; Ciani, Elisabetta; Contestabile, Andrea

    2008-02-01

    The so-called "cholinergic hypothesis" assumes that degenerative dysfunction of the cholinergic system originating in the basal forebrain and innervating several cortical regions and the hippocampus, is related to memory impairment and neurodegeneration found in several forms of dementia and in brain aging. Biochemical methods measuring the activity of the key enzyme for acetylcholine synthesis, choline acetyltransferase, have been used for many years as a reliable marker of the integrity or the damage of the cholinergic pathways. Stereologic counting of the basal forebrain cholinergic cell bodies, has been additionally used to assess neurodegenerative changes of the forebrain cholinergic system. While initially believed to mark relatively early stages of disease, cholinergic dysfunction is at present considered to occur in advanced dementia of Alzheimer's type, while its involvement in mild and prodromal stages of the disease has been questioned. The issue is relevant to better understand the neuropathological basis of the diseases, but it is also of primary importance for therapy. During the last few years, indeed, cholinergic replacement therapies, mainly based on the use of acetylcholinesterase inhibitors to increase synaptic availability of acetylcholine, have been exploited on the assumption that they could ameliorate the progression of the dementia from its initial stages. In the present paper, we review data from human studies, as well as from animal models of Alzheimer's and Down's diseases, focusing on different ways to evaluate cholinergic dysfunction, also in relation to the time point at which these dysfunctions can be demonstrated, and on some discrepancy arising from the use of different methodological approaches. The reviewed literature, as well as some recent data from our laboratories on a mouse model of Down's syndrome, stress the importance of performing biochemical evaluation of choline acetyltransferase activity to assess cholinergic

  20. 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.

  1. 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

  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.

  3. Identification and analysis of aarP, a transcriptional activator of the 2'-N-acetyltransferase in Providencia stuartii.

    PubMed

    Macinga, D R; Parojcic, M M; Rather, P N

    1995-06-01

    The aarP gene has been identified in a search for activators of the 2-N-acetyltransferase [encoded by aac(2')-Ia] in Providencia stuartii. Introduction of aarP into P. stuartii on a multicopy plasmid resulted in a 9.9-fold increase in the accumulation of beta-galactosidase from an aac(2')-lacZ fusion. Northern (RNA) blot analysis demonstrated that this increased aac(2')-Ia expression occurred at the level of mRNA accumulation. The deduced AarP protein was 15,898 Da in size and exhibited significant homology to a number of transcriptional activators in the AraC/XyIS family, including TetD,Rob, MarA, and SoxS. The similarity of AarP to the MarA and SoxS proteins prompted an investigation to determine whether AarP is involved in activation of genes in either the multiple antibiotic resistance (Mar) phenotype or redox stress (SoxRS) system. Introduction of aarP on a multicopy plasmid into either P. stuartii or Escherichia coli conferred a Mar phenotype with higher levels of resistance to tetracycline, chloramphenicol, and ciprofloxacin. Multiple copies of aarP in E. coli also resulted in activation of the endonuclease IV gene (nfo), a gene in the SoxRS regulon of E. coli. The function of aarP in its single-copy state was addressed by using allelic replacement to construct an aarP::Cm disruption, which resulted in a fivefold reduction in the accumulation of aac(2')-Ia mRNA. Analysis of aarP regulation showed that aarP mRNA accumulation was slightly increased by exposure to tetracycline and dramatically increased in cells containing the aarB3 (aar3) mutation, which was previously shown to increase transcription of the aac(2')-Ia gene. (P.N. Rather, E. Oroz, K.J. Shaw, R. Hare, and G. Miller, J. Bacteriol. 175:6492-6498).

  4. Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases.

    PubMed

    Grauffel, Cédric; Abboud, Angèle; Liszczak, Glen; Marmorstein, Ronen; Arnesen, Thomas; Reuter, Nathalie

    2012-01-01

    Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate=MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate=MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate's backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1' sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions

  5. Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants

    PubMed Central

    Tavares, Sílvia; Wirtz, Markus; Beier, Marcel P.; Bogs, Jochen; Hell, Rüdiger; Amâncio, Sara

    2015-01-01

    In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT gene family of the crop plant Vitis vinifera. The identified four members of the VvSERAT protein family are assigned to three distinct groups upon their sequence similarities to Arabidopsis SERATs. Expression of fluorescently labeled VvSERAT proteins uncover that the sub-cellular localization of VvSERAT1;1 and VvSERAT3;1 is the cytosol and that VvSERAT2;1 and VvSERAT2;2 localize in addition in plastids and mitochondria, respectively. The purified VvSERATs of group 1 and 2 have higher enzymatic activity than VvSERAT3;1, which display a characteristic C-terminal extension also present in AtSERAT3;1. VvSERAT1;1 and VvSERAT2;2 are evidenced to form the CSC. CSC formation activates VvSERAT2;2, by releasing CSC-associated VvSERAT2;2 from cysteine inhibition. Thus, subcellular distribution of SERAT isoforms and CSC formation in cytosol and mitochondria is conserved between Arabidopsis and grapevine. Surprisingly, VvSERAT2;1 lack the canonical C-terminal tail of plant SERATs, does not form the CSC and is almost insensitive to cysteine inhibition (IC50 = 1.9 mM cysteine). Upon sulfate depletion VvSERAT2;1 is strongly induced at the transcriptional level, while transcription of other VvSERATs is almost unaffected in sulfate deprived grapevine cell suspension cultures. Application of abiotic stresses to soil grown grapevine plants revealed isoform-specific induction of VvSERAT2;1 in leaves upon drought, whereas high light- or temperature- stress hardly trigger VvSERAT2;1 transcription. PMID:25741355

  6. Characterization of the serine acetyltransferase gene family of Vitis vinifera uncovers differences in regulation of OAS synthesis in woody plants.

    PubMed

    Tavares, Sílvia; Wirtz, Markus; Beier, Marcel P; Bogs, Jochen; Hell, Rüdiger; Amâncio, Sara

    2015-01-01

    In higher plants cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL) and represents the last step of the assimilatory sulfate reduction pathway. It is mainly regulated by provision of O-acetylserine (OAS), the nitrogen/carbon containing backbone for fixation of reduced sulfur. OAS is synthesized by Serine acetyltransferase (SERAT), which reversibly interacts with OASTL in the cysteine synthase complex (CSC). In this study we identify and characterize the SERAT gene family of the crop plant Vitis vinifera. The identified four members of the VvSERAT protein family are assigned to three distinct groups upon their sequence similarities to Arabidopsis SERATs. Expression of fluorescently labeled VvSERAT proteins uncover that the sub-cellular localization of VvSERAT1;1 and VvSERAT3;1 is the cytosol and that VvSERAT2;1 and VvSERAT2;2 localize in addition in plastids and mitochondria, respectively. The purified VvSERATs of group 1 and 2 have higher enzymatic activity than VvSERAT3;1, which display a characteristic C-terminal extension also present in AtSERAT3;1. VvSERAT1;1 and VvSERAT2;2 are evidenced to form the CSC. CSC formation activates VvSERAT2;2, by releasing CSC-associated VvSERAT2;2 from cysteine inhibition. Thus, subcellular distribution of SERAT isoforms and CSC formation in cytosol and mitochondria is conserved between Arabidopsis and grapevine. Surprisingly, VvSERAT2;1 lack the canonical C-terminal tail of plant SERATs, does not form the CSC and is almost insensitive to cysteine inhibition (IC50 = 1.9 mM cysteine). Upon sulfate depletion VvSERAT2;1 is strongly induced at the transcriptional level, while transcription of other VvSERATs is almost unaffected in sulfate deprived grapevine cell suspension cultures. Application of abiotic stresses to soil grown grapevine plants revealed isoform-specific induction of VvSERAT2;1 in leaves upon drought, whereas high light- or temperature- stress hardly trigger VvSERAT2;1 transcription.

  7. Whole-exome-sequencing identifies mutations in histone acetyltransferase gene KAT6B in individuals with the Say-Barber-Biesecker variant of Ohdo syndrome.

    PubMed

    Clayton-Smith, Jill; O'Sullivan, James; Daly, Sarah; Bhaskar, Sanjeev; Day, Ruth; Anderson, Beverley; Voss, Anne K; Thomas, Tim; Biesecker, Leslie G; Smith, Philip; Fryer, Alan; Chandler, Kate E; Kerr, Bronwyn; Tassabehji, May; Lynch, Sally-Ann; Krajewska-Walasek, Malgorzata; McKee, Shane; Smith, Janine; Sweeney, Elizabeth; Mansour, Sahar; Mohammed, Shehla; Donnai, Dian; Black, Graeme

    2011-11-11

    Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS or Ohdo syndrome) is a multiple anomaly syndrome characterized by severe intellectual disability, blepharophimosis, and a mask-like facial appearance. A number of individuals with SBBYSS also have thyroid abnormalities and cleft palate. The condition usually occurs sporadically and is therefore presumed to be due in most cases to new dominant mutations. In individuals with SBBYSS, a whole-exome sequencing approach was used to demonstrate de novo protein-truncating mutations in the highly conserved histone acetyltransferase gene KAT6B (MYST4/MORF)) in three out of four individuals sequenced. Sanger sequencing was used to confirm truncating mutations of KAT6B, clustering in the final exon of the gene in all four individuals and in a further nine persons with typical SBBYSS. Where parental samples were available, the mutations were shown to have occurred de novo. During mammalian development KAT6B is upregulated specifically in the developing central nervous system, facial structures, and limb buds. The phenotypic features seen in the Qkf mouse, a hypomorphic Kat6b mutant, include small eyes, ventrally placed ears and long first digits that mirror the human phenotype. This is a further example of how perturbation of a protein involved in chromatin modification might give rise to a multisystem developmental disorder.

  8. Circadian dynamics of the cone-rod homeobox (CRX) transcription factor in the rat pineal gland and its role in regulation of arylalkylamine N-acetyltransferase (AANAT).

    PubMed

    Rohde, Kristian; Rovsing, Louise; Ho, Anthony K; Møller, Morten; Rath, Martin F

    2014-08-01

    The cone-rod homeobox (Crx) gene encodes a transcription factor in the retina and pineal gland. Crx deficiency influences the pineal transcriptome, including a reduced expression of arylalkylamine N-acetyltransferase (Aanat), a key enzyme in nocturnal pineal melatonin production. However, previous functional studies on pineal Crx have been performed in melatonin-deficient mice. In this study, we have investigated the role of Crx in the melatonin-proficient rat pineal gland. The current study shows that pineal Crx transcript levels exhibit a circadian rhythm with a peak in the middle of the night, which is transferred into daily changes in CRX protein. The study further shows that the sympathetic innervation of the pineal gland controls the Crx rhythm. By use of adenovirus-mediated short hairpin RNA gene knockdown targeting Crx mRNA in primary rat pinealocyte cell culture, we here show that intact levels of Crx mRNA are required to obtain high levels of Aanat expression, whereas overexpression of Crx induces Aanat transcription in vitro. This regulatory function of Crx is further supported by circadian analysis of Aanat in the pineal gland of the Crx-knockout mouse. Our data indicate that the rhythmic nature of pineal CRX protein may directly modulate the daily profile of Aanat expression by inducing nighttime expression of this enzyme, thus facilitating nocturnal melatonin synthesis in addition to its role in ensuring a correct tissue distribution of Aanat expression.

  9. Perinatal Choline Supplementation Reduces Amyloidosis and Increases Choline Acetyltransferase Expression in the Hippocampus of the APPswePS1dE9 Alzheimer's Disease Model Mice

    PubMed Central

    Mellott, Tiffany J.; Huleatt, Olivia M.; Shade, Bethany N.; Pender, Sarah M.; Liu, Yi B.; Slack, Barbara E.; Blusztajn, Jan K.

    2017-01-01

    Prevention of Alzheimer's disease (AD) is a major goal of biomedical sciences. In previous studies we showed that high intake of the essential nutrient, choline, during gestation prevented age-related memory decline in a rat model. In this study we investigated the effects of a similar treatment on AD-related phenotypes in a mouse model of AD. We crossed wild type (WT) female mice with hemizygous APPswe/PS1dE9 (APP.PS1) AD model male mice and maintained the pregnant and lactating dams on a control AIN76A diet containing 1.1 g/kg of choline or a choline-supplemented (5 g/kg) diet. After weaning all offspring consumed the control diet. As compared to APP.PS1 mice reared on the control diet, the hippocampus of the perinatally choline-supplemented APP.PS1 mice exhibited: 1) altered levels of amyloid precursor protein (APP) metabolites–specifically elevated amounts of β-C-terminal fragment (β-CTF) and reduced levels of solubilized amyloid Aβ40 and Aβ42 peptides; 2) reduced number and total area of amyloid plaques; 3) preserved levels of choline acetyltransferase protein (CHAT) and insulin-like growth factor II (IGF2) and 4) absence of astrogliosis. The data suggest that dietary supplementation of choline during fetal development and early postnatal life may constitute a preventive strategy for AD. PMID:28103298

  10. Effect of inhibition of aloe-emodin on N-acetyltransferase activity and gene expression in human malignant melanoma cells (A375.S2).

    PubMed

    Lin, Shuw-Yuan; Yang, Jen-Hung; Hsia, Te-Chun; Lee, Jau-Hong; Chiu, Tsan-Hung; Wei, Yau-Huei; Chung, Jing-Gung

    2005-12-01

    Arylamine carcinogens and drugs are N-acetylated by cytosolic N-acetyltransferase (NAT), which uses acetyl-coenzyme A as a cofactor. NAT plays an initial role in the metabolism of these arylamine compounds. 2-Aminofluorene is one of the arylamine carcinogens which have been demonstrated to undergo N-acetylation in laboratory animals and humans. Our previous study showed that human cancer cell lines (colon cancer, colo 205; liver cancer, Hep G2; bladder cancer, T24; leukemia, HL-60; prostate cancer, LNCaP; osteogenic sarcoma, U-2 OS; malignant melanoma, A375.S2) displayed NAT activity, which was affected by aloe-emodin in human leukemia cells. The purpose of this study was to determine whether aloe-emodin could affect the enzyme activity and gene expression of NAT at the mRNA and protein levels in malignant human melanoma A375.S2 cells. The results showed that aloe-emodin inhibited NAT1 activity (decreased N-acetylation of 2-aminofluorene) in intact cells in a dose-dependent manner. The effect of aloe-emodin on NAT1 at the protein level was determined by Western blotting and the mRNA levels were examined by polymerase chain reaction (PCR) and cDNA microarray. These results clearly indicate that aloe-emodin inhibits the mRNA expression and enzyme activity of NAT1 in A375.S2 cells.

  11. A rice chloroplast transit peptide sequence does not alter the cytoplasmic localization of sheep serotonin N-acetyltransferase expressed in transgenic rice plants.

    PubMed

    Byeon, Yeong; Lee, Hyoung Yool; Lee, Kyungjin; Back, Kyoungwhan

    2014-09-01

    Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin-rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N-acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild-type plants, but lower activity than transgenic rice plants expressing the wild-type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild-type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed-specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared with seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter.

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

    PubMed Central

    Huang, Fu

    2016-01-01

    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

  13. Molecular Structure of WlbB, a Bacterial N-Acetyltransferase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid

    SciTech Connect

    Thoden, James B.; Holden, Hazel M.

    2010-09-08

    The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 {angstrom} resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimeric quaternary structure and belongs to the L{beta}H superfamily of N-acyltransferases. Each subunit contains 27 {beta}-strands, 23 of which form the canonical left-handed {beta}-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O{sup {delta}1} of Asn 84 and the sugar C-3{prime} amino group and the second between the backbone amide group of Arg 94 and the sugar C-5{prime} carboxylate. The sugar C-3{prime} amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.

  14. aarC, an essential gene involved in density-dependent regulation of the 2'-N-acetyltransferase in Providencia stuartii.

    PubMed

    Rather, P N; Solinsky, K A; Paradise, M R; Parojcic, M M

    1997-04-01

    The 2'-N-acetyltransferase [AAC(2')-Ia] in Providencia stuartii has a dual function where it is involved in the acetylation of peptidoglycan and certain aminoglycosides. A search for negative regulators of the aac(2')-Ia gene has resulted in the identification of aarC. A missense allele (aarC1) resulted in an 8.9-fold increase in beta-galactosidase accumulation from an aac(2')-lacZ transcriptional fusion. Northern blot analysis demonstrated an increase in aac(2')-Ia mRNA accumulation that was specific to cells at high density. In addition, the aarC1 allele also resulted in a substantial increase in the expression of aarP, a transcriptional activator of the aac(2')-Ia gene. The wild-type aarC gene was isolated by complementation and encodes a predicted protein of 365 amino acids with a molecular mass of 39,815 Da. The predicted AarC protein exhibited 88% amino acid homology to the previously identified GcpE protein of Escherichia coli and 86% homology to a gene product from Haemophilus influenzae. The E. coli gcpE gene was able to functionally complement the aarC1 allele in P. stuartii. The aarC1 allele was identified as a T to G transversion that resulted in a valine to glycine substitution at position 136 in the AarC protein. The aarC gene appears to be essential for cell viability as construction of a disrupted copy (aarC::lacZ) was possible only in cells that carried an episomal copy of aarC or gcpE.

  15. A missense mutation in the glucosamine-6-phosphate N-acetyltransferase-encoding gene causes temperature-dependent growth defects and ectopic lignin deposition in Arabidopsis.

    PubMed

    Nozaki, Mamoru; Sugiyama, Munetaka; Duan, Jun; Uematsu, Hiroshi; Genda, Tatsuya; Sato, Yasushi

    2012-08-01

    To study the regulatory mechanisms underlying lignin biosynthesis, we isolated and characterized lignescens (lig), a previously undescribed temperature-sensitive mutant of Arabidopsis thaliana that exhibits ectopic lignin deposition and growth defects under high-temperature conditions. The lig mutation was identified as a single base transition in GNA1 encoding glucosamine-6-phosphate N-acetyltransferase (GNA), a critical enzyme of UDP-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. lig harbors a glycine-to-serine substitution at residue 68 (G68S) of GNA1. Enzyme activity assays of the mutant protein (GNA1(G68S)) showed its thermolability relative to the wild-type protein. The lig mutant exposed to the restrictive temperature contained a significantly smaller amount of UDP-GlcNAc than did the wild type. The growth defects and ectopic lignification of lig were suppressed by the addition of UDP-GlcNAc. Since UDP-GlcNAc is an initial sugar donor of N-glycan synthesis and impaired N-glycan synthesis is known to induce the unfolded protein response (UPR), we examined possible relationships between N-glycan synthesis, UPR, and the lig phenotype. N-glycans were reduced and LUMINAL BINDING PROTEIN3, a typical UPR gene, was expressed in lig at the restrictive temperature. Furthermore, treatment with UPR-inducing reagents phenocopied the lig mutant. Our data collectively suggest that impairment of N-glycan synthesis due to a shortage of UDP-GlcNAc leads to ectopic lignin accumulation, mostly through the UPR.

  16. Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells

    PubMed Central

    2010-01-01

    Background CREB and CREM are closely related factors that regulate transcription in response to various stress, metabolic and developmental signals. The CREMτ activator isoform is selectively expressed in haploid spermatids and plays an essential role in murine spermiogenesis. Results We have used chromatin immunoprecipitation coupled to sequencing (ChIP-seq) to map CREM and CREB target loci in round spermatids from adult mouse testis and spermatogonia derived GC1-spg cells respectively. We identify more than 9000 genomic loci most of which are cell-specifically occupied. Despite the fact that round spermatids correspond to a highly specialised differentiated state, our results show that they have a remarkably accessible chromatin environment as CREM occupies more than 6700 target loci corresponding not only to the promoters of genes selectively expressed in spermiogenesis, but also of genes involved in functions specific to other cell types. The expression of only a small subset of these target genes are affected in the round spermatids of CREM knockout animals. We also identify a set of intergenic binding loci some of which are associated with H3K4 trimethylation and elongating RNA polymerase II suggesting the existence of novel CREB and CREM regulated transcripts. Conclusions We demonstrate that CREM and CREB occupy a large number of promoters in highly cell specific manner. This is the first study of CREM target promoters directly in a physiologically relevant tissue in vivo and represents the most comprehensive experimental analysis of CREB/CREM regulatory potential to date. PMID:20920259

  17. Chloroplastic and cytoplasmic overexpression of sheep serotonin N-acetyltransferase in transgenic rice plants is associated with low melatonin production despite high enzyme activity.

    PubMed

    Byeon, Yeong; Lee, Hyoung Yool; Back, Kyoungwhan

    2015-05-01

    Serotonin N-acetyltransferase (SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N-acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep (Ovis aries) SNAT (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast-targeted CTS:OaSNAT lines and cytoplasm-expressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N-acetylserotonin, melatonin, or 2-hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis.

  18. Overexpression of rice serotonin N-acetyltransferase 1 in transgenic rice plants confers resistance to cadmium and senescence and increases grain yield.

    PubMed

    Lee, Kyungjin; Back, Kyoungwhan

    2017-04-01

    While ectopic overexpression of serotonin N-acetyltransferase (SNAT) in plants has been accomplished using animal SNAT genes, ectopic overexpression of plant SNAT genes in plants has not been investigated. Because the plant SNAT protein differs from that of animals in its subcellular localization and enzyme kinetics, its ectopic overexpression in plants would be expected to give outcomes distinct from those observed from overexpression of animal SNAT genes in transgenic plants. Consistent with our expectations, we found that transgenic rice plants overexpressing rice (Oryza sativa) SNAT1 (OsSNAT1) did not show enhanced seedling growth like that observed in ovine SNAT-overexpressing transgenic rice plants, although both types of plants exhibited increased melatonin levels. OsSNAT1-overexpressing rice plants did show significant resistance to cadmium and senescence stresses relative to wild-type controls. In contrast to tomato, melatonin synthesis in rice seedlings was not induced by selenium and OsSNAT1 transgenic rice plants did not show tolerance to selenium. T2 homozygous OsSNAT1 transgenic rice plants exhibited increased grain yield due to increased panicle number per plant under paddy field conditions. These benefits conferred by ectopic overexpression of OsSNAT1 had not been observed in transgenic rice plants overexpressing ovine SNAT, suggesting that plant SNAT functions differently from animal SNAT in plants.

  19. The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair.

    PubMed

    Robert, Flavie; Hardy, Sara; Nagy, Zita; Baldeyron, Céline; Murr, Rabih; Déry, Ugo; Masson, Jean-Yves; Papadopoulo, Dora; Herceg, Zdenko; Tora, Làszlò

    2006-01-01

    Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

  20. Broad-substrate screen as a tool to identify substrates for bacterial Gcn5-related N-acetyltransferases with unknown substrate specificity.

    PubMed

    Kuhn, Misty L; Majorek, Karolina A; Minor, Wladek; Anderson, Wayne F

    2013-02-01

    Due to a combination of efforts from individual laboratories and structural genomics centers, there has been a surge in the number of members of the Gcn5-related acetyltransferasesuperfamily that have been structurally determined within the past decade. Although the number of three-dimensional structures is increasing steadily, we know little about the individual functions of these enzymes. Part of the difficulty in assigning functions for members of this superfamily is the lack of information regarding how substrates bind to the active site of the protein. The majority of the structures do not show ligand bound in the active site, and since the substrate-binding domain is not strictly conserved, it is difficult to predict the function based on structure alone. Additionally, the enzymes are capable of acetylating a wide variety of metabolites and many may exhibit promiscuity regarding their ability to acetylate multiple classes of substrates, possibly having multiple functions for the same enzyme. Herein, we present an approach to identify potential substrates for previously uncharacterized members of the Gcn5-related acetyltransferase superfamily using a variety of metabolites including polyamines, amino acids, antibiotics, peptides, vitamins, catecholamines, and other metabolites. We have identified potential substrates for eight bacterial enzymes of this superfamily. This information will be used to further structurally and functionally characterize them.

  1. Arabidopsis serotonin N-acetyltransferase knockout mutant plants exhibit decreased melatonin and salicylic acid levels resulting in susceptibility to an avirulent pathogen.

    PubMed

    Lee, Hyoung Yool; Byeon, Yeong; Tan, Dun-Xian; Reiter, Russel J; Back, Kyoungwhan

    2015-04-01

    Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthesis pathway in plants. We examined the effects of SNAT gene inactivation in two Arabidopsis T-DNA insertion mutant lines. After inoculation with the avirulent pathogen Pseudomonas syringe pv. tomato DC3000 harboring the elicitor avrRpt2 (Pst-avrRpt2), melatonin levels in the snat knockout mutant lines were 50% less than in wild-type Arabidopsis Col-0 plants. The snat knockout mutant lines exhibited susceptibility to pathogen infection that coincided with decreased induction of defense genes including PR1, ICS1, and PDF1.2. Because melatonin acts upstream of salicylic acid (SA) synthesis, the reduced melatonin levels in the snat mutant lines led to decreased SA levels compared to wild-type, suggesting that the increased pathogen susceptibility of the snat mutant lines could be attributed to decreased SA levels and subsequent attenuation of defense gene induction. Exogenous melatonin treatment failed to induce defense gene expression in nahG Arabidopsis plants, but restored the induction of defense gene expression in the snat mutant lines. In addition, melatonin caused translocation of NPR1 (nonexpressor of PR1) protein from the cytoplasm into the nucleus indicating that melatonin-elicited pathogen resistance in response to avirulent pathogen attack is SA-dependent in Arabidopsis.

  2. A silk peptide fraction restores cognitive function in AF64A-induced Alzheimer disease model rats by increasing expression of choline acetyltransferase gene.

    PubMed

    Cha, Yeseul; Lee, Sang Hoon; Jang, Su Kil; Guo, Haiyu; Ban, Young-Hwan; Park, Dongsun; Jang, Gwi Yeong; Yeon, Sungho; Lee, Jeong-Yong; Choi, Ehn-Kyoung; Joo, Seong Soo; Jeong, Heon-Sang; Kim, Yun-Bae

    2017-01-01

    This study investigated the effects of a silk peptide fraction obtained by incubating silk proteins with Protease N and Neutrase (SP-NN) on cognitive dysfunction of Alzheimer disease model rats. In order to elucidate underlying mechanisms, the effect of SP-NN on the expression of choline acetyltransferase (ChAT) mRNA was assessed in F3.ChAT neural stem cells and Neuro2a neuroblastoma cells; active amino acid sequence was identified using HPLC-MS. The expression of ChAT mRNA in F3.ChAT cells increased by 3.79-fold of the control level by treatment with SP-NN fraction. The active peptide in SP-NN was identified as tyrosine-glycine with 238.1 of molecular weight. Male rats were orally administered with SP-NN (50 or 300mg/kg) and challenged with a cholinotoxin AF64A. As a result of brain injury and decreased brain acetylcholine level, AF64A induced astrocytic activation, resulting in impairment of learning and memory function. Treatment with SP-NN exerted recovering activities on acetylcholine depletion and brain injury, as well as cognitive deficit induced by AF64A. The results indicate that, in addition to a neuroprotective activity, the SP-NN preparation restores cognitive function of Alzheimer disease model rats by increasing the release of acetylcholine.

  3. Yng1p Modulates the Activity of Sas3p as a Component of the Yeast NuA3 Histone Acetyltransferase Complex

    PubMed Central

    Howe, LeAnn; Kusch, Thomas; Muster, Nemone; Chaterji, Ranjana; Yates III, John R.; Workman, Jerry L.

