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Sample records for acetyltransferase tip60 targets

  1. The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma

    van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars;

    2008-01-01

    proteins with which this nuclear receptor interacts under specific conditions. Here we identify the HIV-1 Tat-interacting protein 60 (Tip60) as a novel positive regulator of PPARgamma transcriptional activity. Using tandem mass spectrometry, we found that PPARgamma and the acetyltransferase Tip60 interact......-mediated reduction of Tip60 protein impairs differentiation of 3T3-L1 preadipocytes. Taken together, these findings qualify the acetyltransferase Tip60 as a novel adipogenic factor....

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

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

    2014-06-01

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

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

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

    2016-07-01

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

  4. Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

    Yamagata, Kazutsune, E-mail: kyamagat@ncc.go.jp [Department of Molecular Oncology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Kitabayashi, Issay [Department of Molecular Oncology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2009-12-25

    Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of {gamma}-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.

  5. Allele compensation in tip60+/- mice rescues white adipose tissue function in vivo.

    Yuan Gao

    Full Text Available Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis, a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/- mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/- displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice.

  6. Tip60 HAT Action Mediates Environmental Enrichment Induced Cognitive Restoration.

    Xu, Songjun; Panikker, Priyalakshmi; Iqbal, Sahira; Elefant, Felice

    2016-01-01

    Environmental enrichment (EE) conditions have beneficial effects for reinstating cognitive ability in neuropathological disorders like Alzheimer's disease (AD). While EE benefits involve epigenetic gene control mechanisms that comprise histone acetylation, the histone acetyltransferases (HATs) involved remain largely unknown. Here, we examine a role for Tip60 HAT action in mediating activity- dependent beneficial neuroadaptations to EE using the Drosophila CNS mushroom body (MB) as a well-characterized cognition model. We show that flies raised under EE conditions display enhanced MB axonal outgrowth, synaptic marker protein production, histone acetylation induction and transcriptional activation of cognition linked genes when compared to their genotypically identical siblings raised under isolated conditions. Further, these beneficial changes are impaired in both Tip60 HAT mutant flies and APP neurodegenerative flies. While EE conditions provide some beneficial neuroadaptive changes in the APP neurodegenerative fly MB, such positive changes are significantly enhanced by increasing MB Tip60 HAT levels. Our results implicate Tip60 as a critical mediator of EE-induced benefits, and provide broad insights into synergistic behavioral and epigenetic based therapeutic approaches for treatment of cognitive disorder. PMID:27454757

  7. Deficiency of DNA double-strand break repair and enhanced radiosensitivity in Tip60 silenced cells

    Objective: To investigate the effect of Tip60 on the cellular radiosensitivity,and to explore the related mechanism. Methods: siRNA and anacardic acid (AA, an inhibitor of Tip60 acetyltransferase) were used to inhibit Tip60 expression and its acetyltransferase activity, respectively. Radiosensitivity was analyzed by colony-forming ability assay. γ-H2AX foci were detected to analyze the DNA double-strand break (DSB). Immunoprecipitation was used to determine the interaction of proteins. Results: siRNA-mediated silencing of Tip60 led to enhanced sensitivity of U2OS cells at 1, 2 Gy after γ-ray irradiation, but had no significant effect at 4 Gy post-irradiation (t=3.364, 3.979, P<0.05).γ-H2AX foci detection indicated that Tip60 silencing resulted in a decreased capability of DNA double-strand break repair at 1, 4 and 8 h after irradiation (t=3.875, 3.183 and 3.175, respectively, P<0.05). The interaction of Tip60 and DNA-PKcs was prompted by ionizing radiation. Anacardic acid largely abrogated the phosphorylation of DNA-PKcs at T2609 site induced by irradiation. Conclusions: Tip60 plays a role in the cellular response to ionizing radiation-induced DNA damage through, at least in part, interacting with DNA-PKcs and regulating its phosphorylation. (authors)

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

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

    2016-03-01

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

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

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

    2016-01-01

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

  10. Andrographolide: A potent antituberculosis compound that targets Aminoglycoside 2'-N-acetyltransferase in Mycobacterium tuberculosis.

    Prabu, Amudha; Hassan, Sameer; Prabuseenivasan; Shainaba, A S; Hanna, L E; Kumar, Vanaja

    2015-09-01

    Tuberculosis (TB) still remains a major challenging infectious disease. The increased rate of emergence of multi-drug resistant and extensively-drug resistant strains of the organism has further complicated the situation, resulting in an urgent need for new anti-TB drugs. Antimycobacterial activity of Andrographis paniculata was evaluated using a rapid LRP assay and the probable targets were identified by docking analysis. The methanolic extract of A. paniculata showed maximum antimycobacterial activity at 250μg/ml against all the tested strains of M. tuberculosis (H37Rv, MDR, and drug sensitive). Based on bioassay guided fractionation, andrographolide was identified as the potent molecule. With the docking analysis, both ICDH (Isocitrate Dehydrogenase) and AAC (Aminoglycoside 2'-N-acetyltransferase) were predicted as targets of andrographolide in M. tuberculosis. Molecular simulation revealed that, ICDH showed low binding affinity to andrographolide. However, for AAC, the andrographolide was observed to be well within the active site after 10ns of molecular simulation. This suggests that ACC (PDB ID 1M4I) could be the probable target for andrographolide. PMID:26245695

  11. The effects of over-expressing Tip60 on cellular DNA damage repair and cell cycle progression

    To investigate the effects of Tip60 on DNA damage repair, cell cycle and the related mechanism as well, the proliferative activity, DNA double strand break (DSB) repair competency and cell cycle arrest were analyzed in stable Tip60-overexpression U2OS cells established by transfecting with exogenous Tip60 gene. It was found that the overexpression of Tip60 inhibited the proliferative activity but increased the DNA damage repair competency. The radiation-induced G2/M arrest was prolonged in Tip60 over-expressed U2OS cells, which was associated with a decreasing level of cell cycle checkpoint protein Cyclin B/CDC2 complex. (authors)

  12. SMCX and components of the TIP60 complex contribute to E2 regulation of the HPV E6/E7 promoter

    Smith, Jennifer A.; Haberstroh, Friederike S.; White, Elizabeth A.; Livingston, David M.; DeCaprio, James A.; Howley, Peter M.

    2014-01-01

    An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/TRRAP histone acetyltransferase complex, also contributes to the E2 repression function, and we extend our studies on SMCX. Di- and tri-methyl marks on histone H3K4 are reduced in the presence of E2 and SMCX, suggesting a mechanism by which SMCX contributes to E2-mediated repression of the HPV LCR. Together, these findings lead us to hypothesize that E2 recruits histone-modifying cellular proteins to the HPV LCR, resulting in transcriptional repression of E6 and E7. PMID:25222147

  13. SMCX and components of the TIP60 complex contribute to E2 regulation of the HPV E6/E7 promoter.

    Smith, Jennifer A; Haberstroh, Friederike S; White, Elizabeth A; Livingston, David M; DeCaprio, James A; Howley, Peter M

    2014-11-01

    An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/TRRAP histone acetyltransferase complex, also contributes to the E2 repression function, and we extend our studies on SMCX. Di- and tri-methyl marks on histone H3K4 are reduced in the presence of E2 and SMCX, suggesting a mechanism by which SMCX contributes to E2-mediated repression of the HPV LCR. Together, these findings lead us to hypothesize that E2 recruits histone-modifying cellular proteins to the HPV LCR, resulting in transcriptional repression of E6 and E7. PMID:25222147

  14. MDR1 mediated chemoresistance: BMI1 and TIP60 in action.

    Banerjee Mustafi, Soumyajit; Chakraborty, Prabir Kumar; Naz, Sarwat; Dwivedi, Shailendra Kumar Dhar; Street, Mark; Basak, Rumki; Yang, Da; Ding, Kai; Mukherjee, Priyabrata; Bhattacharya, Resham

    2016-08-01

    Chemotherapy-induced emergence of drug resistant cells is frequently observed and is exemplified by the expression of family of drug resistance proteins including, multidrug resistance protein 1 (MDR1). However, a concise mechanism for chemotherapy-induced MDR1 expression is unclear. Mechanistically, mutational selection, epigenetic alteration, activation of the Wnt pathway or impaired p53 function have been implicated. The present study describes that the surviving fraction of cisplatin resistant cells co- upregulate MDR1, BMI1 and acetyl transferase activity of TIP60. Using complementary gain and loss of function approaches, we demonstrate that the expression of MDR1 is positively regulated by BMI1, a stem-cell factor classically known as a transcriptional repressor. Our study establishes a functional interaction between TIP60 and BMI-1 resulting in upregulation of MDR1 expression. Chromatin immunoprecipitation (ChIP) assays further establish that the proximal MDR1 promoter responds to cisplatin in a BMI1 dependent manner. BMI1 interacts with a cluster of E-box elements on the MDR1 promoter and recruits TIP60 resulting in acetylation of histone H2A and H3. Collectively, our data establish a hitherto unknown liaison among MDR1, BMI1 and TIP60 and provide mechanistic insights into cisplatin-induced MDR1 expression resulting in acquired cross-resistance against paclitaxel, doxorubicin and likely other drugs. In conclusion, our results advocate utilizing anti-BMI1 strategies to alleviate acquired resistance to chemotherapy. PMID:27295567

  15. Tip60-mediated acetylation activates transcription independent apoptotic activity of Abl

    Pandita Tej K

    2011-07-01

    Full Text Available Abstract Background The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR, is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown. Results We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (KXXK--X3-5--SGS is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase. Conclusion Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function.

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

    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...

  17. FOXP3 interactions with histone acetyltransferase and class II histone deacetylases are required for repression

    Li, Bin; Samanta, Arabinda; Song, Xiaomin; Iacono, Kathryn T.; Bembas, Kathryn; Tao, Ran; Basu, Samik; Riley, James L.; Hancock, Wayne W.; Shen, Yuan; Saouaf, Sandra J.; Mark I. Greene

    2007-01-01

    The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase–deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacety...

  18. Targeting cancer using KAT inhibitors to mimic lethal knockouts

    Brown, James A.L.; Bourke, Emer; Eriksson, Leif A.; Kerin, Michael J.

    2016-01-01

    Two opposing enzyme classes regulate fundamental elements of genome maintenance, gene regulation and metabolism, either through addition of an acetyl moiety by histone acetyltransferases (HATs) or its removal by histone de-acetyltransferases (HDAC), and are exciting targets for drug development. Importantly, dysfunctional acetylation has been implicated in numerous diseases, including cancer. Within the HAT superfamily the MYST family holds particular interest, as its members are directly involved in the DNA damage response and repair pathways and crucially, several members have been shown to be down-regulated in common cancers (such as breast and prostate). In the present study we focus on the development of lysine (K) acetyltransferase inhibitors (KATi) targeting the MYST family member Tip60 (Kat5), an essential protein, designed or discovered through screening libraries. Importantly, Tip60 has been demonstrated to be significantly down-regulated in many cancers which urgently require new treatment options. We highlight current and future efforts employing these KATi as cancer treatments and their ability to synergize and enhance current cancer treatments. We investigate the different methods of KATi production or discovery, their mechanisms and their validation models. Importantly, the utility of KATi is based on a key concept: using KATi to abrogate the activity of an already down-regulated essential protein (effectively creating a lethal knockout) provides another innovative mechanism for targeting cancer cells, while significantly minimizing any off-target effects to normal cells. This approach, combined with the rapidly developing interest in KATi, suggests that KATi have a bright future for providing truly personalized therapies. PMID:27528742

  19. Expression Analysis of cPLA2 Alpha Interacting TIP60 in Diabetic KKAy and Non-Diabetic C57BL Wild-Type Mice: No Impact of Transient and Stable TIP60 Overexpression on Glucose-Stimulated Insulin Secretion in Pancreatic Beta-Cells

    Nordentoft, Iver; Jeppesen, Per B; Nielsen, Anders L;

    2007-01-01

    In the present study we investigate the expression levels of cytosolic phospholipase A2 alpha (cPLA2alpha) interacting histone acetyl transferase proteins TIP60alpha and TIP60beta in non-diabetic C57BL wild-type mice and obese type 2 diabetic KKAy model mice. The aim was to test our hypothesis that...

  20. SMCX and components of the TIP60 complex contribute to E2 regulation of the HPV E6/E7 promoter

    Smith, Jennifer A.; Haberstroh, Friederike S.; White, Elizabeth A.; Livingston, David M.; DeCaprio, James A.; Howley, Peter M.

    2014-01-01

    An important step in the malignant progression of HPV-associated lesions is the dysregulation of expression of the viral E6 and E7 oncogenes. This is often achieved through the loss of expression of E2, which represses the HPV LCR promoter and E6/E7 expression. Our previous studies confirmed a role for Brd4 in mediating the E2 transcriptional repression function, and identified JARID1C/SMCX and EP400 as contributors to E2-mediated repression. Here we show that TIP60, a component of the TIP60/...

  1. Human red cell acetyltransferase

    Acetyltransferase was isolated by histone-Sepharose affinity chromatography from human cord blood red cells. The enzyme was detected only in very young red cells. The semipurified enzyme and [14C]acetyl-CoA were used to acetylate isolated Hb F tetramer and α and γ subunits. The in vitro acetylated products were characterized by globin chain separation by CM-cellulose chromatography and tryptic peptide analysis by reverse-phase HPLC. Acetylation of both the γ-chains and the α-chains could occur within the Hb F tetramer. Acetylation also could take place on intact subunits. It appears that some Hb F/sub Ic/ could be formed in the cells by utilizing Hb F or free γ-chains as acetylation substrate

  2. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis

    Jiaming Su

    2016-01-01

    Full Text Available 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.

  3. The Functional Analysis of Histone Acetyltransferase MOF in Tumorigenesis

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

    2016-01-01

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

  4. Regulation of the histone acetyltransferase activity of hMOF via autoacetylation of Lys274

    Bingfa Sun; Shunling Guo; Qingyu Tang; Chen Li; Rong Zeng; Zhiqi Xiong; Chen Zhong; Jianping Ding

    2011-01-01

    Dear Editor, Males-absent-on-the-first (MOF, also called MYST1 or KAT8) is a histone acetyltransferase (HAT) belonging to the MOZ, Ybf2/Sas3, Sas2 and Tip60 (MYST) family.MOF has been shown to possess a specific HAT activity towards Lysl6 of histone H4 (H4K16) [1].Homozygous knockout of MOF in mice results in loss of H4K16 acetylation and embryonic lethality, indicating that MOF and H4K16 acetylation are essential for embryogenesis and genome stability in mammals [2].Downregulation of human MOF (hMOF) leads to dramatic nuclear morphological deformation and inhibition of cell cycle progression [3], and has recently been correlated with primary breast carcinoma and medulloblastoma [4].