    2002-01-01

    The mammalian ING1 gene encodes a tumor suppressor required for the function of p53. In this study we report a novel function for YNG1, a yeast homolog of ING1. Yng1p is a stable component of the NuA3 histone acetyltransferase complex, which contains Sas3p, the yeast homolog of the mammalian MOZ proto-oncogene product, as its catalytic subunit. Yng1p is required for NuA3 function in vivo but surprisingly is not required for the integrity of the complex. Instead, we find that Yng1p mediates the interaction of Sas3p with nucleosomes and is thus required for the ability of NuA3 to modify histone tails. These data, and the observations that other ING1 homologs are found in additional yeast complexes that posttranslationally modify histones, suggest that members of the ING1 class of proteins may have broad roles in enhancing or modifying the activities of chromatin-modifying complexes, thereby regulating their activities in transcription control. PMID:12077334

  4. 82-kDa choline acetyltransferase is in nuclei of cholinergic neurons in human CNS and altered in aging and Alzheimer disease.

    PubMed

    Gill, Sandeep K; Ishak, Margaret; Dobransky, Tomas; Haroutunian, Vahram; Davis, Kenneth L; Rylett, R Jane

    2007-07-01

    Cholinergic neurons express choline acetyltransferase (ChAT) which synthesizes acetylcholine. We show here for the first time that primate-specific 82-kDa ChAT is expressed in nuclei of cholinergic neurons in human brain and spinal cord; isoform-specific antibodies were used to compare localization patterns and temporal expression of the more abundant 69-kDa ChAT and primate-specific 82-kDa ChAT in necropsy tissues. The 82-kDa ChAT co-localizes with 69-kDa ChAT in well-characterized cholinergic areas, but is also found in the claustrum which does not contain 69-kDa ChAT. Cholinergic neuron function changes with increasing age and are targeted in neurodegenerative diseases such as AD, thus we compared expression and subcellular localization of 69- and 82-kDa ChAT in necropsy brain samples from control subjects of varying ages and from Alzheimer disease (AD) subjects. The 82-kDa ChAT protein was expressed in cholinergic neurons in brain from birth until the eighth decade of life and in AD, but the subcellular staining pattern and proportion of neurons that were immunopositive changed with increasing age and in AD.

  5. Functional cooperation between Smad proteins and activator protein-1 regulates transforming growth factor-beta-mediated induction of endothelin-1 expression.

    PubMed

    Rodríguez-Pascual, Fernando; Redondo-Horcajo, Mariano; Lamas, Santiago

    2003-06-27

    Endothelin-1 (ET-1) is a 21-amino-acid potent vasoconstrictor peptide that is mainly produced by vascular endothelial cells. Expression of the ET-1 gene is subject to complex regulation by numerous factors, among which transforming growth factor-beta (TGF-beta) is one of the most important. It has been widely documented that TGF-beta increases ET-1 mRNA and peptide levels. We have explored the mechanism by which TGF-beta upregulates ET-1 expression in endothelial cells. Transcriptional activation of the ET-1 promoter accounted for the TGF-beta-induced increase in ET-1 mRNA levels. We have identified within the ET-1 promoter two DNA elements indispensable for TGF-beta-mediated induction of ET-1: an activator protein-1 (AP-1) site at -108/-102, known to be important for constitutive and induced expression, and a novel regulatory sequence located at -193/-171, which constitutes a specific binding site for Smad transcription factors. Mutation of both elements abolished TGF-beta responsiveness. Binding of Smad3/Smad4 and c-Jun to their corresponding DNA elements was evidenced by electrophoretic mobility shift assays. Furthermore, the coactivator CREB-binding protein (CBP)/p300 was found to play an essential role in the induction of the gene. The simultaneous requirement for two distinct and independent DNA elements suggests that Smads and activator protein-1 functionally cooperate through CBP/p300 to mediate TGF-beta-induced transcriptional activation of the ET-1 gene.

  6. [Evaluation of a caffeine test for determining the phenotype of N-acetyltransferase].

    PubMed

    Gascon, M P; Leemann, T; Dayer, P

    1987-12-05

    Xenobiotic acetylation by N-acetyltransferase is genetically controlled. This polymorphism governs the intestinal and liver metabolism of numerous amines. The use of caffeine, a ubiquitous and nontoxic amine, has been proposed as a probe for phenotyping. The aim of the present study is to evaluate this test and to identify the metabolite of caffeine used as substrate by the polymorphic enzyme. - A cup of coffee, tea or Coca-Cola is administered to fasting subjects. The molar ratio of two metabolites of caffeine (AFMU and 1X) is determined on a spot urine sample 4-6 hours later by means of a UV liquid chromatographic assay. In a reference population (n = 63), the distribution of molar ratios is trimodal with frequencies of 0.14, 0.35 and 0.51. These results correlate with those obtained by the classic isoniazid test. However, in vitro experiments in human liver subcellular fractions did not lead to the identification of a xanthine as the precursor of the acetylated metabolite.

  7. Synthesis of isothiazol-3-one derivatives as inhibitors of histone acetyltransferases (HATs).

    PubMed

    Gorsuch, Stephen; Bavetsias, Vassilios; Rowlands, Martin G; Aherne, G Wynne; Workman, Paul; Jarman, Michael; McDonald, Edward

    2009-01-15

    High-throughput screening led to the identification of isothiazolones 1 and 2 as inhibitors of histone acetyltransferase (HAT) with IC50s of 3 microM and 5 microM, respectively. Analogues of these hit compounds with variations of the N-phenyl group, and with variety of substituents at C-4, C-5 of the thiazolone ring, were prepared and assayed for inhibition of the HAT enzyme PCAF. Potency is modestly favoured when the N-aryl group is electron deficient (4-pyridyl derivative 10 has IC(50)=1.5 microM); alkyl substitution at C-4 has little effect, whilst similar substitution at C-5 causes a significant drop in potency. The ring-fused compound 38 has activity (IC(50)=6.1 microM) to encourage further exploration of this bicyclic structure. The foregoing SAR is consistent with an inhibitory mechanism involving cleavage of the S-N bond of the isothiazolone ring by a catalytically important thiol residue.

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

    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.

  10. Contribution of gentamicin 2'-N-acetyltransferase to the O acetylation of peptidoglycan in Providencia stuartii.

    PubMed

    Payie, K G; Rather, P N; Clarke, A J

    1995-08-01

    A collection of Providencia stuartii mutants which either underexpress or overexpress aac(2')-Ia, the chromosomal gene coding for gentamicin 2'-N-acetyltransferase (EC 2.3.1.59), have been characterized phenotypically as possessing either lower or higher levels of peptidoglycan O acetylation, respectively, than the wild type. These mutants were subjected to both negative-staining and thin-section electron microscopy. P. stuartii PR100, with 42% O acetylation of peptidoglycan compared with 52% O acetylation in the wild type, appeared as irregular rods. In direct contrast, P. stuartii strains PR50.LM3 and PR51, with increased levels of peptidoglycan O acetylation (65 and 63%, respectively), appeared as coccobacilli and chain formers, respectively. Membrane blebbing was also observed with the chain-forming strain PR51. Thin sectioning of this mutant indicated that it was capable of proper constriction and separation. P. stuartii PM1, when grown to mid-exponential phase, did not have altered peptidoglycan O-acetylation levels, and cellular morphology remained similar to that of wild-type strains. However, continued growth into stationary phase resulted in a 15% increase in peptidoglycan O acetylation concomitant with a change of some cells from a rod-shaped to a coccobacillus-shaped morphology. The fact that these apparent morphological changes were directly related to levels of O acetylation support the view that this modification plays a role in the maintenance of peptidoglycan structure, presumably through the control of autolytic activity.

  11. Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene

    PubMed Central

    Knowles, Joshua W.; Xie, Weijia; Zhang, Zhongyang; Chennemsetty, Indumathi; Assimes, Themistocles L.; Paananen, Jussi; Hansson, Ola; Pankow, James; Goodarzi, Mark O.; Carcamo-Orive, Ivan; Morris, Andrew P.; Chen, Yii-Der I.; Mäkinen, Ville-Petteri; Ganna, Andrea; Mahajan, Anubha; Guo, Xiuqing; Abbasi, Fahim; Greenawalt, Danielle M.; Lum, Pek; Molony, Cliona; Lind, Lars; Lindgren, Cecilia; Raffel, Leslie J.; Tsao, Philip S.; Schadt, Eric E.; Rotter, Jerome I.; Sinaiko, Alan; Reaven, Gerald; Yang, Xia; Hsiung, Chao A.; Groop, Leif; Cordell, Heather J.; Laakso, Markku; Hao, Ke; Ingelsson, Erik; Frayling, Timothy M.; Weedon, Michael N.; Walker, Mark; Quertermous, Thomas

    2015-01-01

    Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 “A” allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity. PMID:25798622

  12. Effects of humic acid-metal complexes on hepatic carnitine palmitoyltransferase, carnitine acetyltransferase and catalase activities

    SciTech Connect

    Fungjou Lu; Youngshin Chen . Dept. of Biochemistry); Tienshang Huang . Dept. of Medicine)

    1994-03-01

    A significant increase in activities of hepatic carnitine palmitoyltransferase and carnitine acetyltransferase was observed in male Balb/c mice intraperitoneally injected for 40 d with 0.125 mg/0.1 ml/d humic acid-metal complexes. Among these complexes, the humic acid-As complex was relatively effective, whereas humic acid-25 metal complex was more effective, and humic acid-26 metal complex was most effective. However, humic acid or metal mixtures, or metal such as As alone, was not effective. Humic acid-metal complexes also significantly decreased hepatic catalase activity. A marked decrease of 60-kDa polypeptide in liver cytoplasm was also observed on SDS-polyacrylamide gel electrophoresis after the mice had been injected with the complexes. Morphological analysis of a histopathological biopsy of such treated mice revealed several changes in hepatocytes, including focal necrosis and cell infiltration, mild fatty changes, reactive nuclei, and hypertrophy. Humic acid-metal complexes affect activities of metabolic enzymes of fatty acids, and this results in accumulation of hydrogen peroxide and increase of the lipid peroxidation. The products of lipid peroxidation may be responsible for liver damage and possible carcinogenesis. Previous studies in this laboratory had shown that humic acid-metal complex altered the coagulation system and that humic acid, per se, caused vasculopathy. Therefore, humic acid-metal complexes may be main causal factors of not only so-called blackfoot disease, but also the liver cancer prevailing on the southwestern coast of Taiwan.

  13. 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.

  14. Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase.

    PubMed

    Tao, Weixin; Lee, Myung Hwan; Yoon, Mi-Young; Kim, Jin-Cheol; Malhotra, Shweta; Wu, Jing; Hwang, Eul Chul; Lee, Seon-Woo

    2011-12-01

    Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.

  15. The Aspergillus flavus Histone Acetyltransferase AflGcnE Regulates Morphogenesis, Aflatoxin Biosynthesis, and Pathogenicity

    PubMed Central

    Lan, Huahui; Sun, Ruilin; Fan, Kun; Yang, Kunlong; Zhang, Feng; Nie, Xin Y.; Wang, Xiunai; Zhuang, Zhenhong; Wang, Shihua

    2016-01-01

    Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops. PMID:27625637

  16. Application of the chloramphenicol acetyltransferase (CAT) diffusion assay to transgenic plant tissues.

    PubMed

    Peach, C; Velten, J

    1992-02-01

    Chloramphenicol acetyltransferase (CAT) activity was quantified in crude extracts from tobacco callus tissues using a modification of a previously reported diffusion assay. We describe here the alterations necessary in applying this rapid and simple assay procedure to plant materials. Due to the high concentration of nonspecific oxidases present in most plant tissues, some type of protective agent is required to maintain enzyme activity. We have tested beta-mercaptoethanol, cysteine, dithiothreitol, ascorbic acid and polyvinyl pyrrolidone as protective agents within the initial extraction buffer. We also investigated the effect of heat (60 degrees C, 10 min) and 5 mM EDTA on CAT activity. The highest CAT activity was obtained using 5 mM cysteine plus 5 mM EDTA in 40 mM Tris-HCl (pH 7.8) as the initial extraction buffer followed by a heat treatment. Using this buffer, CAT activity was stable on ice for more than two hours. In our hands, total acetyl-coenzyme A concentration within the assay mixture was found to be saturating at 250 microM and the Km determined to be 100 microM. Assays performed using the same crude plant extract indicate that 1) duplicate assays show less than 1.5% variation in activities and 2) CAT activity increases linearly with respect to volume of extract used.

  17. Environmental History Modulates Arabidopsis Pattern-Triggered Immunity in a HISTONE ACETYLTRANSFERASE1-Dependent Manner.

    PubMed

    Singh, Prashant; Yekondi, Shweta; Chen, Po-Wen; Tsai, Chia-Hong; Yu, Chun-Wei; Wu, Keqiang; Zimmerli, Laurent

    2014-06-01

    In nature, plants are exposed to a fluctuating environment, and individuals exposed to contrasting environmental factors develop different environmental histories. Whether different environmental histories alter plant responses to a current stress remains elusive. Here, we show that environmental history modulates the plant response to microbial pathogens. Arabidopsis thaliana plants exposed to repetitive heat, cold, or salt stress were more resistant to virulent bacteria than Arabidopsis grown in a more stable environment. By contrast, long-term exposure to heat, cold, or exposure to high concentrations of NaCl did not provide enhanced protection against bacteria. Enhanced resistance occurred with priming of Arabidopsis pattern-triggered immunity (PTI)-responsive genes and the potentiation of PTI-mediated callose deposition. In repetitively stress-challenged Arabidopsis, PTI-responsive genes showed enrichment for epigenetic marks associated with transcriptional activation. Upon bacterial infection, enrichment of RNA polymerase II at primed PTI marker genes was observed in environmentally challenged Arabidopsis. Finally, repetitively stress-challenged histone acetyltransferase1-1 (hac1-1) mutants failed to demonstrate enhanced resistance to bacteria, priming of PTI, and increased open chromatin states. These findings reveal that environmental history shapes the plant response to bacteria through the development of a HAC1-dependent epigenetic mark characteristic of a primed PTI response, demonstrating a mechanistic link between the primed state in plants and epigenetics.

  18. A series of shuttle vectors using chloramphenicol acetyltransferase as a reporter enzyme in yeast.

    PubMed

    Mannhaupt, G; Pilz, U; Feldmann, H

    1988-07-30

    Reports from numerous laboratories have shown that the gene coding for the bacterial enzyme chloramphenicol-3-O-acetyltransferase can be used as a reporter gene (cat) in mammalian and plant systems to analyze gene activity at the transcriptional level when combined with endogenous regulatory signals; the enzyme activity can be quantified by a chromatographic or a photometric assay. To adapt this simple and highly sensitive test for the yeast system, we constructed a series of yeast vectors containing the cat gene together with selectable markers for Escherichia coli and yeast; integrating, autonomously replicating and centromere-carrying plasmids were used. We show that the cat gene lacking the endogenous promoter is expressed at low levels in yeast transformants. To demonstrate functional expression of the cat gene placed under the control of a yeast promoter, we chose the PHO5 regulatory region. We found that cat expression was induced via the PHO5 promoter in a manner as observed for the endogenous PHO5 gene, whereas in the repressed state cat expression remained low. Using these vectors, it should be feasible to analyze other sequences conferring promoter activity or other control functions in yeast.

  19. Identification and validation of N-acetyltransferase 2 as an insulin sensitivity gene.

    PubMed

    Knowles, Joshua W; Xie, Weijia; Zhang, Zhongyang; Chennamsetty, Indumathi; Chennemsetty, Indumathi; Assimes, Themistocles L; Paananen, Jussi; Hansson, Ola; Pankow, James; Goodarzi, Mark O; Carcamo-Orive, Ivan; Morris, Andrew P; Chen, Yii-Der I; Mäkinen, Ville-Petteri; Ganna, Andrea; Mahajan, Anubha; Guo, Xiuqing; Abbasi, Fahim; Greenawalt, Danielle M; Lum, Pek; Molony, Cliona; Lind, Lars; Lindgren, Cecilia; Raffel, Leslie J; Tsao, Philip S; Schadt, Eric E; Rotter, Jerome I; Sinaiko, Alan; Reaven, Gerald; Yang, Xia; Hsiung, Chao A; Groop, Leif; Cordell, Heather J; Laakso, Markku; Hao, Ke; Ingelsson, Erik; Frayling, Timothy M; Weedon, Michael N; Walker, Mark; Quertermous, Thomas

    2015-04-01

    Decreased insulin sensitivity, also referred to as insulin resistance (IR), is a fundamental abnormality in patients with type 2 diabetes and a risk factor for cardiovascular disease. While IR predisposition is heritable, the genetic basis remains largely unknown. The GENEticS of Insulin Sensitivity consortium conducted a genome-wide association study (GWAS) for direct measures of insulin sensitivity, such as euglycemic clamp or insulin suppression test, in 2,764 European individuals, with replication in an additional 2,860 individuals. The presence of a nonsynonymous variant of N-acetyltransferase 2 (NAT2) [rs1208 (803A>G, K268R)] was strongly associated with decreased insulin sensitivity that was independent of BMI. The rs1208 "A" allele was nominally associated with IR-related traits, including increased fasting glucose, hemoglobin A1C, total and LDL cholesterol, triglycerides, and coronary artery disease. NAT2 acetylates arylamine and hydrazine drugs and carcinogens, but predicted acetylator NAT2 phenotypes were not associated with insulin sensitivity. In a murine adipocyte cell line, silencing of NAT2 ortholog Nat1 decreased insulin-mediated glucose uptake, increased basal and isoproterenol-stimulated lipolysis, and decreased adipocyte differentiation, while Nat1 overexpression produced opposite effects. Nat1-deficient mice had elevations in fasting blood glucose, insulin, and triglycerides and decreased insulin sensitivity, as measured by glucose and insulin tolerance tests, with intermediate effects in Nat1 heterozygote mice. Our results support a role for NAT2 in insulin sensitivity.

  20. Variation of the N-acetyltransferase 2 gene in a Romanian and a Kyrgyz population.

    PubMed

    Rabstein, Sylvia; Unfried, Klaus; Ranft, Ulrich; Illig, Thomas; Kolz, Melanie; Rihs, Hans-Peter; Mambetova, Chinara; Vlad, Mariana; Brüning, Thomas; Pesch, Beate

    2006-01-01

    As part of a project on environmental disasters in minority populations, this study aimed to evaluate differences in the sequence of N-acetyltransferase 2 (NAT2) as a metabolic susceptibility gene in yet unexplored ethnicities. Eight single nucleotide polymorphisms (SNP) in the NAT2 coding region and a variant in the 3' flanking region were analyzed in 290 unrelated Kyrgyz and 140 unrelated Romanians by SNP-specific PCR analysis. The variants 341C, 481T, and 803G were less and 857A more prevalent in Kyrgyz (P < 0.0001). The variant at site 857 indicates Asian descent. 282C>T and 590G>A showed no significant variation by ethnicity. 364G>A and 411A>T turned out to be monomorphic. Database comparisons of the NAT2 minor allele frequencies support that Romanians belong to Caucasians and Kyrgyz are in between Caucasians and East Asians. The distributions of predicted haplotypes differed significantly between the two ethnicities where the Kyrgyz showed a higher genetic diversity. The haplotype without mutations was more common in Kyrgyz (40.1% in Kyrgyz, 29.3% in Romanians). Accordingly, the imputed slow acetylator phenotype was less prevalent in Kyrgyz (35.2% versus 51.4% in Romanians). We found pronounced ethnic differences in NAT2 genotypes with yet unknown effect on the health risks for environmental or occupational exposures in minority populations.

  1. Muscle-specific Deletion of Carnitine Acetyltransferase Compromises Glucose Tolerance and Metabolic Flexibility

    PubMed Central

    Muoio, Deborah M.; Noland, Robert C.; Kovalik, Jean-Paul; Seiler, Sarah E.; Davies, Michael N.; DeBalsi, Karen L.; Ilkayeva, Olga R.; Stevens, Robert D.; Kheterpal, Indu; Zhang, Jingying; Covington, Jeffrey D.; Bajpeyi, Sudip; Ravussin, Eric; Kraus, William; Koves, Timothy R.; Mynatt, Randall L.

    2012-01-01

    Summary The concept of “metabolic inflexibility” was first introduced to describe the failure of insulin resistant human subjects to appropriately adjust mitochondrial fuel selection in response to nutritional cues. This phenomenon has since gained increasing recognition as a core component of the metabolic syndrome, but the underlying mechanisms have remained elusive. Here, we identify an essential role for the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT), in regulating substrate switching and glucose tolerance. By converting acetyl-CoA to its membrane permeant acetylcarnitine ester, CrAT regulates mitochondrial and intracellular carbon trafficking. Studies in muscle-specific Crat knockout mice, primary human skeletal myocytes and human subjects undergoing L-carnitine supplementation support a model wherein CrAT combats nutrient stress, promotes metabolic flexibility and enhances insulin action by permitting mitochondrial efflux of excess acetyl moieties that otherwise inhibit key regulatory enzymes such as pyruvate dehydrogenase. These findings offer therapeutically relevant insights into the molecular basis of metabolic inflexibility. PMID:22560225

  2. 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

  3. Role of Saccharomyces cerevisiae serine O-acetyltransferase in cysteine biosynthesis.

    PubMed

    Takagi, Hiroshi; Yoshioka, Kenji; Awano, Naoki; Nakamori, Shigeru; Ono, Bun ichiro

    2003-01-28

    Some strains of Saccharomyces cerevisiae have detectable activities of L-serine O-acetyltransferase (SATase) and O-acetyl-L-serine/O-acetyl-L-homoserine sulfhydrylase (OAS/OAH-SHLase), but synthesize L-cysteine exclusively via cystathionine by cystathionine beta-synthase and cystathionine gamma-lyase. To untangle this peculiar feature in sulfur metabolism, we introduced Escherichia coli genes encoding SATase and OAS-SHLase into S. cerevisiae L-cysteine auxotrophs. While the cells expressing SATase grew on medium lacking L-cysteine, those expressing OAS-SHLase did not grow at all. The cells expressing both enzymes grew very well without L-cysteine. These results indicate that S. cerevisiae SATase cannot support L-cysteine biosynthesis and that S. cerevisiae OAS/OAH-SHLase produces L-cysteine if enough OAS is provided by E. coli SATase. It appears as if S. cerevisiae SATase does not possess a metabolic role in vivo either because of very low activity or localization. For example, S. cerevisiae SATase may be localized in the nucleus, thus controlling the level of OAS required for regulation of sulfate assimilation, but playing no role in the direct synthesis of L-cysteine.

  4. Radiosensitizing effect of the histone acetyltransferase inhibitor anacardic acid on various mammalian cell lines

    PubMed Central

    CATE, ROSEMARIE TEN; KRAWCZYK, PRZEMEK; STAP, JAN; ATEN, JACOB A.; FRANKEN, NICOLAAS A.P.

    2010-01-01

    Agents that enhance the effectiveness of ionizing radiation have been investigated over many decades. A relatively new group of potential radiosensitizers consists of agents that inhibit histone acetyltransferases (HATs). This study evaluated the radiosensitizing properties of the HAT inhibitor anacardic acid (AA), used at a low-toxic concentration of 100 μM in V79, SW1573 and U2OS cells. Radiation survival curves were analyzed according to the linear quadratic model. Significant radiosensitization by AA was only obtained in U2OS cells. AA significantly increased the value of the linear parameter α, but not of the quadratic parameter β, indicating fixation of potentially lethal damage and an intact repair function of sublethal damage. The increase of the α value was also observed in SW1573 cells, but was not accompanied by a significant radiosensitization. A likely explanation for the enhancement of the α value may be an increase in the amount of lethal lesions due to the compacted chromatin structure. Despite the conflicting results of the radiosensitizing effect of AA in the three cell lines tested, the ability of AA to increase the α value suggests potential advantages for clinical application. PMID:22966377

  5. Deletion of host histone acetyltransferases and deacetylases strongly affects Agrobacterium-mediated transformation of Saccharomyces cerevisiae.

    PubMed

    Soltani, Jalal; van Heusden, Gerard Paul H; Hooykaas, Paul J J

    2009-09-01

    Agrobacterium tumefaciens is a plant pathogen that genetically transforms plant cells by transferring a part of its Ti-plasmid, the T-strand, to the host cell. Under laboratory conditions, it can also transform cells from many different nonplant organisms, including the yeast Saccharomyces cerevisiae. Collections of S. cerevisiae strains have been developed with systematic deletion of all coding sequences. Here, we used these collections to identify genes involved in the Agrobacterium-mediated transformation (AMT) of S. cerevisiae. We found that deletion of genes (GCN5, NGG1, YAF9 and EAF7) encoding subunits of the SAGA, SLIK, ADA and NuA4 histone acetyltransferase complexes highly increased the efficiency of AMT, while deletion of genes (HDA2, HDA3 and HST4) encoding subunits of histone deacetylase complexes decreased AMT. These effects are specific for AMT as the efficiency of chemical (lithium acetate) transformation was not or only slightly affected by these deletions. Our data are consistent with a positive role of host histone deacetylation in AMT.

  6. Histone acetyltransferase Enok regulates oocyte polarization by promoting expression of the actin nucleation factor spire

    PubMed Central

    Huang, Fu; Paulson, Ariel; Dutta, Arnob; Venkatesh, Swaminathan; Smolle, Michaela

    2014-01-01

    KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and have been shown to play important roles in transcriptional regulation. Here, we demonstrate that the Drosophila KAT6 Enok acetylates histone H3 Lys 23 (H3K23) in vitro and in vivo. Mutants lacking functional Enok exhibited defects in the localization of Oskar (Osk) to the posterior end of the oocyte, resulting in loss of germline formation and abdominal segments in the embryo. RNA sequencing (RNA-seq) analysis revealed that spire (spir) and maelstrom (mael), both required for the posterior localization of Osk in the oocyte, were down-regulated in enok mutants. Chromatin immunoprecipitation showed that Enok is localized to and acetylates H3K23 at the spir and mael genes. Furthermore, Gal4-driven expression of spir in the germline can largely rescue the defective Osk localization in enok mutant ovaries. Our results suggest that the Enok-mediated H3K23 acetylation (H3K23Ac) promotes the expression of spir, providing a specific mechanism linking oocyte polarization to histone modification. PMID:25512562

  7. 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.

  8. Crystal Structures of Murine Carnitine Acetyltransferase in Ternary Complexes with Its Substrates

    SciTech Connect

    Hsiao,Y.; Jogl, G.; Tong, L.

    2006-01-01

    Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general.

  9. 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.

  10. N-Acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species.

    PubMed

    Du, Xiaoyi; Takagi, Hiroshi

    2007-07-01

    N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H(2)O(2), heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H(2)O(2) or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H(2)O(2). Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains.