  5. Spermidine induces autophagy by inhibiting the acetyltransferase EP300.

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

    2015-03-01

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

  6. Structure of Patt1 human proapoptotic histone acetyltransferase

    Jędrzejewski, Roch Paweł; Kaźmierkiewicz, Rajmund

    2013-01-01

    The results of modeling of a novel human histone acetyltransferase Patt1 are presented here. This protein belongs to the GNAT GCN5 family and shows proapoptotic activity in human hepatocellular carcinoma cells. Patt1 is an attractive therapeutic target. The sequence analysis, fold recognition predictions and homology modeling of Patt1 protein structure were performed. N- and C- termini of Patt1 were unstructured. Central part revealed classical GNAT fold–central 7-stranded beta sheet core sur...

  7. MYST Family Histone Acetyltransferases in the Protozoan Parasite Toxoplasma gondii

    Smith, Aaron T.; Tucker-Samaras, Samantha D.; Fairlamb, Alan H; Sullivan, William J.

    2005-01-01

    The restructuring of chromatin precedes tightly regulated events such as DNA transcription, replication, and repair. One type of chromatin remodeling involves the covalent modification of nucleosomes by histone acetyltransferase (HAT) complexes. The observation that apicidin exerts antiprotozoal activity by targeting a histone deacetyltransferase has prompted our search for more components of the histone modifying machinery in parasitic protozoa. We have previously identified GNAT family HATs...

  8. Inhibition of Lysine Acetyltransferase KAT3B/p300 Activity by a Naturally Occurring Hydroxynaphthoquinone, Plumbagin*

    Ravindra, Kodihalli C.; Selvi, B. Ruthrotha; Arif, Mohammed; Reddy, B. A. Ashok; Thanuja, Gali R.; Agrawal, Shipra; Pradhan, Suman Kalyan; Nagashayana, Natesh; Dasgupta, Dipak; Tapas K. Kundu

    2009-01-01

    Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, ...

  9. Acetyltransferase and human hemoglobin acetylation

    A minor component of human fetal hemoglobin (Hb F) is acetylated at the amino-terminus of the γ-globin chains. A similar minor component of Hb F is formed during translation of cord blood mRNA in the rabbit reticulocyte lysate system. The acetylation appeared to be enzymatic. This system contains an acetyltransferase capable of acetylating histones and hemoglobins. The enzyme, partially purified by histone-Sepharose affinity chromatography was capable of incorporating labeled acetyl- group from 1-[14C-acetyl]-CoA into both human Hb F0 and HB A0, but at a lower rate than for histones. Characterization of the labeled products indicated that the α-chains of both hemoglobins were being acetylated presumably at a lysyl-residue, but in the case of Hb F0 the amino-terminus of the γ-globin chains was acetylated as well. While histone-Sepharose bound more than 95% of the enzyme, Sepharose linked Hb F0, γ-globin chains, and Hb Bart's bound 14, 5, and 12% of the activity, respectively. Enzyme bound to these resins was not any more active on the hemoglobins than was the enzyme bound to the histone-Sepharose. The histone-Sepharose was also used to detect the enzyme in human cord blood red cells separated by dextran 40 density gradient centrifugation. Activity was found mostly in the young cells, and was directly related to the number of reticulocytes present in any one fraction

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

    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.

  11. Cloning of Drosophila choline acetyltransferase cDNA.

    Itoh, N; Slemmon, J.R.; Hawke, D.H.; Williamson, R.; Morita, E.; Itakura, K; Roberts, E; Shively, J. E.; Crawford, G D; Salvaterra, P M

    1986-01-01

    Choline acetyltransferase (EC 2.3.1.6) is the biosynthetic enzyme for the neurotransmitter acetylcholine. To isolate choline acetyltransferase cDNA clones, a cDNA library was constructed from poly(A)+ RNA of Drosophila melanogaster heads, these being one of the richest known sources of the enzyme. By screening the cDNA library with a mixture of three different monoclonal antibodies to Drosophila choline acetyltransferase, we isolated 14 positive clones. Only 1 of these clones was identified t...

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

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

    2016-04-01

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

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

    Watanabe, M.(Niigata University, 950-2181, Niigata, Japan); 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 readi...

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

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

    2008-01-01

    The crystal structure of an acetyltransferase encoded by the gene PA1377 from Pseudomonas aeruginosa has been determined at 2.25 Å resolution. Comparison with a related acetyltransferase revealed a structural difference in the active site that was taken to reflect a difference in substrate binding and/or specificity between the two enzymes.

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

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

    2016-04-01

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

  16. Pseudosecretion of Escherichia coli chloramphenicol acetyltransferase by Bacillus subtilis.

    Le Grice, S F; Gentz, R; Bannwarth, W; Kocher, H. P.

    1987-01-01

    Bacillus subtilis harboring the vector 25RBSII secrets an Escherichia coli-derived chloramphenicol acetyltransferase into culture supernatants. The secreted enzyme lacks 18 amino acids; these are removed externally rather than during secretion.

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

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

    2016-07-01

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

  18. Rapid quantitative assay for chloramphenicol acetyltransferase

    Measuring the expression of exogenous genetic material in mammalian cells is commonly done by fusing the DNA of interest to a gene encoding an easily-detected enzyme. Chloramphenicol acetyltransferase(CAT) is a convenient marker because it is not normally found in eukaryotes. CAT activity has usually been detected using a thin-layer chromatographic separation followed by autoradiography. An organic solvent extraction-based method for CAT detection has also been described, as well as a procedure utilizing HPLC analysis. Building on the extraction technique, they developed a rapid sensitive kinetic method for measuring CAT activity in cell homogenates. The method exploits the differential organic solubility of the substrate ([3H] or [14C]acetyl CoA) and the product (labeled acetylchloramphenicol). The assay is a simple one-vial, two-phase procedure and requires no tedious manipulations after the initial setup. Briefly, a 0.25 ml reaction with 100mM Tris-HCL, 1mM chloramphenicol, 0.1mM [14C]acetyl CoA and variable amounts of cell homogenate is pipetted into a miniscintillation vial, overlaid with 5 ml of a water-immiscible fluor, and incubated at 370C. At suitable intervals the vial is counted and the CAT level is quantitatively determined as the rate of increase in counts/min of the labeled product as it diffuses into the fluor phase, compared to a standard curve. When used to measure CAT in transfected Balb 3T3 cells the method correlated well with the other techniques

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

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

  20. 40 CFR 174.522 - Phosphinothricin Acetyltransferase (PAT); exemption from the requirement of a tolerance.

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Phosphinothricin Acetyltransferase...-INCORPORATED PROTECTANTS Tolerances and Tolerance Exemptions § 174.522 Phosphinothricin Acetyltransferase (PAT); exemption from the requirement of a tolerance. Residues of the Phosphinothricin Acetyltransferase...

  1. Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa

    Sim Edith

    2007-03-01

    Full Text Available Abstract Background Arylamine N-acetyltransferases (NATs are important drug- and carcinogen-metabolising enzymes that catalyse the transfer of an acetyl group from a donor, such as acetyl coenzyme A, to an aromatic or heterocyclic amine, hydrazine, hydrazide or N-hydroxylamine acceptor substrate. NATs are found in eukaryotes and prokaryotes, and they may also have an endogenous function in addition to drug metabolism. For example, NAT from Mycobacterium tuberculosis has been proposed to have a role in cell wall lipid biosynthesis, and is therefore of interest as a potential drug target. To date there have been no studies investigating the kinetic mechanism of a bacterial NAT enzyme. Results We have determined that NAT from Pseudomonas aeruginosa, which has been described as a model for NAT from M. tuberculosis, follows a Ping Pong Bi Bi kinetic mechanism. We also describe substrate inhibition by 5-aminosalicylic acid, in which the substrate binds both to the free form of the enzyme and the acetyl coenzyme A-enzyme complex in non-productive reaction pathways. The true kinetic parameters for the NAT-catalysed acetylation of 5-aminosalicylic acid with acetyl coenzyme A as the co-factor have been established, validating earlier approximations. Conclusion This is the first reported study investigating the kinetic mechanism of a bacterial NAT enzyme. Additionally, the methods used herein can be applied to investigations of the interactions of NAT enzymes with new chemical entities which are NAT ligands. This is likely to be useful in the design of novel potential anti-tubercular agents.

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

    Malatesta, Martina; Steinhauer, Cornelia; Mohammad, Faizaan;

    2013-01-01

    The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well...... of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.......The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well...... show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased...

  3. Retinal rhythms in chicks: circadian variation in melantonin and serotonin N-acetyltransferase activity.

    Hamm, H E; Menaker, M

    1980-01-01

    There is a large-amplitude circadian rhythm of indoleamine metabolism in the retina-pigment epithelium of the chicken. N-Acetyltransferase activity (arylamine acetyltransferase; acetyl-CoA:arylamine N-acetyltransferase, EC 2.3.1.5) and melatonin content are 15-fold higher at night than during the day in a cycle of a 4-fold increase during the subjective night. Light at midnight inactivates N-acetyltransferase and lowers melatonin. N-Acetyltransferase activity is found predominantly in the ret...

  4. Erythromycin induces expression of the chloramphenicol acetyltransferase gene cat-86.

    Rogers, E J; Lovett, P S

    1990-01-01

    The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. This gene, like the erythromycin-inducible erm genes, is regulated by translational attenuation. Here we show that cat-86 is also inducibly regulated by erythromycin. cat-86 does not confer resistance to erythromycin.

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

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

    2009-09-02

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

  6. Chloramphenicol acetyltransferase should not provide methanogens with resistance to chloramphenicol.

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

    1984-01-01

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

  7. N-acetyltransferase in human skin and keratinocytes

    Vogel, Tanja; Bonifas, Jutta; Wiegman, Marjon; Pas, Hendrikus; Blömeke, Brunhilde; Coenraads, Pieter Jan; Schuttelaar, Marie-Louise

    2014-01-01

    Background: N-acetyltransferase 1 (NAT1) mediated Nacetylation in human skin and keratinocytes is an important detoxification pathway for aromatic amines including the strong sensitizer para-phenylenediamine (PPD), an important component of oxidative hair dyes. Objectives: Human skin and keratinocyt

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

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

  9. Evolution of arylalkylamine N-acetyltransferase: Emergence and divergence

    Coon, Steven L.; Klein, David C.

    2006-01-01

    The melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase (AANAT) is known to have recognizable ancient homologs in bacteria and fungi, but not in other eukaryotes. Analysis of new cDNA and genomic sequences has identified several additional homologs in other groupings. First, an AANAT homolog has been found in the genome of the cephalochordate amphioxus, representing the oldest homolog in chordates. Second, two AANAT homologs have been identified in unicellular green algae. ...

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

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

    1985-01-01

    To facilitate the rapid and accurate quantitation of parenterally administered aminoglycosides, we defined the optimum conditions (pH, duration of incubation, and cofactor concentrations) 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...

  11. Dysregulation of Histone Acetyltransferases and Deacetylases in Cardiovascular Diseases

    Yonggang Wang; Xiao Miao; Yucheng Liu; Fengsheng Li; Quan Liu; Jian Sun; Lu Cai

    2014-01-01

    Cardiovascular disease (CVD) remains a leading cause of mortality worldwide despite advances in its prevention and management. A comprehensive understanding of factors which contribute to CVD is required in order to develop more effective treatment options. Dysregulation of epigenetic posttranscriptional modifications of histones in chromatin is thought to be associated with the pathology of many disease models, including CVD. Histone acetyltransferases (HATs) and deacetylases (HDACs) are reg...

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

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

    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.

  13. Density Functional Theory Study on the Histidine-assisted Mechanism of Arylamine N-Acetyltransferase Acetylation

    QIAO Qing-An; GAO Shan-Min; JIN Yue-Qing; CHEN Xin; SUN Xiao-Min; YANG Chuan-Lu

    2008-01-01

    Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) catalyze the N-acetylation of primary arylamines, and play a key role in the biotransformation and metabolism of drugs, carcinogens, etc.In this paper, three possible reaction mechanisms are investigated and the results indicate that if the acetyl group directly transfers from the donor to the acceptor, the high activation energies will make it hard to obtain the target products.When using histidine to mediate the acetylation process, these energies will drop in the 15~45 kJ/mol range.If the histidine residue is protonated, the corresponding energies will be decreased by about 35~87 kJ/mol.The calculations predict an enzymatic acetylation mechanism that undergoes a thiolate-imidazolium pair, which agrees with the experimental results very well.

  14. Development of highly glyphosate-tolerant tobacco by coexpression of glyphosate acetyltransferase gat and EPSPS G2-aroA genes

    Baoqing Dun; Xujing Wang; Wei Lu; Ming Chen; Wei Zhang; Shuzhen Ping; Zhixing Wang; Baoming Zhang; Min Lin

    2014-01-01

    The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glyphosate acetyltransferase (GAT) effectively detoxifies glyphosate by N-acetylation. With the aim of identifying a new strategy for development of glyphosate-tolerant crops, the plant expression vector pG2-GAT harboring gat and G2-aroA (encoding EPSPS) has been transformed into tobacco (Nicotiana tabacum) to develop novel plants with higher tolerance to glyphosate. Results from Southern and Wes...

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

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

    2010-05-14

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

  16. Two different subcellular-localized Acetoacetyl-CoA acetyltransferases differentiate diverse functions in Magnaporthe oryzae.

    Zhong, Zhenhui; Norvienyeku, Justice; Yu, Jie; Chen, Meilian; Cai, Renli; Hong, Yonghe; Chen, Liqiong; Zhang, Dongmei; Wang, Baohua; Zhou, Jie; Lu, Guodong; Chen, Xiaofeng; Wang, Zonghua

    2015-10-01

    The mevalonate pathway is an efficient biosynthesis pathway that yields isoprenoids for promoting different crucial cellular functions, including ergosterol synthesis and growth regulation. Acetoacetyl-CoA acetyltransferase (EC2.3.1.9) is the first major catalytic enzyme constituting the mevalonate pathway and catalyzes the transformation of Acetoacetyl-CoA from two molecules of acetyl-CoA enroute ergosterol production in fungi. We identified two homologous genes encoding Acetoacetyl-CoA acetyltransferase (MoAcat1 and MoAcat2) in Magnaporthe oryzae, the rice blast fungus. Phylogenetic analysis indicates these two genes have different evolutionary history. We subsequently, conducted targeted gene deletion using homologous recombination technology to ascertain the unique roles of the two MoAcat homologues during the fungal morphogenesis and pathogenesis. The findings from our investigations showed that the activity of MoAcat1 promoted virulence in the rice blast fungus as such, the ΔMoacat1 mutants generated exhibited defect in virulence, whilst ΔMoacat1 mutants did not portray growth defects. ΔMoacat2 mutants on the other hand were characterized by reduction in growth and virulence. Furthermore, MoAcat1 and MoAcat2 showed different expression patterns and subcellular localizations in M. oryzae. From our investigations we came to the conclusion that, different subcellular localization contributes to the diverse functions of MoAcat1 and MoAcat2, which helps the successful establishment of blast disease by promoting efficient development of cell morphology and effective colonization of host tissue. PMID:26318870

  17. Structural and functional characterization of an arylamine N-acetyltransferase from the pathogen Mycobacterium abscessus

    Cocaign, Angélique; Kubiak, Xavier Jean Philippe; Xu, Ximing;

    2014-01-01

    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...