  11. The histone acetyltransferase MOF is a key regulator of the embryonic stem cell core transcriptional network.

    PubMed

    Li, Xiangzhi; Li, Li; Pandey, Ruchi; Byun, Jung S; Gardner, Kevin; Qin, Zhaohui; Dou, Yali

    2012-08-03

    Pluripotent embryonic stem cells (ESCs) maintain self-renewal and the potential for rapid response to differentiation cues. Both ESC features are subject to epigenetic regulation. Here we show that the histone acetyltransferase Mof plays an essential role in the maintenance of ESC self-renewal and pluripotency. ESCs with Mof deletion lose characteristic morphology, alkaline phosphatase (AP) staining, and differentiation potential. They also have aberrant expression of the core transcription factors Nanog, Oct4, and Sox2. Importantly, the phenotypes of Mof null ESCs can be partially suppressed by Nanog overexpression, supporting the idea that Mof functions as an upstream regulator of Nanog in ESCs. Genome-wide ChIP-sequencing and transcriptome analyses further demonstrate that Mof is an integral component of the ESC core transcriptional network and that Mof primes genes for diverse developmental programs. Mof is also required for Wdr5 recruitment and H3K4 methylation at key regulatory loci, highlighting the complexity and interconnectivity of various chromatin regulators in ESCs.

  12. 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.

  13. 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

  14. Bacterial protein acetylation: new discoveries unanswered questions.

    PubMed

    Wolfe, Alan J

    2016-05-01

    Nε-acetylation is emerging as an abundant post-translational modification of bacterial proteins. Two mechanisms have been identified: one is enzymatic, dependent on an acetyltransferase and acetyl-coenzyme A; the other is non-enzymatic and depends on the reactivity of acetyl phosphate. Some, but not most, of those acetylations are reversed by deacetylases. This review will briefly describe the current status of the field and raise questions that need answering.

  15. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  16. Ex Vivo Expansion of Human Hematopoietic Stem Cells by Garcinol, a Potent Inhibitor of Histone Acetyltransferase

    PubMed Central

    Nishino, Taito; Wang, Changshan; Mochizuki-Kashio, Makiko; Osawa, Mitsujiro; Nakauchi, Hiromitsu; Iwama, Atsushi

    2011-01-01

    Background Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However, methods suitable for clinical practice have yet to be fully established. Methodology/Principal Findings In this study, we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo, and identified Garcinol, a plant-derived histone acetyltransferase (HAT) inhibitor, as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34+CD38– HSCs supplemented with stem cell factor and thrombopoietin, Garcinol increased numbers of CD34+CD38– HSCs/PCs more than 4.5-fold and Isogarcinol, a derivative of Garcinol, 7.4-fold. Furthermore, during a 7-day culture of CD34+ HSCs/PCs, Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones, while inactive derivatives did not. Conclusions/Significance Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs. PMID:21931675

  17. Melatonin and serotonin N-acetyltransferase activity in developing eggs of the cricket Gryllus bimaculatus.

    PubMed

    Itoh, M T; Sumi, Y

    1998-01-19

    Melatonin (N-acetyl-5-methoxytryptamine) and serotonin N-acetyltransferase (NAT), a key regulatory enzyme in melatonin synthesis, are present in the adults and larvae of several insect species, as well as in vertebrates. To determine when melatonin and NAT first appear in insects ontogenetically, melatonin levels and NAT-like activity were measured in developing eggs of the cricket Gryllus bimaculatus. When the eggs were incubated under a 12-h light/12-h dark (LD) cycle at 24-26 degrees C, melatonin was detected in the egg extracts at all of the developmental stages examined. NAT-like activity was first found in the eggs 3 days after oviposition. From 5 to 11 days after oviposition, both NAT-like activity and melatonin levels showed significant day/night changes with the high levels occurring during the dark period of the LD cycle. By contrast, significant day/night changes were not detected in eggs just before hatching. To determine more detailed temporal changes, NAT-like activity was assayed in eggs 6 to 7 days after oviposition at 2- or 4-h intervals over a 48-h period. The activity in the eggs clearly exhibited a diurnal rhythm, peaking in the dark period of the LD cycle, and the rhythm persisted in constant darkness. These results suggest that the cricket egg (probably the embryo) synthesizes melatonin, and that its melatonin synthesis may fluctuate with a circadian rhythm. In addition, the results of the present study strongly suggest that a circadian clock controlling NAT activity functions in the cricket at the embryonic stage.

  18. Ocimum sanctum Linn. stimulate the expression of choline acetyltransferase on the human cerebral microvascular endothelial cells

    PubMed Central

    Kusindarta, Dwi Liliek; Wihadmadyatami, Hevi; Haryanto, Aris

    2016-01-01

    Aim: This research was conducted to identify the expression of choline acetyltransferase (ChAT) in human cerebral microvascular endothelial cells (HCMECs) and to clarify the capability of Ocimum sanctum Linn. ethanolic extract to stimulate the presence of ChAT in the aging HCMECs. Materials and Methods: In this study, we perform an in vitro analysis some in the presence of an ethanolic extract of O. sanctum Linn. as a stimulator for the ChAT expression. HCMECs are divided become two groups, the first is in low passage cells as a model of young aged and the second is in a high passage as a model of aging. Furthermore to analysis the expression of ChAT without and with extract treatments, immunocytochemistry and flow cytometry analysis were performed. In addition, ChAT sandwich enzyme-linked immunosorbent assay is developed to detect the increasing activity of the ChAT under normal, and aging HCMECs on the condition treated and untreated cells. Results: In our in vitro models using HCMECs, we found that ChAT is expressed throughout intracytoplasmic areas. On the status of aging, the ethanolic extract from O. sanctum Linn. is capable to stimulate and restore the expression of ChAT. The increasing of ChAT expression is in line with the increasing activity of this enzyme on the aging treated HCMECs. Conclusions: Our observation indicates that HCMECs is one of the noncholinergic cells which is produced ChAT. The administrated of O. sanctum Linn. ethanolic extract may stimulate and restore the expression of ChAT on the deteriorating cells of HCMECs, thus its may give nerve protection and help the production of acetylcholine. PMID:28096604

  19. Expression of a streptomycete leaderless mRNA encoding chloramphenicol acetyltransferase in Escherichia coli.

    PubMed Central

    Wu, C J; Janssen, G R

    1997-01-01

    The chloramphenicol acetyltransferase (cat) gene from Streptomyces acrimycini encodes a leaderless mRNA. Expression of the cat coding sequence as a leaderless mRNA from a modified lac promoter resulted in chloramphenicol resistance in Escherichia coli. Transcript mapping with nuclease S1 confirmed that the 5' end of the cat message initiated at the A of the AUG translational start codon. Site-directed mutagenesis of the lac promoter or the cat start codon abolished chloramphenicol resistance, indicating that E. coli initiated translation at the 5' terminal AUG of the cat leaderless mRNA. Addition of 5'-AUGC-3' to the 5' end of the cat mRNA resulted in translation occurring also from the reading frame defined by the added AUG triplet, suggesting that a 5'-terminal start codon is an important recognition feature for initiation and establishing reading frame during translation of leaderless mRNA. Addition of an untranslated leader and Shine-Dalgarno sequence to the cat coding sequence increased cat expression in a cat:lacZ fusion; however, the level of expression was significantly lower than when a fragment of the bacteriophage lambda cI gene, also encoding a leaderless mRNA, was fused to lacZ. These results indicate that in the absence of an untranslated leader and Shine-Dalgarno sequence, the streptomycete cat mRNA is translated by E. coli; however, the cat translation signals, or other features of the cat mRNA, provide for only a low level of expression in E. coli. PMID:9352935

  20. Influence of photoperiod on N-acetyltransferase activity and melatonin in the fiddler crab Uca pugilator.

    PubMed

    Tilden, A R; Alt, J; Brummer, K; Groth, R; Herwig, K; Wilson, A; Wilson, S

    2001-06-01

    Melatonin and N-acetyltransferase (NAT) activity were measured in the eyestalks of fiddler crabs acclimated to various photoperiods: constant light, a L:D 12:12 h photoperiod, or constant dark. Following acclimation, eyestalks were collected every 3 h over a 24-h period; they were assayed for melatonin with a radioimmunoassay and for NAT activity with a radioenzymatic assay. In constant light, melatonin levels increased at 1300 h, from 142 to 431 pg x mg(-1) eyestalk; NAT activity increased concurrently, from 97 to 203 pmol x h(-1) x mg(-1) eyestalk, and both remained elevated until 0400 h. In the L:D 12:12 h photoperiod, melatonin levels increased at 1300 h from 28 to 230 pg x mg(-1) eyestalk, and though NAT activity increased significantly, from 80 to 122 pmol x h(-1) x mg(-1) eyestalk, an even greater increase occurred at 0400 h, when melatonin levels were low. In constant dark, melatonin levels increased at 1600 h, from 22 to 196 pg x mg(-1) eyestalk, with a concurrent increase in NAT activity from 93 to 140 pmol x mg(-1) x h(-1) eyestalk. However, the second peak in melatonin (111 pg x mg(-1)), occurring at 0400 h, was out of phase with the second peak of NAT activity (113 pmol x mg(-1) x h(-1) eyestalk) which occurred at 0700 h. NAT may be a rate-limiting step in melatonin synthesis in fiddler crabs under some conditions (constant light and the 1300 h peak in constant dark); however, NAT activity correlates poorly with melatonin levels in a L:D 12:12 h photoperiod and in constant dark relative to the 0400 h melatonin peak.

  1. Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer.

    PubMed

    Mahasneh, Amjad; Jubaili, Amal; El Bateiha, Ahmed; Al-Ghazo, Mohammad; Matalka, Ismail; Malkawi, Mousa

    2012-12-01

    The arylamine N-acetyltransferase 2 (NAT2) enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A), M2 (NAT2*6A), M3 (NAT2*7) and M4 (NAT2 *14A) from the wild-type WT (NAT2 *4) allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were significant differences in allele frequencies between lung cancer patients and controls in the WT, M2 and M3 alleles (p < 0.05). However, only M2 and M3 allele frequencies were different between colorectal cancer patients and controls (p < 0.05). There was a marginal significant difference in the distribution of rapid and slow acetylator genotypes between lung cancer patients and controls (p = 0.06 and p = 0.05, respectively), but not between colorectal cancer patients and controls (p = 1.0 and p = 0.95, respectively). Risk of lung cancer development was found to be lower in slow acetylators [odds ratio (OR): 0.51, 95% confidence interval (95% CI): 0.25, 1.02, p-value = 0.07]. No effect was observed in case of colorectal cancer. Our results showed that NAT2 genotypes and phenotypes might be involved in lung cancer but not colorectal cancer susceptibility in Jordan.

  2. Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer

    PubMed Central

    Mahasneh, Amjad; Jubaili, Amal; El Bateiha, Ahmed; Al-Ghazo, Mohammad; Matalka, Ismail; Malkawi, Mousa

    2012-01-01

    The arylamine N-acetyltransferase 2 (NAT2) enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A), M2 (NAT2*6A), M3 (NAT2*7) and M4 (NAT2 *14A) from the wild-type WT (NAT2 *4) allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were significant differences in allele frequencies between lung cancer patients and controls in the WT, M2 and M3 alleles (p < 0.05). However, only M2 and M3 allele frequencies were different between colorectal cancer patients and controls (p < 0.05). There was a marginal significant difference in the distribution of rapid and slow acetylator genotypes between lung cancer patients and controls (p = 0.06 and p = 0.05, respectively), but not between colorectal cancer patients and controls (p = 1.0 and p = 0.95, respectively). Risk of lung cancer development was found to be lower in slow acetylators [odds ratio (OR): 0.51, 95% confidence interval (95% CI): 0.25, 1.02, p-value = 0.07]. No effect was observed in case of colorectal cancer. Our results showed that NAT2 genotypes and phenotypes might be involved in lung cancer but not colorectal cancer susceptibility in Jordan. PMID:23271930

  3. N-acetyltransferase 2, exposure to aromatic and heterocyclic amines, and receptor-defined breast cancer.

    PubMed

    Rabstein, Sylvia; Brüning, Thomas; Harth, Volker; Fischer, Hans-Peter; Haas, Susanne; Weiss, Tobias; Spickenheuer, Anne; Pierl, Christiane; Justenhoven, Christina; Illig, Thomas; Vollmert, Caren; Baisch, Christian; Ko, Yon-Dschun; Hamann, Ute; Brauch, Hiltrud; Pesch, Beate

    2010-03-01

    The role of N-acetyltransferase 2 (NAT2) polymorphism in breast cancer is still unclear. We explored the associations between potential sources of exposure to aromatic and heterocyclic amines (AHA), acetylation status and receptor-defined breast cancer in 1020 incident cases and 1047 population controls of the German GENICA study. Acetylation status was assessed as slow or fast. Therefore, NAT2 haplotypes were estimated using genotype information from six NAT2 polymorphisms. Most probable haplotypes served as alleles for the deduction of NAT2 acetylation status. The risks of developing estrogen receptor alpha (ER) and progesterone receptor (PR)-positive or negative tumors were estimated for tobacco smoking, consumption of red meat, grilled food, coffee, and tea, as well as expert-rated occupational exposure to AHA with logistic regression conditional on age and adjusted for potential confounders. Joint effects of these factors and NAT2 acetylation status were investigated. Frequent consumption of grilled food and coffee showed higher risks in slow acetylators for receptor-negative tumors [grilled food: ER-: odds ratio (OR) 2.57, 95% confidence interval (CI) 1.07-6.14 for regular vs. rare; coffee: ER-: OR 2.55, 95% CI 1.22-5.33 for >or=4 vs. 0 cups/day]. We observed slightly higher risks for never smokers that are fast acetylators for receptor-positive tumors compared with slow acetylators (ER-: OR 1.32, 95% CI 1.00-1.73). Our results support differing risk patterns for receptor-defined breast cancer. However, the modifying role of NAT2 for receptor-defined breast cancer is difficult to interpret in the light of complex mixtures of exposure to AHA.

  4. Histone Acetyltransferase Activity of MOF Is Required for MLL-AF9 Leukemogenesis.

    PubMed

    Valerio, Daria G; Xu, Haiming; Chen, Chun-Wei; Hoshii, Takayuki; Eisold, Meghan E; Delaney, Christopher; Cusan, Monica; Deshpande, Aniruddha J; Huang, Chun-Hao; Lujambio, Amaia; Zheng, YuJun George; Zuber, Johannes; Pandita, Tej K; Lowe, Scott W; Armstrong, Scott A

    2017-02-15

    Chromatin-based mechanisms offer therapeutic targets in acute myeloid leukemia (AML) that are of great current interest. In this study, we conducted an RNAi-based screen to identify druggable chromatin regulator-based targets in leukemias marked by oncogenic rearrangements of the MLL gene. In this manner, we discovered the H4K16 histone acetyltransferase (HAT) MOF to be important for leukemia cell growth. Conditional deletion of Mof in a mouse model of MLL-AF9-driven leukemogenesis reduced tumor burden and prolonged host survival. RNA sequencing showed an expected downregulation of genes within DNA damage repair pathways that are controlled by MOF, as correlated with a significant increase in yH2AX nuclear foci in Mof-deficient MLL-AF9 tumor cells. In parallel, Mof loss also impaired global H4K16 acetylation in the tumor cell genome. Rescue experiments with catalytically inactive mutants of MOF showed that its enzymatic activity was required to maintain cancer pathogenicity. In support of the role of MOF in sustaining H4K16 acetylation, a small-molecule inhibitor of the HAT component MYST blocked the growth of both murine and human MLL-AF9 leukemia cell lines. Furthermore, Mof inactivation suppressed leukemia development in an NUP98-HOXA9-driven AML model. Taken together, our results establish that the HAT activity of MOF is required to sustain MLL-AF9 leukemia and may be important for multiple AML subtypes. Blocking this activity is sufficient to stimulate DNA damage, offering a rationale to pursue MOF inhibitors as a targeted approach to treat MLL-rearranged leukemias. Cancer Res; 77(7); 1-10. ©2017 AACR.

  5. NolL of Rhizobium sp. Strain NGR234 Is Required for O-Acetyltransferase Activity

    PubMed Central

    Berck, S.; Perret, X.; Quesada-Vincens, D.; Promé, J.-C.; Broughton, W. J.; Jabbouri, S.

    1999-01-01

    Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium Rhizobium sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible nolL gene, which is preceded by a nod box, results in the synthesis of NodNGR factors that lack the 3-O- or 4-O-acetate groups. Interestingly, the nodulation capacity of the mutant NGRΩnolL is not impaired, whereas introduction of the nod box::nolL construct into the related strain Rhizobium fredii USDA257 extends the host range of this bacterium to Calopogonium caeruleum, Leucaena leucocephala, and Lotus halophilus. Nod factors produced by a USDA257(pnolL) transconjugant were also acetylated. The nod box::nolL construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the nodD1 gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not. PMID:9922261

  6. A Novel H2A/H4 Nucleosomal Histone Acetyltransferase in Tetrahymena thermophila

    PubMed Central

    Ohba, Reiko; Steger, David J.; Brownell, James E.; Mizzen, Craig A.; Cook, Richard G.; Côté, Jacques; Workman, Jerry L.; Allis, C. David

    1999-01-01

    Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be ∼80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena. PMID:10022893

  7. Epigenetic regulation of spermidine/spermine N1-acetyltransferase (SAT1) in suicide.

    PubMed

    Fiori, Laura M; Turecki, Gustavo

    2011-09-01

    We have recently shown that the expression of spermidine/spermine N1-acetyltransferase (SAT1) is downregulated across the brains of suicide completers, and that its expression is influenced by genetic variations in the promoter. Several promoter polymorphisms in SAT1, including rs6526342, have been associated with suicide and other psychiatric disorders, and display haplotype-specific effects on expression. However, these effects cannot explain total variability in SAT1 expression, and other regulatory mechanisms, such as epigenetic factors, may also be at play. In this study, we assessed the involvement of epigenetic factors in controlling SAT1 expression in the prefrontal cortex of suicide completers by mapping CpG methylation across a 1880-bp region of the SAT1 promoter, and measuring levels of tri-methylated histone-3-lysine 27 (H3K27me3) at the promoter in suicide completers and controls. Our results demonstrated that CpG methylation was significantly negatively correlated with SAT1 expression. Although overall or site-specific CpG methylation was not associated with suicide or SAT1 expression, we observed high levels of methylation at the polymorphic CpG site created by rs6526342, indicating a relationship between promoter haplotypes and methylation. There was no association between H3K27me3 and suicide, nor was this modification associated with SAT1 expression. Overall, our results indicate that epigenetic factors in the promoter region of SAT1 influence gene expression levels, and may provide a mechanism for both our previous findings of haplotype-specific effects of promoter variations on SAT1 expression, as well as the widespread downregulation of SAT1 expression observed in the brains of suicide completers.

  8. Probing the Catalytic Potential of the Hamster Arylamine N-Acetyltransferase 2 Catalytic Triad by Site-directed Mutagenesis of the Proximal Conserved Residue, Tyrosine 190

    PubMed Central

    Zhou, Xin; Zhang, Naixia; Liu, Li; Walters, Kylie J.; Hanna, Patrick E.; Wagner, Carston R.

    2009-01-01

    Summary Arylamine N-acetyltransferases (NATs) play an important role in both detoxification of arylamine and hydrazine drugs and activation of arylamine carcinogens. Since the catalytic triad, Cys-His-Asp, of mammalian NATs has been shown to be essential for maintaining protein stability, rendering it impossible to assess alterations of the triad on catalysis, we explored the impact of the highly conserved proximal residue, Tyr-190, which forms a direct hydrogen bond interaction with one of the triad residues, Asp-122, as well as a potential pi-pi stacking interaction with the active site His-107. Replacement of Hamster NAT2 Tyr-190 by either phenylalanine, isoleucine, or alanine was well tolerated and did not result in significant alterations in the overall fold of the protein. Nevertheless, stopped-flow and steady-state kinetic analysis revealed that Tyr-190 was critical for maximizing the acetylation rate of NAT2 and the transacetylation rate of p-aminobenzoic acid (PABA) when compared to wild type. Tyr-190 was also shown to play an important role in determining the pKa of the active site cysteine during acetylation, as well as the pH versus rate profile for transacetylation. We hypothesized that the pH-dependence was associated with global changes in the active site structure, which was revealed by the superposition of [1H, 15N] HSQC spectra for wild type and Y190A. These results suggest that NAT2 catalytic efficiency is partially governed by the ability of Tyr-190 to mediate the collective impact of multiple side chains on the electrostatic potential and local conformation of active site. PMID:19860825

  9. Characterization of arylalkylamine N-acetyltransferase (AANAT) activities and action spectrum for suppression in the band-legged cricket, Dianemobius nigrofasciatus (Orthoptera: Gryllidae).

    PubMed

    Izawa, Norimitsu; Suzuki, Takeshi; Watanabe, Masakatsu; Takeda, Makio

    2009-04-01

    Arylalkylamine N-acetyltransferase (AANAT), constituting a large family of enzymes, catalyzes the transacetylation from acetyl-CoA to monoamine substrates, although homology among species is not very high. AANAT in vertebrates is photosensitive and mediates circadian regulation. Here, we analyzed AANAT of the cricket, Dianemobius nigrofasciatus. The central nervous system contained AANAT activity. The optimum pHs were 6.0 (a minor peak) and 10.5 (a major peak) with crude enzyme solution. We analyzed the kinetics at pH 10.5 using the sample containing collective AANAT activities, which we term AANAT. Lineweaver-Burk plot and secondary plot yielded a K(m) for tryptamine as substrate of 0.42 microM, and a V(max) of 9.39 nmol/mg protein/min. The apparent K(m) for acetyl-CoA was 59.9 microM and the V(max) was 8.14 nmol/mg protein/min. AANAT of D. nigrofasciatus was light-sensitive. The activity was higher at night-time than at day-time as in vertebrates. To investigate most effective wavelengths on AANAT activity, a series of monochromatic lights was applied (350, 400, 450, 500, 550, 600 and 650 nm). AANAT showed the highest sensitivity to around 450 nm and 550 nm. 450 nm light was more effective than 550 nm light. Therefore, the most effective light affecting AANAT activity is blue light, which corresponds to the absorption spectrum of blue wave (BW)-opsin.

  10. Heparanase-mediated Loss of Nuclear Syndecan-1 Enhances Histone Acetyltransferase (HAT) Activity to Promote Expression of Genes That Drive an Aggressive Tumor Phenotype*

    PubMed Central

    Purushothaman, Anurag; Hurst, Douglas R.; Pisano, Claudio; Mizumoto, Shuji; Sugahara, Kazuyuki; Sanderson, Ralph D.

    2011-01-01

    Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins known to drive tumor progression (e.g. VEGF, MMP-9, hepatocyte growth factor (HGF), and RANKL). However, the mechanism whereby this enzyme regulates gene expression remains unknown. We previously reported that elevation of heparanase levels in myeloma cells causes a dramatic reduction in the amount of syndecan-1 in the nucleus. Because syndecan-1 has heparan sulfate chains and because exogenous heparan sulfate has been shown to inhibit the activity of histone acetyltransferase (HAT) enzymes in vitro, we hypothesized that the reduction in nuclear syndecan-1 in cells expressing high levels of heparanase would result in increased HAT activity leading to stimulation of protein transcription. We found that myeloma cells or tumors expressing high levels of heparanase and low levels of nuclear syndecan-1 had significantly higher levels of HAT activity when compared with cells or tumors expressing low levels of heparanase. High levels of HAT activity in heparanase-high cells were blocked by SST0001, an inhibitor of heparanase. Restoration of high syndecan-1 levels in heparanase-high cells diminished nuclear HAT activity, establishing syndecan-1 as a potent inhibitor of HAT. Exposure of heparanase-high cells to anacardic acid, an inhibitor of HAT activity, significantly suppressed their expression of VEGF and MMP-9, two genes known to be up-regulated following elevation of heparanase. These results reveal a novel mechanistic pathway driven by heparanase expression, which leads to decreased nuclear syndecan-1, increased HAT activity, and up-regulation of transcription of multiple genes that drive an aggressive tumor phenotype. PMID:21757697

  11. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  12. Protein

    MedlinePlus

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  13. Non-enzymatic protein acylation as a carbon stress regulated by sirtuin deacylases

    PubMed Central

    Wagner, Gregory R.; Hirschey, Matthew D.

    2014-01-01

    Cellular proteins are decorated with a wide range of acetyl and other acyl modifications. Many studies have demonstrated regulation of site-specific acetylation by acetyltransferases and deacetylases. Acylation is emerging as a new type of lysine modification, but less is known about its overall regulatory role. Furthermore, the mechanisms of lysine acylation, its overlap with protein acetylation, and how it influences cellular function are major unanswered questions in the field. In this review, we discuss the known roles of acetyltransferases and deacetylases, and the sirtuins as a conserved family of NAD+-dependent protein deacylases that are important for response to cellular stress and homeostasis. We also consider the evidence for an emerging idea of non-enzymatic protein acylation. Finally, we put forward the hypothesis that protein acylation is a form of protein “carbon stress”, that the deacylases evolved to remove as a part of a global protein quality control network. PMID:24725594

  14. Inhibition of cytosolic human forebrain choline acetyltransferase activity by phospho-L-serine: a phosphomonoester that accumulates during early stages of Alzheimer's disease.