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

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

  19. Requirement for TAFII250 Acetyltransferase Activity in Cell Cycle Progression

    Dunphy, Elizabeth L.; Johnson, Theron; Auerbach, Scott s.; Wang, Edith H.

    2000-01-01

    The TATA-binding protein (TBP)-associated factor TAFII250 is the largest component of the basal transcription factor IID (TFIID). A missense mutation that maps to the acetyltransferase domain of TAFII250 induces the temperature-sensitive (ts) mutant hamster cell lines ts13 and tsBN462 to arrest in late G1. At the nonpermissive temperature (39.5°C), transcription from only a subset of protein encoding genes, including the G1 cyclins, is dramatically reduced in the mutant cells. Here we demonst...

  20. Histone acetyltransferase Rtt109 is required for Candida albicans pathogenesis

    Lopes da Rosa, Jessica; Boyartchuk, Victor L.; Zhu, Lihua Julie; Kaufman, Paul D.

    2010-01-01

    Candida albicans is a ubiquitous opportunistic pathogen that is the most prevalent cause of hospital-acquired fungal infections. In mammalian hosts, C. albicans is engulfed by phagocytes that attack the pathogen with DNA-damaging reactive oxygen species (ROS). Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 is important for yeast model organisms to survive DNA damage and maintain genome integrity. To assess the importance of Rtt109 for C. al...

  1. Enzyme kinetics and inhibition of histone acetyltransferase KAT8.

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

    2015-11-13

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

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

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

    2016-07-01

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

  3. Characterization of an acetyltransferase that detoxifies aromatic chemicals in Legionella pneumophila

    Kubiak, Xavier Jean Philippe; Dervins-Ravault, Delphine; Pluvinage, Benjamin;

    2012-01-01

    molecular and functional levels. In the present paper we report the identification and biochemical and functional characterization of a unique acetyltransferase that metabolizes aromatic amine chemicals in three characterized clinical strains of L. pneumophila (Paris, Lens and Philadelphia). Strain......-specific sequence variations in this enzyme, an atypical member of the arylamine N-acetyltransferase family (EC 2.3.1.5), produce enzymatic variants with different structural and catalytic properties. Functional inactivation and complementation experiments showed that this acetyltransferase allows L. pneumophila to...

  4. Choline acetyltransferase-containing neurons in the human parietal neocortex

    V Benagiano

    2009-06-01

    Full Text Available A number of immunocytochemical studies have indicated the presence of cholinergic neurons in the cerebral cortex of various species of mammals. Whether such cholinergic neurons in the human cerebral cortex are exclusively of subcortical origin is still debated. In this immunocytochemical study, the existence of cortical cholinergic neurons was investigated on surgical samples of human parietal association neocortex using a highly specific monoclonal antibody against choline acetyltransferase (ChAT, the acetylcholine biosynthesising enzyme. ChAT immunoreactivity was detected in a subpopulation of neurons located in layers II and III. These were small or medium-sized pyramidal neurons which showed cytoplasmic immunoreactivity in the perikarya and processes, often in close association to blood microvessels. This study, providing demonstration of ChAT neurons in the human parietal neocortex, strongly supports the existence of intrinsic cholinergic innervation of the human neocortex. It is likely that these neurons contribute to the cholinergic innervation of the intracortical microvessels.

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

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

  6. Reduction of choline acetyltransferase activities in APP770 transgenic mice

    2000-01-01

    Transgenic mice overexpressing the 770-amino acid isoform of human Alzheimer amyloid precursor protein exhibit extracellular b -amyloid deposits in brain regions including cerebral cortex and hippocampus, which are severely affected in Alzheimer's disease patients. Significant reduction in choline acetyltransferase (ChAT) activities has been observed in both cortical and hippocampal brain regions in the transgenic mice at the age of 10 months compared with the age-matched non-transgenic mice, but such changes have not been observed in any brain regions of the transgenic mice under the age of 5 months. These results suggest that deposition of b -amyloid can induce changes in the brain cholinergic system of the transgenic mice.

  7. Structure of Mesorhizobium loti arylamine N-acetyltransferase 1

    The crystal structure of a M. loti arylamine N-acetyltransferase 1 has been determined at 2.0 Å resolution. The arylamine N-acetyltransferase (NAT) enzymes have been found in a broad range of both eukaryotic and prokaryotic organisms. The NAT enzymes catalyse the transfer of an acetyl group from acetyl Co-enzyme A onto the terminal nitrogen of a range of arylamine, hydrazine and arylhydrazine compounds. Recently, several NAT structures have been reported from different prokaryotic sources including Salmonella typhimurium, Mycobacterium smegmatis and Pseudomonas aeruginosa. Bioinformatics analysis of the Mesorhizobium loti genome revealed two NAT paralogues, the first example of multiple NAT isoenzymes in a eubacterial organism. The M. loti NAT 1 enzyme was recombinantly expressed and purified for X-ray crystallographic studies. The purified enzyme was crystallized in 0.5 M Ca(OAc)2, 16% PEG 3350, 0.1 M Tris–HCl pH 8.5 using the sitting-drop vapour-diffusion method. A data set diffracting to 2.0 Å was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic spacegroup P212121, with unit-cell parameters a = 53.2, b = 97.3, c = 114.3 Å. The structure was refined to a final free-R factor of 24.8%. The structure reveals that despite low sequence homology, M. loti NAT1 shares the common fold as reported in previous NAT structures and exhibits the same catalytic triad of residues (Cys-His-Asp) in the active site

  8. Physiology: Kinetics of Acetyl Coenzyme A: Arylamine N-Acetyltransferase from Human Cumulus Cells

    Chang, Chi-Chen; Hsieh, Yao-Yuan; CHUNG, JING-GUNG; Tsai, Horng-Der; Tsai, Chang-Hai

    2001-01-01

    Purpose:N-acetyltransferase (NAT) activity is involved in the detoxification of exogenous amines. We aimed to evaluate the kinetics of acetyl coenzyme A (AcCoA): arylamine NAT for human cumulus cells.

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

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

  10. Inflammatory cytokines suppress arylamine N-acetyltransferase 1 in cholangiocarcinoma cells

    Buranrat, Benjaporn; Prawan, Auemduan; Sripa, Banchob; Kukongviriyapan, Veerapol

    2007-01-01

    AIM: To evaluate the effect of inflammatory cytokines on arylamine N-acetyltransferase 1 (NAT1), which is a phase-II enzyme involved in the biotransformation of aromatic and heterocyclic amines found in food, drugs and the environment.

  11. Leukemia Inhibitory Factor Decreases the Arylamine N-Acetyltransferase Activity in Human Cumulus Granulosa Cells

    Chang, Chi-Chen; Hsieh, Yao-Yuan; CHUNG, JING-GUNG; Tsai, Horng-Der; Tsai, Chang-Hai

    2001-01-01

    Purpose: To evaluate the activities of acetyl coenzyme A (AcCoA): arylamine N-acetyltransferase (NAT) of intact cumulus granulosa cells and the role of leukemia inhibitory factor (LIF) upon their NAT activities.

  12. Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels

    Timmers Marc HTH

    2010-12-01

    Full Text Available Abstract Background The addition of an acetyl group to protein N-termini is a widespread co-translational modification. NatB is one of the main N-acetyltransferases that targets a subset of proteins possessing an N-terminal methionine, but so far only a handful of substrates have been reported. Using a yeast nat3Δ strain, deficient for the catalytic subunit of NatB, we employed a quantitative proteomics strategy to identify NatB substrates and to characterize downstream effects in nat3Δ. Results Comparing by proteomics WT and nat3Δ strains, using metabolic 15N isotope labeling, we confidently identified 59 NatB substrates, out of a total of 756 detected acetylated protein N-termini. We acquired in-depth proteome wide measurements of expression levels of about 2580 proteins. Most remarkably, NatB deletion led to a very significant change in protein phosphorylation. Conclusions Protein expression levels change only marginally in between WT and nat3Δ. A comparison of the detected NatB substrates with their orthologous revealed remarkably little conservation throughout the phylogenetic tree. We further present evidence of post-translational N-acetylation on protein variants at non-annotated N-termini. Moreover, analysis of downstream effects in nat3Δ revealed elevated protein phosphorylation levels whereby the kinase Snf1p is likely a key element in this process.

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

    Agarwal, Vinayak; Metlitskaya, Anastasiya; Severinov, Konstantin; Nair, Satish K. (Rutgers); (Russ. Acad. Sci.); (UIUC)

    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.

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

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

    2016-03-18

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

  15. An aminoglycoside sensing riboswitch controls the expression of aminoglycoside resistance acetyltransferase and adenyltransferases.

    Chen, Dongrong; Murchie, Alastair I H

    2014-10-01

    The emergence of antibiotic resistance in human pathogens is an increasing threat to public health. The fundamental mechanisms that control the high levels of expression of antibiotic resistance genes are not yet completely understood. The aminoglycosides are one of the earliest classes of antibiotics that were introduced in the 1940s. In the clinic aminoglycoside resistance is conferred most commonly through enzymatic modification of the drug although resistance through enzymatic modification of the target rRNA through methylation or the overexpression of efflux pumps is also appearing. An aminoglycoside sensing riboswitch has been identified that controls expression of the aminoglycoside resistance genes that encode the aminoglycoside acetyltransferase (AAC) and aminoglycoside nucleotidyltransferase (ANT) (adenyltransferase (AAD)) enzymes. AAC and ANT cause resistance to aminoglycoside antibiotics through modification of the drugs. Expression of the AAC and ANT resistance genes is regulated by aminoglycoside binding to the 5' leader RNA of the aac/aad genes. The aminoglycoside sensing RNA is also associated with the integron cassette system that captures antibiotic resistance genes. Specific aminoglycoside binding to the leader RNA induces a structural transition in the leader RNA, and consequently induction of resistance protein expression. Reporter gene expression, direct measurements of drug RNA binding, chemical probing and UV cross-linking combined with mutational analysis demonstrated that the leader RNA functioned as an aminoglycoside sensing riboswitch in which drug binding to the leader RNA leads to the induction of aminoglycoside antibiotic resistance. This article is part of a Special Issue entitled: Riboswitches. PMID:24631585

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

    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.

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

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

    2001-01-01

    Arylamine N-acetyltransferase activity has been described in various bacterial species. Bacterial N-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects. We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora. We report for the first time N-acetyltransferase activity in...

  18. Serum Aminoglycoside Assay by Enzyme-Mediated Immunoassay (EMIT): Correlation with Radioimmunoassay, Fluoroimmunoassay, and Acetyltransferase and Microbiological Assays

    White, L O; Scammell, L. M.; Reeves, D S

    1981-01-01

    Enzyme-mediated immunoassay (EMIT) serum aminoglycoside assay results were accurate and precise and correlated well with radioimmunoassay, fluoroimmunoassay, and acetyltransferase and microbiological assay determinations.

  19. C646, a Novel p300/CREB-Binding Protein-Specific Inhibitor of Histone Acetyltransferase, Attenuates Influenza A Virus Infection.

    Zhao, Dongming; Fukuyama, Satoshi; Sakai-Tagawa, Yuko; Takashita, Emi; Shoemaker, Jason E; Kawaoka, Yoshihiro

    2016-03-01

    New strategies to develop novel broad-spectrum antiviral drugs against influenza virus infections are needed due to the emergence of antigenic variants and drug-resistant viruses. Here, we evaluated C646, a novel p300/CREB-binding protein-specific inhibitor of histone acetyltransferase (HAT), as an anti-influenza virus agent in vitro and in vivo and explored how C646 affects the viral life cycle and host response. Our studies highlight the value of targeting HAT activity for anti-influenza drug development. PMID:26711748

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

    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

    2015-07-01

    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. PMID:26154055

  1. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells

    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.

  2. Obesity and lipid stress inhibit carnitine acetyltransferase activity.

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

    2014-04-01

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

  3. Dysregulation of Histone Acetyltransferases and Deacetylases in Cardiovascular Diseases

    Yonggang Wang

    2014-01-01

    Full Text Available Cardiovascular disease (CVD remains a leading cause of mortality worldwide despite advances in its prevention and management. A comprehensive understanding of factors which contribute to CVD is required in order to develop more effective treatment options. Dysregulation of epigenetic posttranscriptional modifications of histones in chromatin is thought to be associated with the pathology of many disease models, including CVD. Histone acetyltransferases (HATs and deacetylases (HDACs are regulators of histone lysine acetylation. Recent studies have implicated a fundamental role of reversible protein acetylation in the regulation of CVDs such as hypertension, pulmonary hypertension, diabetic cardiomyopathy, coronary artery disease, arrhythmia, and heart failure. This reversible acetylation is governed by enzymes that HATs add or HDACs remove acetyl groups respectively. New evidence has revealed that histone acetylation regulators blunt cardiovascular and related disease states in certain cellular processes including myocyte hypertrophy, apoptosis, fibrosis, oxidative stress, and inflammation. The accumulating evidence of the detrimental role of histone acetylation in cardiac disease combined with the cardioprotective role of histone acetylation regulators suggests that the use of histone acetylation regulators may serve as a novel approach to treating the millions of patients afflicted by cardiac diseases worldwide.

  4. Characterization of the Saccharomyces cerevisiae ARG7 gene encoding ornithine acetyltransferase, an enzyme also endowed with acetylglutamate synthase activity.