    PubMed

    Andriamampandry, C; Kanfer, J N

    1993-01-01

    There is no satisfactory explanation for the cholinergic deficit characteristic of Alzheimer's disease. We have performed a series of experiments which demonstrate that (a) an inhibitor of cytosolic human brain choline acetyltransferase is present in the cytosol of Alzheimer brain tissue, (b) human brain cytosolic choline acetyltransferase activity is inhibited by phospho-L-serine in a competitive manner. Cytosol was prepared from human forebrain or amygdala and the Km for choline and acetyl CoA of the choline acetyltransferase were 750 microM and 12.5 microM, respectively. Phospho-L-serine was found to be a competitive inhibitor of this enzyme with respect to choline but not with respect to acetyl CoA with a Ki of 750 microM for the human forebrain and 3 mM for human amygdala. These concentrations of phospho-L-serine are present in brain tissue at early stages of Alzheimer's disease. Several other phosphomonoesters and phosphodiesters that are increased in Alzheimer's disease were either less inhibitory or without effect. The addition of heat denatured and non-heat denatured cytosol from Alzheimers forebrain inhibited the choline acetyltransferase activity present in control human brain cytosol. The inhibitory activity of the Alzheimers cytosol was retained in TCA deproteinized samples and removed by dialysis or by alkaline phosphatase treatment. Dialysis of the cytosol increased the choline acetyltransferase activity of 5 of 8 Alzheimer's disease samples from 21 to 118% with p values of < 0.025 or < 0.001, respectively. These observations provide evidence that an endogenous non-proteinaceous, dialyzable, phosphomonoester, present in Alzheimers brain inhibits the choline acetyltransferase of both control and Alzheimers brain.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Association between polymorphisms at N-acetyltransferase 1 (NAT1) & risk of oral leukoplakia & cancer

    PubMed Central

    Majumder, Mousumi; Ghosh, Saurabh; Roy, Bidyut

    2012-01-01

    Background & objectives: N-acetyltransferases 1 and 2 (NAT1 and NAT2) are important enzymes for metabolism of tobacco carcinogens. Due to polymorphisms, improper activities of these enzymes might lead to the formation of DNA adducts that may modulate risk of tobacco related oral precancer and cancer. Previously, it was shown that NAT2 polymorphisms did not modulate the risk of oral precancer and cancer. We undertook this study to check whether polymorphisms at NAT1 can modulate the risk of oral leukoplakia and cancer either alone or in combination with NAT2. Methods: Genotypes at four SNPs on NAT1 were determined by TaqMan method in 389 controls, 224 leukoplakia and 310 cancer patients. Genotype data were analyzed to know haplotypes and acetylation status of individuals and, then to estimate the risk of diseases. Using our previously published NAT2 data, combination of NAT1 and NAT2 acetylation genotypes of patients and controls were also analyzed to estimate the risk of diseases. Results: Analysis of NAT1 genotype data revealed that 1088T and 1095C alleles exist in strong linkage disequilibrium (r2=0.97, P<0.0001) and SNPs are in Hardy-Weinberg Equilibrium (P=0.1). Wild type or normal acetylating and variant or rapid acetylating alleles were two major alleles (frequencies 0.62 and 0.36, respectively) present in the control population. NAT1 rapid acetylation could not modulate the risk of leukoplakia and cancer (OR=0.9, 95% CI: 0.6-1.3; OR=1.0, 95% CI: 0.7-1.4, respectively). Analysis of combined NAT1 and NAT2 acetylating data also showed no significant enhancement of the risk of diseases. Interpretation & conclusions: NAT1 rapid acetylation alone as well as combination of NAT1 rapid-NAT2 slow acetylation did not modulate the risk of oral precancer and cancer in our patient population. So, NAT1/NAT2 metabolized carcinogen products may not be involved in tobacco related oral precancer and cancer. It may be interpreted that large sample size as well as combination of

  16. Crystallization and preliminary X-ray characterization of arylamine N-acetyltransferase C (BanatC) from Bacillus anthracis

    SciTech Connect

    Pluvinage, Benjamin; Li de la Sierra-Gallay, Inés; Martins, Marta; Ragunathan, Nilusha; Dupret, Jean-Marie; Rodrigues-Lima, Fernando

    2007-10-01

    Bacillus anthracis arylamine N-acetyltransferase C (BanatC) is an enzyme that metabolizes the drug sulfamethoxazole. Crystals of the purified enzyme that diffract at 1.95 Å are reported. The arylamine N-acetyltransferase (NAT) enzymes are xenobiotic metabolizing enzymes that have been found in a large range of eukaryotes and prokaryotes. These enzymes catalyse the acetylation of arylamine drugs and/or pollutants. Recently, a Bacillus anthracis NAT isoform (BanatC) has been cloned and shown to acetylate the sulfonamide antimicrobial sulfamethoxazole (SMX). Subsequently, it was shown that BanatC contributes to the resistance of this bacterium to SMX. Here, the crystallization and the X-ray characterization of BanatC (Y38F mutant) are reported. The crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 53.70, c = 172.40 Å, and diffract to 1.95 Å resolution on a synchrotron source.

  17. Structural and Biochemical Characterization of Acinetobacter spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes.

    PubMed

    Stogios, Peter J; Kuhn, Misty L; Evdokimova, Elena; Law, Melissa; Courvalin, Patrice; Savchenko, Alexei

    2017-02-10

    Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

  18. The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner

    PubMed Central

    Hou, Fajian; Chu, Chih-Wen; Kong, Xiangduo; Yokomori, Kyoko; Zou, Hui

    2007-01-01

    Proper sister chromatid cohesion is critical for maintaining genetic stability. San is a putative acetyltransferase that is important for sister chromatid cohesion in Drosophila melanogaster, but not in budding yeast. We showed that San is critical for sister chromatid cohesion in HeLa cells, suggesting that this mechanism may be conserved in metazoans. Furthermore, although a small fraction of San interacts with the NatA complex, San appears to mediate cohesion independently. San exhibits acetyltransferase activity in vitro, and its activity is required for sister chromatid cohesion in vivo. In the absence of San, Sgo1 localizes correctly throughout the cell cycle. However, cohesin is no longer detected at the mitotic centromeres. Furthermore, San localizes to the cytoplasm in interphase cells; thus, it may not gain access to chromosomes until mitosis. Moreover, in San-depleted cells, further depletion of Plk1 rescues the cohesion along the chromosome arms, but not at the centromeres. Collectively, San may be specifically required for the maintenance of the centromeric cohesion in mitosis. PMID:17502424

  19. Histone acetyltransferase HAT4 modulates navigation across G2/M and re-entry into G1 in Leishmania donovani

    PubMed Central

    Yadav, Aarti; Chandra, Udita; Saha, Swati

    2016-01-01

    Histone acetyltransferases impact multiple processes. This study investigates the role of histone acetyltransferase HAT4 in Leishmania donovani. Though HAT4 was dispensable for survival, its elimination decreased cell viability and caused cell cycle defects, with HAT4-nulls experiencing an unusually long G2/M. Survival of HAT4-nulls in macrophages was also substantially compromised. DNA microarray analysis revealed that HAT4 modestly regulated the expression of only a select number of genes, thus not being a major modulator of global gene expression. Significantly, cdc20 was among the downregulated genes. To ascertain if decreased expression of cdc20 was responsible for HAT4-null growth and cell cycle defects we expressed LdCdc20 ectopically in HAT4-nulls. We found this to alleviate the aberrant growth and cell cycle progression patterns displayed by HAT4-nulls, with cells navigating G2/M phase and re-entering G1 phase smoothly. HAT4-nulls expressing LdCdc20 ectopically showed survival rates comparable to wild type within macrophages, suggesting that G2/M defects were responsible for poor survival of HAT4-nulls within host cells also. These are the first data analyzing the in vivo functional role of HAT4 in any trypanosomatid. Our results directly demonstrate for the first time a role for Cdc20 in regulating trypanosomatid G2/M events, opening avenues for further research in this area. PMID:27272906

  20. Biochemical characteristics of a novel vegetative tissue geraniol acetyltransferase from a monoterpene oil grass (Palmarosa, Cymbopogon martinii var. Motia) leaf.

    PubMed

    Sharma, Pankaj K; Sangwan, Neelam S; Bose, Subir K; Sangwan, Rajender S

    2013-04-01

    Plants synthesize volatile alcohol esters on environmental insult or as metabolic induction during flower/fruit development. However, essential oil plants constitutively produce them as the oil constituents. Their synthesis is catalyzed by BAHD family enzymes called alcohol acyltransferases (AATs). However, no AAT has been characterized from plant foliage synthesizing acyclic monoterpenoids containing essential oils. Therefore, we have purified and biochemically characterized a geraniol: acetyl coenzyme A acetyltransferase (GAAT) from Palmarosa aroma grass (Cymbopogon martinii) leaf. MALDI-assisted proteomic study of the 43kDa monomeric enzyme revealed its sequence motif novelties e.g. relaxed conservation at Phe and Trp in DFGWG'. This suggests permissiveness of variations in the conserved motif without loss of catalytic ability. Also, some new conserved/semi-conserved motifs of AATs were recognized. The GAAT k(cat)/K(m) values (300-700M(-1)s(-1)) were low (a generic characteristic for secondary metabolism enzyme) but higher than those of some floral AATs. Wide substrate acceptability for catalyzing acetylation of diverse primary alcohols (chain of ≥C(6)) implied its catalytic description as a 'primary aliphatic alcohol acetyltransferase'. It signifies metabolic ability to deliver diverse aroma esters, should the acceptor alcohols be available in planta. To our knowledge, this is the first report of detailed kinetics of a vegetal monoterpenol acyltransferase.

  1. N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi

    PubMed Central

    Su, Chang; Lu, Yang; Liu, Haoping

    2016-01-01

    N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription. PMID:27694804

  2. Proximal Tubule Epithelial Cell Specific Ablation of the Spermidine/Spermine N1-Acetyltransferase Gene Reduces the Severity of Renal Ischemia/Reperfusion Injury

    PubMed Central

    Zahedi, Kamyar; Barone, Sharon; Wang, Yang; Murray-Stewart, Tracy; Roy-Chaudhury, Prabir; Smith, Roger D.; Casero, Robert A.; Soleimani, Manoocher

    2014-01-01

    Background Expression and activity of spermidine/spermine N1-acetyltransferase (SSAT) increases in kidneys subjected to ischemia/reperfusion (I/R) injury, while its ablation reduces the severity of such injuries. These results suggest that increased SSAT levels contribute to organ injury; however, the role of SSAT specifically expressed in proximal tubule epithelial cells, which are the primary targets of I/R injury, in the mediation of renal damage remains unresolved. Methods Severity of I/R injury in wt and renal proximal tubule specific SSAT-ko mice (PT-SSAT-Cko) subjected to bilateral renal I/R injury was assessed using cellular and molecular biological approaches. Results Severity of the loss of kidney function and tubular damage are reduced in PT-SSAT-Cko- compared to wt-mice after I/R injury. In addition, animals treated with MDL72527, an inhibitor of polyamine oxidases, had less severe renal damage than their vehicle treated counter-parts. The renal expression of HMGB 1 and Toll like receptors (TLR) 2 and 4 were also reduced in PT-SSAT-Cko- compared to wt mice after I/R injury. Furthermore, infiltration of neutrophils, as well as expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) transcripts were lower in the kidneys of PT-SSAT-Cko compared to wt mice after I/R injury. Finally, the activation of caspase3 was more pronounced in the wt compared to PT-SSAT-Cko animals. Conclusions Enhanced SSAT expression by proximal tubule epithelial cells leads to tubular damage, and its deficiency reduces the severity of renal I/R injury through reduction of cellular damage and modulation of the innate immune response. PMID:25390069

  3. Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct.

    PubMed

    Murray-Stewart, Tracy; Applegren, Nancy B; Devereux, Wendy; Hacker, Amy; Smith, Renee; Wang, Yanlin; Casero, Robert A

    2003-07-15

    Spermidine/spermine N (1)-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N (1), N (11)-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.

  4. Tumor necrosis factor alpha induces spermidine/spermine N1-acetyltransferase through nuclear factor kappaB in non-small cell lung cancer cells.

    PubMed

    Babbar, Naveen; Hacker, Amy; Huang, Yi; Casero, Robert A

    2006-08-25

    Tumor necrosis factor alpha (TNFalpha) is a potent pleiotropic cytokine produced by many cells in response to inflammatory stress. The molecular mechanisms responsible for the multiple biological activities of TNFalpha are due to its ability to activate multiple signal transduction pathways, including nuclear factor kappaB (NFkappaB), which plays critical roles in cell proliferation and survival. TNFalpha displays both apoptotic and antiapoptotic properties, depending on the nature of the stimulus and the activation status of certain signaling pathways. Here we show that TNFalpha can lead to the induction of NFkappaB signaling with a concomitant increase in spermidine/spermine N(1)-acetyltransferase (SSAT) expression in A549 and H157 non-small cell lung cancer cells. Induction of SSAT, a stress-inducible gene that encodes a rate-limiting polyamine catabolic enzyme, leads to lower intracellular polyamine contents and has been associated with decreased cell growth and increased apoptosis. Stable overexpression of a mutant, dominant negative IkappaBalpha protein led to the suppression of SSAT induction by TNFalpha in these cells, thereby substantiating a role of NFkappaB in the induction of SSAT by TNFalpha. SSAT promoter deletion constructs led to the identification of three potential NFkappaB response elements in the SSAT gene. Electromobility shift assays, chromatin immunoprecipitation experiments and mutational studies confirmed that two of the three NFkappaB response elements play an important role in the regulation of SSAT in response to TNFalpha. The results of these studies indicate that a common mediator of inflammation can lead to the induction of SSAT expression by activating the NFkappaB signaling pathway in non-small cell lung cancer cells.

  5. Heart‐Specific Overexpression of Choline Acetyltransferase Gene Protects Murine Heart Against Ischemia Through Hypoxia‐Inducible Factor‐1α–Related Defense Mechanisms

    PubMed Central

    Kakinuma, Yoshihiko; Tsuda, Masayuki; Okazaki, Kayo; Akiyama, Tsuyoshi; Arikawa, Mikihiko; Noguchi, Tatsuya; Sato, Takayuki

    2013-01-01

    Background Murine and human ventricular cardiomyocytes rich in acetylcholine (Ach) receptors are poorly innervated by the vagus, compared with whole ventricular innervation by the adrenergic nerve. However, vagal nerve stimulation produces a favorable outcome even in the murine heart, despite relatively low ventricular cholinergic nerve density. Such a mismatch and missing link suggest the existence of a nonneuronal cholinergic system in ventricular myocardium. Methods and Results To examine the role of the nonneuronal cardiac cholinergic system, we generated choline acetyltransferase (ChAT)–expressing cells and heart‐specific ChAT transgenic (ChAT‐tg) mice. Compared with cardiomyocytes of wild‐type (WT) mice, those of the ChAT‐tg mice had high levels of ACh and hypoxia‐inducible factor (HIF)‐1α protein and augmented glucose uptake. These phenotypes were also reproduced by ChAT‐overexpressing cells, which utilized oxygen less. Before myocardial infarction (MI), the WT and ChAT‐tg mice showed similar hemodynamics; after MI, however, the ChAT‐tg mice had better survival than did the WT mice. In the ChAT‐tg hearts, accelerated angiogenesis at the ischemic area, and accentuated glucose utilization prevented post‐MI remodeling. The ChAT‐tg heart was more resistant to ischemia–reperfusion injury than was the WT heart. Conclusions These results suggest that the activated cardiac ACh‐HIF‐1α cascade improves survival after MI. We conclude that de novo synthesis of ACh in cardiomyocytes is a pivotal mechanism for self‐defense against ischemia. PMID:23525439

  6. Protective Immunity Elicited by Oral Immunization of Mice with Salmonella enterica Serovar Typhimurium Braun Lipoprotein (Lpp) and Acetyltransferase (MsbB) Mutants

    PubMed Central

    Erova, Tatiana E.; Kirtley, Michelle L.; Fitts, Eric C.; Ponnusamy, Duraisamy; Baze, Wallace B.; Andersson, Jourdan A.; Cong, Yingzi; Tiner, Bethany L.; Sha, Jian; Chopra, Ashok K.

    2016-01-01

    We evaluated the extent of attenuation and immunogenicity of the ΔlppAB and ΔlppAB ΔmsbB mutants of Salmonella enterica serovar Typhimurium when delivered to mice by the oral route. These mutants were deleted either for the Braun lipoprotein genes (lppA and lppB) or in combination with the msbB gene, which encodes an acetyltransferase required for lipid A modification of lipopolysaccharide. Both the mutants were attenuated (100% animal survival) and triggered robust innate and adaptive immune responses. Comparable levels of IgG and its isotypes were produced in mice infected with wild-type (WT) S. typhimurium or its aforementioned mutant strains. The ΔlppAB ΔmsbB mutant-immunized animals resulted in the production of higher levels of fecal IgA and serum cytokines during later stages of vaccination (adaptive response). A significant production of interleukin-6 from T-cells was also noted in the ΔlppAB ΔmsbB mutant-immunized mice when compared to that of the ΔlppAB mutant. On the other hand, IL-17A production was significantly more in the serum of ΔlppAB mutant-immunized mice (innate response) with a stronger splenic T-cell proliferative and tumor-necrosis factor-α production. Based on 2-dimensional gel analysis, alterations in the levels of several proteins were observed in both the mutant strains when compared to that in WT S. typhimurium and could be associated with the higher immunogenicity of the mutants. Finally, both ΔlppAB and ΔlppAB ΔmsbB mutants provided complete protection to immunized mice against a lethal oral challenge dose of WT S. typhimurium. Thus, these mutants may serve as excellent vaccine candidates and also provide a platform for delivering heterologous antigens. PMID:27891321

  7. Molecular Cloning, Characterization, and Functional Analysis of Acetyl-CoA C-Acetyltransferase and Mevalonate Kinase Genes Involved in Terpene Trilactone Biosynthesis from Ginkgo biloba.

    PubMed

    Chen, Qiangwen; Yan, Jiaping; Meng, Xiangxiang; Xu, Feng; Zhang, Weiwei; Liao, Yongling; Qu, Jinwang

    2017-01-02

    Ginkgolides and bilobalide, collectively termed terpene trilactones (TTLs), are terpenoids that form the main active substance of Ginkgo biloba. Terpenoids in the mevalonate (MVA) biosynthetic pathway include acetyl-CoA C-acetyltransferase (AACT) and mevalonate kinase (MVK) as core enzymes. In this study, two full-length (cDNAs) encoding AACT (GbAACT, GenBank Accession No. KX904942) and MVK (GbMVK, GenBank Accession No. KX904944) were cloned from G. biloba. The deduced GbAACT and GbMVK proteins contain 404 and 396 amino acids with the corresponding open-reading frame (ORF) sizes of 1215 bp and 1194 bp, respectively. Tissue expression pattern analysis revealed that GbAACT was highly expressed in ginkgo fruits and leaves, and GbMVK was highly expressed in leaves and roots. The functional complementation of GbAACT in AACT-deficient Saccharomyces cerevisiae strain Δerg10 and GbMVK in MVK-deficient strain Δerg12 confirmed that GbAACT mediated the conversion of mevalonate acetyl-CoA to acetoacetyl-CoA and GbMVK mediated the conversion of mevalonate to mevalonate phosphate. This observation indicated that GbAACT and GbMVK are functional genes in the cytosolic mevalonate (MVA) biosynthesis pathway. After G. biloba seedlings were treated with methyl jasmonate and salicylic acid, the expression levels of GbAACT and GbMVK increased, and TTL production was enhanced. The cloning, characterization, expression and functional analysis of GbAACT and GbMVK will be helpful to understand more about the role of these two genes involved in TTL biosynthesis.

  8. Piperidinols that show anti-tubercular activity as inhibitors of arylamine N-acetyltransferase: an essential enzyme for mycobacterial survival inside macrophages.

    PubMed

    Abuhammad, Areej; Fullam, Elizabeth; Lowe, Edward D; Staunton, David; Kawamura, Akane; Westwood, Isaac M; Bhakta, Sanjib; Garner, Alun Christopher; Wilson, David L; Seden, Peter T; Davies, Stephen G; Russell, Angela J; Garman, Elspeth F; Sim, Edith

    2012-01-01

    Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from

  9. Moonlight affects mRNA abundance of arylalkylamine N-acetyltransferase in the retina of a lunar-synchronized spawner, the goldlined spinefoot.

    PubMed

    Kashiwagi, Tomomi; Park, Yong-Ju; Park, Ji-Gweon; Imamura, Satoshi; Takeuchi, Yuki; Hur, Sung-Pyo; Takemura, Akihiro

    2013-11-01

    Melatonin synthesis in the pineal gland and retina shows a rhythmic fashion with high levels at night and is controlled by a rate-limiting enzyme, arylalkylamine N-acetyltransferase (AANAT). A previous study revealed that moonlight suppresses the plasma melatonin levels of the goldlined spinefoot (Siganus guttatus), which exhibits a lunar cycle in its reproductive activity and repeats gonadal development toward and spawning around the first quarter moon. Whether the retina of this species responds to moonlight is unknown. To clarify the photoperceptive ability of this species, we aimed to clone the full-length cDNA of Aanat1 (sgAanat1) from the retina and examine its transcriptional pattern under several daylight and moonlight regimes. The full-length sgAanat1 cDNA (1,038 bp) contained a reading frame encoding a protein of 225 amino acids, which was highly homologous to AANAT1 of other teleosts. Reverse transcription-polymerase chain reaction (PCR) analysis revealed that among the tissues tested, sgAanat1 fragments were expressed exclusively in the retina. Real-time quantitative PCR analysis revealed that sgAanat1 fluctuated with high abundance at night under light-dark cycle and at subjective night under constant darkness, but not under constant light. These results suggest that sgAanat1 is regulated by both the external light signal and internal clock system. The abundance of sgAanat1 in the retina was higher at the culmination time around new moon than full moon phase. Additionally, exposing fish to brightness around the full moon period suppressed sgAanat1 mRNA abundance. Thus, moonlight is perceived by fish and has an impact on melatonin fluctuation in the retina.

  10. Arsenic Trioxide Reduces Global Histone H4 Acetylation at Lysine 16 through Direct Binding to Histone Acetyltransferase hMOF in Human Cells

    PubMed Central

    Liu, Da; Wu, Donglu; Zhao, Linhong; Yang, Yang; Ding, Jian; Dong, Liguo; Hu, Lianghai; Wang, Fei; Zhao, Xiaoming; Cai, Yong; Jin, Jingji

    2015-01-01

    Histone post-translational modification heritably regulates gene expression involved in most cellular biological processes. Experimental studies suggest that alteration of histone modifications affects gene expression by changing chromatin structure, causing various cellular responses to environmental influences. Arsenic (As), a naturally occurring element and environmental pollutant, is an established human carcinogen. Recently, increasing evidence suggests that As-mediated epigenetic mechanisms may be involved in its toxicity and carcinogenicity, but how this occurs is still unclear. Here we present evidence that suggests As-induced global histone H4K16 acetylation (H4K16ac) partly due to the direct physical interaction between As and histone acetyltransferase (HAT) hMOF (human male absent on first) protein, leading to the loss of hMOF HAT activity. Our data show that decreased global H4K16ac and increased deacetyltransferase HDAC4 expression occurred in arsenic trioxide (As2O3)-exposed HeLa or HEK293T cells. However, depletion of HDAC4 did not affect global H4K16ac, and it could not raise H4K16ac in cells exposed to As2O3, suggesting that HDAC4 might not directly be involved in histone H4K16 de-acetylation. Using As-immobilized agarose, we confirmed that As binds directly to hMOF, and that this interaction was competitively inhibited by free As2O3. Also, the direct interaction of As and C2CH zinc finger peptide was verified by MAIDI-TOF mass and UV absorption. In an in vitro HAT assay, As2O3 directly inhibited hMOF activity. hMOF over-expression not only increased resistance to As and caused less toxicity, but also effectively reversed reduced H4K16ac caused by As exposure. These data suggest a theoretical basis for elucidating the mechanism of As toxicity. PMID:26473953

  11. Piperidinols That Show Anti-Tubercular Activity as Inhibitors of Arylamine N-Acetyltransferase: An Essential Enzyme for Mycobacterial Survival Inside Macrophages

    PubMed Central

    Abuhammad, Areej; Fullam, Elizabeth; Lowe, Edward D.; Staunton, David; Kawamura, Akane; Westwood, Isaac M.; Bhakta, Sanjib; Garner, Alun Christopher; Wilson, David L.; Seden, Peter T.; Davies, Stephen G.; Russell, Angela J.; Garman, Elspeth F.; Sim, Edith

    2012-01-01

    Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3–16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different

  12. Protective Immunity Elicited by Oral Immunization of Mice with Salmonella enterica Serovar Typhimurium Braun Lipoprotein (Lpp) and Acetyltransferase (MsbB) Mutants.

    PubMed

    Erova, Tatiana E; Kirtley, Michelle L; Fitts, Eric C; Ponnusamy, Duraisamy; Baze, Wallace B; Andersson, Jourdan A; Cong, Yingzi; Tiner, Bethany L; Sha, Jian; Chopra, Ashok K

    2016-01-01

    We evaluated the extent of attenuation and immunogenicity of the ΔlppAB and ΔlppAB ΔmsbB mutants of Salmonella enterica serovar Typhimurium when delivered to mice by the oral route. These mutants were deleted either for the Braun lipoprotein genes (lppA and lppB) or in combination with the msbB gene, which encodes an acetyltransferase required for lipid A modification of lipopolysaccharide. Both the mutants were attenuated (100% animal survival) and triggered robust innate and adaptive immune responses. Comparable levels of IgG and its isotypes were produced in mice infected with wild-type (WT) S. typhimurium or its aforementioned mutant strains. The ΔlppAB ΔmsbB mutant-immunized animals resulted in the production of higher levels of fecal IgA and serum cytokines during later stages of vaccination (adaptive response). A significant production of interleukin-6 from T-cells was also noted in the ΔlppAB ΔmsbB mutant-immunized mice when compared to that of the ΔlppAB mutant. On the other hand, IL-17A production was significantly more in the serum of ΔlppAB mutant-immunized mice (innate response) with a stronger splenic T-cell proliferative and tumor-necrosis factor-α production. Based on 2-dimensional gel analysis, alterations in the levels of several proteins were observed in both the mutant strains when compared to that in WT S. typhimurium and could be associated with the higher immunogenicity of the mutants. Finally, both ΔlppAB and ΔlppAB ΔmsbB mutants provided complete protection to immunized mice against a lethal oral challenge dose of WT S. typhimurium. Thus, these mutants may serve as excellent vaccine candidates and also provide a platform for delivering heterologous antigens.

  13. Spermidine/spermine N1-acetyltransferase regulates cell growth and metastasis via AKT/β-catenin signaling pathways in hepatocellular and colorectal carcinoma cells

    PubMed Central

    Zhao, Ying; Li, Xiaomin; Wang, Jun; Wu, Xiaoxiao; Liu, Tong; Wang, Shasha; Hou, Jiuzhou; Li, Wei; Li, Qian; Li, Jinghua; Dai, Fujun; Fang, Dong; Wang, Chaojie; Xie, Songqiang

    2017-01-01

    Hepatocellular carcinoma (HCC) and colorectal cancer (CRC) are among the most common cancers across the world. Therefore, identifying the potential molecular mechanisms that promote HCC and CRC progression and metastasis are urgently needed. Spermidine/spermine N1-acetyltransferase (SSAT) is a catabolic enzyme that acetylates the high-order polyamines spermine and spermidine, thus decreasing the cellular content of polyamines. Several publications have suggested that depletion of intracellular polyamines inhibited tumor progression and metastasis in various cancer cells. However, whether and how SSAT regulates cell growth, migration and invasion in hepatocellular and colorectal carcinoma cells remains unclear. In this study, depletion of polyamines mediated by SSAT not only attenuated the tumor cell proliferation but also dramatically inhibited cell migration and invasion in hepatocellular and colorectal carcinoma cells. Subsequent investigations revealed introduction of SSAT into HepG2, SMMC7721 hepatocellular carcinoma cells and HCT116 colorectal carcinoma cells significantly suppressed p-AKT, p-GSK3β expression as well as β-catenin nuclear translocation, while inhibition of GSK3β activity or exogenous polyamines could restore SSAT-induced decreases in the protein expression of p-AKT, p-GSK3β and β-catenin. Conversely, knockdown of SSAT in Bel7402 hepatocellular carcinoma cells and HT-29 colorectal carcinoma cells which expressed high levels of SSAT endogenously significantly promoted the expression of p-AKT, p-GSK3β as well as β-catenin nuclear translocation. Taken together, our results indicated depletion of polyamines by SSAT significantly inhibited cell proliferation, migration and invasion through AKT/GSK3β/β-catenin signaling pathway in hepatocellular carcinoma and colorectal cancer cells. PMID:27901475

  14. Human T-cell leukemia virus type 2 antisense viral protein 2 is dispensable for in vitro immortalization but functions to repress early virus replication in vivo.