    Crabeel, M; Abadjieva, A; Hilven, P; Desimpelaere, J; Soetens, O

    1997-12-01

    We have cloned by functional complementation and characterized the yeast ARG7 gene encoding mitochondrial ornithine acetyltransferase, the enzyme catalyzing the fifth step in arginine biosynthesis. While forming ornithine, this enzyme regenerates acetylglutamate, also produced in the first step by the ARG2-encoded acetylglutamate synthase. Interestingly, total deletion of the genomic ARG7 ORF resulted in an arginine-leaky phenotype, indicating that yeast cells possess an alternative route for generating ornithine from acetylornithine. Yeast ornithine acetyltransferase has been purified and characterized previously as a heterodimer of two subunits proposed to derive from a single precursor protein [Liu, Y-S., Van Heeswijck R., Hoj, P. & Hoogenraad, N. (1995) Eur. J. Biochem. 228, 291-296]; those authors further suggested that the internal processing of Arg7p, which is a mitochondrial enzyme, might occur in the matrix, while the leader peptide would be of the non-cleavable-type. The characterization of the gene (a) establishes that Arg7p is indeed encoded by a single gene, (b) demonstrates the existence of a cleaved mitochondrial prepeptide of eight residues, and (c) shows that the predicted internal processing site is unlike the mitochondrial proteolytic peptidase target sequence. Yeast Arg7p shares between 32-43% identity in pairwise comparisons with the ten analogous bacterial ArgJ enzymes characterized. Among these evolutionarily related enzymes, some but not all appear bifunctional, being able to produce acetylglutamate not only from acetylornithine but also from acetyl-CoA, thus catalyzing the same reaction as the apparently unrelated acetylglutamate synthase. We have addressed the question of the bifunctionality of the eucaryotic enzyme, showing that overexpressed ARG7 can complement yeast arg2 and Escherichia coli argA mutations (affecting acetylglutamate synthase). Furthermore, Arg7p-linked acetylglutamate synthase activity was measurable in an assay. The

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

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

    2016-01-01

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

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

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

    2005-01-01

    The overexpression of serine acetyltransferase from the Ni-hyperaccumulating plant Thlaspi goesingense causes enhanced nickel and cobalt resistance in Escherichia coli. Furthermore, overexpression of T. goesingense serine acetyltransferase results in enhanced sensitivity to cadmium and has no significant effect on resistance to zinc. Enhanced nickel resistance is directly related to the constitutive overactivation of sulfur assimilation and glutathione biosynthesis, driven by the overproducti...

  7. DNA hybridization and phosphinothricin acetyltransferase gene sequence detection based on zirconia/nanogold film modified electrode

    Zhang, Wei; Yang, Tao; Jiang, Chen; Jiao, Kui

    2008-05-01

    This study reports a novel electrochemical DNA biosensor based on zirconia (ZrO 2) and gold nanoparticles (NG) film modified glassy carbon electrode (GCE). NG was electrodeposited onto the glassy carbon electrode at 1.5 V, and then zirconia thin film on the NG/GCE was fabricated by cyclic voltammetric method (CV) in an aqueous electrolyte of ZrOCl 2 and KCl at a scan rate of 20 mV/s. DNA probes were attached onto the ZrO 2/NG/GCE due to the strong binding of the phosphate group of DNA with the zirconia film and the excellent biocompatibility of nanogold with DNA. CV and electrochemical impedance spectroscopy (EIS) were used to characterize the modification of the electrode and the probe DNA immobilization. The electrochemical response of the DNA hybridization was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. After the hybridization of DNA probe (ssDNA) with the complementary DNA (cDNA), the cathodic peak current of MB decreased obviously. The difference of the cathodic peak currents of MB between before and after the hybridization of the probe DNA was used as the signal for the detection of the target DNA. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene in the transgenic plants was detected with a detection range from 1.0 × 10 -10 to 1.0 × 10 -6 mol/L, and a detection limit of 3.1 × 10 -11 mol/L.

  8. Arylamine N-acetyltransferase 2 (NAT2 genetic diversity and traditional subsistence: a worldwide population survey.

    Audrey Sabbagh

    Full Text Available Arylamine N-acetyltransferase 2 (NAT2 is involved in human physiological responses to a variety of xenobiotic compounds, including common therapeutic drugs and exogenous chemicals present in the diet and the environment. Many questions remain about the evolutionary mechanisms that have led to the high prevalence of slow acetylators in the human species. Evidence from recent surveys of NAT2 gene variation suggests that NAT2 slow-causing variants might have become targets of positive selection as a consequence of the shift in modes of subsistence and lifestyle in human populations in the last 10,000 years. We aimed to test more extensively the hypothesis that slow acetylation prevalence in humans is related to the subsistence strategy adopted by the past populations. To this end, published frequency data on the most relevant genetic variants of NAT2 were collected from 128 population samples (14,679 individuals representing different subsistence modes and dietary habits, allowing a thorough analysis at both a worldwide and continent scale. A significantly higher prevalence of the slow acetylation phenotype was observed in populations practicing farming (45.4% and herding (48.2% as compared to populations mostly relying on hunting and gathering (22.4% (P = 0.0007. This was closely mirrored by the frequency of the slow 590A variant that was found to occur at a three-fold higher frequency in food producers (25% as compared to hunter-gatherers (8%. These findings are consistent with the hypothesis that the Neolithic transition to subsistence economies based on agricultural and pastoral resources modified the selective regime affecting the NAT2 acetylation pathway. Furthermore, the vast amount of data collected enabled us to provide a comprehensive and up-to-date description of NAT2 worldwide genetic diversity, thus building up a useful resource of frequency data for further studies interested in epidemiological or anthropological research

  9. Suppression of Exogenous Gene Expression by Spermidine/Spermine N1-Acetyltransferase 1 (SSAT1) Cotransfection*

    Lee, Seung Bum; Park, Jong Hwan; Woster, Patrick M.; CASERO, ROBERT A.; Park, Myung Hee

    2010-01-01

    Spermidine/spermine N1-acetyltransferase 1 (SSAT1), which catalyzes the N1-acetylation of spermidine and spermine to form acetyl derivatives, is a rate-limiting enzyme in polyamine catabolism. We now report a novel activity of transiently transfected SSAT1 in suppressing the exogenous expression of other proteins, i.e. green fluorescent protein (GFP) or GFP-eIF5A. Spermidine/spermine N1-acetyltransferase 2 (SSAT2) or inactive SSAT1 mutant enzymes (R101A or R101K) were without effect. The loss...

  10. Cigarette Smoking, N-Acetyltransferase 2 Acetylation Status, and Bladder Cancer Risk

    Marcus, P.M.; Hayes, R.B.; Vineis, P.;

    2000-01-01

    Tobacco use is an established cause of bladder cancer. The ability to detoxify aromatic amines, which are present in tobacco and are potent bladder carcinogens, is compromised in persons with the N-acetyltransferase 2 slow acetylation polymorphism. The relationship of cigarette smoking with bladder...... interaction between smoking and N-acetyltransferase 2 slow acetylation (OR, 1.3; 95% confidence interval, 1.0-1.6) that was somewhat stronger when analyses were restricted to studies conducted in Europe (OR, 1.5; confidence interval, 1.1-1.9), a pooling that included nearly 80% of the collected data. Using...

  11. Crystallization and preliminary X-ray diffraction analysis of PAT, an acetyltransferase from Sulfolobus solfataricus

    PAT, an acetyltransferase from the archaeon S. solfataricus that specifically acetylates the chromatin protein Alba, was expressed, purified and crystallized. PAT is an acetyltransferase from the archaeon Sulfolobus solfataricus that specifically acetylates the chromatin protein Alba. The enzyme was expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data were collected to 1.70 Å resolution on the BL13C1 beamline of NSRRC from a flash-frozen crystal at 100 K. The crystals belonged to space group P212121, with unit-cell parameters a = 44.30, b = 46.59, c = 68.39 Å

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

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

    2006-01-01

    A study 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.

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

    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...

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

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

  15. Chloramphenicol acetyltransferase may confer resistance to fusidic acid by sequestering the drug.

    Proctor, G N; McKell, J.; Rownd, R H

    1983-01-01

    Enterobacterial chloramphenicol acetyltransferase bound fusidic acid with high affinity, but did not acetylate the drug at an experimentally detectable rate. The enzyme may therefore confer resistance to fusidic acid by sequestering the drug and thereby preventing the drug from binding to translational elongation factor G.

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

    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...

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

    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...

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

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

    2014-01-01

    The structure of homoserine O-acetyltransferase (HTA) from the human pathogen Staphylococcus aureus has been determined. Despite a similar overall fold and active site architecture to other α/β-hydrolases, this more compact HTA structure has a more narrow access to the active site than can confer important specificity differences.

  19. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-{kappa}B acetylation in fibroblast-like synoviocyte MH7A cells

    Seong, Ah-Reum; Yoo, Jung-Yoon; Choi, KyungChul [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Lee, Mee-Hee [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University, College of Medicine, Seoul (Korea, Republic of); Lee, Yoo-Hyun [Department of Food Science and Nutrition, The University of Suwon, Kyunggi-do (Korea, Republic of); Lee, Jeongmin [Department of Medical Nutrition, Kyung Hee University, Kyunggi-do (Korea, Republic of); Jun, Woojin [Department of Food and Nutrition, Chonnam National University, Gwangju (Korea, Republic of); Kim, Sunoh, E-mail: sunoh@korea.ac.kr [Jeollanamdo Institute of Natural Resources Research, Jeonnam (Korea, Republic of); Yoon, Ho-Geun, E-mail: yhgeun@yuhs.ac [Department of Biochemistry and Molecular Biology, Center for Chronic Metabolic Disease Research, College of Medicine, Yonsei University, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University, College of Medicine, Seoul (Korea, Republic of)

    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.

  20. Delphinidin, a specific inhibitor of histone acetyltransferase, suppresses inflammatory signaling via prevention of NF-κB acetylation in fibroblast-like synoviocyte MH7A cells

    Highlights: → Delphinidin is a novel inhibitor of p300/CBP histone acetyltransferase. → Delphinidin prevents the hyperacetylation of p65 by inhibiting the HAT activity of p300/CBP. → Delphinidin efficiently suppresses the expression of inflammatory cytokines in MH7A cells via hypoacetylation of NF-κB. → 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 IKBα. Accordingly, DP treatment inhibited TNFα-stimulated increases in NF-κB function and expression of NF-κB 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.

  1. A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion

    The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae...

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

    Belikoff Esther J

    2010-11-01

    Full Text Available Abstract 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

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

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

    2005-12-01

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

  4. Choline acetyltransferase expressed by radial neuroglia cells in the development of telencephalon: A validated study

    Li Zhou; Lingling Ding; Zhisuo Xiao; Yuanyuan Qin; Guibin Li

    2007-01-01

    BACKGROUND: Cholinergic neuron directly participants in human motion, learning and memory and is a target cell for multiple degenerative diseases of central nervous system.OBJECTIVE: To investigate whether the mitotic cell is the radial glial cell expressing choline acetyltransferase (ChAT) in ventricle zone (VZ) of telencephalon and whether cholinergic neuron is derived from radial glial cell in ventricle zone of telencephalon.DESIGN: Observational study.SETTING: Department of Histology and Embryology, Basic Medical College of Jilin University.MATERIALS: Nine healthy Wistar rats included 6 females and 3 male. Male and female rats were mated routinely, and the day when spermatozoa or vaginal plug were found was regarded as embryonic 0 (E0).Primary monoclonal antibodies ChAT and vimentin were provided respectively by Wuhan Boster Company,and Biogenex Company, USA.METHODS: The experiment was carried out in the Laboratory of Cell Culture and Immunohistochemistry,Department of Histology and Embryology from march 2002 to January 2003. Firstly, fluorescence-activated cell sorting (FACS) was used to confirm the time of generation of cholinergic neuron; secondly,telencephalons of rats at embryonic 14 days (E14) were performed coronary sections, then immunohistochemistry double staining for vimentin (a protein marker of radial neuroglia cell) and ChAT (a protein marker of cholinergic neuron) were used to test whether ChAT was expressed in the radial neuroglia cells.results of immunohistochemistry double staining.RESULTS: It is confirmed using by flow cytometer that embryogenesis time of cholinergic neuron was at E12, and shown the population of cells in VZ of dorsal telencephalon of E14 rat co-expressed vimentin and ChAT through immunohistochemistry double staining. A lot of vimentin-positive cells and ChAT-positive cells respectively were observed in VZ of lateral ganglionic eminence.CONCLUSION: Cholinergic neuron in cerebral cortex is derived from radial glial cells in VZ

  5. Glycyl-L-glutamine opposes the fall in choline acetyltransferase in the denervated superior cervical ganglion of the cat.

    Koelle, G B; O'Neill, J J; Thampi, N S; Han, M S; Caccese, R

    1989-01-01

    Intracarotid infusion of 3 microM glycyl-L-glutamine was found to oppose the fall in the choline acetyl-transferase content of the preganglionically denervated cat superior cervical ganglion; this same effect has been demonstrated previously for acetylcholinesterase content. Because choline acetyltransferase, in contrast to acetylcholinesterase, occurs exclusively in the preganglionic axons and their terminals, this finding raises the possibility that glycyl-L-glutamine opposes postsectional ...

  6. Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a Confocal Scanning Optical Microscopy Analysis

    NILSON NUNES-TAVARES; NARCISA LEAL CUNHA-E-SILVA; AÍDA HASSÓN-VOLOCH

    2000-01-01

    Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We ...

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

    Crosby, Heidi A [University of Wisconsin, Madison; Pelletier, Dale A [ORNL; Hurst, Gregory {Greg} B [ORNL; Escalante-Semerena, Jorge C [University of Wisconsin, Madison

    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.

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

    Kubiak, Xavier Jean Philippe; Pluvinage, Benjamin; Li de la Sierra-Gallay, Inès;

    2012-01-01

    Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X-ray characterizat......Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X...

  9. Neuropeptide Y-like immunoreactivity in rat cranial parasympathetic neurons: coexistence with vasoactive intestinal peptide and choline acetyltransferase

    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

  10. G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression.

    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

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

    Michael N. Davies

    2016-01-01

    Full Text Available 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.

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

    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

    2016-01-12

    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

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

    Bingfu eGuo

    2015-10-01

    Full Text Available 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 four-fold 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.

  14. G9a-mediated methylation of ERα links the PHF20/MOF histone acetyltransferase complex to hormonal gene expression

    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

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

    Maes, Dominique, E-mail: dominique.maes@vub.ac.be; Crabeel, Marjolaine [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium); Van de Weerdt, Cécile; Martial, Joseph [Laboratoire de Biologie Moléculaire et de Génie Génétique, Université de Liège, Allée de la Chimie 3, B-4000 Liège (Belgium); Peeters, Eveline; Charlier, Daniël [Erfelijkheidsleer en Microbiologie, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels (Belgium); Decanniere, Klaas; Vanhee, Celine; Wyns, Lode; Zegers, Ingrid [Laboratorium voor Ultrastructuur, Vrije Universiteit Brussel (VUB) and Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Pleinlaan 2, B-1050 Brussels (Belgium)

    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.