    PubMed

    Yin, Han; Kannian, Priya; Dissinger, Nathan; Haines, Robyn; Niewiesk, Stefan; Green, Patrick L

    2012-08-01

    Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are closely related but pathogenically distinct human retroviruses. The antisense strand of the HTLV-1 genome encodes HTLV-1 basic leucine zipper (b-ZIP) protein (HBZ), a protein that inhibits Tax-mediated viral transcription, enhances T-cell proliferation, and promotes viral persistence. Recently, an HTLV-2 antisense viral protein (APH-2) was identified. Despite its lack of a typical b-ZIP domain, APH-2, like HBZ, interacts with cyclic AMP response element binding protein (CREB) and downregulates Tax-mediated viral transcription. Here, we provide evidence that the APH-2 C-terminal LXXLL motif is important for CREB binding and Tax repression. In order to investigate the functional role of APH-2 in the HTLV-2-mediated immortalization of primary T lymphocytes in vitro and in HTLV-2 infection in vivo, we generated APH-2 mutant viruses. In cell cultures, the immortalization capacities of APH-2 mutant viruses were indistinguishable from that of wild-type HTLV-2 (wtHTLV-2), indicating that, like HBZ, APH-2 is dispensable for viral infection and cellular transformation. In vivo, rabbits inoculated with either wtHTLV-2 or APH-2 mutant viruses established a persistent infection. However, the APH-2 knockout virus displayed an increased replication rate, as measured by an increased viral antibody response and a higher proviral load. In contrast to HTLV-1 HBZ, we show that APH-2 is dispensable for the establishment of an efficient infection and persistence in a rabbit animal model. Therefore, antisense proteins of HTLV-1 and HTLV-2 have evolved different functions in vivo, and further comparative studies will provide fundamental insights into the distinct pathobiologies of these two viruses.

  15. Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® Cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    LibertyLink® cotton cultivars are engineered for glufosinate resistance by overexpressing the bar gene that encodes phosphinothricin acetyltransferase (PAT), whereas the insect-resistant WideStrike® cultivars were obtained by using the similar pat gene as a selectable marker. The latter cultivars ca...

  16. Bioprospecting for Trichothecene 3-O-acetyltransferases in the fungal genus Fusarium yields functional enzymes that vary in their Aaility to modify the mycotoxin deoxynivalenol

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to mod...

  17. Expression of Iron Regulatory Protein 1 Is Regulated not only by HIF-1 but also pCREB under Hypoxia

    PubMed Central

    Luo, Qian-Qian; Qian, Zhong-Ming; Zhou, Yu-Fu; Zhang, Meng-Wan; Wang, Dang; Zhu, Li; Ke, Ya

    2016-01-01

    The inconsistent of responses of IRP1 and HIF-1 alpha to hypoxia and the similar tendencies in the changes of IRP1 and pCREB contents led us to hypothesize that pCREB might be involved in the regulation of IRP1 under hypoxia. Here, we investigated the role of pCREB in IRP1 expression in HepG2 cells under hypoxia using quantitative PCR, western blot, immunofluorescence, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). We demonstrated that 1) Hypoxia increased pCREB levels inside of the nucleus; 2) Putative CREs were found in the IRP1 gene; 3) Nuclear extracts of HepG2 cells treated with hypoxia could bind to CRE1 and CRE3, and 100-fold competitor of putative CREs could abolish the binding activity to varying degrees; 4) pCREB was found in the CRE1 and CRE3 DNA-protein complexes of EMSA; 5) CRE1 and CRE3 binding activity of IRP1 depended on CREB activation but not on HIF-1; 6) Increased IRP1 expression under hypoxia could be prevented by LY294002; 7) ChIP assays demonstrated that pCREB binds to IRP1 promoter; and 8) HIF-1 and/or HIF-2 siRNA had no effect on the expression of pCREB and IRP1 proteins in cells treated with hypoxia for 8 hours. Our findings evidenced for the involvement of pCREB in IRP1 expression and revealed a dominant role of PI3K/Akt pathway in CREB activation under hypoxia and also suggested that dual-regulation of IRP1 expression by HIF-1 and pCERB or other transcription factor(s) under hypoxia might be a common mechanism in most if not all of hypoxia-inducible genes. PMID:27766034

  18. Chromatographic separation of reaction products from the choline acetyltransferase and carnitine acetyltransferase assay: differential ChAT and CrAT activity in brain extracts from Alzheimer's disease versus controls.

    PubMed

    Bailey, Jason A; Lahiri, Debomoy K

    2012-08-01

    Choline acetyltransferase (ChAT) catalyzes the reaction between choline and acetylcoenzyme A (AcCoA) to form acetylcholine (ACh) in nerve terminals. ACh metabolism has implications in numerous aspects of physiology and varied disease states, such as Alzheimer's disease. Therefore a specific, sensitive, and reliable method for detecting ChAT enzyme activity is of great utility in a number of situations. Using an existing radionuclide-based enzyme activity assay, we have observed detectable ChAT signals from non-cholinergic cells, suggesting a contaminant in the assay producing an artifactual signal. Previous reports have suggested that L-acetylcarnitine (LAC) contaminates many assays of ChAT activity, because of difficulties in separating LAC from ACh by organic extraction. To determine the source of this hypothesized artifact and to rectify the problem, we have developed a paper chromatography-based assay for the detection of acetylcholine and other contaminating reaction products of this assay, including LAC. Our first goal was to develop a simple and economical method for resolving and verifying the identities of various reaction products or contaminants that could be performed in most laboratories without specialized equipment. Our second goal was to apply this separation method in postmortem human brain tissue samples. Our assay successfully detected several contaminants, especially in assays using brain tissue, and allowed the separation of the intended ACh product from these contaminants. We further demonstrate that this assay can be used to measure carnitine acetyltransferase (CrAT) activity in the same samples, and assays comparing ChAT and CrAT show that CrAT is highly active in neuronal tissues and in neuronal cell cultures relative to ChAT. Thus, the simple chromatography-based assay we describe allows the measurement of specific reaction products separated from contaminants using commonly available and inexpensive materials. Further, we show that Ch

  19. Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate.

    PubMed

    Fukuda, K; Yamamoto, N; Kiyokawa, Y; Yanagiuchi, T; Wakai, Y; Kitamoto, K; Inoue, Y; Kimura, A

    1998-10-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

  20. Balance of Activities of Alcohol Acetyltransferase and Esterase in Saccharomyces cerevisiae Is Important for Production of Isoamyl Acetate

    PubMed Central

    Fukuda, Kiyoshi; Yamamoto, Nagi; Kiyokawa, Yoshifumi; Yanagiuchi, Toshiyasu; Wakai, Yoshinori; Kitamoto, Katsuhiko; Inoue, Yoshiharu; Kimura, Akira

    1998-01-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash. PMID:9758847

  1. Is There a Link Between Expression Levels of Histone Deacetylase/Acetyltransferase in Mouse Sperm and Subsequent Blastocyst Development?

    PubMed

    Kim, Jayeon; Kim, Ji-Hee; Jee, Byung-Chul; Suh, Chang-Suk; Kim, Seok-Hyun

    2015-11-01

    Histone acetylation has been known to be significant in spermatogenesis. Histone acetylation is regulated by the act of histone deacetylases (HDACs) and histone acetyltransferases (HATs). We investigated the link between expression levels of HDACs and HATs in mouse sperm and subsequent blastocyst formation rate. In the univariate analysis, expression levels of HDAC1 and HAT were generally not associated with the blastocyst formation rate. When divided by the mature oocyte number category, a significant positive association was observed between the expression levels of HDAC1 and the blastocyst-forming rate in the highest (> 75th) percentile group (a group with ≥34 mature oocytes). In conclusion, expression of sperm HDAC1 could be considered as a possible predictor of embryo development in mice with high ovarian response.

  2. Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis

    SciTech Connect

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

    2016-12-08

    A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28–37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

  3. HnRNPA2 is a novel histone acetyltransferase that mediates mitochondrial stress-induced nuclear gene expression

    PubMed Central

    Guha, Manti; Srinivasan, Satish; Guja, Kip; Mejia, Edison; Garcia-Diaz, Miguel; Johnson, F Brad; Ruthel, Gordon; Kaufman, Brett A; Rappaport, Eric F; Glineburg, M Rebecca; Fang, Ji-Kang; Szanto, Andres Klein; Nakagawa, Hiroshi; Basha, Jeelan; Kundu, Tapas; Avadhani, Narayan G

    2016-01-01

    Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies. PMID:27990297

  4. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B).

    PubMed

    Haigney, Allison; Ricketts, M Daniel; Marmorstein, Ronen

    2015-12-18

    The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

  5. Structural analysis of PseH, the Campylobacter jejuni N-acetyltransferase involved in bacterial O-linked glycosylation.

    PubMed

    Song, Wan Seok; Nam, Mi Sun; Namgung, Byeol; Yoon, Sung-il

    2015-03-20

    Campylobacter jejuni is a bacterium that uses flagella for motility and causes worldwide acute gastroenteritis in humans. The C. jejuni N-acetyltransferase PseH (cjPseH) is responsible for the third step in flagellin O-linked glycosylation and plays a key role in flagellar formation and motility. cjPseH transfers an acetyl group from an acetyl donor, acetyl coenzyme A (AcCoA), to the amino group of UDP-4-amino-4,6-dideoxy-N-acetyl-β-L-altrosamine to produce UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. To elucidate the catalytic mechanism of cjPseH, crystal structures of cjPseH alone and in complex with AcCoA were determined at 1.95 Å resolution. cjPseH folds into a single-domain structure of a central β-sheet decorated by four α-helices with two continuously connected grooves. A deep groove (groove-A) accommodates the AcCoA molecule. Interestingly, the acetyl end of AcCoA points toward an open space in a neighboring shallow groove (groove-S), which is occupied by extra electron density that potentially serves as a pseudosubstrate, suggesting that the groove-S may provide a substrate-binding site. Structure-based comparative analysis suggests that cjPseH utilizes a unique catalytic mechanism of acetylation that has not been observed in other glycosylation-associated acetyltransferases. Thus, our studies on cjPseH will provide valuable information for the design of new antibiotics to treat C. jejuni-induced gastroenteritis.

  6. The use of choline acetyltransferase for measuring the synthesis of acetyl-coenzyme A and its release from brain mitochondria.

    PubMed

    Tucek, S

    1967-09-01

    1. A method for measuring small amounts of acetyl-CoA synthesized in subcellular fractions of the brain from pyruvate and released from particles into the incubation medium has been developed by using placental choline acetyltransferase and choline in the incubation medium to transform acetyl-CoA into acetylcholine. Acetylcholine is measured by biological assay. Optimum conditions of incubation are described. 2. With fresh mitochondria, a decrease of acetyl-CoA output into the medium is observed in the presence of ATP or ADP, and an increase in the presence of calcium chloride or 2,4-dinitrophenol. Fluorocitrate and malonate have little or no effect. 3. After the mitochondria had been treated with ether, the release of acetyl-CoA into the medium is much larger; presumably, nearly all acetyl-CoA synthesized is then released and transformed into acetylcholine under the conditions used. The release of acetyl-CoA is diminished in the presence of Krebs-cycle intermediates and ADP. 4. Of all subcellular fractions, the highest acetyl-CoA production from pyruvate is found in the crude mitochondria; rates up to 51 mumoles of acetyl-CoA/g. of original tissue/hr. are observed in ether-treated samples. 5. The activities of acetyl-CoA synthetase and ATP citrate lyase found in homogenates and nerve-ending fractions of brain tissue are considerably lower than those of pyruvate oxidase complex and choline acetyltransferase. 6. The bearing of some of the findings on the question of the source of acetyl radicals for the synthesis of acetylcholine in vivo is discussed.

  7. p300/β-Catenin Interactions Regulate Adult Progenitor Cell Differentiation Downstream of WNT5a/Protein Kinase C (PKC)*

    PubMed Central

    Rieger, Megan E.; Zhou, Beiyun; Solomon, Nicola; Sunohara, Mitsuhiro; Li, Changgong; Nguyen, Cu; Liu, Yixin; Pan, Jie-hong; Minoo, Parviz; Crandall, Edward D.; Brody, Steven L.; Kahn, Michael; Borok, Zea

    2016-01-01

    Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of β-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential β-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific β-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/β-catenin but not CBP/β-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/β-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/β-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/β-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting β-catenin to modulate adult progenitor cell

  8. Isoform-level brain expression profiling of the spermidine/spermine N1-Acetyltransferase1 (SAT1) gene in major depression and suicide.

    PubMed

    Pantazatos, Spiro P; Andrews, Stuart J; Dunning-Broadbent, Jane; Pang, Jiuhong; Huang, Yung-Yu; Arango, Victoria; Nagy, Peter L; John Mann, J

    2015-07-01

    Low brain expression of the spermidine/spermine N-1 acetyltransferase (SAT1) gene, the rate-limiting enzyme involved in catabolism of polyamines that mediate the polyamine stress response (PSR), has been reported in depressed suicides. However, it is unknown whether this effect is associated with depression or with suicide and whether all or only specific isoforms expressed by SAT1, such as the primary 171 amino acid protein-encoding transcript (SSAT), or an alternative splice variant (SSATX) that is involved in SAT1 regulated unproductive splicing and transcription (RUST), are involved. We applied next generation sequencing (RNA-seq) to assess gene-level, isoform-level, and exon-level SAT1 expression differences between healthy controls (HC, N = 29), DSM-IV major depressive disorder suicides (MDD-S, N = 21) and MDD non-suicides (MDD, N = 9) in the dorsal lateral prefrontal cortex (Brodmann Area 9, BA9) of medication-free individuals postmortem. Using small RNA-seq, we also examined miRNA species putatively involved in SAT1 post-transcriptional regulation. A DSM-IV diagnosis was made by structured interview. Toxicology and history ruled out recent psychotropic medication. At the gene-level, we found low SAT1 expression in both MDD-S (vs. HC, p = 0.002) and MDD (vs. HC, p = 0.002). At the isoform-level, reductions in MDD-S (vs. HC) were most pronounced in four transcripts including SSAT and SSATX, while reductions in MDD (vs. HC) were pronounced in three transcripts, one of which was reduced in MDD relative to MDD-S (all p < 0.1 FDR corrected). We did not observe evidence for differential exon-usage (i.e. splicing) nor differences in miRNA expression. Results replicate the finding of low SAT1 brain expression in depressed suicides in an independent sample and implicate low SAT1 brain expression in MDD independent of suicide. Low expressions of both SSAT and SATX isoforms suggest that shared transcriptional mechanisms involved in RUST may account for low SAT1 brain

  9. Regulation of platelet activating factor synthesis: modulation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase by phosphorylation and dephosphorylation in rat spleen microsomes

    SciTech Connect

    Lenihan, D.J.; Lee, T.C.

    1984-05-16

    1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase plays an important regulatory role in the biosynthesis of platelet activating factor, a potent bioactive mediator. The authors tested the hypothesis that the activity of acetyltransferase may be modulated by enzymatic phosphorylation and dephosphorylation. The results showed that acetyltransferase activity in rat spleens was 2- to 3-fold higher in microsomes isolated in the presence of F/sup -/ than in those isolated in the presence of Cl/sup -/. The microsomal acetyltransferase could be activated by preincubation of microsomes, isolated in the presence of Cl/sup -/, with ATP, Mg/sup 2 +/, and the soluble fraction from rat spleen. Addition of phosphatidylserine, diacylglycerols, plus Ca/sup 2 +/ further enhanced the activity. The increase in the activity of acetyltranferase was abolished by treatment of the activated microsomes with alkaline phosphatase. Conversely, the activity of acetyltransferase can be reactivated in the alkaline phosphatase-treated microsomes with incubation conditions that favor phosphorylation. Therefore, the findings suggest that acetyltransferase activity is regulated by reversible activation/inactivation through phosphorylation/dephosphorylation.

  10. Activation of the 2'-N-acetyltransferase gene [aac(2')-Ia] in Providencia stuartii by an interaction of AarP with the promoter region.

    PubMed

    Macinga, D R; Paradise, M R; Parojcic, M M; Rather, P N

    1999-07-01

    The aac(2')-Ia gene in Providencia stuartii encodes a 2'-N-acetyltransferase capable of acetylating both peptidoglycan and certain aminoglycoside antibiotics. Regulation of the aac(2')-Ia gene is influenced in a positive manner by the product of the aarP gene, which encodes a small transcriptional activator of the AraC (XylS) family. In this study, we demonstrate the sequence requirements at the aac(2')-Ia promoter for AarP binding and activation.

  11. Role of CBP/p300 and SRC-1 in transcriptional regulation of the pulmonary surfactant protein-A (SP-A) gene by thyroid transcription factor-1 (TTF-1).

    PubMed

    Yi, Ming; Tong, Guo-Xia; Murry, Barbara; Mendelson, Carole R

    2002-01-25

    Surfactant protein-A (SP-A) gene expression is developmentally regulated in fetal lung type II cells and is enhanced by cAMP. cAMP stimulation of SP-A gene expression is mediated by protein kinase A (PKA) phosphorylation of thyroid transcription factor 1 (TTF-1), expressed selectively in developing lung epithelium. In this study, we analyzed roles of CREB-binding protein (CBP) and steroid receptor coactivator-1 (SRC-1) in TTF-1 regulation of SP-A expression. Upon differentiation of human fetal lung in culture, nuclear localization of CBP, SRC-1, and TTF-1 increased in ductular epithelium in association with type II cell differentiation and induction of SP-A expression. In transient transfections, CBP and SRC-1 acted synergistically with TTF-1 to increase SP-A promoter activity. Overexpression of PKA catalytic subunit enhanced hSP-A promoter activation by SRC-1 plus TTF-1. Adenoviral E1A overexpression reduced TTF-1 +/- SRC-1 induction of SP-A promoter activity, suggesting a role of endogenous CBP/p300. TTF-1 interacted with SRC-1 and CBP in vitro. SRC-1 immunodepletion from type II cell nuclear extracts reduced binding to the TTF-1 binding element upstream of SP-A gene. In cultured type II cells, cAMP increased TTF-1 acetylation. This suggests that cAMP-mediated TTF-1 phosphorylation facilitates interaction with CBP and SRC-1, resulting in its hyperacetylation, further enhancing TTF-1 DNA-binding and transcriptional activity.

  12. Herpes simplex virus 1-encoded tegument protein VP16 abrogates the production of beta interferon (IFN) by inhibiting NF-κB activation and blocking IFN regulatory factor 3 to recruit its coactivator CBP.

    PubMed

    Xing, Junji; Ni, Liwen; Wang, Shuai; Wang, Kezhen; Lin, Rongtuan; Zheng, Chunfu

    2013-09-01

    Host cells activate innate immune signaling pathways to defend against invading pathogens. To survive within an infected host, viruses have evolved intricate strategies to counteract host immune responses. Herpesviruses, including herpes simplex virus type 1 (HSV-1), have large genomes and therefore have the capacity to encode numerous proteins that modulate host innate immune responses. Here we define the contribution of HSV-1 tegument protein VP16 in the inhibition of beta interferon (IFN-β) production. VP16 was demonstrated to significantly inhibit Sendai virus (SeV)-induced IFN-β production, and its transcriptional activation domain was not responsible for this inhibition activity. Additionally, VP16 blocked the activation of the NF-κB promoter induced by SeV or tumor necrosis factor alpha treatment and expression of NF-κB-dependent genes through interaction with p65. Coexpression analysis revealed that VP16 selectively blocked IFN regulatory factor 3 (IRF-3)-mediated but not IRF-7-mediated transactivation. VP16 was able to bind to IRF-3 but not IRF-7 in vivo, based on coimmunoprecipitation analysis, but it did not affect IRF-3 dimerization, nuclear translocation, or DNA binding activity. Rather, VP16 interacted with the CREB binding protein (CBP) coactivator and efficiently inhibited the formation of the transcriptional complexes IRF-3-CBP in the context of HSV-1 infection. These results illustrate that VP16 is able to block the production of IFN-β by inhibiting NF-κB activation and interfering with IRF-3 to recruit its coactivator CBP, which may be important to the early events leading to HSV-1 infection.

  13. Oridonin, a novel lysine acetyltransferases inhibitor, inhibits proliferation and induces apoptosis in gastric cancer cells through p53- and caspase-3-mediated mechanisms

    PubMed Central

    Zhang, Juan; Diao, Hua; Li, Guangming; Xu, Ling; Wang, Ting; Wei, Jue; Meng, Wenying; Ma, Jia-Li; Yu, Heguo; Wang, Yu-Gang

    2016-01-01

    Lysine acetylation has been reported to involve in the pathogenesis of multiple diseases including cancer. In our screening study to identify natural compounds with lysine acetyltransferase inhibitor (KATi) activity, oridonin was found to possess acetyltransferase-inhibitory effects on multiple acetyltransferases including P300, GCN5, Tip60, and pCAF. In gastric cancer cells, oridonin treatment inhibited cell proliferation in a concentration-dependent manner and down-regulated the expression of p53 downstream genes, whereas p53 inhibition by PFT-α reversed the antiproliferative effects of oridonin. Moreover, oridonin treatment induced cell apoptosis, increased the levels of activated caspase-3 and caspase-9, and decreased the mitochondrial membrane potential in gastric cancer cells in a concentration-dependent manner. Caspase-3 inhibition by Ac-DEVD-CHO reversed the proapoptosis effect of oridonin. In conclusion, our study identified oridonin as a novel KATi and demonstrated its tumor suppressive effects in gastric cancer cells at least partially through p53-and caspase-3-mediated mechanisms. PMID:26980707

  14. Small-Angle X-Ray Scattering Analysis of the Bifunctional Antibiotic Resistance Enzyme Aminoglycoside (6′) Acetyltransferase-Ie/Aminoglycoside (2″) Phosphotransferase-Ia Reveals a Rigid Solution Structure

    PubMed Central

    Caldwell, Shane J.

    2012-01-01

    Aminoglycoside (6′) acetyltransferase-Ie/aminoglycoside (2″) phosphotransferase-Ia [AAC(6′)-Ie/APH(2″)-Ia] is one of the most problematic aminoglycoside resistance factors in clinical pathogens, conferring resistance to almost every aminoglycoside antibiotic available to modern medicine. Despite 3 decades of research, our understanding of the structure of this bifunctional enzyme remains limited. We used small-angle X-ray scattering (SAXS) to model the structure of this bifunctional enzyme in solution and to study the impact of substrate binding on the enzyme. It was observed that the enzyme adopts a rigid conformation in solution, where the N-terminal AAC domain is fixed to the C-terminal APH domain and not loosely tethered. The addition of acetyl-coenzyme A, coenzyme A, GDP, guanosine 5′-[β,γ-imido]triphosphate (GMPPNP), and combinations thereof to the protein resulted in only modest changes to the radius of gyration (RG) of the enzyme, which were not consistent with any large changes in enzyme structure upon binding. These results imply some selective advantage to the bifunctional enzyme beyond coexpression as a single polypeptide, likely linked to an improvement in enzymatic properties. We propose that the rigid structure contributes to improved electrostatic steering of aminoglycoside substrates toward the two active sites, which may provide such an advantage. PMID:22290965

  15. G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression

    PubMed Central

    Zhang, Xi; Peng, Danni; Xi, Yuanxin; Yuan, Chao; Sagum, Cari A.; Klein, Brianna J.; Tanaka, Kaori; Wen, Hong; Kutateladze, Tatiana G.; Li, Wei; Bedford, Mark T.; Shi, Xiaobing

    2016-01-01

    The euchromatin histone methyltransferase 2 (also known as G9a) methylates histone H3K9 to repress gene expression, but it also acts as a coactivator for some nuclear receptors. The molecular mechanisms underlying this activation remain elusive. Here we show that G9a functions as a coactivator of the endogenous oestrogen receptor α (ERα) in breast cancer cells in a histone methylation-independent manner. G9a dimethylates ERα at K235 both in vitro and in cells. Dimethylation of ERαK235 is recognized by the Tudor domain of PHF20, which recruits the MOF histone acetyltransferase (HAT) complex to ERα target gene promoters to deposit histone H4K16 acetylation promoting active transcription. Together, our data suggest the molecular mechanism by which G9a functions as an ERα coactivator. Along with the PHF20/MOF complex, G9a links the crosstalk between ERα methylation and histone acetylation that governs the epigenetic regulation of hormonal gene expression. PMID:26960573

  16. Emergence of aminoglycoside 3-N-acetyltransferase IV in Escherichia coli and Salmonella typhimurium isolated from animals in France.

    PubMed Central

    Chaslus-Dancla, E; Martel, J L; Carlier, C; Lafont, J P; Courvalin, P

    1986-01-01

    We studied two outbreaks of calf salmonellosis caused by apramycin and gentamicin-resistant Salmonella typhimurium strains. In both cases, the responsible strains were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, tetracycline, and trimethoprim; one strain was also resistant to nalidixic acid in one outbreak. A systematic survey of the intestinal Escherichia coli strains of calves from the two affected flocks showed that 11 of 24 animals sampled were also colonized by apramycin- and gentamicin-resistant E. coli strains. These isolates belonged to four biotypes and were resistant to ampicillin, chloramphenicol, kanamycin, streptomycin, tetracycline, trimethoprim, and nalidixic acid. All of the strains were resistant to high levels of apramycin (MICs, 512 to 1,024 micrograms/ml) and to gentamicin (MICs, 8 to 32 micrograms/ml), and these resistances were always transferred en bloc. In S. typhimurium, this coresistance was borne by plasmids that were approximately 39 kilobases long (outbreak 1) or 90 kilobases long (outbreak 2), whereas in E. coli, the coresistance was due to plasmids that were approximately 110 kilobases long in both outbreaks. The two plasmids of Salmonella and four plasmids of E. coli encoded type IV aminoglycoside 3-N-acetyltransferases. The intensive use of curative and preventive treatments in calf production could be responsible for the emergence of enzymic resistance to apramycin and gentamicin. Images PMID:3521474

  17. Serotonin N-acetyltransferase activity as a target for temperature in the regulation of melatonin production by frog retina.

    PubMed

    Valenciano, A I; Alonso-Gómez, A L; De Pedro, N; Alonso-Bedate, M; Delgado, M J

    1994-12-01

    The adaptive mechanisms of serotonin N-acetyltransferase (NAT) activity in the regulation of melatonin synthesis in frog retina in the face of chronic and acute temperature changes have been investigated. We performed thermal acclimation experiments to test different environmental temperatures at two seasons of the year (summer and winter), followed by the set-up of an eyecup culture system to investigate the acute effects of temperature on NAT activity and melatonin production daily rhythms. Low temperature induced a significant increase in NAT activity, independent of both the time of the photocycle (midday or midnight) and the season of the year (winter or summer). Acute cold-induced stimulation of NAT activity may be associated with lower decreases in the enzyme synthesis rate, rather than decreases in the degradation rate. In contrast, acclimation to warm temperature (25 degrees C) stimulated ocular melatonin production. Nocturnal melatonin production in eyecups cultured at 25 degrees C was significantly higher than in eyecups cultured at 5 degrees C. We suggest that this discrepancy in thermal regulation of melatonin synthesis can be justified by a seasonal variation in serotonin content within the photoreceptor cells, which determines the thermal response of melatonin production through changes in NAT kinetics.

  18. Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.

    PubMed

    Byeon, Yeong; Back, Kyoungwhan

    2016-08-01

    Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. Two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of N-acetylserotonin O-methyltransferase (ASMT), resulting in the synthesis of O-methylated NAS, also known as melatonin. Attempts to document melatonin production in Escherichia coli have been unsuccessful to date due to either low enzyme activity or inactive ASMT expression. Here, we employed caffeic acid O-methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that has ASMT activity as well. Among several combinations of dual expression cassettes, recombinant E. coli that expressed sheep SNAT with rice COMT produced a high quantity of melatonin, which was measured in a culture medium (1.46 mg/L in response to 1 mM serotonin). This level was several orders of magnitude higher than that produced in transgenic rice and tomato overexpressing sheep SNAT and ASMT, respectively. This heterologous expression system can be widely employed to screen various putative SNAT or ASMT genes from animals and plants as well as to overproduce melatonin in various useful microorganisms.

  19. Stress induction of the spermidine/spermine N1-acetyltransferase by a post-transcriptional mechanism in mammalian cells.

    PubMed Central

    Gerner, E W; Kurtts, T A; Fuller, D J; Casero, R A

    1993-01-01

    Heat shock and diethyldithiocarbamate stimulate polyamine catabolism in animal cells by a mechanism involving the induction of spermidine/spermine N1-acetyltransferase (N1-SSAT) activity. Steady-state levels of RNA encoding this enzyme remain essentially unchanged during periods after these stresses when N1-SSAT activity is increased by 3.5-10-fold or more in three different cell lines of hamster and human origin. Depletion of intracellular spermidine pools by alpha-difluoromethylornithine (DFMO) inhibits stress induction of N1-SSAT activity. Exogenous spermidine can restore stress inducibility of N1-SSAT to DFMO-treated cells, and induce this enzyme activity in non-heat-shocked but polyamine-depleted cells. Acetylation at N1 suppresses the ability of spermidine to induce N1-SSAT activity, relative to this same modification at N8. Fluorinated spermidine analogues, which decrease the pKa values of the amine groups at positions 4 and 8, neither induce nor inhibit N1-SSAT activity in DFMO-treated cells. These data demonstrate that certain stresses induce N1-SSAT by a spermidine-dependent post-transcriptional mechanism. The mode of induction is affected by both the propyl and butyl moieties of spermidine. Images Figure 2 PMID:8396915

  20. Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening.

    PubMed

    Ma, Yi; Ghoshdastider, Umesh; Wang, Jufang; Ye, Wei; Dötsch, Volker; Filipek, Slawomir; Bernhard, Frank; Wang, Xiaoning

    2012-12-01

    Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.

  1. Arginine vasotocin activates phosphoinositide signal transduction system and potentiates N-acetyltransferase activity in the rat pineal gland.

    PubMed

    Novotná, R; Jác, M; Hájek, I; Novotný, I

    1999-03-05

    The pineal gland is innervated by pinealopetal peptidergic fibers originating in the hypothalamic nuclei which release arginine vasopressin (AVP) and arginine vasotocin (AVT) from their endings. Since the mechanism of AVT action on the pineal signal transduction and melatonin synthesis has not been determined so far, we examined the effect of AVT on the phosphoinositide signalling system and the N-acetyltransferase (NAT) activity in the rat pineal gland. The effect of AVP 4-9 fragment and AVP analogue desmopressin was also tested. The phosphoinositide signalling system was studied by measuring 32P labelling of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) which reflects PI cycle activation. AVT (10(-5) and 10(-4) M) induced a significant increase in 32P labelling of PI, PIP and PIP2. The AVT mediated activation of the PI signal cascade was supressed by the vasopressin V1 receptor antagonist. The desmopressin and AVP 4-9 fragment were without the effect on PI signalling. To assess the AVT role in the melatonin synthesis we studied the daily pattern of the pineal NAT activity in rats treated by AVT (10 microg/100 g b.w). AVT application in the dark period of the day significantly increased nocturnal NAT activity. It can be summarized that AVT activates PI signalling system and potentiates NAT activity in the rat pineal gland.

  2. Anti-histone acetyltransferase activity from allspice extracts inhibits androgen receptor-dependent prostate cancer cell growth.

    PubMed

    Lee, Yoo-Hyun; Hong, Soon Won; Jun, Woojin; Cho, Hong Yon; Kim, Han-Cheon; Jung, Myung Gu; Wong, Jiemin; Kim, Ha-Il; Kim, Chang-Hoon; Yoon, Ho-Geun

    2007-11-01

    Histone acetylation depends on the activity of two enzyme families, histone acetyltransferase (HAT) and deacetylase (HDAC). In this study, we screened various plant extracts to find potent HAT inhibitors. Hot water extracts of allspice inhibited HAT activity, especially p300 and CBP (40% at 100 microg/ml). The mRNA levels of two androgen receptor (AR) regulated genes, PSA and TSC22, decreased with allspice treatment (100 microg/ml). Importantly, in IP western analysis, AR acetylation was dramatically decreased by allspice treatment.Furthermore, chromatin immunoprecipitation indicated that the acetylation of histone H3 in the PSA and B2M promoter regions was also repressed. Finally, allspice treatment reduced the growth of human prostate cancer cells, LNCaP (50% growth inhibition at 200 microg/ml). Taken together, our data indicate that the potent HAT inhibitory activity of allspice reduced AR and histone acetylation and led to decreased transcription of AR target genes, resulting in inhibition of prostate cancer cell growth.

  3. Reduction in choline acetyltransferase immunoreactivity but not muscarinic-m2 receptor immunoreactivity in the brainstem of SIDS infants.

    PubMed

    Mallard, C; Tolcos, M; Leditschke, J; Campbell, P; Rees, S

    1999-03-01

    The cholinergic neurotransmitter system is vital for several brainstem functions including cardiorespiratory control and central chemosensitivity. This study has examined aspects of the cholinergic neurotransmitter system in the brainstem of sudden infant death syndrome (SIDS) and control infants. The cellular localisation and the optical density of the immunoreactivity of the cholinergic enzyme choline acetyltransferase (CHAT-IR) and the muscarinic acetylcholine receptor m2 (m2-IR) in the medulla was described in 14 SIDS and 9 control cases. There was a reduction in the number of CHAT-IR neurons in the hypoglossal nucleus (control: 71.2+/-8.3% vs SIDS: 46.1+/-5.3%) and the dorsal motor nucleus of the vagus (DMV) (control: 77.2+/-5.0% vs SIDS: 52.5+/-7.4%) and reduced optical density of CHAT-IR in the hypoglossal nucleus (control: 0.20+/-0.01 vs SIDS; 0.14+/-0.02) in SIDS infants. In contrast there were no changes in the optical density of m2-IR in the hypoglossal nucleus, the DMV, or the arcuate nucleus. Hypoplasia of the arcuate nucleus was observed in one SIDS infant. These results suggest that there is a specific defect in some cholinergic motor neurons in the medulla of SIDS infants. This could lead to abnormal control of cardiovascular and respiratory function and airway patency and may be one of the contributing factors in the etiology of SIDS.

  4. A Naturally-Occurring Histone Acetyltransferase Inhibitor Derived from Garcinia indica Impairs Newly Acquired and Reactivated Fear Memories

    PubMed Central

    Maddox, Stephanie A.; Watts, Casey S.; Doyère, Valérie; Schafe, Glenn E.

    2013-01-01

    The study of the cellular and molecular mechanisms underlying the consolidation and reconsolidation of traumatic fear memories has progressed rapidly in recent years, yet few compounds have emerged that are readily useful in a clinical setting for the treatment of anxiety disorders such as post-traumatic stress disorder (PTSD). Here, we use a combination of biochemical, behavioral, and neurophysiological methods to systematically investigate the ability of garcinol, a naturally-occurring histone acetyltransferase (HAT) inhibitor derived from the rind of the fruit of the Kokum tree (Garcina indica), to disrupt the consolidation and reconsolidation of Pavlovian fear conditioning, a widely studied rodent model of PTSD. We show that local infusion of garcinol into the rat lateral amygdala (LA) impairs the training and retrieval-related acetylation of histone H3 in the LA. Further, we show that either intra-LA or systemic administration of garcinol within a narrow window after either fear conditioning or fear memory retrieval significantly impairs the consolidation and reconsolidation of a Pavlovian fear memory and associated neural plasticity in the LA. Our findings suggest that a naturally-occurring compound derived from the diet that regulates chromatin function may be useful in the treatment of newly acquired or recently reactivated traumatic memories. PMID:23349897

  5. Depletion of the human N-terminal acetyltransferase hNaa30 disrupts Golgi integrity and ARFRP1-localization.

    PubMed

    Kobbenes Starheim, Kristian; Kalvik, Thomas Vikestad; Bjørkøy, Geir; Arnesen, Thomas

    2017-03-29

    The organization of the Golgi apparatus is tightly regulated. Golgi stack scattering is observed in cellular processes such as apoptosis and mitosis, and has also been associated with disruption of cellular lipid metabolism, and neurodegenerative diseases. Our studies show that depletion of the human N-α-acetyltransferase 30 (hNaa30) induces fragmentation of the Golgi stack in HeLa and CAL-62 cell lines. The Golgi apparatus-associated GTPase ARFRP1 was previously shown to require N-terminal acetylation for membrane association, and based on its N-terminal sequence it is likely to be a substrate of hNaa30. ARFRP1 is involved in endosome-to- trans- Golgi Network traffic. We observed that ARFRP1 shifted from a predominantly cis- Golgi and trans- Golgi Network localization to localizing both to the Golgi and to non-Golgi vesicular structures in hNaa30-depleted cells. However, we did not observe loss of membrane association of ARFRP1. We conclude that hNaa30-depletion induces Golgi-scattering, and induces aberrant ARFRP1 Golgi-localization.

  6. Histone acetyltransferase AtGCN5/HAG1 is a versatile regulator of developmental and inducible gene expression in Arabidopsis.

    PubMed

    Servet, Caroline; Conde e Silva, Natalia; Zhou, Dao-Xiu

    2010-07-01

    Histone acetylation/deacetylation is a dynamic process and plays an important role in gene regulation. Histone acetylation homeostasis is regulated by antagonist actions of histone acetyltransferases (HAT) and deacetylases (HDAC). Plant genome encodes multiple HATs and HDACs. The Arabidopsis HAT gene AtGCN5/HAG1plays an essential role in many plant development processes, such as meristem function, cell differentiation, leaf and floral organogenesis, and responses to environmental conditions such as light and cold, indicating an important role of this HAT in the regulation of both long-term developmental switches and short-term inducible gene expression. AtGCN5 targets to a large number of promoters and is required for acetylation of several histone H3 lysine residues. Recruitment of AtGCN5 to target promoters is likely to be mediated by direct or indirect interaction with DNA-binding transcription factors and/or by interaction with acetylated histone lysine residues on the targets. Interplay between AtGCN5 and other HAT and HDAC is demonstrated to control specific regulatory pathways. Analysis of the role of AtGCN5 in light-inducible gene expression suggests a function of AtGCN5 in preparing chromatin commitment for priming inducible gene activation in plants.

  7. Implication of an Aldehyde Dehydrogenase Gene and a Phosphinothricin N-Acetyltransferase Gene in the Diversity of Pseudomonas cichorii Virulence

    PubMed Central

    Tanaka, Masayuki; Wali, Ullah Md; Nakayashiki, Hitoshi; Fukuda, Tatsuya; Mizumoto, Hiroyuki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2011-01-01

    Pseudomonas cichorii harbors the hrp genes. hrp-mutants lose their virulence on eggplant but not on lettuce. A phosphinothricin N-acetyltransferase gene (pat) is located between hrpL and an aldehyde dehydrogenase gene (aldH) in the genome of P. cichorii. Comparison of nucleotide sequences and composition of the genes among pseudomonads suggests a common ancestor of hrp and pat between P. cichorii strains and P. viridiflava strains harboring the single hrp pathogenicity island. In contrast, phylogenetic diversification of aldH corresponded to species diversification amongst pseudomonads. In this study, the involvement of aldH and pat in P. cichorii virulence was analyzed. An aldH-deleted mutant (ΔaldH) and a pat-deleted mutant (Δpat) lost their virulence on eggplant but not on lettuce. P. cichorii expressed both genes in eggplant leaves, independent of HrpL, the transcriptional activator for the hrp. Inoculation into Asteraceae species susceptible to P. cichorii showed that the involvement of hrp, pat and aldH in P. cichorii virulence is independent of each other and has no relationship with the phylogeny of Asteraceae species based on the nucleotide sequences of ndhF and rbcL. It is thus thought that not only the hrp genes but also pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible host plant species. PMID:24704843

  8. Choline acetyltransferase-like immunoreactivity in a physiologically distinct subtype of olfactory nonspiking local interneurons in the cockroach (periplaneta americana).

    PubMed

    Fusca, Debora; Husch, Andreas; Baumann, Arnd; Kloppenburg, Peter

    2013-10-15

    Behavioral and physiological studies have shown that local interneurons are pivotal for processing odor information in the insect antennal lobe. They mediate inhibitory and excitatory interactions between the glomerular pathways and ultimately shape the tuning profile of projection neurons. To identify putative cholinergic local interneurons in the antennal lobe of Periplaneta americana, an antibody raised against the biosynthetic enzyme choline acetyltransferase (ChAT) was applied to individual morphologically and electrophysiologically characterized local interneurons. In nonspiking type IIa1 local interneurons, which were classified in this study, we found ChAT-like immunoreactivity suggesting that they are most likely excitatory. This is a well-defined population of neurons that generates Ca(2+) -driven spikelets upon depolarization and stimulation with odorants, but not Na(+) -driven action potentials, because they lack voltage-activated transient Na(+) currents. The nonspiking type IIa2 and type IIb local interneurons, in which Ca(2+) -driven spikelets were absent, had no ChAT-like immunoreactivity. The GABA-like immunoreactive, spiking type I local interneurons had no ChAT-like immunoreactivity. In addition, we showed that uniglomerular projection neurons with cell bodies located in the ventral portion of the ventrolateral somata group and projections along the inner antennocerebral tract exhibited ChAT-like immunoreactivity. Assigning potential transmitters and neuromodulators to distinct morphological and electrophysiological types of antennal lobe neurons is an important prerequisite for a detailed understanding of odor information processing in insects.

  9. The dihydrolipoamide acetyltransferase is a novel metabolic longevity factor and is required for calorie restriction-mediated life span extension.

    PubMed

    Easlon, Erin; Tsang, Felicia; Dilova, Ivanka; Wang, Chen; Lu, Shu-Ping; Skinner, Craig; Lin, Su-Ju

    2007-03-02

    Calorie restriction (CR) extends life span in a wide variety of species. Recent studies suggest that an increase in mitochondrial metabolism mediates CR-induced life span extension. Here we present evidence that Lat1 (dihydrolipoamide acetyltransferase), the E2 component of the mitochondrial pyruvate dehydrogenase complex, is a novel metabolic longevity factor in the CR pathway. Deleting the LAT1 gene abolishes life span extension induced by CR. Overexpressing Lat1 extends life span, and this life span extension is not further increased by CR. Similar to CR, life span extension by Lat1 overexpression largely requires mitochondrial respiration, indicating that mitochondrial metabolism plays an important role in CR. Interestingly, Lat1 overexpression does not require the Sir2 family to extend life span, suggesting that Lat1 mediates a branch of the CR pathway that functions in parallel to the Sir2 family. Lat1 is also a limiting longevity factor in nondividing cells in that overexpressing Lat1 extends cell survival during prolonged culture at stationary phase. Our studies suggest that Lat1 overexpression extends life span by increasing metabolic fitness of the cell. CR may therefore also extend life span and ameliorate age-associated diseases by increasing metabolic fitness through regulating central metabolic enzymes.

  10. Adolescent, but not adult, binge ethanol exposure leads to persistent global reductions of choline acetyltransferase expressing neurons in brain.

    PubMed

    Vetreno, Ryan P; Broadwater, Margaret; Liu, Wen; Spear, Linda P; Crews, Fulton T

    2014-01-01

    During the adolescent transition from childhood to adulthood, notable maturational changes occur in brain neurotransmitter systems. The cholinergic system is composed of several distinct nuclei that exert neuromodulatory control over cognition, arousal, and reward. Binge drinking and alcohol abuse are common during this stage, which might alter the developmental trajectory of this system leading to long-term changes in adult neurobiology. In Experiment 1, adolescent intermittent ethanol (AIE; 5.0 g/kg, i.g., 2-day on/2-day off from postnatal day [P] 25 to P55) treatment led to persistent, global reductions of choline acetyltransferase (ChAT) expression. Administration of the Toll-like receptor 4 agonist lipopolysaccharide to young adult rats (P70) produced a reduction in ChAT+IR that mimicked AIE. To determine if the binge ethanol-induced ChAT decline was unique to the adolescent, Experiment 2 examined ChAT+IR in the basal forebrain following adolescent (P28-P48) and adult (P70-P90) binge ethanol exposure. Twenty-five days later, ChAT expression was reduced in adolescent, but not adult, binge ethanol-exposed animals. In Experiment 3, expression of ChAT and vesicular acetylcholine transporter expression was found to be significantly reduced in the alcoholic basal forebrain relative to moderate drinking controls. Together, these data suggest that adolescent binge ethanol decreases adult ChAT expression, possibly through neuroimmune mechanisms, which might impact adult cognition, arousal, or reward sensitivity.

  11. Structure and nucleosome interaction of the yeast NuA4 and Piccolo-NuA4 histone acetyltransferase complexes

    PubMed Central

    Chittuluru, Johnathan R.; Chaban, Yuriy; Monnet-Saksouk, Julie; Carrozza, Michael J.; Sapountzi, Vasileia; Selleck, William; Huang, Jiehuan; Utley, Rhea T.; Cramet, Myriam; Allard, Stephane; Cai, Gang; Workman, Jerry L.; Fried, Michael G.; Tan, Song; Côté, Jacques; Asturias, Francisco J.

    2011-01-01

    We have used electron microscopy (EM) and biochemistry to characterize the structure and nucleosome core particle (NCP) interaction of NuA4, an essential yeast histone acetyltransferase (HAT) complex conserved throughout eukaryotes. The ATM-related Tra1 subunit, shared with the SAGA coactivator, forms a large domain joined to a second portion that accommodates the Piccolo catalytic subcomplex and other NuA4 subunits. EM analysis of an NuA4–NCP complex shows the NCP bound at NuA4's periphery. EM characterization of Piccolo and Piccolo–NCP provided further information about subunit organization and confirmed that histone acetylation requires minimal contact with the NCP. A small conserved region at the N-terminus of Piccolo subunit Epl1 is essential for NCP interaction, whereas subunit Yng2 apparently positions Piccolo for efficient acetylation of H4 or H2A tails. Taken together, these results provide an understanding of NuA4 subunit organization and NCP interactions. PMID:21984211

  12. The histone acetyltransferases CBP and Chameau integrate developmental and DNA replication programs in Drosophila ovarian follicle cells.

    PubMed

    McConnell, Kristopher H; Dixon, Michael; Calvi, Brian R

    2012-10-01

    DNA replication origin activity changes during development. Chromatin modifications are known to influence the genomic location of origins and the time during S phase that they initiate replication in different cells. However, how chromatin regulates origins in concert with cell differentiation remains poorly understood. Here, we use developmental gene amplification in Drosophila ovarian follicle cells as a model to investigate how chromatin modifiers regulate origins in a developmental context. We find that the histone acetyltransferase (HAT) Chameau (Chm) binds to amplicon origins and is partially required for their function. Depletion of Chm had relatively mild effects on origins during gene amplification and genomic replication compared with previous knockdown of its ortholog HBO1 in human cells, which has severe effects on origin function. We show that another HAT, CBP (Nejire), also binds amplicon origins and is partially required for amplification. Knockdown of Chm and CBP together had a more severe effect on nucleosome acetylation and amplicon origin activity than knockdown of either HAT alone, suggesting that these HATs collaborate in origin regulation. In addition to their local function at the origin, we show that Chm and CBP also globally regulate the developmental transition of follicle cells into the amplification stages of oogenesis. Our results reveal a complexity of origin epigenetic regulation by multiple HATs during development and suggest that chromatin modifiers are a nexus that integrates differentiation and DNA replication programs.

  13. Acetate ester production by Chinese yellow rice wine yeast overexpressing the alcohol acetyltransferase-encoding gene ATF2.

    PubMed

    Zhang, J; Zhang, C; Qi, Y; Dai, L; Ma, H; Guo, X; Xiao, D

    2014-11-27

    Acetate ester, which are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction, are responsible for the fruity character of fermented alcoholic beverages such as Chinese yellow rice wine. Alcohol acetyltransferase (AATase) is currently believed to be the key enzyme responsible for the production of acetate ester. In order to determine the precise role of the ATF2 gene in acetate ester production, an ATF2 gene encoding a type of AATase was overexpressed and the ability of the mutant to form acetate esters (including ethyl acetate, isoamyl acetate, and isobutyl acetate) was investigated. The results showed that after 5 days of fermentation, the concentrations of ethyl acetate, isoamyl acetate, and isobutyl acetate in yellow rice wines fermented with EY2 (pUC-PIA2K) increased to 137.79 mg/L (an approximate 4.9-fold increase relative to the parent cell RY1), 26.68 mg/L, and 7.60 mg/L, respectively. This study confirms that the ATF2 gene plays an important role in the production of acetate ester production during Chinese yellow rice wine fermentation, thereby offering prospects for the development of yellow rice wine yeast starter strains with optimized ester-producing capabilities.

  14. Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with vasoactive intestinal peptide and choline acetyltransferase

    SciTech Connect

    Leblanc, G.C.; Trimmer, B.A.; Landis, S.C.

    1987-05-01

    Neuropeptide Y (NPY) is widely distributed in the sympathetic nervous system, where it is colocalized with norepinephrine. The authors report here that NPY-immunoreactive neurons are also abundant in three cranial parasympathetic ganglia, the otic, sphenopalatine, and ciliary, in the rat measured by radioimmunoassay. High-performance liquid chromatographic analysis of the immunoreactive material present in the otic ganglion indicates that this material is very similar to porcine NPY and indistinguishable from the NPY-like immunoreactivity present in rat sympathetic neurons. These findings raise the possibility that NPY acts as a neuromodulator in the parasympathetic as well as the sympathetic nervous system. In contrast to what had been observed for sympathetic neurons, NPY-immunoreactive neurons in cranial parasympathetic ganglia do not contain detectable catecholamines or tyrosine hydroxylase immunoreactivity, and many do contain immunoreactivity for vasoactive intestinal peptide and/or choline acetyltransferase. These findings suggest that there is no simple rule governing coexpression of NPY with norepinephrine, acetylcholine, or vasoactive intestinal peptide in autonomic neurons. Further, while functional studies have indicated that NPY exerts actions on the peripheral vasculature which are antagonistic to those of acetylcholine and vasoactive intestinal peptide, the present results raise the possibility that these three substances may have complementary effects on other target tissues.

  15. Synergistic action of histone acetyltransferase GCN5 and receptor CLAVATA1 negatively affects ethylene responses in Arabidopsis thaliana.

    PubMed

    Poulios, Stylianos; Vlachonasios, Konstantinos E

    2016-02-01

    GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5) is a histone acetyltransferase (HAT) and the catalytic subunit of several multicomponent HAT complexes that acetylate lysine residues of histone H3. Mutants in AtGCN5 display pleiotropic developmental defects including aberrant meristem function. Shoot apical meristem (SAM) maintenance is regulated by CLAVATA1 (CLV1), a receptor kinase that controls the size of the shoot and floral meristems. Upon activation through CLV3 binding, CLV1 signals to the transcription factor WUSCHEL (WUS), restricting WUS expression and thus the meristem size. We hypothesized that GCN5 and CLV1 act together to affect SAM function. Using genetic and molecular approaches, we generated and characterized clv gcn5 mutants. Surprisingly, the clv1-1 gcn5-1 double mutant exhibited constitutive ethylene responses, suggesting that GCN5 and CLV signaling act synergistically to inhibit ethylene responses in Arabidopsis. This genetic and molecular interaction was mediated by ETHYLENE INSENSITIVE 3/ EIN3-LIKE1 (EIN3/EIL1) transcription factors. Our data suggest that signals from the CLV transduction pathway reach the GCN5-containing complexes in the nucleus and alter the histone acetylation status of ethylene-responsive genes, thus translating the CLV information to transcriptional activity and uncovering a link between histone acetylation and SAM maintenance in the complex mode of ethylene signaling.

  16. Absence of Rtt109p, a fungal-specific histone acetyltransferase, results in improved acetic acid tolerance of Saccharomyces cerevisiae.

    PubMed

    Cheng, Cheng; Zhao, Xinqing; Zhang, Mingming; Bai, Fengwu

    2016-03-01

    RTT109 is a histone acetyltransferase for the acetylation of histone H3. It is still not clear whether RTT109 plays a role in regulation of gene expression under environmental stresses. In this study, the involvement of RTT109 in acetic acid stress tolerance of Saccharomyces cerevisiae was investigated. It was revealed that the absence of RTT109 enhanced resistance to 5.5 g L(-1) acetic acid, which was indicated by improved growth of RTT109Δ mutant compared with that of the wild-type BY4741 strain. Meanwhile, the lag phase was shortened for 48 h and glucose consumption completed 36 h in advance for RTT109Δ mutant compared to the wild-type strain, with ethanol production rate increased from 0.39 to 0.60 g L(-1) h(-1). Significantly, elevated transcription levels of HSP12, CTT1 and GSH1, as well as increased activities of antioxidant enzymes were observed in RTT109Δ under acetic acid stress. Improved flocculation of RTT109Δ compared to that of the control strain BY4741 under the acetic acid stress was also observed. These results suggest that the absence of RTT109 not only activates transcription of stress responsive genes, but also improves resistance to oxidative stress, which ultimately contributes to improved acetic acid tolerance in S. cerevisiae.

  17. The Enok acetyltransferase complex interacts with Elg1 and negatively regulates PCNA unloading to promote the G1/S transition

    PubMed Central

    Huang, Fu; Saraf, Anita; Florens, Laurence; Kusch, Thomas; Swanson, Selene K.; Szerszen, Leanne T.; Li, Ge; Dutta, Arnob; Washburn, Michael P.; Abmayr, Susan M.; Workman, Jerry L.

    2016-01-01

    KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and are involved in cell cycle regulation. However, information regarding their roles in regulating cell cycle progression is limited. Here, we report the identification of subunits of the Drosophila Enok complex and demonstrate that all subunits are important for its HAT activity. We further report a novel interaction between the Enok complex and the Elg1 proliferating cell nuclear antigen (PCNA)-unloader complex. Depletion of Enok in S2 cells resulted in a G1/S cell cycle block, and this block can be partially relieved by depleting Elg1. Furthermore, depletion of Enok reduced the chromatin-bound levels of PCNA in both S2 cells and early embryos, suggesting that the Enok complex may interact with the Elg1 complex and down-regulate its PCNA-unloading function to promote the G1/S transition. Supporting this hypothesis, depletion of Enok also partially rescued the endoreplication defects in Elg1-depleted nurse cells. Taken together, our study provides novel insights into the roles of KAT6 HATs in cell cycle regulation through modulating PCNA levels on chromatin. PMID:27198229

  18. Subfunctionalization of arylalkylamine N-acetyltransferases in the sea bass Dicentrarchus labrax: two-ones for one two.