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

    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

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

    Acetyl-CoA:α-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 α-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 [3H]CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with [3H]acetyl-CoA. The acetyl group can be transferred to glucosamine, forming [3H]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

  18. Cryptococcus neoformans Histone Acetyltransferase Gcn5 Regulates Fungal Adaptation to the Host ▿ † ‡

    O'Meara, Teresa R.; Hay, Christie; Price, Michael S.; Giles, Steve; Alspaugh, J. Andrew

    2010-01-01

    Cryptococcus neoformans is an environmental fungus and an opportunistic human pathogen. Previous studies have demonstrated major alterations in its transcriptional profile as this microorganism enters the hostile environment of the human host. To assess the role of chromatin remodeling in host-induced transcriptional responses, we identified the C. neoformans Gcn5 histone acetyltransferase and demonstrated its function by complementation studies of Saccharomyces cerevisiae. The C. neoformans ...

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

    Campeau, Philippe M.; Kim, Jaeseung C.; Lu, James T.; Schwartzentruber, Jeremy A.; Abdul-Rahman, Omar A.; Schlaubitz, Silke; Murdock, David M.; Jiang, Ming-Ming; Lammer, Edward J.; Enns, Gregory M.; Rhead, William J.; Rowland, Jon; Robertson, Stephen P.; Cormier-Daire, Valérie; Bainbridge, Matthew N.

    2012-01-01

    Genitopatellar syndrome (GPS) is a skeletal dysplasia with cerebral and genital anomalies for which the molecular basis has not yet been determined. By exome sequencing, we found de novo heterozygous truncating mutations in KAT6B (lysine acetyltransferase 6B, formerly known as MYST4 and MORF) in three subjects; then by Sanger sequencing of KAT6B, we found similar mutations in three additional subjects. The mutant transcripts do not undergo nonsense-mediated decay in cells from subjects with G...

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

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

    2011-01-01

    A convenient synthesis of 4′-aminopantetheine from commercial D-pantethine is reported. The amino group was introduced by reductive amination in order to avoid substitution at a sterically congested position. Derivatives of 4′-aminopantetheine were also prepared to evaluate the effect of O-to-N substitution on inhibitors of the resistance-causing enzyme aminoglycoside N-6′-acetyltransferase. The biological results combined with docking studies indicate that in spite of its reported unusual fl...

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

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

    2009-01-01

    We studied the responses of the activities of adenosine-triphosphate (ATP) sulfurylase (ATPS) and serine acetyltransferase (SAT) to cadmium (Cd) levels and treatment time in hyperaccumulating ecotype (HE) Sedum alfredii Hance, as compared with its non-hyperaccumulating ecotype (NHE). The results show that plant growth was inhibited in NHE but promoted in HE when exposed to high Cd level. Cd concentrations in leaves and shoots rapidly increased in HE rather than in NHE, and they became much hi...

  2. Cryptococcus neoformans histone acetyltransferase Gcn5 regulates fungal adaptation to the host.

    O'Meara, Teresa R; Hay, Christie; Price, Michael S; Giles, Steve; Alspaugh, J Andrew

    2010-08-01

    Cryptococcus neoformans is an environmental fungus and an opportunistic human pathogen. Previous studies have demonstrated major alterations in its transcriptional profile as this microorganism enters the hostile environment of the human host. To assess the role of chromatin remodeling in host-induced transcriptional responses, we identified the C. neoformans Gcn5 histone acetyltransferase and demonstrated its function by complementation studies of Saccharomyces cerevisiae. The C. neoformans gcn5Delta mutant strain has defects in high-temperature growth and capsule attachment to the cell surface, in addition to increased sensitivity to FK506 and oxidative stress. Treatment of wild-type cells with the histone acetyltransferase inhibitor garcinol mimics cellular effects of the gcn5Delta mutation. Gcn5 regulates the expression of many genes that are important in responding to the specific environmental conditions encountered by C. neoformans inside the host. Accordingly, the gcn5Delta mutant is avirulent in animal models of cryptococcosis. Our study demonstrates the importance of chromatin remodeling by the conserved histone acetyltransferase Gcn5 in regulating the expression of specific genes that allow C. neoformans to respond appropriately to the human host. PMID:20581290

  3. GCN5 Acetyltransferase Inhibits PGC1α-induced Hepatitis B Virus Biosynthesis

    Xiaohui Tian; Fei Zhao; Zhikui Cheng; Ming Zhou; Xiaoguang Zhi; Jiafu Li; Kanghong Hu

    2013-01-01

    Hepatitis B virus (HBV) biosynthesis is primarily restricted to hepatocytes due to the goveming of liver-enriched nuclear receptors (NRs) on viral RNA synthesis.The liver-enriched NR hepatocyte nuclear factor 4α (HNF4α),the key regulator of genes implicated in hepatic glucose metabolism,is also a primary determinant of HBV pregenomic RNA synthesis and HBV replication.Peroxisome proliferator-activated receptor-γ coactivator lα (PGC1α) coactivates and further enhances the effect of HNF4α on HBV biosynthesis.Here,we showed that the acetyltransferase General Control Non-repressed Protein 5 (GCN5) acetylated PGC1α,leading to alteration of PGC1α from a transcriptionally active state into an inactive state.As a result,the coactivation activity of PGClα on HBV transcription and replication was suppressed.Apparently,an acetylation site mutant of PGC 1α (PGC1αR13) still had coactivation activity as GCN5 could not suppress the coactivation activity of the mutant.Moreover,a catalytically inactive acetyltransferase mutant GCN5m,due to the loss of acetylation activity,failed to inhibit the coactivation function of PGC 1α in HBV biosynthesis.Our results demonstrate that GCN5,through its acetyltransferase activity,inhibits PGClα-induced enhancement of HBV transcription and replication both in vitro and in vivo.

  4. Chromatin-regulating proteins as targets for cancer therapy

    Oike, Takahiro; Ogiwara, Hideaki; Amornwichet, Napapat; Nakano, Takashi; Kohno, Takashi

    2014-01-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium...

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

    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

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

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

    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.

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

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

    2010-04-01

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

  8. Untersuchung lysosomaler Membranproteine mit dem Schwerpunkt der Charakterisierung der Acetyl-Coenzym A: a-Glucosaminid N-Acetyltransferase aus lysosomalen Membranpräparationen aus humaner Plazenta

    Wätzig, Kristin Irene

    2007-01-01

    Die Acetyl-CoA-Glucosaminid-N-Acetyltransferase ist ein lysosomales Protein, dessen Ausfall das Sanfilippo C-Syndrom (Mukopolysaccharidose Typ IIIC, OMIM 252930) bedingt. Dabei handelt es sich um eine von elf durch distinkte Enzymdefekte bedingten Mukopolysaccharidosen. Durch den Ausfall der lysosomalen Acetyltransferase kommt es zu einer Störung des Heparansulfatabbaus mit einer Ansammlung von Stoffwechselprodukten im Ly...

  9. Expression, crystallization and preliminary X-ray crystallographic analyses of two N-terminal acetyltransferase-related proteins from Thermoplasma acidophilum

    Han, Sang Hee; Ha, Jun Yong; Kim, Kyoung Hoon; Oh, Sung Jin; Kim, Do Jin; Kang, Ji Yong; Yoon, Hye Jin; Kim, Se-Hee; Seo, Ji Hae; Kim, Kyu-Won; Suh, Se Won

    2006-01-01

    An N-terminal acetyltransferase ARD1 subunit-related protein (Ta0058) and an N-terminal acetyltransferase-related protein (Ta1140) from T. acidophilum were crystallized. X-ray diffraction data were collected to 2.17 and 2.40 Å, respectively.

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

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

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

    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.

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

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

    2011-01-01

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

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

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

    2011-03-01

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

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

    Wang Yong; Zhang Rui; Wu Donglu; Lu Zhihua; Sun Wentao; Cai Yong; Wang Chunxi; Jin Jingji

    2013-01-01

    Abstract Background MYST1 (also known as hMOF), a member of the MYST family of histone acetyltransferases (HATs) as an epigenetic mark of active genes, is mainly responsible for histone H4K16 acetylation in the cells. Recent studies have shown that the abnormal gene expression of hMOF is involved in certain primary cancers. Here we examined the involvement of hMOF expression and histone H4K16 acetylation in primary renal cell carcinoma (RCC). Simultaneously, we investigated the correlation be...

  15. Horizontal gene transfer of acetyltransferases, invertases and chorismate mutases from different bacteria to diverse recipients

    Noon, Jason B.; Baum, Thomas J.

    2016-01-01

    Background Hoplolaimina plant-parasitic nematodes (PPN) are a lineage of animals with many documented cases of horizontal gene transfer (HGT). In a recent study, we reported on three likely HGT candidate genes in the soybean cyst nematode Heterodera glycines, all of which encode secreted candidate effectors with putative functions in the host plant. Hg-GLAND1 is a putative GCN5-related N-acetyltransferase (GNAT), Hg-GLAND13 is a putative invertase (INV), and Hg-GLAND16 is a putative chorismat...

  16. Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

    Lee, K. F.; Atiee, S H; Rosen, J. M.

    1989-01-01

    Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of tr...

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

    Morrison, Michael J; Imperiali, Barbara

    2013-11-01

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

  18. Epigenetic regulation of proliferation and invasion in hepatocellular carcinoma cells by CBP/p300 histone acetyltransferase activity.

    Inagaki, Yuji; Shiraki, Katsuya; Sugimoto, Kazushi; Yada, Takazumi; Tameda, Masahiko; Ogura, Suguru; Yamamoto, Norihiko; Takei, Yoshiyuki; Ito, Masaaki

    2016-02-01

    Altered epigenetic control of gene expression plays a substantial role in tumor development and progression. Accumulating studies suggest that somatic mutations of CREB binding proteins (CBP)/p300 occur in some cancer cells. CBP/p300 possess histone acetyltransferase (HAT) activity, and are involved in many cellular processes. In this study, we investigated the expression and functional role of CBP/p300 in hepatocellular carcinoma (HCC) using the specific inhibitor C646 of CBP/p300 HAT activity. We examined its effect on several apoptosis-related proteins and invasion-related genes. The results showed that CBP/p300 were highly expressed in HCC tissues and that expression of p300, but not of CBP, was strongly correlated with the malignant character of HCC. C646 inhibited proliferation of HCC cell lines in a dose dependent manner. C646 significantly augmented TRAIL-induced apoptotic sensitivity, which was accompanied by reduced levels of survivin, in HepG2, HLE and SK-HEP1 cells. C646 significantly inhibited invasion of Huh7, HLE and SK-HEP1 cells. The level of matrix metallopeptidase 15 (MMP15) mRNA expression was significantly reduced, whereas the level of laminin alpha 3 (LAMA3) and secreted phosphoprotein 1 (SPP1) mRNA expression was significantly increased in Huh7 cells following exposure to C646. In conclusion, our results suggest that CBP/p300 HAT activity has an important role in malignant transformation, proliferation, apoptotic sensitivity and invasion in HCC. CBP/p300 could be a promising therapeutic target in HCC. PMID:26676548

  19. Effects of human arylamine N-acetyltransferase I knockdown in triple-negative breast cancer cell lines

    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

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

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

    2012-02-01

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

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

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

    2015-02-01

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

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

    Rv1653, an ornithine acetyltransferase from M. tuberculosis, has been crystallized and diffraction data have been collected to 1.7 Å resolution. The gene product of open reading frame Rv1653 from Mycobacterium tuberculosis is annotated as encoding a probable ornithine acetyltransferase (OATase; EC 2.3.1.35), an enzyme that catalyzes two steps in the arginine-biosynthesis pathway. It transfers an acetyl group from N-acetylornithine to l-glutamate to produce N-acetylglutamate and l-ornithine. Rv1653 was crystallized using the sitting-drop vapour-diffusion method. The native crystals diffracted to a resolution of 1.7 Å and belonged to space group P212121, with unit-cell parameters a = 60.1, b = 99.7, c = 155.3 Å. The preliminary X-ray study showed the presence of a dimer in the asymmetric unit of the crystals, which had a Matthews coefficient VM of 2.8 Å3 Da−1

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

    Takahara, Kentaro; Akashi, Kinya; Yokota, Akiho

    2005-10-01

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

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

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

    2012-02-10

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

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

    Abu Iftiaf Md Salah Ud-Din

    2016-06-01

    Full Text Available 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.

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

    Wainwright, S D

    1980-06-12

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

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

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

    1989-05-01

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

  8. Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6′-acetyltransferase AAC(6′)-Im

    Toth, Marta; Vakulenko, Sergei B.; Smith, Clyde A.

    2012-01-01

    AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken.

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

    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...

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

    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...

  11. Genetic variants in the choline acetyltransferase (ChAT) gene are modestly associated with normal cognitive function in the elderly

    Mengel-From, J; Christensen, K; Thinggaard, M; McGue, M; Christiansen, L

    2011-01-01

    Genetic variants in the choline acetyltransferase (ChAT) gene have been suggested as risk factors for neurodegenerative Alzheimer's disease (AD). Here we tested the importance of genetic variants in the ChAT gene in normal cognitive function of elderly in a study sample of Danish twins and single...

  12. Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p

    Greenblatt Jack F

    2007-09-01

    Full Text Available Abstract Background Histone modifications have been implicated in the regulation of transcription and, more recently, in DNA replication and repair. In yeast, a major conserved histone acetyltransferase, Hat1p, preferentially acetylates lysine residues 5 and 12 on histone H4. Results Here, we report that a nuclear sub-complex consisting of Hat1p and its partner Hat2p interacts physically and functionally with the origin recognition complex (ORC. While mutational inactivation of the histone acetyltransferase (HAT gene HAT1 alone does not compromise origin firing or initiation of DNA replication, a deletion in HAT1 (or HAT2 exacerbates the growth defects of conditional orc-ts mutants. Thus, the ORC-associated Hat1p-dependent histone acetyltransferase activity suggests a novel linkage between histone modification and DNA replication. Additional genetic and biochemical evidence points to the existence of partly overlapping histone H3 acetyltransferase activities in addition to Hat1p/Hat2p for proper DNA replication efficiency. Furthermore, we demonstrated a dynamic association of Hat1p with chromatin during S-phase that suggests a role of this enzyme at the replication fork. Conclusion We have found an intriguing new association of the Hat1p-dependent histone acetyltransferase in addition to its previously known role in nuclear chromatin assembly (Hat1p/Hat2p-Hif1p. The participation of a distinct Hat1p/Hat2p sub-complex suggests a linkage of histone H4 modification with ORC-dependent DNA replication.

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

    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. PMID:26801935

  14. RNAi-mediated knock-down of arylamine N-acetyltransferase-1 expression induces E-cadherin up-regulation and cell-cell contact growth inhibition.

    Tiang, Jacky M; Butcher, Neville J; Cullinane, Carleen; Humbert, Patrick O; Minchin, Rodney F

    2011-01-01

    Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics. PMID:21347396

  15. RNAi-mediated knock-down of arylamine N-acetyltransferase-1 expression induces E-cadherin up-regulation and cell-cell contact growth inhibition.

    Jacky M Tiang

    Full Text Available Arylamine N-acetyltransferase-1 (NAT1 is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics.

  16. Development of highly glyphosate-tolerant tobacco by coexpression of glyphosate acetyltransferase gat and EPSPS G2-aroA genes

    Baoqing; Dun; Xujing; Wang; Wei; Lu; Ming; Chen; Wei; Zhang; Shuzhen; Ping; Zhixing; Wang; Baoming; Zhang; Min; Lin

    2014-01-01

    The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).Glyphosate acetyltransferase(GAT)effectively detoxifies glyphosate by N-acetylation.With the aim of identifying a new strategy for development of glyphosate-tolerant crops,the plant expression vector pG2-GAT harboring gat and G2-aroA(encoding EPSPS)has been transformed into tobacco(Nicotiana tabacum)to develop novel plants with higher tolerance to glyphosate.Results from Southern and Western blotting analyses indicated that the target genes were integrated into tobacco chromosomes and expressed effectively at the protein level.Glyphosate tolerance was compared among transgenic tobacco plants containing gat,G2-aroA,or both genes.Plants containing both gat and G2-aroA genes were the most glyphosate-tolerant.This study has shown that a combination of different strategies may result in higher tolerance in transgenic crops,providing a new approach for development of glyphosate-tolerant crops.

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

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

    2009-02-01

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

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

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

  19. Changes in pineal N-acetyltransferase activity in gamma irradiated rats

    Male Wistar rats were exposed to whole-body irradiation with 14.35 Gy gamma rays after adaptation to the light/dark cycle (LD 12:12). Three groups of rats were studied: A) rats irradiated at night and placed in the 12 h LD cycle again, B) rats irradiated at daytime and placed in the 12 LD cycle, and C) rats irradiated at night and kept in constant darkness. All analyses were carried out in the dark. Radiation enhanced the activity of pineal N-acetyltransferase 3-4 days after exposure in all groups, in the C group significantly on the 4th day. Different light regimes during and after irradiation did not substantially affect the activity of this key enzyme of melatonin synthesis. (author) 1 fig., 8 refs

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

    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

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

    Hu, Xiaohu [ORNL; Norris, Adrianne [University of Tennessee, Knoxville (UTK); Baudry, Jerome Y [ORNL; Serpersu, Engin H [University of Tennessee, Knoxville (UTK)

    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.

  2. Depression of nocturnal pineal serotonin N-acetyltransferase activity in castrate male rats

    Pineal serotonin N-acetyltransferase (NAT) activity was examined in intact rats, castrated rats, and in rats that had been castrated and had received testosterone proprionate. Castration resulted in significantly depressing nocturnal levels of pineal NAT (p<0.05) when compared to enzyme activity in intact rats. Testosterone proprionate administration restored plasma LH levels to normal values in castrate rats but did not induce nocturnal pineal enzyme activity to levels seen in the pineal glands of intact rats. The data substantiate the existence of a feedback control of pineal biosynthetic activity by the hypophyseal-gonadal system, but the identity of the hormone(s) responsible for regulation of pineal NAT activity is not known. (author)

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

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

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

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

    2016-06-17

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

  5. Mechanism of p300 specific histone acetyltransferase inhibition by small molecules.

    Arif, M; Pradhan, Suman Kalyan; Thanuja, G R; Vedamurthy, B M; Agrawal, Shipra; Dasgupta, Dipak; Kundu, Tapas K

    2009-01-22

    Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, diabetes, and asthma. Therefore, small molecule inhibitors and activators of HATs are being considered as new generation therapeutics. Here, we report the molecular mechanisms of p300 HAT inhibition by specific and nonspecific HAT inhibitors: garcinol, isogarcinol, and 1 (LTK14). The p300 specific HAT inhibitor 1 behaves as a noncompetitive inhibitor for both acetyl-CoA and histone, unlike nonspecific HAT inhibitors garcinol and isogarcinol. The isothermal calorimetric data suggest that there is a high affinity enthalpy driven single binding site for 1 on p300HAT domain in contrast to two binding sites for garcinol and isogarcinol. Furthermore, the precise nature of molecular interactions was determined by using fluorescence, docking, and mutational studies. On the basis of these observations, we have proposed the mechanisms of specific versus nonspecific HAT inhibition by these small molecule compounds, which may be useful to design therapeutically favorable HAT inhibitors. PMID:19086895

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

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

    2016-08-10

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

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

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

    2015-12-01

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

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

    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 (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. PMID:22210605

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

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

    2014-10-01

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

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

    Garvey, Graeme S.; McCormick, Susan P.; Alexander, Nancy J.; Rayment, Ivan; (US-Agriculture); (UW)

    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.

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

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

    2003-01-15

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

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

    Wang Yong

    2013-02-01

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

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

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

    2012-01-01

    B. cereus arylamine N-acetyltransferase 3 was expressed, purified and crystallized. X-ray diffraction data were collected to 2.42 Å resolution and the crystals belonged to the monoclinic space group C121.

  14. Effects of F171 Mutations in the 6′-N-Acetyltransferase Type Ib [AAC(6′)-Ib] Enzyme on Susceptibility to Aminoglycosides

    Chavideh, Ramona; Sholly, Steven; Panaite, Doina; Tolmasky, Marcelo E.

    1999-01-01

    Substitutions at position F171 of 6′-N-acetyltransferase type Ib cause variable loss of aminoglycoside resistance, indicating that this residue plays an important role in the structure and/or function of the enzyme.

  15. Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

    de la Fuente, A.; Martín, J F; Rodríguez-García, A.; Liras, P

    2004-01-01

    The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and ...

  16. Aminoglycoside 6′-N-Acetyltransferase Variants of the Ib Type with Altered Substrate Profile in Clinical Isolates of Enterobacter cloacae and Citrobacter freundii

    Casin, Isabelle; Bordon, Florence; Bertin, Philippe; Coutrot, Anne; Podglajen, Isabelle; Brasseur, Robert; Collatz, Ekkehard

    1998-01-01

    Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Citrobacter freundii CFr564, displayed an aminoglycoside resistance profile evocative of low-level 6′-N acetyltransferase type II [AAC(6′)-II] production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin. Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6′) which acetylated amikacin practically as well as it acetylated gentamicin in vitro. Bot...

  17. Small molecule inhibition of arylamine N-acetyltransferase Type I inhibits proliferation and invasiveness of MDA-MB-231 breast cancer cells

    Tiang, Jacky M. [School of Biomedical Sciences, University of Queensland, St. Lucia, Qld 4072 (Australia); Butcher, Neville J., E-mail: n.butcher@uq.edu.au [School of Biomedical Sciences, University of Queensland, St. Lucia, Qld 4072 (Australia); Minchin, Rodney F. [School of Biomedical Sciences, University of Queensland, St. Lucia, Qld 4072 (Australia)

    2010-02-26

    Arylamine N-acetyltransferase 1 is a phase II metabolizing enzyme that has been associated with certain breast cancer subtypes. While it has been linked to breast cancer risk because of its role in the metabolic activation and detoxification of carcinogens, recent studies have suggested it may be important in cell growth and survival. To address the possible importance of NAT1 in breast cancer, we have used a novel small molecule inhibitor (Rhod-o-hp) of the enzyme to examine growth and invasion of the breast adenocarcinoma line MDA-MB-231. The inhibitor significantly reduced cell growth by increasing the percent of cells in G2/M phase of the cell cycle. Rhod-o-hp also reduced the ability of the MDA-MB-231 cells to grow in soft agar. Using an in vitro invasion assay, the inhibitor significantly reduced the invasiveness of the cells. To test whether this effect was due to inhibition of NAT1, the enzyme was knocked down using a lentivirus-based shRNA approach and invasion potential was significantly reduced. Taken together, the results of this study demonstrate that NAT1 activity may be important in breast cancer growth and metastasis. The study suggests that NAT1 is a novel target for breast cancer treatment.

  18. Small molecule inhibition of arylamine N-acetyltransferase Type I inhibits proliferation and invasiveness of MDA-MB-231 breast cancer cells

    Arylamine N-acetyltransferase 1 is a phase II metabolizing enzyme that has been associated with certain breast cancer subtypes. While it has been linked to breast cancer risk because of its role in the metabolic activation and detoxification of carcinogens, recent studies have suggested it may be important in cell growth and survival. To address the possible importance of NAT1 in breast cancer, we have used a novel small molecule inhibitor (Rhod-o-hp) of the enzyme to examine growth and invasion of the breast adenocarcinoma line MDA-MB-231. The inhibitor significantly reduced cell growth by increasing the percent of cells in G2/M phase of the cell cycle. Rhod-o-hp also reduced the ability of the MDA-MB-231 cells to grow in soft agar. Using an in vitro invasion assay, the inhibitor significantly reduced the invasiveness of the cells. To test whether this effect was due to inhibition of NAT1, the enzyme was knocked down using a lentivirus-based shRNA approach and invasion potential was significantly reduced. Taken together, the results of this study demonstrate that NAT1 activity may be important in breast cancer growth and metastasis. The study suggests that NAT1 is a novel target for breast cancer treatment.

  19. Genetic variants in microsomal epoxide hydrolase and N-acetyltransferase 2 in susceptibility of IBD in the Danish population

    Ernst, Anja; Andersen, Vibeke; Østergaard, Mette;

    induce or sustain an immune response. Changes in detoxification of substances that causes epithelial damage may confer susceptibility to IBD. Hence, polymorphic enzymes involved in the detoxification processes may be risk factors of IBD. Methods. The two biotransformation enzymes microsomal epoxide...... hydrolase and N-acetyltransferase 2 were genotyped using TaqMan based Real-Time PCR in 388 patients with Crohn's disease (CD), 565 patients with ulcerative colitis (UC) and 796 healthy Danish controls. Results. No association was found between low microsomal epoxide hydrolase activity or slow N......-acetyltransferase 2 acetylator status and IBD. An association between high activity of microsomal epoxide hydrolase and disease diagnosis before age 40 in CD with an OR of 2.2(1.1- 4.2) P=0.02) was found. No other phenotypic associations were found for the two enzymes and IBD, regarding age at onset, disease location...

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

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

    2016-07-15

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

  1. Purification, crystallization and preliminary X-ray analysis of the glucosamine-6-phosphate N-acetyltransferase from human liver

    Glucosamine-6-phosphate N-acetyltransferase from human liver was expressed, purified and crystallized. Diffraction data have been collected to 2.6 Å resolution. Glucosamine-6-phosphate N-acetyltransferase from human liver, which catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to the primary amine of d-glucosamine 6-phosphate to form N-acetyl-d-glucosamine 6-phosphate, was expressed in a soluble form from Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity using Ni2+-chelating chromatography followed by size-exclusion chromatography. Crystals of the protein were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.6 Å resolution. The crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 50.08, c = 142.88 Å

  2. Maintenance of Neuronal Laterality in Caenorhabditis elegans Through MYST Histone Acetyltransferase Complex Components LSY-12, LSY-13 and LIN-49

    O'Meara, M. Maggie; Zhang, Feifan; Hobert, Oliver

    2010-01-01

    Left/right asymmetrically expressed genes permit an animal to perform distinct tasks with the right vs. left side of its brain. Once established during development, lateralized gene expression patterns need to be maintained during the life of the animal. We show here that a histone modifying complex, composed of the LSY-12 MYST-type histone acetyltransferase, the ING-family PHD domain protein LSY-13, and PHD/bromodomain protein LIN-49, is required to first initiate and then actively maintain ...

  3. Sequence analysis and complementation studies of the argJ gene encoding ornithine acetyltransferase from Neisseria gonorrhoeae.

    Martin, P R; Mulks, M H

    1992-01-01

    Clinical isolates of Neisseria gonorrhoeae frequently are deficient in arginine biosynthesis. These auxotrophs often have defects in the fifth step of the arginine biosynthetic pathway, the conversion of acetylornithine to ornithine. This reaction is catalyzed by the enzyme ornithine acetyltransferase, which is a product of the argJ gene. We have cloned and sequenced the gonococcal argJ gene and found that it contains an open reading frame of 1,218 nucleotides and encodes a peptide with a ded...

  4. Crystal structure of the histone acetyltransferase domain of the human PCAF transcriptional regulator bound to coenzyme A.

    Clements, A.; Rojas, J R; Trievel, R C; Wang, L.; Berger, S L; Marmorstein, R

    1999-01-01

    The human p300/CBP-associating factor, PCAF, mediates transcriptional activation through its ability to acetylate nucleosomal histone substrates as well as transcriptional activators such as p53. We have determined the 2.3 A crystal structure of the histone acetyltransferase (HAT) domain of PCAF bound to coenzyme A. The structure reveals a central protein core associated with coenzyme A binding and a pronounced cleft that sits over the protein core and is flanked on opposite sides by the N- a...

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

    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-...

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

    Fei Zheng; Lawryn H Kasper; Bedford, David C.; Stephanie Lerach; 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 ...

  7. Cohesin acetyltransferase Esco2 is a cell viability factor and is required for cohesion in pericentric heterochromatin

    Whelan, Gabriela; Kreidl, Emanuel; Wutz, Gordana; Egner, Alexander; Peters, Jan-Michael; Eichele, Gregor

    2012-01-01

    Sister chromatid cohesion, mediated by cohesin and regulated by Sororin, is essential for chromosome segregation. In mammalian cells, cohesion establishment and Sororin recruitment to chromatin‐bound cohesin depends on the acetyltransferases Esco1 and Esco2. Mutations in Esco2 cause Roberts syndrome, a developmental disease in which mitotic chromosomes have a ‘railroad’ track morphology. Here, we show that Esco2 deficiency leads to termination of mouse development at pre‐ and post‐implantatio...

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

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

    2016-01-01

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

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

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

    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...

  10. N-Acetyltransferase 1 (NAT1) Genotype: A Risk Factor for Urinary Bladder Cancer in a Lebanese Population

    Yassine, Ibrahim A.; Loulou Kobeissi; Jabbour, Michel E.; Dhaini, Hassan R

    2012-01-01

    In Lebanon, bladder cancer is the second most incident cancer among men. This study investigates a possible association between N-acetyltransferase 1 (NAT1) genotype, a drug-metabolizing enzyme coding gene, and bladder cancer in Lebanese men. A case-control study (54 cases and 105 hospital-based controls) was conducted in two major hospitals in Beirut. Cases were randomly selected from patients diagnosed in the period of 2002–2008. Controls were conveniently identified and selected from the s...

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

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

    1996-12-01

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

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

    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

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

    Fungjou Lu; Youngshin Chen (National Taiwan Univ., Taipei (Taiwan, Province of China). Dept. of Biochemistry); Tienshang Huang (National Taiwan Univ., Taipei (Taiwan, Province of China). 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.

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

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

    2016-01-01

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

  15. Comparison of Protein Acetyltransferase Action of CRTAase with the Prototypes of HAT

    Prija Ponnan

    2014-01-01

    Full Text Available 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.

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

    Berndsen, Christopher E; Tsubota, Toshiaki; Lindner, Scott E; Lee, Susan; Holton, James M; Kaufman, Paul D; Keck, James L; Denu, John M [UMASS, MED; (UCB); (UW-MED)

    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.

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

    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 β-naphthylamine (β-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 H2O2 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.

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

    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 14C-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 κ-σ, as well as steric properties of the substituted benzene ring. Additional radiometric and spectrophotometric were performed with 2-(3'-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

  19. In Silico Identification and Characterization of N-Terminal Acetyltransferase Genes of Poplar (Populus trichocarpa

    Hang-Yong Zhu

    2014-01-01

    Full Text Available 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. Distinct Localization of Peripheral and Central Types of Choline Acetyltransferase in the Rat Cochlea

    We previously discovered a splice variant of choline acetyltransferase (ChAT) mRNA, and designated the variant protein pChAT because of its preferential expression in peripheral neuronal structures. In this study, we examined the immunohistochemical localization of pChAT in rat cochlea and compared the distribution pattern to those of common ChAT (cChAT) and acetylcholinesterase. Some neuronal cell bodies and fibers in the spiral ganglia showed immunoreactivity for pChAT, predominantly the small spiral ganglion cells, indicating outer hair cell type II neurons. In contrast, cChAT- and acetylcholinesterase-positive structures were localized to fibers and not apparent in ganglion cells. After ablation of the cochlear nuclei, many pChAT-positive cochlear nerve fibers became clearly visible, whereas fibers immunopositive for cChAT and acetylcholine esterase disappeared. These results suggested that pChAT and cChAT are localized in different systems of the rat cochlea; pChAT in the afferent and cChAT in the efferent structures

  1. Insulin stimulates choline acetyltransferase activity in cultured embryonic chicken retina neurons

    The effect of insulin on the appearance of the enzyme choline acetyltransferase in embryonic chicken retina neurons cultured in defined medium was studied. In the presence of a minimal level of insulin (1 ng/ml), ChoAcT activity increased with time in culture. A correspondence between the insulin concentration in the defined medium (1-100 ng/ml) and both the rate of increase and maximum attained level of ChoAcT activity was observed. Maximal ChoAcT activity was 2- to 3-fold greater in cells cultured in the presence of 100 ng of insulin per ml than in cells cultured in the presence of 1 ng of insulin per ml. To elicit maximum ChoAcT activity, insulin at 100 ng/ml was required in the medium for only the first 4 days of the culture period, at which time insulin could be reduced to maintenance levels (10 ng/ml) without affecting ChoAcT activity. Insulin binding assays performed during a 7-day culture period revealed that irrespective of the 125I-insulin concentration in the medium during culture, cell-surface insulin receptors decreased by ≅ 90% between 4 and 7 days in culture. This decrease in insulin binding corresponded to the observed decrease in the sensitivity of ChoAcT activity to insulin. The findings suggest that insulin plays a role in mediating cholinergic differentiation in the embryonic chicken retina

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

    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 (SC); (Toronto); (UV)

    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.

  3. The lysine acetyltransferase activator Brpf1 governs dentate gyrus development through neural stem cells and progenitors.

    Linya You

    2015-03-01

    Full Text Available 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.

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

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

    2015-09-01

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

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

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

    2016-04-01

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

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

    Jeffers, Victoria; Gao, Hongyu; Checkley, Lisa A; Liu, Yunlong; Ferdig, Michael T; Sullivan, William J

    2016-04-01

    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 parasiteToxoplasma 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 forToxoplasmatachyzoite 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 inhibitsToxoplasmatachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibitsPlasmodium falciparumasexual replication with a 50% inhibitory concentration (IC50) similar to that forToxoplasma Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication. PMID:26810649

  7. N-acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species

    Du, Xiaoyi [Fukui Prefectural Univ., Fukui (Japan). Dept. of Bioscience; Takagi, Hiroshi [Nara Inst. of Science and Technology, Ikoma, Nara (Japan). Graduate School of Biological Sciences

    2007-07-15

    N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H{sub 2}O{sub 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{sub 2}O{sub 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{sub 2}O{sub 2}. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains. (orig.)

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

    Jia Li

    2015-12-01

    Full Text Available 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.

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

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

    2014-03-01

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

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

    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. PMID:1616705

  11. Molecular Evolution of Multiple Arylalkylamine N-Acetyltransferase (AANAT in Fish

    Bina Zilberman-Peled

    2011-05-01

    Full Text Available Arylalkylamine N-acetyltransferase (AANAT catalyzes the transfer of an acetyl group from acetyl coenzyme A (AcCoA to arylalkylamines, including indolethylamines and phenylethylamines. Multiple aanats are present in teleost fish as a result of whole genome and gene duplications. Fish aanat1a and aanat2 paralogs display different patterns of tissue expression and encode proteins with different substrate preference: AANAT1a is expressed in the retina, and acetylates both indolethylamines and phenylethylamines; while AANAT2 is expressed in the pineal gland, and preferentially acetylates indolethylamines. The two enzymes are therefore thought to serve different roles. Here, the molecular changes that led to their specialization were studied by investigating the structure-function relationships of AANATs in the gilthead seabream (sb, Sperus aurata. Acetylation activity of reciprocal mutated enzymes pointed to specific residues that contribute to substrate specificity of the enzymes. Inhibition tests followed by complementary analyses of the predicted three-dimensional models of the enzymes, suggested that both phenylethylamines and indolethylamines bind to the catalytic pocket of both enzymes. These results suggest that substrate selectivity of AANAT1a and AANAT2 is determined by the positioning of the substrate within the catalytic pocket, and its accessibility to catalysis. This illustrates the evolutionary process by which enzymes encoded by duplicated genes acquire different activities and play different biological roles.

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

    Kyungha Shin

    2016-01-01

    Full Text Available 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.

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

    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

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

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

    2009-04-01

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

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

    Wei-dong GUO; Jun LIANG; Xiao-e YANG; Yue-en CHAO; Ying FENG

    2009-01-01

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

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

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

    2009-01-01

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

  17. Improvement of L-arginine production by overexpression of a bifunctional ornithine acetyltransferase in Corynebacterium crenatum.

    Dou, Wenfang; Xu, Meijuan; Cai, Dongmei; Zhang, Xiaomei; Rao, Zhiming; Xu, Zhenghong

    2011-10-01

    Ornithine acetyltransferase (EC 2.3.1.35; OATase) gene (argJ) from the L-arginine-producing mutant Corynebacterium crenatum SYPA5-5 was cloned, sequenced, and expressed in Escherichia coli BL21 (DE3). Analysis of the argJ sequence revealed that the argJ coded a polypeptide of 388 amino acids with a calculated molecular weight of 39.7 kDa. In this study, the function of the OATase (argJ) of C. crenatum SYPA5-5 has been identified as a conserved ATML sequence for the autolysis of the protein to α- and β-subunits. When the argJ regions corresponding to the α- and β-subunits were cloned and expressed separately in E. coli BL21, OATase activities were abolished. At the same time, a functional study revealed that OATase from C. crenatum SYPA5-5 was a bifunctional enzyme with the functions of acetylglutamate synthase (EC 2.3.1.1, NAGS) and acetylornithine deacetylase (EC 3.5.1.16, AOase) activities. In order to investigate the effects of the overexpression of the argJ gene on L: -arginine production, the argJ gene was inserted into pJCtac to yield the recombinant shuttle plasmid pJCtac-argJ and then transformed into C. crenatum SYPA5-5. The results showed that the engineered strains could not only express more OATase (90.9%) but also increase the production of L: -arginine significantly (16.8%). PMID:21785983

  18. Acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism

    The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [3H]CoA were found to produce acetyl-[3H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [3H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [3H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate

  19. The Spt-Ada-Gcn5 Acetyltransferase (SAGA complex in Aspergillus nidulans.

    Paraskevi Georgakopoulos

    Full Text Available A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.

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

    Amjad Mahasneh

    2012-01-01

    Full Text Available 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.

  1. Characterization and kinetic mechanism of mono- and bifunctional ornithine acetyltransferases from thermophilic microorganisms.

    Marc, F; Weigel, P; Legrain, C; Almeras, Y; Santrot, M; Glansdorff, N; Sakanyan, V

    2000-08-01

    The argJ gene coding for N2-acetyl-L-ornithine: L-glutamate N-acetyltransferase, the key enzyme involved in the acetyl cycle of L-arginine biosynthesis, has been cloned from thermophilic procaryotes: the archaeon Methanoccocus jannaschii, and the bacteria Thermotoga neapolitana and Bacillus stearothermophilus. Archaeal argJ only complements an Escherichia coli argE mutant (deficient in acetylornithinase, which catalyzes the fifth step in the linear biosynthetic pathway), whereas bacterial genes additionally complement an argA mutant (deficient in N-acetylglutamate synthetase, the first enzyme of the pathway). In keeping with these in vivo data the purified His-tagged ArgJ enzyme of M. jannaschii only catalyzes N2-acetylornithine conversion to ornithine, whereas T. neapolitana and B. stearothermophilus ArgJ also catalyze the conversion of glutamate to N-acetylglutamate using acetylCoA as the acetyl donor. M. jannaschii ArgJ is therefore a monofunctional enzyme, whereas T. neapolitana and B. stearothermophilus encoded ArgJ are bifunctional. Kinetic data demonstrate that in all three thermophilic organisms ArgJ-mediated catalysis follows ping-pong bi-bi kinetic mechanism. Acetylated ArgJ intermediates were detected in semireactions using [14C]acetylCoA or [14C]N2-acetyl-L-glutamate as acetyl donors. In this catalysis L-ornithine acts as an inhibitor; this amino acid therefore appears to be a key regulatory molecule in the acetyl cycle of L-arginine synthesis. Thermophilic ArgJ are synthesized as protein precursors undergoing internal cleavage to generate alpha and beta subunits which appear to assemble to alpha2beta2 heterotetramers in E. coli. The cleavage occurs between alanine and threonine residues within the highly conserved PXM-ATML motif detected in all available ArgJ sequences. PMID:10931207

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

    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.

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

    Studies of [3H]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 [3H]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

  4. N-Acetyltransferase 2 genetic polymorphisms and risk of colorectal cancer

    Tiago Donizetti da Silva; Aledson Vitor Felipe; Jacqueline Miranda de Lima; Celina Tizuko Fujiyama Oshima; Nora Manoukian Forones

    2011-01-01

    AIM: To investigate the possible association between meat intake, cigarette smoking and N-acetyltransferase 2 (NAT2) genetic polymorphisms on colorectal cancer (CRC) risk.METHODS: Patients with CRC were matched for gender and age to healthy controls. Meat intake and cigarette smoking were assessed using a specific frequency questionnaire. DNA was extracted from peripheral blood and the genotypes of the polymorphism were assessed by polymerase chain reaction-restriction fragment length polymorphism. Five NAT2 alleles were studied (WT, M1,M2, M3 and M4) using specific digestion enzymes.RESULTS: A total of 147 patients with colorectal cancer (76 women and 90 men with colon cancer) and 212 controls were studied. The mean age of the two groups was 62 years. More than half the subjects (59.8% in the case group and 51.9% in the control group) were NAT2 slow acetylators. The odds ratio for colorectal cancer was 1.38 (95% CI: 0.90-2.12) in slow acetylators. Although the number of women was small (n = 76 in the case group),the cancer risk was found to be lower in intermediate (W/Mx) acetylators [odds ratio (OR): 0.55, 95% confidence interval (95% CI): 0.29-1.02]. This difference was not observed in men (OR: 0.56, 95% CI: 0.16-2.00). Among NAT2 fast acetylators (W/W or W/Mx), meat consumption more than 3 times a week increased the risk of colorectal cancer (OR: 2.05, 95% CI: 1.01-4.16). In contrast,cigarette smoking increased the risk of CRC among slow acetylators (OR: 1.97, 95% CI: 1.02-3.79).CONCLUSION: The risk of CRC was higher among fast acetylators who reported a higher meat intake. Slow NAT2 acetylation was associated with an increased risk of CRC.

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

    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 β-galactosidase (SA-β-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 inhibits NHEJ

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

    Oike, Takahiro [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Ogiwara, Hideaki [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Torikai, Kohta [Gunma University Heavy Ion Medical Center, Maebashi, Gunma (Japan); Nakano, Takashi [Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi, Gunma (Japan); Yokota, Jun [Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan); Kohno, Takashi, E-mail: tkkohno@ncc.go.jp [Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo (Japan)

    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

  7. N-Acetyltransferase 2 genetic polymorphisms and risk of colorectal cancer

    Tiago Donizetti da Silva

    2011-02-01

    Full Text Available AIM: To investigate the possible association between meat intake, cigarette smoking and N-acetyltransferase 2 (NAT2 genetic polymorphisms on colorectal cancer (CRC risk.METHODS: Patients with CRC were matched for gender and age to healthy controls. Meat intake and cigarette smoking were assessed using a specific frequency questionnaire. DNA was extracted from peripheral blood and the genotypes of the polymorphism were assessed by polymerase chain reaction-restriction fragment length polymorphism. Five NAT2 alleles were studied (WT, M1, M2, M3 and M4 using specific digestion enzymes.RESULTS: A total of 147 patients with colorectal cancer (76 women and 90 men with colon cancer and 212 controls were studied. The mean age of the two groups was 62 years. More than half the subjects (59.8% in the case group and 51.9% in the control group were NAT2 slow acetylators. The odds ratio for colorectal cancer was 1.38 (95% CI: 0.90-2.12 in slow acetylators. Although the number of women was small (n = 76 in the case group, the cancer risk was found to be lower in intermediate (W/Mx acetylators [odds ratio (OR: 0.55, 95% confidence interval (95% CI: 0.29-1.02]. This difference was not observed in men (OR: 0.56, 95% CI: 0.16-2.00. Among NAT2 fast acetylators (W/W or W/Mx, meat consumption more than 3 times a week increased the risk of colorectal cancer (OR: 2.05, 95% CI: 1.01-4.16. In contrast, cigarette smoking increased the risk of CRC among slow acetylators (OR: 1.97, 95% CI: 1.02-3.79.CONCLUSION: The risk of CRC was higher among fast acetylators who reported a higher meat intake. Slow NAT2 acetylation was associated with an increased risk of CRC.

  8. N-Acetyltransferase 2 gene polymorphism in a group of senile dementia patients in Shanghai suburb

    Wei-chao GUO; Guo-fang LIN; Yong-lin ZHA; Ke-jian LOU; Qing-wen MA; Jian-hua SHEN

    2004-01-01

    AIM: To investigate the possible association of hereditary polymorphism of N-acetyltransferase 2 (NAT2) gene with the susceptibility towards senile dementia in farmer population of Shanghai suburb. METHODS: NAT2 gene genotyping was performed at 7 major polymorphic loci (G191A, C282T, T341C, C481T, G590A, A803G, and .G857A) with a polymerase chain reaction-based restriction fragment length polymorphism based procedure in 2 groups of farmer subjects in Shanghai suburb. A group of 51 diagnosed dementia patients [comprising 29 sporadic Alzheimer disease(AD) patients and 22 sporadic vascular dementia (VD) patients] and a group of 112 healthy individuals were in the same area. RESULTS: The homogenous rapid genotypes (R/R, including*4/*4, *13/*13, and *4/*13) was found over-present in both groups of patients, compared with healthy individuals, for all farmer dementia patients, 52.9 %vs 33.0 %, P=0.016, OR (95 % CI): 2.28(1.16-4.48); for AD group only, 51.7 % vs 33.0 %, P=0.063, OR (95 %CI): 2.18 (0.95-4.97); for VD group 54.5 % vs 33.0 %, P=0.055, OR (95 % CI): 2.43 (0.96-2.43). The significant frequency difference of genotype *4/* 7B between farmer dementia patients and healthy individuals, and that of solo-alleles *13, and *7B were observed between the healthy individuals and both groups of dementia patients.CONCLUSION: Our data suggest the involvement of various NAT2 rapid-acetylating genotypes in the individual susceptibility to senile dementia. Variant genotypes of NAT2 might serve as a hereditary risk factor for AD and VD in Chinese population.

  9. The MYST histone acetyltransferases are essential for gametophyte development in Arabidopsis

    Zhou Dao-Xiu

    2008-11-01

    Full Text Available Abstract Background Histone acetyltransferases (HATs play critical roles in the regulation of chromatin structure and gene expression. Arabidopsis genome contains 12 HAT genes, but the biological functions of many of them are still unknown. In this work, we studied the evolutionary relationship and cellular functions of the two Arabidopsis HAT genes homologous to the MYST family members. Results An extensive phylogenetic analysis of 105 MYST proteins revealed that they can be divided into 5 classes, each of which contains a specific combination of protein modules. The two Arabidopsis MYST proteins, HAM1 and HAM2, belong to a "green clade", clearly separated from other families of HATs. Using a reverse genetic approach, we show that HAM1 and HAM2 are a functionally redundant pair of genes, as single Arabidopsis ham1 and ham2 mutants displayed a wild-type phenotype, while no double mutant seedling could be recovered. Genetic analysis and cytological study revealed that ham1ham2 double mutation induced severe defects in the formation of male and female gametophyte, resulting in an arrest of mitotic cell cycle at early stages of gametogenesis. RT-PCR experiments and the analysis of transgenic plants expressing the GUS reporter gene under the HAM1 or the HAM2 promoter showed that both genes displayed an overlapping expression pattern, mainly in growing organs such as shoots and flower buds. Conclusion The work presented here reveals novel properties for MYST HATs in Arabidopsis. In addition to providing an evolutionary relationship of this large protein family, we show the evidence of a link between MYST and gamete formation as previously suggested in mammalian cells. A possible function of the Arabidopsis MYST protein-mediated histone acetylation during cell division is suggested.

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

    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. PMID:19996973

  11. Estrogen intervention in microvascular morphology and choline acetyltransferase expression in rat hippocampal neurons in chronic cerebral ischemia

    Zhenjun Yang; Hongwei Yan; Guomin Zhang; Zhihong Chen; Jingfeng Xue

    2011-01-01

    We observed dynamic changes in microvessels and a protective effect of estrogen on chronic cerebral ischemia ovariectomized rat models established through permanent occlusion of bilateral carotid arteries at 7, 14 and 21 days. The results revealed that estrogen improved microvasculature in the hippocampus of chronic cerebral ischemic rats, upregulated Bcl-2 protein expression, downregulated Bax protein expression, increased choline acetyltransferase expression in hippocampal cholinergic neurons, and suppressed hippocampal neuronal apoptosis. These findings indicate that estrogen can protect hippocampal neurons in rats with chronic cerebral ischemia.

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

    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

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

    He, Yuan [Northwest University, Xi’an 710069 (China); The University of York, York YO10 5DD (United Kingdom); Roth, Christian; Turkenburg, Johan P.; Davies, Gideon J., E-mail: gideon.davies@york.ac.uk [The University of York, York YO10 5DD (United Kingdom); Northwest University, Xi’an 710069 (China)

    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.

  14. A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase family 3 β-N-acetylglucosaminidase.

    Qin, Zhen; Xiao, Yibei; Yang, Xinbin; Mesters, Jeroen R; Yang, Shaoqing; Jiang, Zhengqiang

    2015-01-01

    Glycoside hydrolase (GH) family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 Å resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions--a GH family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH family 3 proteins. PMID:26669854

  15. The chromatin remodeling factor CSB recruits histone acetyltransferase PCAF to rRNA gene promoters in active state for transcription initiation.

    Meili Shen

    Full Text Available The promoters of poised rRNA genes (rDNA are marked by both euchromatic and heterochromatic histone modifications and are associated with two transcription factors, UBF and SL1 that nucleate transcription complex formation. Active rRNA genes contain only euchromatic histone modifications and are loaded with all components of transcriptional initiation complex including RNA polymerase I. Coupled with histone acetylation and RNA polymerase I targeting, poised promoters can be converted to active ones by ATP-dependent chromatin remodeling factor CSB for initiation of rDNA transcription. However, it is not clear how dynamic histone modifications induce the assembly of polymerase I transcription initiation complex to active promoters during such conversion. Here we show that a complex consisting of CSB, RNA polymerase I and histone acetyltransferase PCAF is present at the rDNA promoters in active state. CSB is required for the association of PCAF with rDNA, which induces acetylation of histone H4 and histone H3K9. Overexpression of CSB promotes the association of PCAF with rDNA. Knockdown of PCAF leads to decreased levels of H4ac and H3K9ac at rDNA promoters, prevents the association of RNA polymerase I and inhibits pre-rRNA synthesis. The results demonstrate that CSB recruits PCAF to rDNA, which allows histone acetylation that is required for the assembly of polymerase I transcription initiation complex during the transition from poised to active state of rRNA genes, suggesting that CSB and PCAF play cooperative roles to establish the active state of rRNA genes by histone acetylation.

  16. Three-dimensional collagen I promotes gemcitabine resistance in vitro in pancreatic cancer cells through HMGA2-dependent histone acetyltransferase expression.

    Surabhi Dangi-Garimella

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is associated with a pronounced collagen-rich stromal reaction that has been shown to contribute to chemo-resistance. We have previously shown that PDAC cells are resistant to gemcitabine chemotherapy in the collagen microenvironment because of increased expression of the chromatin remodeling protein high mobility group A2 (HMGA2. We have now found that human PDAC tumors display higher levels of histone H3K9 and H3K27 acetylation in fibrotic regions. We show that relative to cells grown on tissue culture plastic, PDAC cells grown in three-dimensional collagen gels demonstrate increased histone H3K9 and H3K27 acetylation, along with increased expression of p300, PCAF and GCN5 histone acetyltransferases (HATs. Knocking down HMGA2 attenuates the effect of collagen on histone H3K9 and H3K27 acetylation and on collagen-induced p300, PCAF and GCN5 expression. We also show that human PDAC tumors with HMGA2 demonstrate increased histone H3K9 and H3K27 acetylation. Additionally, we show that cells in three-dimensional collagen gels demonstrate increased protection against gemcitabine. Significantly, down-regulation of HMGA2 or p300, PCAF and GCN5 HATs sensitizes the cells to gemcitabine in three-dimensional collagen. Overall, our results increase our understanding of how the collagen microenvironment contributes to chemo-resistance in vitro and identify HATs as potential therapeutic targets against this deadly cancer.

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

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

    2016-01-01

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

  18. Crystallization and preliminary X-ray characterization of arylamine N-acetyltransferase C (BanatC) from Bacillus anthracis

    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 P41212 or P43212, with unit-cell parameters a = b = 53.70, c = 172.40 Å, and diffract to 1.95 Å resolution on a synchrotron source

  19. Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6′-acetyltransferase AAC(6′)-Im

    AAC(6′)-Im is an N-acetyltransferase enzyme responsible for aminoglycoside resistance in E. faecium and E. coli isolates. Crystals of the kanamycin complex of this enzyme have been prepared and preliminary X-ray diffraction experiments have been undertaken. Bacterial resistance to the aminoglycoside antibiotics is primarily the result of enzymatic deactivation of the drugs. The aminoglycoside N-acetyltransferases (AACs) are a large family of bacterial enzymes that are responsible for coenzyme-A-facilitated acetylation of aminoglycosides. The gene encoding one of these enzymes, AAC(6′)-Im, has been cloned and the protein (comprising 178 amino-acid residues) was expressed in Escherichia coli, purified and crystallized as the kanamycin complex. Synchrotron diffraction data to approximately 2.0 Å resolution were collected from a crystal of this complex on beamline BL12-2 at SSRL (Stanford, California, USA). The crystals belonged to the hexagonal space group P65, with approximate unit-cell parameters a = 107.75, c = 37.33 Å, and contained one molecule in the asymmetric unit. Structure determination is under way using molecular replacement

  20. Histone acetyltransferase HAT4 modulates navigation across G2/M and re-entry into G1 in Leishmania donovani.

    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

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

    Rim Khlifi

    2013-01-01

    Full Text Available 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.

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

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

    Coon, S.L.; Bernard, M.; Roseboom, P.H. [National Institutes of Health, Bethesda, MD (United States)] [and others

    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.

  4. Over-expression, purification, and characterization of recombinant human arylamine N-acetyltransferase 1.

    Wang, Haiqing; Vath, Gregory M; Kawamura, Akane; Bates, Caleb A; Sim, Edith; Hanna, Patrick E; Wagner, Carston R

    2005-02-01

    Human arylamine N-acetyltransferase 1 (NAT1) has been overexpressed in E. coli as a mutant dihydrofolic acid reductase (DHFR) fusion protein with a thrombin sensitive linker. An initial DEAE anion-exchange chromatography resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin, and human rNAT1 was purified with a second DEAE column. A total of 8 mg of human rNAT1 from 2 1 of cell culture was purified to homogeneity with this methodology. Arylamine substrate specificities were determined for human rNATI and hamster rNAT2. With both NATs, the second order rate constants (k(cat)/ Kmb) for p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were several thousand-fold higher than those for procainamide (PA), consistent with the expected substrate specificities of the enzymes. However, p-aminosalicylic acid (PAS), previously reported to be a human NAT1 and hamster NAT2 selective substrate, exhibits 20-fold higher specificity for hamster rNAT2 (k(cat)/Kmb 3410 microM(-1) s(-1)) than for human rNAT1 (k(cat)/Kmb 169.4 microM(-1) s(-1)). p-aminobenzoyl-glutamic acid (pABglu) was acetylated 10-fold more efficiently by human rNAT1 than by hamster rNAT2. Inhibition studies of human rNAT1 and hamster rNAT2 revealed that folic acid and methotrexate (MTX) are competitive inhibitors of both the unacetylated and acetylated forms of the enzymes, with K(I) values in 50 - 300 micro range. Dihydrofolic acid (DHF) was a much poorer inhibitor of human rNAT1 than of hamster rNAT2. The combined results demonstrate that human rNAT1 and hamster rNAT2 have similar but distinct kinetic properties with certain substrates, and suggest that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo. PMID:16003948

  5. Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling

    QiFan; LijiaAn; LiwangCui

    2005-01-01

    The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain. The Plasmodiumfalclparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in P. falciparum, PfADA2. Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P. falciparum.

  6. Bioprospecting for Trichothecene 3-O-acetyltransferases in the fungal genus Fusarium yields functional enzymes that vary in their Aaility to modify the mycotoxin deoxynivalenol

    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...

  7. Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® Cotton

    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...

  8. Some properties of acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from rat pineal gland

    N-acetylation of serotonin to N-acetylserotonin in the pineal gland is catalysed by acetyl-CoA:arylamine N-acetyltransferase (SNAT). The present investigation was an attempt to design an assay technique which would permit sensitive evaluation of SNAT in order to evaluate some kinetic properties of the enzyme

  9. Effects of chronic renal failure rat serum on histone acetyltransferase p300 and activation of activating transcription factor 4 of arterial smooth muscle cells cultured in vitro

    张耀全

    2014-01-01

    Objective To investigate the effects of the rat serum with chronic renal failure(CRF)on ubiquitin-proteasome pathway,histone acetyltransferase p300 and activation of activating transcription factor 4(ATF4)of rat arterial vascular smooth muscle cells(VSMCs)cultured in vitro,and explore the possible mechanism.Methods Objective To establish the rat model of

  10. Urinary acetylated metabolites and N-acetyltransferase-2 genotype in human subjects treated with a para-phenylenediamine-containing oxidative hair dye

    Nohynek, G.J.; Skare, J.A.; Meuling, W.J.A.; Hein, D.W.; Bie, A.T.H.J. de; Toutain, H.

    2004-01-01

    In the organism of mammals, important detoxification pathways of arylamines are catalysed by N-acetyltransferase 2 (NAT2). A recent case-control epidemiology study suggested that human NAT2 slow acetylators exposed to oxidative hair dyes may be at greater risk to develop bladder cancer. We therefore