    PubMed

    Paulin, Charles-Hubert; Cazaméa-Catalan, Damien; Zilberman-Peled, Bina; Herrera-Perez, Patricia; Sauzet, Sandrine; Magnanou, Elodie; Fuentès, Michael; Gothilf, Yoav; Muñoz-Cueto, Jose Antonio; Falcón, Jack; Besseau, Laurence

    2015-10-01

    Melatonin is an important component of the vertebrates circadian system, synthetized from serotonin by the successive action of the arylalkylamine N-acetyltransferase (Aanat: serotonin→N-acetylserotonin) and acetylserotonin-O-methyltransferase (Asmt: N-acetylserotonin→melatonin). Aanat is responsible for the daily rhythm in melatonin production. Teleost fish are unique because they express two Aanat genes, aanat1 and aanat2, mainly expressed in the retina and pineal gland, respectively. In silico analysis indicated that the teleost-specific whole-genome duplication generated Aanat1 duplicates (aanat1a and aanat1b); some fish express both of them, while others express either one of the isoforms. Here, we bring the first information on the structure, function, and distribution of Aanat1a and Aanat1b in a teleost, the sea bass Dicentrarchus labrax. Aanat1a and Aanat1b displayed a wide and distinct distribution in the nervous system and peripheral tissues, while Aanat2 appeared as a pineal enzyme. Co-expression of Aanats with asmt was found in the pineal gland and the three retinal nuclear layers. Enzyme kinetics indicated subtle differences in the affinity and catalytic efficiency of Aanat1a and Aanat1b for indolethylamines and phenylethylamines, respectively. Our data are consistent with the idea that Aanat2 is a pineal enzyme involved in melatonin production, while Aanat1 enzymes have a broader range of functions including melatonin synthesis in the retina, and catabolism of serotonin and dopamine in the retina and other tissues. The data are discussed in light of the recently uncovered roles of N-acetylserotonin and N-acetyldopamine as antioxidants, neuroprotectants, and modulators of cell proliferation and enzyme activities.

  19. 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

  20. MicroRNAs in the pineal gland: miR-483 regulates melatonin synthesis by targeting arylalkylamine N-acetyltransferase.

    PubMed

    Clokie, Samuel J H; Lau, Pierre; Kim, Hyun Hee; Coon, Steven L; Klein, David C

    2012-07-20

    MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.

  1. Regulation of NuA4 histone acetyltransferase activity in transcription and DNA repair by phosphorylation of histone H4.

    PubMed

    Utley, Rhea T; Lacoste, Nicolas; Jobin-Robitaille, Olivier; Allard, Stéphane; Côté, Jacques

    2005-09-01

    The NuA4 complex is a histone H4/H2A acetyltransferase involved in transcription and DNA repair. While histone acetylation is important in many processes, it has become increasingly clear that additional histone modifications also play a crucial interrelated role. To understand how NuA4 action is regulated, we tested various H4 tail peptides harboring known modifications in HAT assays. While dimethylation at arginine 3 (R3M) had little effect on NuA4 activity, phosphorylation of serine 1 (S1P) strongly decreased the ability of the complex to acetylate H4 peptides. However, R3M in combination with S1P alleviates the repression of NuA4 activity. Chromatin from cells treated with DNA damage-inducing agents shows an increase in phosphorylation of serine 1 and a concomitant decrease in H4 acetylation. We found that casein kinase 2 phosphorylates histone H4 and associates with the Rpd3 deacetylase complex, demonstrating a physical connection between phosphorylation of serine 1 and unacetylated H4 tails. Chromatin immunoprecipitation experiments also link local phosphorylation of H4 with its deacetylation, during both transcription and DNA repair. Time course chromatin immunoprecipitation data support a model in which histone H4 phosphorylation occurs after NuA4 action during double-strand break repair at the step of chromatin restoration and deacetylation. These findings demonstrate that H4 phospho-serine 1 regulates chromatin acetylation by the NuA4 complex and that this process is important for normal gene expression and DNA repair.

  2. Differential association for N-acetyltransferase 2 genotype and phenotype with bladder cancer risk in Chinese population

    PubMed Central

    Quan, Lei; Chattopadhyay, Koushik; Nelson, Heather H.; Chan, Kenneth K.; Xiang, Yong-Bing; Zhang, Wei; Wang, Renwei; Gao, Yu-Tang; Yuan, Jian-Min

    2016-01-01

    Background N-acetyltransferase 2 (NAT2) is involved in both carcinogen detoxification through hepatic N-acetylation and carcinogen activation through local O-acetylation. NAT2 slow acetylation status is significantly associated with increased bladder cancer risk among European populations, but its association in Asian populations is inconclusive. Methods NAT2 acetylation status was determined by both single nucleotide polymorphisms (SNPs) and caffeine metabolic ratio (CMR), in a population-based study of 494 bladder cancer patients and 507 control subjects in Shanghai, China. Results The CMR, a functional measure of hepatic N-acetylation, was significantly reduced in a dose-dependent manner among both cases and controls possessing the SNP-inferred NAT2 slow acetylation status (all P-values<5.0×10−10). The CMR-determined slow N-acetylation status (CMR<0.34) was significantly associated with a 50% increased risk of bladder cancer (odds ratio = 1.50, 95% confidence interval = 1.10-2.06) whereas the SNP-inferred slow acetylation statuses were significantly associated with an approximately 50% decreased risk of bladder cancer. The genotype-disease association was strengthened after the adjustment for CMR and was primarily observed among never smokers. Conclusions The apparent differential associations for phenotypic and genetic measures of acetylation statuses with bladder cancer risk may reflect dual functions of NAT2 in bladder carcinogenesis because the former only measures the capacity of carcinogen detoxification pathway while the latter represents both carcinogen activation and detoxification pathways. Future studies are warranted to ascertain the specific role of N- and O-acetylation in bladder carcinogenesis, particularly in populations exposed to different types of bladder carcinogens. PMID:27223070

  3. Cigarette smoking, N-acetyltransferase 2 genotypes, and breast cancer risk: pooled analysis and meta-analysis.

    PubMed

    Ambrosone, Christine B; Kropp, Silke; Yang, Jun; Yao, Song; Shields, Peter G; Chang-Claude, Jenny

    2008-01-01

    Approximately 10 years ago, it was noted that smoking increased risk of breast cancer among women with N-acetyltransferase 2 (NAT2) slow acetylation genotypes. This report was followed by a number of studies to address this question. We pooled data from 10 existing studies and also conducted a meta-analysis of 13 studies published from 1996 to October 2006 that were conducted among women, were published in English, and had adequate information on smoking and NAT2 genotyping. Raw data were requested from authors. Unconditional logistic regression was done for pooled analysis, and random effect models was done for meta-analysis. Study heterogeneity was assessed, and sensitivity tests were done when subgroups were excluded from the analysis. In the pooled analysis, there was a significant interaction between smoking, NAT2 genotype, and risk of breast cancer [pack-years (continuous variable, P(interaction) = 0.03)], with higher pack-years significantly associated with an increased risk of breast cancer among women with NAT2 slow genotypes (pooled analysis relative risk, 1.49; 95% confidence interval, 1.08-2.04). These findings were supported by the meta-analysis including all studies; pack-years were significantly associated with risk among slow acetylators in a dose-dependent fashion (meta-analysis relative risk, 1.44; 95% confidence interval, 1.23-1.68 for > or =20 pack-years versus never smokers), but not among rapid acetylators. Similar relationships were noted for smoking status (ever, never) and duration of smoking. Our results show that cigarette smoking is associated with an increase in breast cancer risk among women with NAT2 slow acetylation genotypes. Because slow NAT2 genotypes are present in 50% to 60% of Caucasian populations, smoking is likely to play an important role in breast cancer etiology.

  4. The role of nitric oxide in the PKA inhibitor induced spatial memory deficits in rat: involvement of choline acetyltransferase.

    PubMed

    Najafi, Sheyda; Payandemehr, Borna; Tabrizian, Kaveh; Shariatpanahi, Marjan; Nassireslami, Ehsan; Azami, Kian; Mohammadi, Mojdeh; Asadi, Farideh; Roghani, Ali; Sharifzadeh, Mohammad

    2013-08-15

    Several lines of evidence show that cAMP-PKA signaling pathway plays critical role in memory functions and suggest nitric oxide as an important modulator in learning and memory. In this study, we assessed the effects of intra-hippocampal infusion of H-89, a selective PKAII inhibitor, and 1400 W, a selective inducible nitric oxide synthase (iNOS) inhibitor, on spatial memory in rats. By using the Morris water maze, spatial memory retention parameters were examined 48 h after the infusions through measuring escape latency, traveled distance, and swimming speed. The rats receiving intra-hippocampal infusions of 1400 W (100 µM/side) showed a significant reduction (*P<0.05) in escape latency and traveled distance in comparison with the control saline group. In contrast, a significant increase (**P<0.01) in escape latency and traveled distance was observed after infusion of 10 µM H-89. Moreover, among combination groups, co-administration of 1400 W (400 µM/side) with 10 µM/side of H-89 caused a significant reduction (*P<0.05) in escape latency and traveled distance in comparison with the H-89 group. Also, we evaluated the molecular effects of 1400 W on the expression of choline acetyltransferase (ChAT), a cholinergic marker, in the CA1 region of the hippocampus and medial septal area (MSA). Immunohistochemical analysis of post-training bilateral intra-hippocampal infusion of 1400 W revealed a significant increase in ChAT immunoreactivity levels in both the CA1 and the MSA regions. Overall, the results suggest that 1400 W has protective effect against H89-induced spatial memory impairment. Moreover, the observed memory improvements caused by 1400 W infusions, might be due to interaction of iNOS with the cholinergic system.

  5. Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated caucasian individuals: Correlation with phenotypic activity

    SciTech Connect

    Cascorbi, I.; Drakoulis, N.; Brockmoeller, J.

    1995-09-01

    The polymorphic arylamine N-acetyltransferase (NAT2; EC2.3.1.5) is supposed to be a susceptibility factor for several drug side effects and certain malignancies. A group of 844 unrelated German subjects was genotyped for their acetylation type, and 563 of them were also phenotyped. Seven mutations of the NAT2 gene were evaluated by allele-specific PCR (mutation 341C to T) and PCR-RFLP for mutations at nt positions 191, 282, 481, 590, 803, and 857. From the mutation pattern eight different alleles, including the wild type coding for rapid acetylation and seven alleles coding for slow phenotype, were determined. Four hundred ninety-seven subjects had a genotype of slow acetylation (58.9%; 95% confidence limits 55.5%-62.2%). Phenotypic acetylation capacity was expressed as the ratio of 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine in urine after caffeine intake. Some 6.7% of the cases deviated in genotype and phenotype, but sequencing DNA of these probands revealed no new mutations. Furthermore, linkage pattern of the mutations was always confirmed, as tested in 533 subjects. In vivo acetylation capacity of homozygous wild-type subjects (NAT2{sup *}4/{sup *}4) was significantly higher than in heterozygous genotypes (P = .001). All mutant alleles showed low in vivo acetylation capacities, including the previously not-yet-defined alleles {sup *}5A, {sup *}5C, and {sup *}13. Moreover, distinct slow genotypes differed significantly among each other, as reflected in lower acetylation capacity of {sup *}6A, {sup *}7B, and {sup *}13 alleles than the group of {sup *}5 alleles. The study demonstrated differential phenotypic activity of various NAT2 genes and gives a solid basis for clinical and molecular-epidemiological investigations. 34 refs., 4 figs., 7 tabs.

  6. Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

    PubMed Central

    Bansal, Sunil; Durrett, Timothy P.

    2016-01-01

    Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants. PMID:27688773

  7. Immunohistochemical localization of two types of choline acetyltransferase in neurons and sensory cells of the octopus arm.

    PubMed

    Sakaue, Yuko; Bellier, Jean-Pierre; Kimura, Shin; D'Este, Loredana; Takeuchi, Yoshihiro; Kimura, Hiroshi

    2014-01-01

    Cholinergic structures in the arm of the cephalopod Octopus vulgaris were studied by immunohistochemistry using specific antisera for two types (common and peripheral) of acetylcholine synthetic enzyme choline acetyltransferase (ChAT): antiserum raised against the rat common type ChAT (cChAT), which is cross-reactive with molluscan cChAT, and antiserum raised against the rat peripheral type ChAT (pChAT), which has been used to delineate peripheral cholinergic structures in vertebrates, but not previously in invertebrates. Western blot analysis of octopus extracts revealed a single pChAT-positive band, suggesting that pChAT antiserum is cross-reactive with an octopus counterpart of rat pChAT. In immunohistochemistry, only neuronal structures of the octopus arm were stained by cChAT and pChAT antisera, although the pattern of distribution clearly differed between the two antisera. cChAT-positive varicose nerve fibers were observed in both the cerebrobrachial tract and neuropil of the axial nerve cord, while pChAT-positive varicose fibers were detected only in the neuropil of the axial nerve cord. After epitope retrieval, pChAT-positive neuronal cells and their processes became visible in all ganglia of the arm, including the axial and intramuscular nerve cords, and in ganglia of suckers. Moreover, pChAT-positive structures also became detectable in nerve fibers connecting the different ganglia, in smooth nerve fibers among muscle layers and dermal connective tissues, and in sensory cells of the suckers. These results suggest that the octopus arm has two types of cholinergic nerves: cChAT-positive nerves from brain ganglia and pChAT-positive nerves that are intrinsic to the arm.

  8. Mechanism of action of peptidoglycan O-acetyltransferase B involves a Ser-His-Asp catalytic triad.

    PubMed

    Moynihan, Patrick J; Clarke, Anthony J

    2014-10-07

    The O-acetylation of the essential cell wall polymer peptidoglycan is essential in many bacteria for their integrity and survival, and it is catalyzed by peptidoglycan O-acetlytransferase B (PatB). Using PatB from Neisseria gonorrhoeae as the model, we have shown previously that the enzyme has specificity for polymeric muropeptides that possess tri- and tetrapeptide stems and that rates of reaction increase with increasing degrees of polymerization. Here, we present the catalytic mechanism of action of PatB, the first to be described for an O-acetyltransferase of any bacterial exopolysaccharide. The influence of pH on PatB activity was investigated, and pKa values of 6.4-6.45 and 6.25-6.35 for the enzyme-substrate complex (kcat vs pH) and the free enzyme (kcat·KM(-1) vs pH), respectively, were determined for the respective cosubstrates. The enzyme is partially inactivated by sulfonyl fluorides but not by EDTA, suggesting the participation of a serine residue in its catalytic mechanism. Alignment of the known and hypothetical PatB amino acid sequences identified Ser133, Asp302, and His305 as three invariant amino acid residues that could potentially serve as a catalytic triad. Replacement of Asp302 with Ala resulted in an enzyme with less than 20% residual activity, whereas activity was barely detectable with (His305 → Ala)PatB and (Ser133 → Ala)PatB was totally inactive. The reaction intermediate of the transferase reaction involving acetyl- and propionyl-acyl donors was trapped on both the wild-type and (Asp302 → Ala) enzymes and LC-MS/MS analysis of tryptic peptides identified Ser133 as the catalytic nucleophile. A transacetylase mechanism is proposed based on the mechanism of action of serine esterases.

  9. Profiling brain expression of the spermidine/spermine N1-acetyltransferase 1 (SAT1) gene in suicide.

    PubMed

    Klempan, Timothy A; Rujescu, Dan; Mérette, Chantal; Himmelman, Carla; Sequeira, Adolfo; Canetti, Lilian; Fiori, Laura M; Schneider, Barbara; Bureau, Alexandre; Turecki, Gustavo

    2009-10-05

    Altered stress reactivity is considered to be a risk factor for both major depressive disorder and suicidal behavior. The authors have sought to expand their previous findings implicating altered expression of spermidine/spermine N(1)-acetyltransferase 1 (SAT1), the rate-limiting enzyme involved in catabolism of the polyamines spermidine and spermine in the polyamine stress response (PSR), across multiple brain regions between control individuals and depressed individuals who have died by suicide. Microarray expression of probesets annotated to SAT1 were examined across 17 brain regions in 13 controls and 26 individuals who have died by suicide (16 with a diagnosis of major depression and 10 without), all of French-Canadian origin. Profiling conducted on the Affymetrix U133A/B chipset was further examined on a second chipset (U133 Plus 2.0) using RT-PCR, and analyzed in a second, independent sample. A reduction in SAT1 expression identified through multiple probesets was observed across 12 cortical regions in depressed individuals who have died by suicide compared with controls. Of these, five cortical regions showed statistically significant reductions which were supported by RT-PCR and analysis on the additional chipset. SAT1 cortical expression levels were also found to be significantly lower in an independent sample of German subjects with major depression who died by suicide in comparison with controls. These findings suggest that downregulation of SAT1 expression may play a role in depression and suicidality, possibly by impeding the normal PSR program or through compensation for the increased polyamine metabolism accompanying the psychological distress associated with depressive disorders.

  10. Effects of human arylamine N-acetyltransferase I knockdown in triple-negative breast cancer cell lines

    PubMed Central

    Tiang, Jacky M; Butcher, Neville J; Minchin, Rodney F

    2015-01-01

    Expression of human arylamine N-acetyltransferase I (NAT1) has been associated with various cancer subtypes and inhibition of this enzyme with small molecule inhibitors or siRNA affects cell growth and survival. Here, we have investigated the role of NAT1 in the invasiveness of breast cancer cells both in vitro and in vivo. We knocked down NAT1 using a lentivirus-based shRNA approach and observed marked changes in cell morphology in the triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-436, and BT-549. Most notable was a reduction in the number and size of the filopodia protrusions on the surface of the cells. The loss of filopodia could be rescued by the reintroduction of NAT1 into the knockdown cells. NAT1 expression was localized to the lamellipodia and extended into the filopodia protrusions. In vitro invasion through Geltrex was significantly inhibited in both the MDA cell lines but not in the BT-549 cells. The expression of Snail increased when NAT1 was knocked down, while other genes associated with mesenchymal to epithelial transition (vimentin, cytokeratin-18, and Twist) did not show any changes. By contrast, both N-cadherin and β-catenin were significantly reduced. When MDA-MB-231 cells expressing shRNA were injected in vivo into BALB/c nu/nu nude mice, a significant reduction in the number of colonies that formed in the lungs was observed. Taken together, the results show that NAT1 can alter the invasion and metastatic properties of some triple-negative breast cancer cells but not all. The study suggests that NAT1 may be a novel therapeutic target in a subset of breast cancers. PMID:25627111

  11. Characterization of a novel spermidine/spermine acetyltransferase, BltD, from Bacillus subtilis.

    PubMed Central

    Woolridge, D P; Martinez, J D; Stringer, D E; Gerner, E W

    1999-01-01

    Overexpression of the BltD gene in Bacillus subtilis causes acetylation of the polyamines spermidine and spermine. BltD is co-regulated with another gene, Blt, which encodes a multidrug export protein whose overexpression facilitates spermidine export [Woolridge, Vazquez-Laslop, Markham, Chevalier, Gerner and Neyfakh (1997) J. Biol. Chem. 272, 8864-8866]. Here we show that BltD acetylates both spermidine and spermine at primary propyl amine moieties, with spermine being the preferred substrate. In the presence of saturating concentrations of acetyl CoA, BltD rapidly acetylates spermine at both the N1 and N12 positions. The Km (app) values for spermine, spermidine and N1-acetylspermine are

  12. TGF-β induces p53/Smads complex formation in the PAI-1 promoter to activate transcription

    PubMed Central

    Kawarada, Yuki; Inoue, Yasumichi; Kawasaki, Fumihiro; Fukuura, Keishi; Sato, Koichi; Tanaka, Takahito; Itoh, Yuka; Hayashi, Hidetoshi

    2016-01-01

    Transforming growth factor β (TGF-β) signaling facilitates tumor development during the advanced stages of tumorigenesis, but induces cell-cycle arrest for tumor suppression during the early stages. However, the mechanism of functional switching of TGF-β is still unknown, and it is unclear whether inhibition of TGF-β signaling results amelioration or exacerbation of cancers. Here we show that the tumor suppressor p53 cooperates with Smad proteins, which are TGF-β signal transducers, to selectively activate plasminogen activator inhibitor type-1 (PAI-1) transcription. p53 forms a complex with Smad2/3 in the PAI-1 promoter to recruit histone acetyltransferase CREB-binding protein (CBP) and enhance histone H3 acetylation, resulting in transcriptional activation of the PAI-1 gene. Importantly, p53 is required for TGF-β-induced cytostasis and PAI-1 is involved in the cytostatic activity of TGF-β in several cell lines. Our results suggest that p53 enhances TGF-β-induced cytostatic effects by activating PAI-1 transcription, and the functional switching of TGF-β is partially caused by p53 mutation or p53 inactivation during cancer progression. It is expected that these findings will contribute to optimization of TGF-β-targeting therapies for cancer. PMID:27759037

  13. p300/CBP acetyl transferases interact with and acetylate the nucleotide excision repair factor XPG.

    PubMed

    Tillhon, Micol; Cazzalini, Ornella; Nardo, Tiziana; Necchi, Daniela; Sommatis, Sabrina; Stivala, Lucia A; Scovassi, A Ivana; Prosperi, Ennio

    2012-10-01

    Nucleotide excision repair (NER) is an important DNA repair mechanism through which cells remove bulky DNA lesions. Following DNA damage, the histone acetyltransferase (HAT) p300 (also referred to as lysine acetyltransferase or KAT) is known to associate with proliferating cell nuclear antigen (PCNA), a master regulator of DNA replication and repair processes. This interaction, which results in HAT inhibition, may be dissociated by the cell cycle inhibitor p21(CDKN1A), thereby restoring p300 activity; however, the role of this protein interplay is still unclear. Here, we report that silencing p300 or its homolog CREB-binding protein (CBP) by RNA interference (RNAi) significantly reduces DNA repair synthesis in human fibroblasts. In addition, we determined whether p300 and CBP may associate with and acetylate specific NER factors such as XPG, the 3'-endonuclease that is involved in the incision/excision step and is known to interact with PCNA. Our results show that p300 and CBP interact with XPG, which has been found to be acetylated in vivo. XPG is acetylated by p300 in vitro, and this reaction is inhibited by PCNA. Knocking down both p300/CBP by RNAi or by chemical inhibition with curcumin greatly reduced XPG acetylation, and a concomitant accumulation of the protein at DNA damage sites was observed. The ability of p21 to bind PCNA was found to regulate the interaction between p300 and XPG, and an abnormal accumulation of XPG at DNA damage sites was also found in p21(-/-) fibroblasts. These results indicate an additional function of p300/CBP in NER through the acetylation of XPG protein in a PCNA-p21 dependent manner.

  14. Human Immunodeficiency Virus Tat-Activated Expression of Poliovirus Protein 2A Inhibits mRNA Translation

    NASA Astrophysics Data System (ADS)

    Sun, Xiao-Hong; Baltimore, David

    1989-04-01

    To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was contransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.

  15. Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases

    PubMed Central

    2011-01-01

    Background The trichothecene mycotoxin deoxynivalenol (DON) may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation) when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm) in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON), and may reduce its toxicity in plants and animals. Results Two Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (Saccharomyces cerevisiae) during a series of small-scale ethanol fermentations using barley (Hordeum vulgare). DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red®, which also consumed galactose when present in the mash. Conclusions This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation. PMID:21888629

  16. Inhibition of aminoglycoside 6'-N-acetyltransferase type Ib-mediated amikacin resistance in Klebsiella pneumoniae by zinc and copper pyrithione.

    PubMed

    Chiem, Kevin; Fuentes, Brooke A; Lin, David L; Tran, Tung; Jackson, Alexis; Ramirez, Maria S; Tolmasky, Marcelo E

    2015-09-01

    The in vitro activity of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] was inhibited by CuCl2 with a 50% inhibitory concentration (IC50) of 2.8 μM. The growth of an amikacin-resistant Klebsiella pneumoniae strain isolated from a neonate with meningitis was inhibited when amikacin was supplemented by the addition of Zn(2+) or Cu(2+) in complex with the ionophore pyrithione. Coordination complexes between cations and ionophores could be developed for their use, in combination with aminoglycosides, to treat resistant infections.

  17. Inhibition of Aminoglycoside 6′-N-Acetyltransferase Type Ib-Mediated Amikacin Resistance in Klebsiella pneumoniae by Zinc and Copper Pyrithione

    PubMed Central

    Chiem, Kevin; Fuentes, Brooke A.; Lin, David L.; Tran, Tung; Jackson, Alexis; Ramirez, Maria S.

    2015-01-01

    The in vitro activity of the aminoglycoside 6′-N-acetyltransferase type Ib [AAC(6′)-Ib] was inhibited by CuCl2 with a 50% inhibitory concentration (IC50) of 2.8 μM. The growth of an amikacin-resistant Klebsiella pneumoniae strain isolated from a neonate with meningitis was inhibited when amikacin was supplemented by the addition of Zn2+ or Cu2+ in complex with the ionophore pyrithione. Coordination complexes between cations and ionophores could be developed for their use, in combination with aminoglycosides, to treat resistant infections. PMID:26169410

  18. Vaccinia virus K1 ankyrin repeat protein inhibits NF-κB activation by preventing RelA acetylation.

    PubMed

    Bravo Cruz, Ariana G; Shisler, Joanna L

    2016-10-01

    The vaccinia virus (VACV) K1 protein inhibits dsRNA-dependent protein kinase (PKR) activation. A consequence of this function is that K1 inhibits PKR-induced NF-κB activation during VACV infection. However, transient expression of K1 also inhibits Toll-like receptor (TLR)-induced NF-κB activation. This suggests that K1 has a second NF-κB inhibitory mechanism that is PKR-independent. This possibility was explored by expressing K1 independently of infection and stimulating NF-κB under conditions that minimized or excluded PKR activation. K1 inhibited both TNF- and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB activation, as detected by transcription of synthetic (e.g. luciferase) and natural (e.g. CXCL8) genes controlled by NF-κB. K1 also inhibited NF-κB activity in PKRkd cells, cells that have greatly decreased amounts of PKR. K1 no longer prevented IκBα degradation or NF-κB nuclear translocation in the absence of PKR, suggesting that K1 acted on a nuclear event. Indeed, K1 was present in the nucleus and cytoplasm of stimulated and unstimulated cells. K1 inhibited acetylation of the RelA (p65) subunit of NF-κB, a nuclear event known to be required for NF-κB activation. Moreover, p65-CBP (CREB-binding protein) interactions were blocked in the presence of K1. However, K1 did not preclude NF-κB binding to oligonucleotides containing κB-binding sites. The current interpretation of these data is that NF-κB-promoter interactions still occur in the presence of K1, but NF-κB cannot properly trigger transcriptional activation because K1 antagonizes acetylation of RelA. Thus, in comparison to all known VACV NF-κB inhibitory proteins, K1 acts at one of the most downstream events of NF-κB activation.

  19. Pancreatic β-cell prosurvival effects of the incretin hormones involve post-translational modification of Kv2.1 delayed rectifier channels

    PubMed Central

    Kim, S-J; Widenmaier, S B; Choi, W S; Nian, C; Ao, Z; Warnock, G; McIntosh, C H S

    2012-01-01

    Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the major incretin hormones that exert insulinotropic and anti-apoptotic actions on pancreatic β-cells. Insulinotropic actions of the incretins involve modulation of voltage-gated potassium (Kv) channels. In multiple cell types, Kv channel activity has been implicated in cell volume changes accompanying initiation of the apoptotic program. Focusing on Kv2.1, we examined whether regulation of Kv channels in β-cells contributes to the prosurvival effects of incretins. Overexpression of Kv2.1 in INS-1 β-cells potentiated apoptosis in response to mitochondrial and ER stress and, conversely, co-stimulation with GIP/GLP-1 uncoupled this potentiation, suppressing apoptosis. In parallel, incretins promoted phosphorylation and acetylation of Kv2.1 via pathways involving protein kinase A (PKA)/mitogen- and stress-activated kinase-1 (MSK-1) and histone acetyltransferase (HAT)/histone deacetylase (HDAC). Further studies demonstrated that acetylation of Kv2.1 was mediated by incretin actions on nuclear/cytoplasmic shuttling of CREB binding protein (CBP) and its interaction with Kv2.1. Regulation of β-cell survival by GIP and GLP-1 therefore involves post-translational modifications (PTMs) of Kv channels by PKA/MSK-1 and HAT/HDAC. This appears to be the first demonstration of modulation of delayed rectifier Kv channels contributing to the β-cell prosurvival effects of incretins and of 7-transmembrane G protein-coupled receptor (GPCR)-stimulated export of a nuclear lysine acetyltransferase that regulates cell surface ion channel function. PMID:21818121

  20. Differential transcription of the human spermidine/spermine N1-acetyltransferase (SSAT) gene in human lung carcinoma cells.

    PubMed Central

    Xiao, L; Casero, R A

    1996-01-01

    The expression of spermidine/spermine N1-acetyltransferase (SSAT), the rate-limiting enzyme in the catabolism of polyamines, is highly regulated by a number of factors including the natural polyamines and their analogues. The phenotype-specific cytotoxicity that occurs in response to a class of polyamine analogues, the diethylpolyamines, is associated with a phenotype-specific superinduction of SSAT in human non-small-cell lung carcinomas, whereas in non-responding cell types, including the small-cell lung carcinomas, the superinduction of SSAT does not occur. In this study, we have investigated the molecular basis of this phenotype-specific SSAT induction in human lung carcinoma cells in response to N1,N12-diethylspermine (BESpm). To facilitate the study of transcriptional regulation, we have cloned and characterized 11 kb of the human SSAT locus, including 3500 bp of the 5' promoter region. Nuclear run-on transcription studies suggest that the initial induction of SSAT results from an increase in the rate of gene transcription. Results from Northern blot analysis and ribonuclease protection assays indicate a differential expression of SSAT mRNA between the analogue-responsive H157 and non-responsive H82 cells. There is no detectable SSAT mRNA in H82 cells, even after a 24-h analogue treatment, whereas SSAT mRNA in H157 cells was detectable by Northern blot analysis and increased more than 100-fold following drug exposure. Furthermore, nuclear run-on transcription assays do not detect any active transcription of SSAT gene in either treated or untreated H82 cells. These results indicate that at least one component of the phenotype-specific induction of SSAT appears to be due to differences in transcriptional regulation of the gene. In addition, mapping of DNase I-hypersensitive sites of the SSAT gene suggest that the cell type-specific promoter/enhancer utilization may control the expression of the SSAT gene in differentially sensitive cell types in vivo. PMID

  1. Role of signal peptides in targeting of proteins in cyanobacteria.

    PubMed Central

    Mackle, M M; Zilinskas, B A

    1994-01-01

    Proteins of cyanobacteria may be transported across one of two membrane systems: the typical eubacterial cell envelope (consisting of an inner membrane, periplasmic space, and an outer membrane) and the photosynthetic thylakoids. To investigate the role of signal peptides in targeting in cyanobacteria, Synechococcus sp. strain PCC 7942 was transformed with vectors carrying the chloramphenicol acetyltransferase reporter gene fused to coding sequences for one of four different signal peptides. These included signal peptides of two proteins of periplasmic space origin (one from Escherichia coli and the other from Synechococcus sp. strain PCC 7942) and two other signal peptides of proteins located in the thylakoid lumen (one from a cyanobacterium and the other from a higher plant). The location of the gene fusion products expressed in Synechococcus sp. strain PCC 7942 was determined by a chloramphenicol acetyltransferase enzyme-linked immunosorbent assay of subcellular fractions. The distribution pattern for gene fusions with periplasmic signal peptides was different from that of gene fusions with thylakoid lumen signal peptides. Primary sequence analysis revealed conserved features in the thylakoid lumen signal peptides that were absent from the periplasmic signal peptides. These results suggest the importance of the signal peptide in protein targeting in cyanobacteria and point to the presence of signal peptide features conserved between chloroplasts and cyanobacteria for targeting of proteins to the thylakoid lumen. Images PMID:8144451

  2. Corepressive Action of CBP on Androgen Receptor Transactivation in Pericentric Heterochromatin in a Drosophila Experimental Model System▿ †

    PubMed Central

    Zhao, Yue; Takeyama, Ken-ichi; Sawatsubashi, Shun; Ito, Saya; Suzuki, Eriko; Yamagata, Kaoru; Tanabe, Masahiko; Kimura, Shuhei; Fujiyama, Sally; Ueda, Takashi; Murata, Takuya; Matsukawa, Hiroyuki; Shirode, Yuko; Kouzmenko, Alexander P.; Li, Feng; Tabata, Testuya; Kato, Shigeaki

    2009-01-01

    Ligand-bound nuclear receptors (NR) activate transcription of the target genes. This activation is coupled with histone modifications and chromatin remodeling through the function of various coregulators. However, the nature of the dependence of a NR coregulator action on the presence of the chromatin environment at the target genes is unclear. To address this issue, we have developed a modified position effect variegation experimental model system that includes an androgen-dependent reporter transgene inserted into either a pericentric heterochromatin region or a euchromatic region of Drosophila chromosome. Human androgen receptor (AR) and its constitutively active truncation mutant (AR AF-1) were transcriptionally functional in both chromosomal regions. Predictably, the level of AR-induced transactivation was lower in the pericentric heterochromatin. In genetic screening for AR AF-1 coregulators, Drosophila CREB binding protein (dCBP) was found to corepress AR transactivation at the pericentric region whereas it led to coactivation in the euchromatic area. Mutations of Sir2 acetylation sites or deletion of the CBP acetyltransferase domain abrogated dCBP corepressive action for AR at heterochromatic areas in vivo. Such a CBP corepressor function for AR was observed in the transcriptionally silent promoter of an AR target gene in cultured mammalian cells. Thus, our findings suggest that the action of NR coregulators may depend on the state of chromatin at the target loci. PMID:19075001

  3. Platelet activating factor-induced expression of p21 is correlated with histone acetylation.

    PubMed

    Damiani, Elisabetta; Puebla-Osorio, Nahum; Lege, Bree M; Liu, Jingwei; Neelapu, Sattva S; Ullrich, Stephen E

    2017-02-03

    Ultraviolet (UV)-irradiated keratinocytes secrete the lipid mediator of inflammation, platelet-activating factor (PAF). PAF plays an essential role in UV-induced immune suppression and skin cancer induction. Dermal mast cell migration from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced PAF activates mast cell migration by up-regulating mast cell CXCR4 surface expression. Recent findings indicate that PAF up-regulates CXCR4 expression via histone acetylation. UV-induced PAF also activates cell cycle arrest and disrupts DNA repair, in part by increasing p21 expression. Do epigenetic alterations play a role in p21 up-regulation? Here we show that PAF increases Acetyl-CREB-binding protein (CBP/p300) histone acetyltransferase expression in a time and dose-dependent fashion. Partial deletion of the HAT domain in the CBP gene, blocked these effects. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the p21 promoter. PAF-treatment had no effect on other acetylating enzymes (GCN5L2, PCAF) indicating it is not a global activator of histone acetylation. This study provides further evidence that PAF activates epigenetic mechanisms to affect important cellular processes, and we suggest this bioactive lipid can serve as a link between the environment and the epigenome.

  4. Platelet activating factor-induced expression of p21 is correlated with histone acetylation

    PubMed Central

    Damiani, Elisabetta; Puebla-Osorio, Nahum; Lege, Bree M.; Liu, Jingwei; Neelapu, Sattva S.; Ullrich, Stephen E.

    2017-01-01

    Ultraviolet (UV)-irradiated keratinocytes secrete the lipid mediator of inflammation, platelet-activating factor (PAF). PAF plays an essential role in UV-induced immune suppression and skin cancer induction. Dermal mast cell migration from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced PAF activates mast cell migration by up-regulating mast cell CXCR4 surface expression. Recent findings indicate that PAF up-regulates CXCR4 expression via histone acetylation. UV-induced PAF also activates cell cycle arrest and disrupts DNA repair, in part by increasing p21 expression. Do epigenetic alterations play a role in p21 up-regulation? Here we show that PAF increases Acetyl-CREB-binding protein (CBP/p300) histone acetyltransferase expression in a time and dose-dependent fashion. Partial deletion of the HAT domain in the CBP gene, blocked these effects. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the p21 promoter. PAF-treatment had no effect on other acetylating enzymes (GCN5L2, PCAF) indicating it is not a global activator of histone acetylation. This study provides further evidence that PAF activates epigenetic mechanisms to affect important cellular processes, and we suggest this bioactive lipid can serve as a link between the environment and the epigenome. PMID:28157211

  5. Progesterone receptor induces bcl-x expression through intragenic binding sites favoring RNA polymerase II elongation

    PubMed Central

    Bertucci, Paola Y.; Nacht, A. Silvina; Alló, Mariano; Rocha-Viegas, Luciana; Ballaré, Cecilia; Soronellas, Daniel; Castellano, Giancarlo; Zaurin, Roser; Kornblihtt, Alberto R.; Beato, Miguel; Vicent, Guillermo P.; Pecci, Adali

    2013-01-01

    Steroid receptors were classically described for regulating transcription by binding to target gene promoters. However, genome-wide studies reveal that steroid receptors-binding sites are mainly located at intragenic regions. To determine the role of these sites, we examined the effect of progestins on the transcription of the bcl-x gene, where only intragenic progesterone receptor-binding sites (PRbs) were identified. We found that in response to hormone treatment, the PR is recruited to these sites along with two histone acetyltransferases CREB-binding protein (CBP) and GCN5, leading to an increase in histone H3 and H4 acetylation and to the binding of the SWI/SNF complex. Concomitant, a more relaxed chromatin was detected along bcl-x gene mainly in the regions surrounding the intragenic PRbs. PR also mediated the recruitment of the positive elongation factor pTEFb, favoring RNA polymerase II (Pol II) elongation activity. Together these events promoted the re-distribution of the active Pol II toward the 3′-end of the gene and a decrease in the ratio between proximal and distal transcription. These results suggest a novel mechanism by which PR regulates gene expression by facilitating the proper passage of the polymerase along hormone-dependent genes. PMID:23640331

  6. ATF1 modulates the heat shock response by regulating the stress-inducible heat shock factor 1 transcription complex.

    PubMed

    Takii, Ryosuke; Fujimoto, Mitsuaki; Tan, Ke; Takaki, Eiichi; Hayashida, Naoki; Nakato, Ryuichiro; Shirahige, Katsuhiko; Nakai, Akira

    2015-01-01

    The heat shock response is an evolutionally conserved adaptive response to high temperatures that controls proteostasis capacity and is regulated mainly by an ancient heat shock factor (HSF). However, the regulation of target genes by the stress-inducible HSF1 transcription complex has not yet been examined in detail in mammalian cells. In the present study, we demonstrated that HSF1 interacted with members of the ATF1/CREB family involved in metabolic homeostasis and recruited them on the HSP70 promoter in response to heat shock. The HSF1 transcription complex, including the chromatin-remodeling factor BRG1 and lysine acetyltransferases p300 and CREB-binding protein (CBP), was formed in a manner that was dependent on the phosphorylation of ATF1. ATF1-BRG1 promoted the establishment of an active chromatin state and HSP70 expression during heat shock, whereas ATF1-p300/CBP accelerated the shutdown of HSF1 DNA-binding activity during recovery from acute stress, possibly through the acetylation of HSF1. Furthermore, ATF1 markedly affected the resistance to heat shock. These results revealed the unanticipated complexity of the primitive heat shock response mechanism, which is connected to metabolic adaptation.

  7. Intrinsic epigenetic factors cooperate with the steroid hormone ecdysone to govern dendrite pruning in Drosophila.

    PubMed

    Kirilly, Daniel; Wong, Jack Jing Lin; Lim, Edwin Kok Hao; Wang, Yan; Zhang, Heng; Wang, Cheng; Liao, Qiuming; Wang, Haifeng; Liou, Yih-Cherng; Wang, Hongyan; Yu, Fengwei

    2011-10-06

    Pruning that selectively removes unnecessary axons/dendrites is crucial for sculpting neural circuits during development. During Drosophila metamorphosis, dendritic arborization sensory neurons, ddaCs, selectively prune their larval dendrites in response to the steroid hormone ecdysone. However, it is unknown whether epigenetic factors are involved in dendrite pruning. Here, we analyzed 81 epigenetic factors, from which a Brahma (Brm)-containing chromatin remodeler and a histone acetyltransferase CREB-binding protein (CBP) were identified for their critical roles in initiating dendrite pruning. Brm and CBP specifically activate a key ecdysone response gene, sox14, but not EcR-B1. Furthermore, the HAT activity of CBP is important for sox14 expression and dendrite pruning. EcR-B1 associates with CBP in the presence of ecdysone, which is facilitated by Brm, resulting in local enrichment of an active chromatin mark H3K27Ac at the sox14 locus. Thus, specific intrinsic epigenetic factors cooperate with steroid hormones to activate selective transcriptional programs, thereby initiating neuronal remodeling.

  8. Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut.

    PubMed Central

    Hintermann, E; Grieder, N C; Amherd, R; Brodbeck, D; Meyer, U A

    1996-01-01

    In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyl-transferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNAT1 mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8901578

  9. Enhanced morphinan alkaloid production in hairy root cultures of Papaver bracteatum by over-expression of salutaridinol 7-o-acetyltransferase gene via Agrobacterium rhizogenes mediated transformation.

    PubMed

    Sharafi, Ali; Hashemi Sohi, Haleh; Mousavi, Amir; Azadi, Pejman; Dehsara, Bahareh; Hosseini Khalifani, Bahman

    2013-11-01

    Papaver bracteatum is an important medicinal plant valued for its high content of thebaine and an alternative to P. somniferum for benzylisoquinoline alkaloid production. Salutaridinol 7-o-acetyltransferase (SalAT) is a key gene in morphinan alkaloids biosynthesis pathway. Over expression of SalAT gene was used for metabolic engineering in P. bracteatum hairy root cultures. Transcript level of the salutaridinol 7-o-acetyltransferase gene in transgenic hairy root lines increased up to 154 and 128 % in comparison with hairy roots without SalAT over expression and wild type roots, respectively. High performance liquid chromatography analysis showed that the transgenic hairy roots relatively improved levels of thebaine (1.28 % dry weight), codeine (0.02 % dry weight) and morphine (0.03 % dry weight) compared to those hairy roots without SalAT over expression. This suggests that P. bracteatum hairy roots expressing the SalAT gene could be potentially used for the production of valuable morphinan alkaloids.

  10. Disruption of the histone acetyltransferase MYST4 leads to a Noonan syndrome–like phenotype and hyperactivated MAPK signaling in humans and mice

    PubMed Central

    Kraft, Michael; Cirstea, Ion Cristian; Voss, Anne Kathrin; Thomas, Tim; Goehring, Ina; Sheikh, Bilal N.; Gordon, Lavinia; Scott, Hamish; Smyth, Gordon K.; Ahmadian, Mohammad Reza; Trautmann, Udo; Zenker, Martin; Tartaglia, Marco; Ekici, Arif; Reis, André; Dörr, Helmuth-Guenther; Rauch, Anita; Thiel, Christian Thomas

    2011-01-01

    Epigenetic regulation of gene expression, through covalent modification of histones, is a key process controlling growth and development. Accordingly, the transcription factors regulating these processes are important targets of genetic diseases. However, surprisingly little is known about the relationship between aberrant epigenetic states, the cellular process affected, and their phenotypic consequences. By chromosomal breakpoint mapping in a patient with a Noonan syndrome–like phenotype that encompassed short stature, blepharoptosis, and attention deficit hyperactivity disorder, we identified haploinsufficiency of the histone acetyltransferase gene MYST histone acetyltransferase (monocytic leukemia) 4 (MYST4), as the underlying cause of the phenotype. Using acetylation, whole genome expression, and ChIP studies in cells from the patient, cell lines in which MYST4 expression was knocked down using siRNA, and the Myst4 querkopf mouse, we found that H3 acetylation is important for neural, craniofacial, and skeletal morphogenesis, mainly through its ability to specifically regulating the MAPK signaling pathway. This finding further elucidates the complex role of histone modifications in mammalian development and adds what we believe to be a new mechanism to the pathogenic phenotypes resulting from misregulation of the RAS signaling pathway. PMID:21804188

  11. Structural and Biochemical Characterization of an Active Arylamine N-Acetyltransferase Possessing a Non-canonical Cys-His-Glu Catalytic Triad*

    PubMed Central

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

    2013-01-01

    Arylamine N-acetyltransferases (NATs), a class of xenobiotic-metabolizing enzymes, catalyze the acetylation of aromatic amine compounds through a strictly conserved Cys-His-Asp catalytic triad. Each residue is essential for catalysis in both prokaryotic and eukaryotic NATs. Indeed, in (HUMAN)NAT2 variants, mutation of the Asp residue to Asn, Gln, or Glu dramatically impairs enzyme activity. However, a putative atypical NAT harboring a catalytic triad Glu residue was recently identified in Bacillus cereus ((BACCR)NAT3) but has not yet been characterized. We report here the crystal structure and functional characterization of this atypical NAT. The overall fold of (BACCR)NAT3 and the geometry of its Cys-His-Glu catalytic triad are similar to those present in functional NATs. Importantly, the enzyme was found to be active and to acetylate prototypic arylamine NAT substrates. In contrast to (HUMAN) NAT2, the presence of a Glu or Asp in the triad of (BACCR)NAT3 did not significantly affect enzyme structure or function. Computational analysis identified differences in residue packing and steric constraints in the active site of (BACCR)NAT3 that allow it to accommodate a Cys-His-Glu triad. These findings overturn the conventional view, demonstrating that the catalytic triad of this family of acetyltransferases is plastic. Moreover, they highlight the need for further study of the evolutionary history of NATs and the functional significance of the predominant Cys-His-Asp triad in both prokaryotic and eukaryotic forms. PMID:23770703

  12. Unc-5 homolog B (UNC5B) is one of the key downstream targets of N-α-Acetyltransferase 10 (Naa10)

    PubMed Central

    Xu, Huiyu; Han, Yong; Liu, Bing; Li, Rong

    2016-01-01

    N-α-acetyltransferase 10 (Naa10) displays alpha (N-terminal) acetyltransferase activity. It functions as a major modulator of cell growth and differentiation. Until now, a few downstream targets were found, but no studies have concerned about which gene is the early event of Naa10 downstream target. As we know, the earlier events may play more significant role in Naa10 pathway. Through construction of Naa10 stably knocked down H1299 cell line, we discovered cell morphological changes induced by Naa10. Moreover, potential function of Naa10 in cell morphogenesis was also indicated using cDNA microarray analysis of the Naa10 stably knock-down cell line. We further discovered that netrin-1 (NTN1) and its receptor UNC-5 Homology B (UNC5B) were the early event among the genes involved in Naa10 stably knocked down induced genes expression changes in cell morphogenesis. This was further validated in caudal half region of E10 mouse embryos. Negative regulation of Naa10 towards NTN1 and its receptor UNC5B were also detected upon treatment of all-trans retinoid acid, which was often used to induce morphological differentiation. PMID:27910960

  13. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-{kappa}B acetylation in fibroblast-like synoviocyte MH7A cells

    SciTech Connect

    Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul; Lee, Mee-Hee; Lee, Yoo-Hyun; Lee, Jeongmin; Jun, Woojin; Kim, Sunoh; Yoon, Ho-Geun

    2011-07-08

    Highlights: {yields} Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. {yields} Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. {yields} Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-{kappa}B. {yields} Delphinidin inhibits cytokine release in the Jurkat T lymphocyte cell line. -- Abstract: Histone acetyltransferase (HAT) inhibitors (HATi) isolated from dietary compounds have been shown to suppress inflammatory signaling, which contributes to rheumatoid arthritis. Here, we identified a novel HATi in Punica granatum L. known as delphinidin (DP). DP did not affect the activity of other epigenetic enzymes (histone deacetylase, histone methyltransferase, or sirtuin1). DP specifically inhibited the HAT activities of p300/CBP. It also inhibited p65 acetylation in MH7A cells, a human rheumatoid arthritis synovial cell line. DP-induced hypoacetylation was accompanied by cytosolic accumulation of p65 and nuclear localization of IKB{alpha}. Accordingly, DP treatment inhibited TNF{alpha}-stimulated increases in NF-{kappa}B function and expression of NF-{kappa}B target genes in these cells. Importantly, DP suppressed lipopolysaccharide-induced pro-inflammatory cytokine expression in Jurkat T lymphocytes, demonstrating that HATi efficiently suppresses cytokine-mediated immune responses. Together, these results show that the HATi activity of DP counters anti-inflammatory signaling by blocking p65 acetylation and that this compound may be useful in preventing inflammatory arthritis.

  14. Serine 133 Phosphorylation Is Not Required for Hippocampal CREB-Mediated Transcription and Behavior

    ERIC Educational Resources Information Center

    Brian, Lisa A.; Lee, Bridgin G.; Lelay, John; Kaestner, Klaus H.; Blendy, Julie A.

    2015-01-01

    The cAMP response element (CRE)-binding protein, CREB, is a transcription factor whose activity in the brain is critical for long-term memory formation. Phosphorylation of Ser133 in the kinase-inducible domain (KID), that in turn leads to the recruitment of the transcriptional coactivator CREB-binding protein (CBP), is thought to mediate the…

  15. Day/night fluctuations in melatonin content, arylalkylamine N-acetyltransferase activity and NAT mRNA expression in the CNS, peripheral tissues and hemolymph of the cockroach, Periplaneta americana.

    PubMed

    Bembenek, Jadwiga; Sehadova, Hana; Ichihara, Naoyuki; Takeda, Makio

    2005-01-01

    Melatonin content measured by a radioenzymatic assay in the brain of the American cockroach (Periplaneta americana) showed a day/night fluctuation with higher levels at night under LD 12:12. The activity of arylalkylamine N-acetyltransferase (NAT) in brain was also higher at night and this pattern continued in constant darkness. The results suggest that the rhythmicity in melatonin content can be caused by NAT. Melatonin content in hemolymph showed an even greater day/night difference, more than 12 times that in brain under LD 12:12. Melatonin levels in retina were also higher at night while NAT activity was not significantly higher at night than at daytime. Using a probe designed from NAT cloned from testes we performed Northern blot analysis of total RNA, which revealed that the level of NAT mRNA was higher in midgut, ovary and female accessory glands than in fat body and brain. The level of transcript in midgut was higher at night, but the levels in ovary and female accessory reproductive gland showed the opposite pattern. We also used the antibody to whole Drosophila melanogaster aaNAT1 protein, seeking a homologous antigen in the cephalic ganglia. NAT-like antigen was detected in several restricted populations of cells in the brain that were partially co-localized with PER-like antigen. The results suggest that NAT exists in multiple forms in various tissues of the cockroach and that its functions and regulations can vary among tissues. The results in the brain led to the conclusion that NAT could be a clock-controlled gene functioning as an output regulator of the circadian clock.

  16. aarD, a Providencia stuartii homologue of cydD: role in 2'-N-acetyltransferase expression, cell morphology and growth in the presence of an extracellular factor.

    PubMed

    Macinga, D R; Rather, P N

    1996-02-01

    In a search for genes involved in regulation of the 2'-N-acetyltransferase in Providencia stuartii, a mini-Tn5Cm insertion has been isolated in a locus designated aarD. The aarD1::mini-Tn5Cm mutation resulted in a 4.7-fold increase in the levels of beta-galactosidase accumulation from an aac(2')-lacZ transcriptional fusion and a 32-fold increase in the levels of gentamicin resistance in P. stuartii. The wild-type aarD locus was cloned on a 5.0 kb Cla I fragment and complemented the aarD1 mutation. Nucleotide sequence analysis of this fragment identified two large open reading frames whose deduced products displayed significant amino acid identity, 64% and 64%, respectively, to the CydD and CydC proteins of Escherichia coli, which are involved in formation of the cytochrome d oxidase complex. Physical mapping indicated the aarD1::mini-Tn5Cm insertion was within the open reading homologous to CydD. The strain containing the aarD1 mutation was unable to grow in the presence of toluidine blue or on glycerol minimal media in the presence of zinc, suggesting that aarD is functionally equivalent to cydD. Additional phenotypes resulting from the aarD1 mutation included: altered cell morphology, a reduced growth rate and the inability of cells to grow beyond early log phase. Further examination of this phenomenon revealed that the aarD1 mutant was unable to grow in the presence of a self-produced extracellular factor(s). This novel phenotype was limited to P. stuartii as E. coli cydD and delta cydAB::kan mutants were also sensitive to a self-produced extracellular factor.

  17. Identification and characterization of aarF, a locus required for production of ubiquinone in Providencia stuartii and Escherichia coli and for expression of 2'-N-acetyltransferase in P. stuartii.

    PubMed

    Macinga, D R; Cook, G M; Poole, R K; Rather, P N

    1998-01-01

    Providencia stuartii contains a chromosomal 2'-N-acetyltransferase [AAC(2')-Ia] involved in the O acetylation of peptidoglycan. The AAC(2')-Ia enzyme is also capable of acetylating and inactivating certain aminoglycosides and confers high-level resistance to these antibiotics when overexpressed. We report the identification of a locus in P. stuartii, designated aarF, that is required for the expression of AAC(2')-Ia. Northern (RNA) analysis demonstrated that aac(2')-Ia mRNA levels were dramatically decreased in a P. stuartii strain carrying an aarF::Cm disruption. The aarF::Cm disruption also resulted in a deficiency in the respiratory cofactor ubiquinone. The aarF locus encoded a protein that had a predicted molecular mass of 62,559 Da and that exhibited extensive amino acid similarity to the products of two adjacent open reading frames of unknown function (YigQ and YigR), located at 86 min on the Escherichia coli chromosome. An E. coli yigR::Kan mutant was also deficient in ubiquinone content. Complementation studies demonstrated that the aarF and the E. coli yigQR loci were functionally equivalent. The aarF or yigQR genes were unable to complement ubiD and ubiE mutations that are also present at 86 min on the E. coli chromosome. This result indicates that aarF (yigQR) represents a novel locus for ubiquinone production and reveals a previously unreported connection between ubiquinone biosynthesis and the regulation of gene expression.

  18. Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892

    SciTech Connect

    Rudolf, Jeffrey D.; Bigelow, Lance; Chang, Changsoo; Cuff, Marianne E.; Lohman, Jeremy R.; Chang, Chin-Yuan; Ma, Ming; Yang, Dong; Clancy, Shonda; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

    2015-11-17

    The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 angstrom, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics.

  19. Expression of Aequorea green fluorescent protein in plant cells.

    PubMed

    Hu, W; Cheng, C L

    1995-08-07

    The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co-transformed with another construct in which GFP has been replaced with chloramphenicol acetyltransferase (CAT). The heat-induced expression of GFP in maize protoplasts parallels that of CAT. While GFP was expressed in both dark-grown and green maize leaf protoplasts, no green fluorescence was observed in similarly transformed Arabidopsis protoplasts.

  20. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    PubMed

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle.