Sample records for o-glcnac transferase regulates

  1. OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A.

    PubMed

    Simon, Dan N; Wriston, Amanda; Fan, Qiong; Shabanowitz, Jeffrey; Florwick, Alyssa; Dharmaraj, Tejas; Peterson, Sherket B; Gruenbaum, Yosef; Carlson, Cathrine R; Grønning-Wang, Line M; Hunt, Donald F; Wilson, Katherine L

    2018-05-17

    The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases ('laminopathies'). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is β- O -linked N -acetylglucosamine- (O -GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified O -GlcNAc transferase (OGT) enzyme showed robust O -GlcNAcylation of recombinant mature lamin A tails (residues 385⁻646), with no detectable modification of lamin B1, lamin C, or 'progerin' (Δ50) tails. Using mass spectrometry, we identified 11 O -GlcNAc sites in a 'sweet spot' unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O -GlcNAc-modified at seven sites. By contrast, O -GlcNAcylation was undetectable on tails bearing deletion Δ50, which causes Hutchinson⁻Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, which does not remove the majority of identified O -GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622⁻625 and 639⁻645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A.

  2. Epigenetic activation of MGAT3 and corresponding bisecting GlcNAc shortens the survival of cancer patients.

    PubMed

    Kohler, Reto S; Anugraham, Merrina; López, Mónica Núñez; Xiao, Christina; Schoetzau, Andreas; Hettich, Timm; Schlotterbeck, Goetz; Fedier, André; Jacob, Francis; Heinzelmann-Schwarz, Viola

    2016-08-09

    Bisecting GlcNAc on N-glycoproteins is described in E-cadherin-, EGF-, Wnt- and integrin- cancer-associated signaling pathways. However, the mechanisms regulating bisecting GlcNAc expression are not clear. Bisecting GlcNAc is attached to N-glycans through beta 1-4 N-acetylglucosaminyl transferase III (MGAT3), which is encoded by two exons flanked by high-density CpG islands. Despite a recently described correlation of MGAT3 and bisecting GlcNAc in ovarian cancer cells, it remains unknown whether DNA methylation is causative for the presence of bisecting GlcNAc. Here, we narrow down the regulatory genomic region and show that reconstitution of MGAT3 expression with 5-Aza coincides with reduced DNA methylation at the MGAT3 transcription start site. The presence of bisecting GlcNAc on released N-glycans was detected by mass spectrometry (LC-ESI-qTOF-MS/MS) in serous ovarian cancer cells upon DNA methyltransferase inhibition. The regulatory impact of DNA methylation on MGAT3 was further evaluated in 18 TCGA cancer types (n = 6118 samples) and the results indicate an improved overall survival in patients with reduced MGAT3 expression, thereby identifying long-term survivors of high-grade serous ovarian cancers (HGSOC). Epigenetic activation of MGAT3 was also confirmed in basal-like breast cancers sharing similar molecular and genetic features with HGSOC. These results provide novel insights into the epigenetic regulation of MGAT3/bisecting GlcNAc and demonstrate the importance of N-glycosylation in cancer progression.

  3. GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system

    PubMed Central

    Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D.; Baba, Hiroko; Ikenaka, Kazuhiro

    2017-01-01

    Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy. PMID:28186137

  4. GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system.

    PubMed

    Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D; Baba, Hiroko; Ikenaka, Kazuhiro

    2017-02-10

    Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P 0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy.

  5. New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro.

    PubMed

    Qi, Jieqiong; Wang, Ruihong; Zeng, Yazhen; Yu, Wengong; Gu, Yuchao

    2017-08-09

    O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3',5,5'-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.

  6. Bisecting GlcNAc restricts conformations of branches in model N-glycans with GlcNAc termini.

    PubMed

    Hanashima, Shinya; Suga, Akitsugu; Yamaguchi, Yoshiki

    2018-02-01

    Bisected N-glycans play significant roles in tumor migration and Alzheimer's disease through modulating the action and localization of their carrier proteins. Such biological functions are often discussed in terms of the conformation of the attached N-glycans with or without bisecting GlcNAc. To obtain insights into the effects of bisecting GlcNAc on glycan conformation, a systematic NMR structural analysis was performed on two pairs of synthetic N-glycans, with and without bisecting GlcNAc. The analysis reveals that terminal GlcNAcs and bisecting GlcNAc cooperate to restrict the conformations of both the α1-3 and α1-6 branches of N-glycans. 1 H and 13 C chemical shift comparisons suggest that bisecting GlcNAc directly modulates local conformation. Unique NOE correlations between core-mannose and the α1-3 branch mannose as well as the 3 J C-H constant of the glycoside linkage indicate that bisecting GlcNAc restricts the conformation of the 1-3 branch. The angles of the glycosidic bonds between core-mannose and α1-6 branch mannose derived from 3 J C-H and 3 J H-H coupling constants show that terminal GlcNAcs restrict the distribution of the ψ angle to 180° and the bisecting GlcNAc increases the distribution of the ω angle +60° in the presence of terminal GlcNAcs. It is feasible that restriction of branch conformations by bisecting GlcNAc has important consequences for protein-glycan interplay and following biological events. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. O-GlcNAc transferase regulates transcriptional activity of human Oct4.

    PubMed

    Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

    2017-10-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Polymorphism in the intron 20 of porcine O-linked N-acetylglucosamine transferase

    USDA-ARS?s Scientific Manuscript database

    Objective: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes the addition of O-GlcNAc and GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling and transcription. Pig OGT is located near the region of chromosome X that affects follicle stimulating hormone...

  9. O-GlcNAc Transferase/Host Cell Factor C1 Complex Regulates Gluconeogenesis by Modulating PGC-1α Stability

    PubMed Central

    Ruan, Hai-Bin; Han, Xuemei; Li, Min-Dian; Singh, Jay Prakash; Qian, Kevin; Azarhoush, Sascha; Zhao, Lin; Bennett, Anton M.; Samuel, Varman T.; Wu, Jing; Yates, John R.; Yang, Xiaoyong

    2012-01-01

    SUMMARY A major cause of hyperglycemia in diabetic patients is inappropriate hepatic gluconeogenesis. PGC-1α is a master regulator of gluconeogenesis, and its activity is controlled by various post-translational modifications. A small portion of glucose metabolizes through the hexosamine biosynthetic pathway, which leads to O-linked β-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins. Using a proteomic approach, we identified a broad variety of proteins associated with O-GlcNAc transferase (OGT), among which host cell factor C1 (HCF-1) is highly abundant. HCF-1 recruits OGT to O-GlcNAcylate PGC-1α and O-GlcNAcylation facilitates the binding of the deubiquitinase BAP1, thus protecting PGC-1α from degradation and promoting gluconeogenesis. Glucose availability modulates gluconeogenesis through the regulation of PGC-1α O-GlcNAcylation and stability by the OGT/HCF1 complex. Hepatic knockdown of OGT and HCF-1 improves glucose homeostasis in diabetic mice. These findings define the OGT/HCF-1 complex as a glucose sensor and key regulator of gluconeogenesis, shedding light on new strategies for treating diabetes. PMID:22883232

  10. MicroRNA-200a/200b Modulate High Glucose-Induced Endothelial Inflammation by Targeting O-linked N-Acetylglucosamine Transferase Expression.

    PubMed

    Lo, Wan-Yu; Yang, Wen-Kai; Peng, Ching-Tien; Pai, Wan-Yu; Wang, Huang-Joe

    2018-01-01

    Background and Aims: Increased O -linked N -acetylglucosamine ( O -GlcNAc) modification of proteins by O -GlcNAc transferase (OGT) is associated with diabetic complications. Furthermore, oxidative stress promotes endothelial inflammation during diabetes. A previous study reported that microRNA-200 (miR-200) family members are sensitive to oxidative stress. In this study, we examined whether miR-200a and miR-200b regulate high-glucose (HG)-induced OGT expression in human aortic endothelial cells (HAECs) and whether miRNA-200a/200b downregulate OGT expression to control HG-induced endothelial inflammation. Methods: HAECs were stimulated with high glucose (25 mM) for 12 and 24 h. Real-time polymerase chain reaction (PCR), western blotting, THP-1 adhesion assay, bioinformatics predication, transfection of miR-200a/200b mimic or inhibitor, luciferase reporter assay, and transfection of siRNA OGT were performed. The aortic endothelium of db/db diabetic mice was evaluated by immunohistochemistry staining. Results: HG upregulated OGT mRNA and protein expression and protein O -GlcNAcylation levels (RL2 antibody) in HAECs, and showed increased intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin gene expression; ICAM-1 expression; and THP-1 adhesion. Bioinformatics analysis revealed homologous sequences between members of the miR-200 family and the 3'-untranslated region (3'-UTR) of OGT mRNA, and real-time PCR analysis confirmed that members of miR-200 family were significantly decreased in HG-stimulated HAECs. This suggests the presence of an impaired feedback restraint on HG-induced endothelial protein O -GlcNAcylation levels because of OGT upregulation. A luciferase reporter assay demonstrated that miR-200a/200b mimics bind to the 3'-UTR of OGT mRNA. Transfection with miR-200a/200b mimics significantly inhibited HG-induced OGT mRNA expression, OGT protein expression; protein O -GlcNAcylation levels; ICAM-1, VCAM-1, and E

  11. Glucose-induced expression of MIP-1 genes requires O-GlcNAc transferase in monocytes

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

    Chikanishi, Toshihiro; ERATO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012; Fujiki, Ryoji

    2010-04-16

    O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1{alpha} and -1{beta}more » genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1{alpha} gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1{alpha} gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.« less

  12. O-GLYCBASE Version 3.0: a revised database of O-glycosylated proteins.

    PubMed Central

    Hansen, J E; Lund, O; Nilsson, J; Rapacki, K; Brunak, S

    1998-01-01

    O-GLYCBASE is a revised database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the literature, and from the sequence databases. Entries include information about species, sequence, glycosylation sites and glycan type and is fully cross-referenced. Compared to version 2.0 the number of entries has increased by 20%. Sequence logos displaying the acceptor specificity patterns for the GalNAc, mannose and GlcNAc transferases are shown. The O-GLYCBASE database is available through the WWW at http://www.cbs.dtu. dk/databases/OGLYCBASE/ PMID:9399880

  13. Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development.

    PubMed Central

    Hartweck, Lynn M; Scott, Cheryl L; Olszewski, Neil E

    2002-01-01

    The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting. PMID:12136030

  14. A Putative O-Linked β-N-Acetylglucosamine Transferase Is Essential for Hormogonium Development and Motility in the Filamentous Cyanobacterium Nostoc punctiforme.

    PubMed

    Khayatan, Behzad; Bains, Divleen K; Cheng, Monica H; Cho, Ye Won; Huynh, Jessica; Kim, Rachelle; Omoruyi, Osagie H; Pantoja, Adriana P; Park, Jun Sang; Peng, Julia K; Splitt, Samantha D; Tian, Mason Y; Risser, Douglas D

    2017-05-01

    Most species of filamentous cyanobacteria are capable of gliding motility, likely via a conserved type IV pilus-like system that may also secrete a motility-associated polysaccharide. In a subset of these organisms, motility is achieved only after the transient differentiation of hormogonia, which are specialized filaments that enter a nongrowth state dedicated to motility. Despite the fundamental importance of hormogonia to the life cycles of many filamentous cyanobacteria, the molecular regulation of hormogonium development is largely undefined. To systematically identify genes essential for hormogonium development and motility in the model heterocyst-forming filamentous cyanobacterium Nostoc punctiforme , a forward genetic screen was employed. The first gene identified using this screen, designated ogtA , encodes a putative O-linked β- N -acetylglucosamine transferase (OGT). The deletion of ogtA abolished motility, while ectopic expression of ogtA induced hormogonium development even under hormogonium-repressing conditions. Transcription of ogtA is rapidly upregulated (1 h) following hormogonium induction, and an OgtA-GFPuv fusion protein localized to the cytoplasm. In developing hormogonia, accumulation of PilA but not HmpD is dependent on ogtA Reverse transcription-quantitative PCR (RT-qPCR) analysis indicated equivalent levels of pilA transcript in the wild-type and Δ ogtA mutant strains, while a reporter construct consisting of the intergenic region in the 5' direction of pilA fused to gfp produced lower levels of fluorescence in the Δ ogtA mutant strain than in the wild type. The production of hormogonium polysaccharide in the Δ ogtA mutant strain is reduced compared to that in the wild type but comparable to that in a pilA deletion strain. Collectively, these results imply that O -GlcNAc protein modification regulates the accumulation of PilA via a posttranscriptional mechanism in developing hormogonia. IMPORTANCE Filamentous cyanobacteria are among

  15. AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7.

    PubMed

    Meyer, Benjamin H; Shams-Eldin, Hosam; Albers, Sonja-Verena

    2017-01-01

    AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D 100 ), IV (F 220 ) and V (F 264 ) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

  16. Too sweet to resist: Control of immune cell function by O-GlcNAcylation.

    PubMed

    de Jesus, Tristan; Shukla, Sudhanshu; Ramakrishnan, Parameswaran

    2018-06-02

    O-linked β-N-acetyl glucosamine modification (O-GlcNAcylation) is a dynamic, reversible posttranslational modification of cytoplasmic and nuclear proteins. O-GlcNAcylation depends on nutrient availability and the hexosamine biosynthetic pathway (HBP), which produces the donor substrate UDP-GlcNAc. O-GlcNAcylation is mediated by a single enzyme, O-GlcNAc transferase (OGT), which adds GlcNAc and another enzyme, O-GlcNAcase (OGA), which removes O-GlcNAc from proteins. O-GlcNAcylation controls vital cellular processes including transcription, translation, the cell cycle, metabolism, and cellular stress. Aberrant O-GlcNAcylation has been implicated in various pathologies including Alzheimer's disease, diabetes, obesity, and cancer. Growing evidences indicate that O-GlcNAcylation plays crucial roles in regulating immunity and inflammatory responses, especially under hyperglycemic conditions. This review will highlight the emerging functions of O-GlcNAcylation in mammalian immunity under physiological and various pathological conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. The O-GlcNAc Transferase Intellectual Disability Mutation L254F Distorts the TPR Helix.

    PubMed

    Gundogdu, Mehmet; Llabrés, Salomé; Gorelik, Andrii; Ferenbach, Andrew T; Zachariae, Ulrich; van Aalten, Daan M F

    2018-05-17

    O-linked β-N-acetyl- D -glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  18. Glutathione transferases, regulators of cellular metabolism and physiology.

    PubMed

    Board, Philip G; Menon, Deepthi

    2013-05-01

    The cytosolic glutathione transferases (GSTs) comprise a super family of proteins that can be categorized into multiple classes with a mixture of highly specific and overlapping functions. The review covers the genetics, structure and function of the human cytosolic GSTs with particular attention to their emerging roles in cellular metabolism. All the catalytically active GSTs contribute to the glutathione conjugation or glutathione dependant-biotransformation of xenobiotics and many catalyze glutathione peroxidase or thiol transferase reactions. GSTs also catalyze glutathione dependent isomerization reactions required for the synthesis of several prostaglandins and steroid hormones and the catabolism of tyrosine. An increasing body of work has implicated several GSTs in the regulation of cell signaling pathways mediated by stress-activated kinases like Jun N-terminal kinase. In addition, some members of the cytosolic GST family have been shown to form ion channels in intracellular membranes and to modulate ryanodine receptor Ca(2+) channels in skeletal and cardiac muscle. In addition to their well established roles in the conjugation and biotransformation of xenobiotics, GSTs have emerged as significant regulators of pathways determining cell proliferation and survival and as regulators of ryanodine receptors that are essential for muscle function. This article is part of a Special Issue entitled Cellular functions of glutathione. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells*

    PubMed Central

    Sacoman, Juliana L.; Dagda, Raul Y.; Burnham-Marusich, Amanda R.; Dagda, Ruben K.; Berninsone, Patricia M.

    2017-01-01

    O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics. PMID:28100784

  20. Stress-induced O-GlcNAcylation: an adaptive process of injured cells.

    PubMed

    Martinez, Marissa R; Dias, Thiago Braido; Natov, Peter S; Zachara, Natasha E

    2017-02-08

    In the 30 years, since the discovery of nucleocytoplasmic glycosylation, O -GlcNAc has been implicated in regulating cellular processes as diverse as protein folding, localization, degradation, activity, post-translational modifications, and interactions. The cell co-ordinates these molecular events, on thousands of cellular proteins, in concert with environmental and physiological cues to fine-tune epigenetics, transcription, translation, signal transduction, cell cycle, and metabolism. The cellular stress response is no exception: diverse forms of injury result in dynamic changes to the O -GlcNAc subproteome that promote survival. In this review, we discuss the biosynthesis of O -GlcNAc, the mechanisms by which O -GlcNAc promotes cytoprotection, and the clinical significance of these data. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  1. Effects of hypo-O-GlcNAcylation on Drosophila development.

    PubMed

    Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

    2018-05-11

    Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc ( O -GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila , null mutants of the Polycomb gene O -GlcNAc transferase ( OGT ; also known as super sex combs ( sxc )) display homeotic phenotypes. To dissect the requirement for O -GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxc K872M , was recessive lethal, whereas a second mutant, the hypomorphic sxc H537A , was homozygous viable. We observed that reduced total protein O -GlcNAcylation in the sxc H537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc H537A and a null allele of Drosophila host cell factor ( dHcf ), encoding an extensively O -GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxc H537A flies lacking a copy of skuld ( skd ), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxc H537A enabled us to identify pleiotropic effects of globally reduced protein O -GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development. © 2018 Mariappa et al.

  2. Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage.

    PubMed

    Goth, Christoffer K; Tuhkanen, Hanna E; Khan, Hamayun; Lackman, Jarkko J; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H; Overall, Christopher M; Clausen, Henrik; Schjoldager, Katrine T; Petäjä-Repo, Ulla E

    2017-03-17

    The β 1 -adrenergic receptor (β 1 AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β 1 AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O -glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O -glycosylates β 1 AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O -glycosylation and proteolytic cleavage assays, a cell line deficient in O -glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β 1 AR. Furthermore, we demonstrate that impaired O -glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O -glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β 1 AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage*

    PubMed Central

    Goth, Christoffer K.; Tuhkanen, Hanna E.; Khan, Hamayun; Lackman, Jarkko J.; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H.; Overall, Christopher M.; Clausen, Henrik; Schjoldager, Katrine T.; Petäjä-Repo, Ulla E.

    2017-01-01

    The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates β1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. PMID:28167537

  4. Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing.

    PubMed

    Byrd, J C; Tarentino, A L; Maley, F; Atkinson, P H; Trimble, R B

    1982-12-25

    Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1

  5. Checkpoint kinase 1-induced phosphorylation of O-linked β-N-acetylglucosamine transferase regulates the intermediate filament network during cytokinesis.

    PubMed

    Li, Zhe; Li, Xueyan; Nai, Shanshan; Geng, Qizhi; Liao, Ji; Xu, Xingzhi; Li, Jing

    2017-12-01

    Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1's pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified O- linked β- N -acetylglucosamine ( O -GlcNAc)-transferase (OGT) as one of Chk1's substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis. Phospho-specific antibodies of OGT-pSer-20 exhibited specific signals at the midbody of the cell, consistent with midbody localization of OGT as reported previously. Moreover, phospho-deficient OGT (S20A) cells attenuated cellular O -GlcNAcylation levels and also reduced phosphorylation of Ser-71 in the cytoskeletal protein vimentin, a modification critical for severing vimentin filament during cytokinesis. Consequently, elongated vimentin bridges were observed in cells depleted of OGT via an si OGT- based approach. Lastly, expression of plasmids resistant to si OGT efficiently rescued the vimentin bridge phenotype, but the OGT-S20A rescue plasmids did not. Our results suggest a Chk1-OGT-vimentin pathway that regulates the intermediate filament network during cytokinesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. O-linked N-acetylglucosamine transferase enhances secretory clusterin expression via liver X receptors and sterol response element binding protein regulation in cervical cancer.

    PubMed

    Kim, Min Jun; Choi, Mee Young; Lee, Dong Hoon; Roh, Gu Seob; Kim, Hyun Joon; Kang, Sang Soo; Cho, Gyeong Jae; Kim, Yoon Sook; Choi, Wan Sung

    2018-01-12

    O-linked N-acetylglucosamine transferase (OGT) expression is increased in various cancer types, indicating the potential importance of O-GlcNAcylation in tumorigenesis. Secretory clusterin (sCLU) is involved in cancer cell proliferation and drug resistance, and recently, liver X receptors (LXRs) and sterol response element binding protein-1 (SREBP-1) were reported to regulate sCLU transcription. Here, we found that sCLU is significantly increased in cervical cancer cell lines, which have higher expression levels of O-GlcNAc and OGT than keratinocytes. OGT knockdown decreased expression of LXRs, SREBP-1 and sCLU through hypo-O-GlcNAcylation of LXRs. Additionally, treatment with Thiamet G, O-GlcNAcase OGA inhibitor, increased expression of O-GlcNAcylation and sCLU, and high glucose increased levels of LXRs, SREBP-1 and sCLU in HeLa cells. Moreover, OGT knockdown induced G 0 /G 1 phase cell cycle arrest and late apoptosis in cisplatin-treated HeLa cells, and decreased viability compared to OGT intact HeLa cells. Taken together, these findings suggest that OGT, O-GlcNAcylated LXRs, and SREBP-1 increase sCLU expression in cervical cancer cells, which contributes to drug resistance.

  7. O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling.

    PubMed

    Shi, Jianhua; Gu, Jin-hua; Dai, Chun-ling; Gu, Jianlan; Jin, Xiaoxia; Sun, Jianming; Iqbal, Khalid; Liu, Fei; Gong, Cheng-Xin

    2015-09-28

    Apoptosis plays an important role in neural development and neurological disorders. In this study, we found that O-GlcNAcylation, a unique protein posttranslational modification with O-linked β-N-acetylglucosamine (GlcNAc), promoted apoptosis through attenuating phosphorylation/activation of AKT and Bad. By using co-immunoprecipitation and mutagenesis techniques, we identified O-GlcNAc modification at both Thr308 and Ser473 of AKT. O-GlcNAcylation-induced apoptosis was attenuated by over-expression of AKT. We also found a dynamic elevation of protein O-GlcNAcylation during the first four hours of cerebral ischemia, followed by continuous decline after middle cerebral artery occlusion (MCAO) in the mouse brain. The elevation of O-GlcNAcylation coincided with activation of cell apoptosis. Finally, we found a negative correlation between AKT phosphorylation and O-GlcNAcylation in ischemic brain tissue. These results indicate that cerebral ischemia induces a rapid increase of O-GlcNAcylation that promotes apoptosis through down-regulation of AKT activity. These findings provide a novel mechanism through which O-GlcNAcylation regulates ischemia-induced neuronal apoptosis through AKT signaling.

  8. GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

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

    Yoo, Jiho; Mashalidis, Ellene H.; Kuk, Alvin C. Y.

    N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structuralmore » information. Here we present crystal structures of human GPT in complex with tunicamycin. In conclusion, structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.« less

  9. O-GlcNAc reports ambient temperature and confers heat resistance on ectotherm development.

    PubMed

    Radermacher, Pablo T; Myachina, Faina; Bosshardt, Fritz; Pandey, Rahul; Mariappa, Daniel; Müller, H-Arno J; Lehner, Christian F

    2014-04-15

    Effects of temperature on biological processes are complex. Diffusion is less affected than the diverse enzymatic reactions that have distinct individual temperature profiles. Hence thermal fluctuations pose a formidable challenge to ectothermic organisms in which body temperature is largely dictated by the ambient temperature. How cells in ectotherms cope with the myriad disruptive effects of temperature variation is poorly understood at the molecular level. Here we show that nucleocytoplasmic posttranslational modification of proteins with O-linked GlcNAc (O-GlcNAc) is closely correlated with ambient temperature during development of distantly related ectotherms ranging from the insect Drosophila melanogaster to the nematode Caenorhabditis elegans to the fish Danio rerio. Regulation seems to occur at the level of activity of the only two enzymes, O-GlcNAc transferase and O-GlcNAcase, that add and remove, respectively, this posttranslational modification in nucleus and cytoplasm. With genetic approaches in D. melanogaster and C. elegans, we demonstrate the importance of high levels of this posttranslational modification for successful development at elevated temperatures. Because many cytoplasmic and nuclear proteins in diverse pathways are O-GlcNAc targets, temperature-dependent regulation of this modification might contribute to an efficient coordinate adjustment of cellular processes in response to thermal change.

  10. O-GlcNAc Transferase Is Essential for Sensory Neuron Survival and Maintenance

    PubMed Central

    Su, Cathy

    2017-01-01

    O-GlcNAc transferase (OGT) regulates a wide range of cellular processes through the addition of the O-GlcNAc sugar moiety to thousands of protein substrates. Because nutrient availability affects the activity of OGT, its role has been broadly studied in metabolic tissues. OGT is enriched in the nervous system, but little is known about its importance in basic neuronal processes in vivo. Here, we show that OGT is essential for sensory neuron survival and maintenance in mice. Sensory neuron-specific knock-out of OGT results in behavioral hyposensitivity to thermal and mechanical stimuli accompanied by decreased epidermal innervation and cell-body loss in the dorsal root ganglia. These effects are observed early in postnatal development and progress as animals age. Cultured sensory neurons lacking OGT also exhibit decreased axonal outgrowth. The effects on neuronal health in vivo are not solely due to disruption of developmental processes, because inducing OGT knock-out in the sensory neurons of adult mice results in a similar decrease in nerve fiber endings and cell bodies. Significant nerve-ending loss occurs before a decrease in cell bodies; this phenotype is indicative of axonal dieback that progresses to neuronal death. Our findings demonstrate that OGT is important in regulating axonal maintenance in the periphery and the overall health and survival of sensory neurons. SIGNIFICANCE STATEMENT We show the importance of O-GlcNAc transferase (OGT) for sensory neuron health and survival in vivo. This study is the first to find that loss of OGT results in neuronal cell death. Moreover, it suggests that aberrant O-GlcNAc signaling can contribute to the development of neuropathy. The sensory neurons lie outside of the blood–brain barrier and therefore, compared to central neurons, may have a greater need for mechanisms of metabolic sensing and compensation. Peripheral sensory neurons in particular are subject to degeneration in diabetes. Our findings provide a

  11. O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat

    PubMed Central

    Ruan, Hai-Bin; Dietrich, Marcelo O.; Liu, Zhong-Wu; Zimmer, Marcelo R.; Li, Min-Dian; Singh, Jay Prakash; Zhang, Kaisi; Yin, Ruonan; Wu, Jing; Horvath, Tamas L.; Yang, Xiaoyong

    2014-01-01

    SUMMARY Induction of beige cells causes the browning of white fat and improves energy metabolism. However, the central mechanism that controls adipose tissue browning and its physiological relevance are largely unknown. Here we demonstrate that fasting and chemical-genetic activation of orexigenic AgRP neurons in the hypothalamus suppress the browning of white fat. O-linked β-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates fundamental cellular processes. The levels of O-GlcNAc transferase (OGT) and O-GlcNAc modification are enriched in AgRP neurons and are elevated by fasting. Genetic ablation of OGT in AgRP neurons inhibits neuronal excitability through the voltage-dependent potassium channel, promotes white adipose tissue browning, and protects mice against diet-induced obesity and insulin resistance. These data reveal adipose tissue browning as a highly dynamic physiological process under central control, in which O-GlcNAc signaling in AgRP neurons is essential for suppressing thermogenesis to conserve energy in response to fasting. PMID:25303527

  12. Effect of bisecting GlcNAc and core fucosylation on conformational properties of biantennary complex-type N-glycans in solution.

    PubMed

    Nishima, Wataru; Miyashita, Naoyuki; Yamaguchi, Yoshiki; Sugita, Yuji; Re, Suyong

    2012-07-26

    The introduction of bisecting GlcNAc and core fucosylation in N-glycans is essential for fine functional regulation of glycoproteins. In this paper, the effect of these modifications on the conformational properties of N-glycans is examined at the atomic level by performing replica-exchange molecular dynamics (REMD) simulations. We simulate four biantennary complex-type N-glycans, namely, unmodified, two single-substituted with either bisecting GlcNAc or core fucose, and disubstituted forms. By using REMD as an enhanced sampling technique, five distinct conformers in solution, each of which is characterized by its local orientation of the Manα1-6Man glycosidic linkage, are observed for all four N-glycans. The chemical modifications significantly change their conformational equilibria. The number of major conformers is reduced from five to two and from five to four upon the introduction of bisecting GlcNAc and core fucosylation, respectively. The population change is attributed to specific inter-residue hydrogen bonds, including water-mediated ones. The experimental NMR data, including nuclear Overhauser enhancement and scalar J-coupling constants, are well reproduced taking the multiple conformers into account. Our structural model supports the concept of "conformer selection", which emphasizes the conformational flexibility of N-glycans in protein-glycan interactions.

  13. Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

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

    Dehennaut, Vanessa; EA 4020, Laboratoire de Regulation des Signaux de Division, USTL, IFR147, Villeneuve d'Ascq; Hanoulle, Xavier

    2008-05-02

    In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads tomore » a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.« less

  14. MOF Acetyl Transferase Regulates Transcription and Respiration in Mitochondria.

    PubMed

    Chatterjee, Aindrila; Seyfferth, Janine; Lucci, Jacopo; Gilsbach, Ralf; Preissl, Sebastian; Böttinger, Lena; Mårtensson, Christoph U; Panhale, Amol; Stehle, Thomas; Kretz, Oliver; Sahyoun, Abdullah H; Avilov, Sergiy; Eimer, Stefan; Hein, Lutz; Pfanner, Nikolaus; Becker, Thomas; Akhtar, Asifa

    2016-10-20

    A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc.

    PubMed

    Chandrasekaran, E V; Jain, R K; Larsen, R D; Wlasichuk, K; Matta, K L

    1996-07-09

    The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in

  16. O-Linked N-Acetylglucosamine Cycling Regulates Mitotic Spindle Organization*

    PubMed Central

    Tan, Ee Phie; Caro, Sarah; Potnis, Anish; Lanza, Christopher; Slawson, Chad

    2013-01-01

    Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events. PMID:23946484

  17. O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

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

    Sakaidani, Yuta; Ichiyanagi, Naoki; Saito, Chika

    2012-03-02

    Highlights: Black-Right-Pointing-Pointer We characterized A130022J15Rik (Eogt1)-a mouse gene homologous to Drosophila Eogt. Black-Right-Pointing-Pointer Eogt1 encodes EGF domain O-GlcNAc transferase. Black-Right-Pointing-Pointer Expression of Eogt1 in Drosophila rescued the cell-adhesion defect in the Eogt mutant. Black-Right-Pointing-Pointer O-GlcNAcylation reaction in the secretory pathway is conserved through evolution. -- Abstract: O-linked-{beta}-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shownmore » whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.« less

  18. O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

    PubMed

    Fan, Qiong; Moen, Anders; Anonsen, Jan Haug; Bindesbøll, Christian; Sæther, Thomas; Carlson, Cathrine Rein; Grønning-Wang, Line M

    2018-05-05

    The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20 LWKPGAQDASSQAQGGSSCILRE 42 . However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. The EGF Repeat-Specific O-GlcNAc-Transferase Eogt Interacts with Notch Signaling and Pyrimidine Metabolism Pathways in Drosophila

    PubMed Central

    Müller, Reto; Jenny, Andreas; Stanley, Pamela

    2013-01-01

    The O-GlcNAc transferase Eogt modifies EGF repeats in proteins that transit the secretory pathway, including Dumpy and Notch. In this paper, we show that the Notch ligands Delta and Serrate are also substrates of Eogt, that mutation of a putative UDP-GlcNAc binding DXD motif greatly reduces enzyme activity, and that Eogt and the cytoplasmic O-GlcNAc transferase Ogt have distinct substrates in Drosophila larvae. Loss of Eogt is larval lethal and disrupts Dumpy functions, but does not obviously perturb Notch signaling. To identify novel genetic interactions with eogt, we investigated dominant modification of wing blister formation caused by knock-down of eogt. Unexpectedly, heterozygosity for several members of the canonical Notch signaling pathway suppressed wing blister formation. And importantly, extensive genetic interactions with mutants in pyrimidine metabolism were identified. Removal of pyrimidine synthesis alleles suppressed wing blister formation, while removal of uracil catabolism alleles was synthetic lethal with eogt knock-down. Therefore, Eogt may regulate protein functions by O-GlcNAc modification of their EGF repeats, and cellular metabolism by affecting pyrimidine synthesis and catabolism. We propose that eogt knock-down in the wing leads to metabolic and signaling perturbations that increase cytosolic uracil levels, thereby causing wing blister formation. PMID:23671640

  20. O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway.

    PubMed

    Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George; Zhang, Lianwen

    2016-03-18

    O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Regulation of Endothelial Permeability by Glutathione S-Transferase Pi Against Actin Polymerization.

    PubMed

    Yang, Yang; Yin, Fangyuan; Hang, Qiyun; Dong, Xiaoliang; Chen, Jiao; Li, Ling; Cao, Peng; Yin, Zhimin; Luo, Lan

    2018-01-01

    Inflammation-induced injury of the endothelial barrier occurs in several pathological conditions, including atherosclerosis, ischemia, and sepsis. Endothelial cytoskeleton rearrangement is an important pathological mechanism by which inflammatory stimulation triggers an increase of vascular endothelial permeability. However, the mechanism maintaining endothelial cell barrier function against inflammatory stress is not fully understood. Glutathione S-transferase pi (GSTpi) exists in various types of cells and protects them against different stresses. In our previous study, GSTpi was found to act as a negative regulator of inflammatory responses. We used a Transwell permeability assay to test the influence of GSTpi and its transferase activity on the increase of endothelial permeability induced by tumor necrosis factor alpha (TNF-α). TNF-α-induced actin remodeling and the influence of GSTpi were observed by using laser confocal microscopy. Western blotting was used to test the influence of GSTpi on TNF-α-activated p38 mitogen-activated protein kinase (MAPK)/MK2/heat shock protein 27 (HSP27). GSTpi reduced TNF-α-induced stress fiber formation and endothelial permeability increase by restraining actin cytoskeleton rearrangement, and this reduction was unrelated to its transferase activity. We found that GSTpi inhibited p38MAPK phosphorylation by directly binding p38 and influenced downstream substrate HSP27-induced actin remodeling. GSTpi inhibited TNF-α-induced actin remodeling, stress fiber formation and endothelial permeability increase by inhibiting the p38MAPK/HSP27 signaling pathway. © 2018 The Author(s). Published by S. Karger AG, Basel.

  2. Insulin/IGF-I Signaling Pathways Enhances Tumor Cell Invasion through Bisecting GlcNAc N-glycans Modulation. An Interplay with E-Cadherin

    PubMed Central

    Dias, Ana M.; Oliveira, Patrícia; Cabral, Joana; Seruca, Raquel; Oliveira, Carla; Morgado-Díaz, José Andrés; Reis, Celso A.; Pinho, Salomé S.

    2013-01-01

    Changes in glycosylation are considered a hallmark of cancer, and one of the key targets of glycosylation modifications is E-cadherin. We and others have previously demonstrated that E-cadherin has a role in the regulation of bisecting GlcNAc N-glycans expression, remaining to be determined the E-cadherin-dependent signaling pathway involved in this N-glycans expression regulation. In this study, we analysed the impact of E-cadherin expression in the activation profile of receptor tyrosine kinases such as insulin receptor (IR) and IGF-I receptor (IGF-IR). We demonstrated that exogenous E-cadherin expression inhibits IR, IGF-IR and ERK 1/2 phosphorylation. Stimulation with insulin and IGF-I in MDA-MD-435 cancer cells overexpressing E-cadherin induces a decrease of bisecting GlcNAc N-glycans that was accompanied with alterations on E-cadherin cellular localization. Concomitantly, IR/IGF-IR signaling activation induced a mesenchymal-like phenotype of cancer cells together with an increased tumor cell invasion capability. Altogether, these results demonstrate an interplay between E-cadherin and IR/IGF-IR signaling as major networking players in the regulation of bisecting N-glycans expression, with important effects in the modulation of epithelial characteristics and tumor cell invasion. Here we provide new insights into the role that Insulin/IGF-I signaling play during cancer progression through glycosylation modifications. PMID:24282611

  3. O-GlcNAc transferase inhibitors: current tools and future challenges.

    PubMed

    Trapannone, Riccardo; Rafie, Karim; van Aalten, Daan M F

    2016-02-01

    The O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of nucleocytoplasmic target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that add and remove sugar moieties respectively. OGT knockout is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc and the dissection of catalytic compared with non-catalytic roles of OGT. Therefore, selective and potent chemical tools are necessary to inhibit OGT activity in the context of biological systems. The present review focuses on the available OGT inhibitors and summarizes advantages, limitations and future challenges. © 2016 Authors; published by Portland Press Limited.

  4. The Biochemistry of O-GlcNAc Transferase: Which Functions Make It Essential in Mammalian Cells?

    PubMed

    Levine, Zebulon G; Walker, Suzanne

    2016-06-02

    O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding β-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells.

  5. A robust protocol for directed aryl sulfotransferase evolution toward the carbohydrate building block GlcNAc.

    PubMed

    Islam, Shohana; Mate, Diana M; Martínez, Ronny; Jakob, Felix; Schwaneberg, Ulrich

    2018-05-01

    Bacterial aryl sulfotransferases (AST) utilize p-nitrophenylsulfate (pNPS) as a phenolic donor to sulfurylate typically a phenolic acceptor. Interest in aryl sulfotransferases is growing because of their broad variety of acceptors and cost-effective sulfuryl-donors. For instance, aryl sulfotransferase A (ASTA) from Desulfitobacterium hafniense was recently reported to sulfurylate d-glucose. In this study, a directed evolution protocol was developed and validated for aryl sulfotransferase B (ASTB). Thereby the well-known pNPS quantification system was advanced to operate efficiently as a continuous screening system in 96-well MTP format with a true coefficient of variation of 14.3%. A random mutagenesis library (SeSaM library) of ASTB was screened (1,760 clones) to improve sulfurylation of the carbohydrate building block N-acetylglucosamine (GlcNAc). The beneficial variant ASTB-V1 (Val579Asp) showed an up to 3.4-fold increased specific activity toward GlcNAc when compared to ASTB-WT. HPLC- and MS-analysis confirmed ASTB-V1's increased GlcNAc monosulfurylation (2.4-fold increased product formation) representing the validation of the first successful directed evolution round of an AST for a saccharide substrate. © 2017 Wiley Periodicals, Inc.

  6. Cysteine S-linked N-acetylglucosamine (S-GlcNAcylation), A New Post-translational Modification in Mammals.

    PubMed

    Maynard, Jason C; Burlingame, Alma L; Medzihradszky, Katalin F

    2016-11-01

    Intracellular GlcNAcylation of Ser and Thr residues is a well-known and widely investigated post-translational modification. This post-translational modification has been shown to play a significant role in cell signaling and in many regulatory processes within cells. O-GlcNAc transferase is the enzyme responsible for glycosylating cytosolic and nuclear proteins with a single GlcNAc residue on Ser and Thr side-chains. Here we report that the same enzyme may also be responsible for S-GlcNAcylation, i.e. for linking the GlcNAc unit to the peptide by modifying a cysteine side-chain. We also report that O-GlcNAcase, the enzyme responsible for removal of O-GlcNAcylation does not appear to remove the S-linked sugar. Such Cys modifications have been detected and identified in mouse and rat samples. This work has established the occurrence of 14 modification sites assigned to 11 proteins unambiguously. We have also identified S-GlcNAcylation from human Host Cell Factor 1 isolated from HEK-cells. Although these site assignments are primarily based on electron-transfer dissociation mass spectra, we also report that S-linked GlcNAc is more stable under collisional activation than O-linked GlcNAc derivatives. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Glucokinase expression is regulated by glucose through O-GlcNAc glycosylation.

    PubMed

    Baldini, Steffi F; Steenackers, Agata; Olivier-Van Stichelen, Stéphanie; Mir, Anne-Marie; Mortuaire, Marlène; Lefebvre, Tony; Guinez, Céline

    2016-09-16

    Blood glucose fluctuates with the fasting-feeding cycle. One of the liver's functions is to maintain blood glucose concentrations within a physiological range. Glucokinase (GCK) or hexokinase IV, is the main enzyme that regulates the flux and the use of glucose in the liver leading to a compensation of hyperglycemia. In hepatocytes, GCK catalyzes the phosphorylation of glucose into glucose-6-phosphate. This critical enzymatic reaction is determinant for the metabolism of glucose in the liver which includes glycogen synthesis, glycolysis, lipogenesis and gluconeogenesis. In liver, simultaneous increase of glucose and insulin enhances GCK activity and gene expression, changes its subcellular location and interaction with regulatory proteins. The post-translational O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) acts as a glucose-sensitive modification and is believed to take part in hepatic glucose sensing by modifying key regulatory proteins. Therefore, we aimed to determine whether GCK is modified by O-GlcNAcylation in the liver of mice and investigated the role that this modification plays in regulating GCK protein expression. We demonstrated that endogenous GCK expression correlated with O-GlcNAc levels in the pathophysiological model ob/ob mice. More specifically, in response to the pharmacological inhibition of O-GlcNAcase (OGA) contents of GCK increased. Using the GlcNAc specific lectin succinylated-WGA and click chemistry labeling approaches, we demonstrated that GCK is modified by O-GlcNAcylation. Further, we demonstrated that siRNA-mediated Ogt knock-down not only decreases O-GlcNAc content but also GCK protein level. Altogether, our in vivo and in vitro results demonstrate that GCK expression is regulated by nutrient-sensing O-GlcNAc cycling in liver. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

    PubMed

    Li, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng George

    2016-11-18

    O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac 3 4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac 3 4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

  9. Genetic studies on the ghrelin, growth hormone secretagogue receptor (GHSR) and ghrelin O-acyl transferase (GOAT) genes.

    PubMed

    Liu, Boyang; Garcia, Edwin A; Korbonits, Márta

    2011-11-01

    Ghrelin is a 28 amino acid peptide hormone that is produced both centrally and peripherally. Regulated by the ghrelin O-acyl transferase enzyme, ghrelin exerts its action through the growth hormone secretagogue receptor, and is implicated in a diverse range of physiological processes. These implications have placed the ghrelin signaling pathway at the center of a large number of candidate gene and genome-wide studies which aim to identify the genetic basis of human heterogeneity. In this review we summarize the available data on the genetic variability of ghrelin, its receptor and its regulatory enzyme, and their association with obesity, stature, type 2 diabetes, cardiovascular disease, eating disorders, and reward seeking behavior. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Candida albicans Adheres to Chitin by Recognizing N-acetylglucosamine (GlcNAc).

    PubMed

    Ishijima, Sanae A; Yamada, Tsuyoshi; Maruyama, Naho; Abe, Shigeru

    2017-01-01

    The binding of Candida albicans cells to chitin was examined in a cell-binding assay. Microscopic observations indicated that both living and heat-killed Candida cells bound to chitin-coated substrates. C. albicans preferentially bound to chitin-coated plastic plates over chitosan-coated and uncoated plates. We prepared 125 I-labeled Candida cells for quantitative analysis of their binding to chitin. Heat-killed 125 I-labeled Candida cells bound to chitin-coated plates in a time-dependent manner until 1.5 hours after start of incubation at 4℃. The binding of 125 I-labeled Candida cells to chitin-coated plates was inhibited by adding unlabeled living or unlabeled heat-killed Candida cells. The binding of Candida to chitin was also reduced by addition of 25 mg/ml chitin or chitosan up to 10%. N-acetylglucosamine (GlcNAc), which is a constituent of chitin, inhibited binding of Candida to chitin in a dose-dependent manner between 12.5 and 200 mM. Glucosamine, which is a constituent of chitosan, showed no such inhibitory effect. These findings suggest that the binding of Candida to chitin may be mediated by recognition of GlcNAc.

  11. O-GlcNAc modification is essential for the regulation of autophagy in Drosophila melanogaster.

    PubMed

    Park, Sujin; Lee, Yangsin; Pak, Jin Won; Kim, Hanbyeol; Choi, Hyeonjin; Kim, Jae-woo; Roth, Jürgen; Cho, Jin Won

    2015-08-01

    O-GlcNAcylation is a dynamic post-translational modification that takes place on ser/thr residues of nucleocytoplasmic proteins. O-GlcNAcylation regulates almost all cellular events as a nutrient sensor, a transcriptional and translational regulator, and a disease-related factor. Although the role of O-GlcNAcylation in insulin signaling and metabolism are well established, the relationship between O-GlcNAcylation and autophagy is largely unknown. Here, we manipulated O-GlcNAcylation in Drosophila and found that it regulates autophagy through Akt/dFOXO signaling. We demonstrate that O-GlcNAcylation and the levels of O-GlcNAc transferase (OGT) are increased during starvation. Furthermore, Atg proteins and autolysosomes are increased in OGT-reduced flies without fasting. Atg proteins and autophagosomes are reduced in OGT-overexpressing flies. Our results suggest that not only autophagy gene expression but also autophagic structures are regulated by OGT through Akt and dFOXO. These data imply that O-GlcNAcylation is important in modulating autophagy as well as insulin signaling in Drosophila.

  12. Functional Analysis of SPINDLY in Gibberellin Signaling in Arabidopsis1[C][W][OA

    PubMed Central

    Silverstone, Aron L.; Tseng, Tong-Seung; Swain, Stephen M.; Dill, Alyssa; Jeong, Sun Yong; Olszewski, Neil E.; Sun, Tai-ping

    2007-01-01

    The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-Δ17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-Δ17 phenotype but does not reduce rga-Δ17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification. PMID:17142481

  13. Glutathione -S-Transferase μ 1 Regulates Vascular Smooth Muscle Cell Proliferation, Migration, and Oxidative Stress

    PubMed Central

    Yang, Yanqiang; Parsons, Kelly K.; Chi, Liqun; Malakauskas, Sandra M.; Le, Thu H.

    2009-01-01

    Glutathione S-transferase μ-1, GSTM1, belongs to a superfamily of glutathione-S-transferases that metabolize a broad range of reactive oxygen species (ROS) and xenobiotics. Across species, genetic variants that result in decreased expression of the Gstm1 gene are associated with increased susceptibility for vascular diseases, including atherosclerosis in humans. We previously identified Gstm1 as a positional candidate in our gene mapping study for susceptibility to renal vascular injury characterized by medial hypertrophy and hyperplasia of the renal vessels. To determine the role of Gstm1 in vascular smooth muscle cells (VSMCs), we isolated VSMCs from mouse aortas. We demonstrate that VSMCs from the susceptible C57BL/6 mice have reduced expression of Gstm1 mRNA and its protein product compared to that of the resistant 129 mice. After serum stimulation, C57BL/6 VSMCs proliferate and migrate at a much faster rate than 129 VSMCs. Furthermore, C57BL/6 VSMCs have higher levels of ROS, and exhibit exaggerated p38 MAPK phosphorylation after exposure to H2O2. To establish causality, we show that knockdown of Gstm1 by siRNA results in increased proliferation of VSMCs in a dose dependent manner, as well as in increased ROS levels and VSM cell migration. Moreover, Gstm1 siRNA causes increased p38 MAPK phosphorylation, and attenuates the anti-proliferative effect of TEMPOL. Our data suggest that Gstm1 is a novel regulator of VSMC proliferation and migration through its role in handling ROS. Genetic variants that cause a decremental change in expression of Gstm1 may permit an environment of exaggerated oxidative stress, leading to susceptibility to vascular remodeling and atherosclerosis. PMID:19822795

  14. Regulation of the Hippo-YAP Pathway by Glucose Sensor O-GlcNAcylation.

    PubMed

    Peng, Changmin; Zhu, Yue; Zhang, Wanjun; Liao, Qinchao; Chen, Yali; Zhao, Xinyuan; Guo, Qiang; Shen, Pan; Zhen, Bei; Qian, Xiaohong; Yang, Dong; Zhang, Jin-San; Xiao, Dongguang; Qin, Weijie; Pei, Huadong

    2017-11-02

    The Hippo pathway is crucial in organ size control and tissue homeostasis, with deregulation leading to cancer. An extracellular nutrition signal, such as glucose, regulates the Hippo pathway activation. However, the mechanisms are still not clear. Here, we found that the Hippo pathway is directly regulated by the hexosamine biosynthesis pathway (HBP) in response to metabolic nutrients. Mechanistically, the core component of Hippo pathway (YAP) is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 109. YAP O-GlcNAcylation disrupts its interaction with upstream kinase LATS1, prevents its phosphorylation, and activates its transcriptional activity. And this activation is not dependent on AMPK. We also identified OGT as a YAP-regulated gene that forms a feedback loop. Finally, we confirmed that glucose-induced YAP O-GlcNAcylation and activation promoted tumorigenesis. Together, our data establish a molecular mechanism and functional significance of the HBP in directly linking extracellular glucose signal to the Hippo-YAP pathway and tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Structure of human O-GlcNAc transferase and its complex with a peptide substrate

    PubMed Central

    Lazarus, Michael B.; Nam, Yunsun; Jiang, Jiaoyang; Sliz, Piotr; Walker, Suzanne

    2010-01-01

    O-GlcNAc transferase (OGT) is an essential mammalian enzyme that couples metabolic status to the regulation of a wide variety of cellular signaling pathways by acting as a nutrient sensor1. OGT catalyzes the transfer of N-acetyl-glucosamine from UDP-GlcNAc to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins2,3, including numerous transcription factors4, tumor suppressors, kinases5, phosphatases1, and histone-modifying proteins6. Aberrant O-GlcNAcylation by OGT has been linked to insulin resistance7, diabetic complications8, cancer9 and neurodegenerative diseases including Alzheimer’s10. Despite the importance of OGT, the details of how it recognizes and glycosylates its protein substrates are largely unknown. We report here two crystal structures of human OGT, as a binary complex with UDP (2.8 A) and a ternary complex with UDP and a peptide substrate (1.95 A). The structures provide clues to the enzyme mechanism, show how OGT recognizes target peptide sequences, and reveal the fold of the unique domain between the two halves of the catalytic region. This information will accelerate the rational design of biological experiments to investigate OGT’s functions and the design of inhibitors for use as cellular probes and to assess its potential as a therapeutic target. PMID:21240259

  16. The active site of O-GlcNAc transferase imposes constraints on substrate sequence

    PubMed Central

    Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.

    2016-01-01

    O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509

  17. Global Regulation of Plant Immunity by Histone Lysine Methyl Transferases

    PubMed Central

    Lee, Sanghun; Xu, Siming; Lee, Sang Yeol; Yun, Dae-Jin; Mengiste, Tesfaye

    2016-01-01

    Posttranslational modification of histones modulates gene expression affecting diverse biological functions. We showed that the Arabidopsis thaliana histone methyl transferases SET DOMAIN GROUP8 (SDG8) and SDG25 regulate pep1-, flg22-, and effector-triggered immunity as well as systemic acquired resistance. Genome-wide basal and induced transcriptome changes regulated by SDG8 and/or SDG25 showed that two genes of the SDG-dependent transcriptome, CAROTENOID ISOMERASE2 (CCR2) and ECERIFERUM3 (CER3), were also required for plant immunity, establishing mechanisms in defense functions for SDG8 and SDG25. CCR2 catalyzes the biosynthesis of carotenoids, whereas CER3 is involved in the biosynthesis of cuticular wax. SDG8 and SDG25 affected distinct and overlapping global and locus-specific histone H3 lysine 4 (H3K4) and histone H3 lysine 36 (H3K36) methylations. Loss of immunity in sdg mutants was attributed to altered global and CCR2- and CER3-specific histone lysine methylation (HLM). Loss of immunity in sdg, ccr2, and cer3 mutants was also associated with diminished accumulation of lipids and loss of cuticle integrity. In addition, sdg8 and sdg25 mutants were impaired in H2B ubiquitination (H2Bubn) at CCR2, CER3, and H2Bubn regulated R gene, SNC1, revealing crosstalk between the two types of histone modifications. In summary, SDG8 and SDG25 contribute to plant immunity directly through HLM or indirectly through H2Bubn and by regulating expression of plant immunity genes, accumulation of lipids, biosynthesis of carotenoids, and maintenance of cuticle integrity. PMID:27354553

  18. O-linked β-N-acetylglucosamine transferase directs cell proliferation in idiopathic pulmonary arterial hypertension.

    PubMed

    Barnes, Jarrod W; Tian, Liping; Heresi, Gustavo A; Farver, Carol F; Asosingh, Kewal; Comhair, Suzy A A; Aulak, Kulwant S; Dweik, Raed A

    2015-04-07

    Idiopathic pulmonary arterial hypertension (IPAH) is a cardiopulmonary disease characterized by cellular proliferation and vascular remodeling. A more recently recognized characteristic of the disease is the dysregulation of glucose metabolism. The primary link between altered glucose metabolism and cell proliferation in IPAH has not been elucidated. We aimed to determine the relationship between glucose metabolism and smooth muscle cell proliferation in IPAH. Human IPAH and control patient lung tissues and pulmonary artery smooth muscle cells (PASMCs) were used to analyze a specific pathway of glucose metabolism, the hexosamine biosynthetic pathway. We measured the levels of O-linked β-N-acetylglucosamine modification, O-linked β-N-acetylglucosamine transferase (OGT), and O-linked β-N-acetylglucosamine hydrolase in control and IPAH cells and tissues. Our data suggest that the activation of the hexosamine biosynthetic pathway directly increased OGT levels and activity, triggering changes in glycosylation and PASMC proliferation. Partial knockdown of OGT in IPAH PASMCs resulted in reduced global O-linked β-N-acetylglucosamine modification levels and abrogated PASMC proliferation. The increased proliferation observed in IPAH PASMCs was directly impacted by proteolytic activation of the cell cycle regulator, host cell factor-1. Our data demonstrate that hexosamine biosynthetic pathway flux is increased in IPAH and drives OGT-facilitated PASMC proliferation through specific proteolysis and direct activation of host cell factor-1. These findings establish a novel regulatory role for OGT in IPAH, shed a new light on our understanding of the disease pathobiology, and provide opportunities to design novel therapeutic strategies for IPAH. © 2015 American Heart Association, Inc.

  19. Understanding the Role of O-GlcNAc Modifications in Plant Development

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

    Olszewski, Neil, E.

    2011-06-16

    This project has contributed towards understanding the role of O-GlcNAc (O-linked N-acetylglucosamine) transferases (OGTs) in plants. Through analyses of single and double mutants, we have investigated the unique and overlapping functions of SECRET AGENT (SEC) and SPINDLY (SPY), the arabidopsis OGTs. This work showed that SEC functions as negative regulators of the long-day flowering pathway. SEC also has a positive role in regulation of rosette. An E. coli co-expression system that allows potential substrates to be co-expressed with and O-GlcNAc modified by SEC was developed. We showed that SEC is a bona fide OGT that modifies itself with single O-linkedmore » GlcNAc(s). Using this system, we tested a number of proteins that were hypothesized to be substrates of SEC and identified a number of substrates include GIGANTEA (GI), a component of the long day flowering pathway. The hypothesis that O-GlcNAc modification controls GI activity was tested by first mapping where E. coli-expressed SEC modifies GI and then assessing the activity of a non-modifiable mutant form of GI. The activity of the mutant form of GI was indistinguishable from that of wild type suggesting that either O-GlcNAc does not regulate GI activity or that additional modification sites exist on GI. In collaboration with Dr. Juan Antonio Garcia at Universidad Autónoma de Madrid the role of O-GlcNAc modification of the plum pox virus coat protein (PPV-CP) was investigated. SEC was shown to O-GlcNAc modify PPV-CP and the modification was shown to facilitate the infection process. E. coli-expressed SEC was shown to modify the same PPV-CP sites that are modified in plants. SEC has a large protein interaction domain called the TPR domain that has been hypothesized to have a role in determining the substrate specificity of the enzyme and/or to regulate its activity. A mutational analysis of the TPR domain did not find evidence for a role in substrate specificity but did obtain evidence that the domain

  20. Calreticulin discriminates the proximal region at the N-glycosylation site of Glc1Man9GlcNAc2 ligand

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

    Hirano, Makoto; Adachi, Yuka; Ito, Yukishige

    Calreticulin (CRT) is well known as a lectin-like chaperone that recognizes Glc1Man9GlcNAc2 (G1M9)-glycoproteins in the endoplasmic reticulum (ER). However, whether CRT can directly interact with the aglycone moiety (protein portion) of the glycoprotein remains controversial. To improve our understanding of CRT interactions, structure-defined G1M9-derivatives with different aglycones (–OH, –Gly–NH{sub 2}, and –Gly–Glu–{sup t}Bu) were used as CRT ligands, and their interactions with recombinant CRT were analyzed using thermal shift analysis. The results showed that CRT binds strongly to a G1M9-ligand in the order –Gly–Glu–{sup t}Bu > –Gly–NH{sub 2} > –OH, which is the same as that of the reglucosylation of Man9GlcNAc2 (M9)-derivatives by themore » folding sensor enzyme UGGT (UDP-glucose: glycoprotein glucosyltransferase). Our results indicate that, similar to UGGT, CRT discriminates the proximal region at the N-glycosylation site, suggesting a similar mechanism mediating the recognition of aglycone moieties in the ER glycoprotein quality control system. - Highlights: • Glc1Man9GlcNAc2 (G1M9) ligands with different aglycones were chemically prepared. • Calreticulin (CRT) discriminates the aglycone of Glc1Man9GlcNAc2 (G1M9) ligand. • CRT binds with G1M9 ligands in a similar manner to folding sensor enzyme.« less

  1. Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER.

    PubMed

    Sobering, Andrew K; Watanabe, Reika; Romeo, Martin J; Yan, Benjamin C; Specht, Charles A; Orlean, Peter; Riezman, Howard; Levin, David E

    2004-05-28

    The yeast ERI1 gene encodes a small ER-localized protein that associates in vivo with GTP bound Ras2 in an effector loop-dependent manner. We showed previously that loss of Eri1 function results in hyperactive Ras phenotypes. Here, we demonstrate that Eri1 is a component of the GPI-GlcNAc transferase (GPI-GnT) complex in the ER, which catalyzes transfer of GlcNAc from UDP-GlcNAc to an acceptor phosphatidylinositol, the first step in the production of GPI-anchors for cell surface proteins. We also show that GTP bound Ras2 associates with the GPI-GnT complex in vivo and inhibits its activity, indicating that yeast Ras uses the ER as a signaling platform from which to negatively regulate the GPI-GnT. We propose that diminished GPI-anchor protein production contributes to hyperactive Ras phenotypes.

  2. The Arabidopsis O-Linked N-Acetylglucosamine Transferase SPINDLY Interacts with Class I TCPs to Facilitate Cytokinin Responses in Leaves and Flowers[C][W

    PubMed Central

    Steiner, Evyatar; Efroni, Idan; Gopalraj, Manjula; Saathoff, Katie; Tseng, Tong-Seung; Kieffer, Martin; Eshed, Yuval; Olszewski, Neil; Weiss, David

    2012-01-01

    O-linked N-acetylglucosamine (O-GlcNAc) modifications regulate the posttranslational fate of target proteins. The Arabidopsis thaliana O-GlcNAc transferase (OGT) SPINDLY (SPY) suppresses gibberellin signaling and promotes cytokinin (CK) responses by unknown mechanisms. Here, we present evidence that two closely related class I TCP transcription factors, TCP14 and TCP15, act with SPY to promote CK responses. TCP14 and TCP15 interacted with SPY in yeast two-hybrid and in vitro pull-down assays and were O-GlcNAc modified in Escherichia coli by the Arabidopsis OGT, SECRET AGENT. Overexpression of TCP14 severely affected plant development in a SPY-dependent manner and stimulated typical CK morphological responses, as well as the expression of the CK-regulated gene RESPONSE REGULATOR5. TCP14 also promoted the transcriptional activity of the CK-induced mitotic factor CYCLIN B1;2. Whereas TCP14-overexpressing plants were hypersensitive to CK, spy and tcp14 tcp15 double mutant leaves and flowers were hyposensitive to the hormone. Reducing CK levels by overexpressing CK OXIDASE/DEHYDROGENASE3 suppressed the TCP14 overexpression phenotypes, and this suppression was reversed when the plants were treated with exogenous CK. Taken together, we suggest that responses of leaves and flowers to CK are mediated by SPY-dependent TCP14 and TCP15 activities. PMID:22267487

  3. Lack of O-GlcNAcylation enhances exercise-dependent glucose utilization potentially through AMP-activated protein kinase activation in skeletal muscle.

    PubMed

    Murata, Koichiro; Morino, Katsutaro; Ida, Shogo; Ohashi, Natsuko; Lemecha, Mengistu; Park, Shi-Young; Ishikado, Atsushi; Kume, Shinji; Choi, Cheol Soo; Sekine, Osamu; Ugi, Satoshi; Maegawa, Hiroshi

    2018-01-08

    O-GlcNAcylation is a post-translational modification that is characterized by the addition of N-acetylglucosamine (GlcNAc) to proteins by O-GlcNAc transferase (Ogt). The degree of O-GlcNAcylation is thought to be associated with glucotoxicity and diabetic complications, because GlcNAc is produced by a branch of the glycolytic pathway. However, its role in skeletal muscle has not been fully elucidated. In this study, we created skeletal muscle-specific Ogt knockout (Ogt-MKO) mice and analyzed their glucose metabolism. During an intraperitoneal glucose tolerance test, blood glucose was slightly lower in Ogt-MKO mice than in control Ogt-flox mice. High fat diet-induced obesity and insulin resistance were reversed in Ogt-MKO mice. In addition, 12-month-old Ogt-MKO mice had lower adipose and body mass. A single bout of exercise significantly reduced blood glucose in Ogt-MKO mice, probably because of higher AMP-activated protein kinase α (AMPKα) protein expression. Furthermore, intraperitoneal injection of 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, resulted in a more marked decrease in blood glucose levels in Ogt-MKO mice than in controls. Finally, Ogt knockdown by siRNA in C2C12 myotubes significantly increased protein expression of AMPKα, glucose uptake and oxidation. In conclusion, loss of O-GlcNAcylation facilitates glucose utilization in skeletal muscle, potentially through AMPK activation. The inhibition of O-GlcNAcylation in skeletal muscle may have an anti-diabetic effect, through an enhancement of glucose utilization during exercise. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Global Regulation of Plant Immunity by Histone Lysine Methyl Transferases.

    PubMed

    Lee, Sanghun; Fu, Fuyou; Xu, Siming; Lee, Sang Yeol; Yun, Dae-Jin; Mengiste, Tesfaye

    2016-07-01

    Posttranslational modification of histones modulates gene expression affecting diverse biological functions. We showed that the Arabidopsis thaliana histone methyl transferases SET DOMAIN GROUP8 (SDG8) and SDG25 regulate pep1-, flg22-, and effector-triggered immunity as well as systemic acquired resistance. Genome-wide basal and induced transcriptome changes regulated by SDG8 and/or SDG25 showed that two genes of the SDG-dependent transcriptome, CAROTENOID ISOMERASE2 (CCR2) and ECERIFERUM3 (CER3), were also required for plant immunity, establishing mechanisms in defense functions for SDG8 and SDG25. CCR2 catalyzes the biosynthesis of carotenoids, whereas CER3 is involved in the biosynthesis of cuticular wax. SDG8 and SDG25 affected distinct and overlapping global and locus-specific histone H3 lysine 4 (H3K4) and histone H3 lysine 36 (H3K36) methylations. Loss of immunity in sdg mutants was attributed to altered global and CCR2- and CER3-specific histone lysine methylation (HLM). Loss of immunity in sdg, ccr2, and cer3 mutants was also associated with diminished accumulation of lipids and loss of cuticle integrity. In addition, sdg8 and sdg25 mutants were impaired in H2B ubiquitination (H2Bubn) at CCR2, CER3, and H2Bubn regulated R gene, SNC1, revealing crosstalk between the two types of histone modifications. In summary, SDG8 and SDG25 contribute to plant immunity directly through HLM or indirectly through H2Bubn and by regulating expression of plant immunity genes, accumulation of lipids, biosynthesis of carotenoids, and maintenance of cuticle integrity. © 2016 American Society of Plant Biologists. All rights reserved.

  5. Thio-Linked UDP–Peptide Conjugates as O-GlcNAc Transferase Inhibitors

    PubMed Central

    2018-01-01

    O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA (Ki = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay. PMID:29723473

  6. Revisiting the substrate specificity of mammalian α1,6-fucosyltransferase reveals that it catalyzes core fucosylation of N-glycans lacking α1,3-arm GlcNAc.

    PubMed

    Yang, Qiang; Zhang, Roushu; Cai, Hui; Wang, Lai-Xi

    2017-09-08

    The mammalian α1,6-fucosyltransferase (FUT8) catalyzes the core fucosylation of N -glycans in the biosynthesis of glycoproteins. Previously, intensive in vitro studies with crude extract or purified enzyme concluded that the attachment of a GlcNAc on the α1,3 mannose arm of N -glycan is essential for FUT8-catalyzed core fucosylation. In contrast, we have recently shown that expression of erythropoietin in a GnTI knock-out, FUT8-overexpressing cell line results in the production of fully core-fucosylated glycoforms of the oligomannose substrate Man 5 GlcNAc 2 , suggesting that FUT8 can catalyze core fucosylation of N -glycans lacking an α1,3-arm GlcNAc in cells. Here, we revisited the substrate specificity of FUT8 by examining its in vitro activity toward an array of selected N -glycans, glycopeptides, and glycoproteins. Consistent with previous studies, we found that free N -glycans lacking an unmasked α1,3-arm GlcNAc moiety are not FUT8 substrates. However, Man 5 GlcNAc 2 glycan could be efficiently core-fucosylated by FUT8 in an appropriate protein/peptide context, such as with the erythropoietin protein, a V3 polypeptide derived from HIV-1 gp120, or a simple 9-fluorenylmethyl chloroformate-protected Asn moiety. Interestingly, when placed in the V3 polypeptide context, a mature bi-antennary complex-type N -glycan also could be core-fucosylated by FUT8, albeit at much lower efficiency than the Man 5 GlcNAc 2 peptide. This study represents the first report of in vitro FUT8-catalyzed core fucosylation of N -glycans lacking the α1,3-arm GlcNAc moiety. Our results suggest that an appropriate polypeptide context or other adequate structural elements in the acceptor substrate could facilitate the core fucosylation by FUT8. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Quantitative time-resolved chemoproteomics reveals that stable O-GlcNAc regulates box C/D snoRNP biogenesis

    PubMed Central

    Qin, Wei; Lv, Pinou; Fan, Xinqi; Quan, Baiyi; Zhu, Yuntao; Qin, Ke; Chen, Ying; Wang, Chu

    2017-01-01

    O-linked GlcNAcylation (O-GlcNAcylation), a ubiquitous posttranslational modification on intracellular proteins, is dynamically regulated in cells. To analyze the turnover dynamics of O-GlcNAcylated proteins, we developed a quantitative time-resolved O-linked GlcNAc proteomics (qTOP) strategy based on metabolic pulse-chase labeling with an O-GlcNAc chemical reporter and stable isotope labeling with amino acids in cell culture (SILAC). Applying qTOP, we quantified the turnover rates of 533 O-GlcNAcylated proteins in NIH 3T3 cells and discovered that about 14% exhibited minimal removal of O-GlcNAc or degradation of protein backbones. The stability of those hyperstable O-GlcNAcylated proteins was more sensitive to O-GlcNAcylation inhibition compared with the more dynamic populations. Among the hyperstable population were three core proteins of box C/D small nucleolar ribonucleoprotein complexes (snoRNPs): fibrillarin (FBL), nucleolar protein 5A (NOP56), and nucleolar protein 5 (NOP58). We showed that O-GlcNAcylation stabilized these proteins and was essential for snoRNP assembly. Blocking O-GlcNAcylation on FBL altered the 2′-O-methylation of rRNAs and impaired cancer cell proliferation and tumor formation in vivo. PMID:28760965

  8. Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

    PubMed

    Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

    2017-06-16

    Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

  9. Molecular and biochemical characterization of tomato farnesyl-protein transferase.

    PubMed

    Schmitt, D; Callan, K; Gruissem, W

    1996-10-01

    The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity. In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified. We partially purified and characterized farnesyl-protein transferase from tomato (Lycopersicon esculentum, LeFTase) to analyze its biochemical and molecular properties. Using Ras- and G gamma-specific peptide substrates and competition assays we showed that tomato protein extracts have both farnesyl-protein transferase and geranylgeranyl-protein transferase 1 activities. Compared with the heterologous synthetic peptide substrates, the plant-specific CaaX sequence of the ANJ1 protein is a less efficient substrate for LeFTase in vitro. LeFTase activity profiles and LeFTase beta-subunit protein (LeFTB) levels differ significantly in various tissues and are regulated during fruit development. Partially purified LeFTase requires Zn2+ and Mg2+ for enzymatic activity and has an apparent molecular mass of 100 kD Immunoprecipitation experiments using anti-alpha LeFTB antibodies confirmed that LeFTB is a component of LeFTase but not of tomato geranylgeranyl-protein transferase 1. Based on their conserved bio-chemical activities, we expect that prenyltransferases are likely integrated with the sterol biosynthesis pathway in the control of plant cell growth.

  10. Down-regulation of Glutathione S-transferase Pi in Asthma Contributes to Enhanced Oxidative Stress

    PubMed Central

    Schroer, Kathy T.; Gibson, Aaron M.; Sivaprasad, Umasundari; Bass, Stacey A.; Ericksen, Mark B.; Wills-Karp, Marsha; LeCras, Tim; Fitzpatrick, Anne M.; Brown, Lou Ann S.; Stringer, Keith F.; Khurana Hershey, Gurjit K.

    2011-01-01

    Background Glutathione S-transferase Pi (GSTPi) is the predominant redox regulator in the lung. While evidence implicates an important role for GSTPi in asthma, the mechanism for this has remained elusive. Objectives To determine how GSTPi is regulated in asthma and to elucidate its role in maintaining redox homeostasis. Methods We elucidated the regulation of GSTPi in children with asthma and utilized murine models of asthma to determine the role of GSTPi in redox homeostasis. Measurements and Main Results Our findings demonstrate that GSTPi transcript levels are markedly down-regulated in allergen and IL-13 treated mouse models of asthma via STAT6 dependent and independent pathways. Nuclear factor-erythroid 2 related factor 2 (Nrf2) was also down-regulated in these models. The decrease in GSTPi expression was associated with decreased total GST activity in the lungs of mice. Examination of cystine intermediates uncovered a functional role for GSTPi in regulating Cys oxidation, whereby GSTPi-deficient mice exhibited increased oxidative stress (increase in % cystine) compared with wild-type mice following allergen challenge. GSTPi expression was similarly down-regulated in children with asthma. Conclusions These data collectively suggest that down-regulation of GSTPi following allergen challenge may contribute to the asthma phenotype due to disruption of redox homeostasis and increased oxidative stress. Furthermore, GSTPi may be an important therapeutic target for asthma, and evaluation of GSTPi expression may prove beneficial in identifying individuals who would benefit from therapy targeting this pathway. PMID:21570714

  11. Cross regulation between mTOR signaling and O-GlcNAcylation.

    PubMed

    Very, Ninon; Steenackers, Agata; Dubuquoy, Caroline; Vermuse, Jeanne; Dubuquoy, Laurent; Lefebvre, Tony; El Yazidi-Belkoura, Ikram

    2018-06-01

    The hexosamine biosynthetic pathway (HBP) integrates glucose, amino acids, fatty acids and nucleotides metabolisms for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is the nucleotide sugar donor for O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) processes. O-GlcNAc transferase (OGT) is the enzyme which transfers the N-acetylglucosamine (O-GlcNAc) residue onto target proteins. Several studies previously showed that glucose metabolism dysregulations associated with obesity, diabetes or cancer correlated with an increase of OGT expression and global O-GlcNAcylation levels. Moreover, these diseases present an increased activation of the nutrient sensing mammalian target of rapamycin (mTOR) pathway. Other works demonstrate that mTOR regulates protein O-GlcNAcylation in cancer cells through stabilization of OGT. In this context, we studied the cross-talk between these two metabolic sensors in vivo in obese mice predisposed to diabetes and in vitro in normal and colon cancer cells. We report that levels of OGT and O-GlcNAcylation are increased in obese mice colon tissues and colon cancer cells and are associated with a higher activation of mTOR signaling. In parallel, treatments with mTOR regulators modulate OGT and O-GlcNAcylation levels in both normal and colon cancer cells. However, deregulation of O-GlcNAcylation affects mTOR signaling activation only in cancer cells. Thus, a crosstalk exists between O-GlcNAcylation and mTOR signaling in contexts of metabolism dysregulation associated to obesity or cancer.

  12. The effect of chemical carcinogenesis on rat glutathione S-transferase P1 gene transcriptional regulation.

    PubMed

    Liu, D; Liao, M; Zuo, J; Henner, W D; Fan, F

    2001-03-01

    To investigate mechanisms of rat glutathione S-transferase P1 gene (rGSTP1) expression regulation during chemical carcinogenesis. we studied enhancer elements located in the region between -2.5 kb to -2.2 kb. The region was upstream from the start site of transcription and was divided into two major fragments, GPEI and GPEII. The GPEII fragment was further divided into two smaller fragments, GPEII- I and GPEII-2. Using a luciferase reporter system, we identified a strong enhancer of GPEI and a weak enhancer of GPEII in HeLa and a rat hepatoma cell line CBRH79 19 cell. The enhancer of GPEII was located within the GPEII-I region. Chemical stimulation by glycidyl methatylate (GMA) and phorbol 12-o-tetradecanoate 13-acetate (TPA) analysis revealed that induction of rGSTP1 expression was mainly through GPEI. Although H2O2 could enhance GPEII enhancer activity, the enhancement is not mediated by the NF-kappaB factor that bound the NF-kappaB site in GPEII. Using electrophoretic mobility shift assays (EMSA) and the UV cross-linking assays, we found that HeLa and CBRH7919 cells had proteins that specifically bound GPEI core sequence and a 64 kDa protein that interacted with GPEII-1. The cells from normal rat liver did not express the binding proteins. Therefore, the trans-acting factors seem to be closely related to GPEI, GPEII enhancer activities and may play an important role in high expression of rGSTPI gene.

  13. Identification of the S-transferase like superfamily bacillithiol transferases encoded by Bacillus subtilis

    PubMed Central

    Perera, Varahenage R.; Lapek, John D.; Newton, Gerald L.; Gonzalez, David J.; Pogliano, Kit

    2018-01-01

    Bacillithiol is a low molecular weight thiol found in Firmicutes that is analogous to glutathione, which is absent in these bacteria. Bacillithiol transferases catalyze the transfer of bacillithiol to various substrates. The S-transferase-like (STL) superfamily contains over 30,000 putative members, including bacillithiol transferases. Proteins in this family are extremely divergent and are related by structural rather than sequence similarity, leaving it unclear if all share the same biochemical activity. Bacillus subtilis encodes eight predicted STL superfamily members, only one of which has been shown to be a bacillithiol transferase. Here we find that the seven remaining proteins show varying levels of metal dependent bacillithiol transferase activity. We have renamed the eight enzymes BstA-H. Mass spectrometry and gene expression studies revealed that all of the enzymes are produced to varying levels during growth and sporulation, with BstB and BstE being the most abundant and BstF and BstH being the least abundant. Interestingly, several bacillithiol transferases are induced in the mother cell during sporulation. A strain lacking all eight bacillithiol transferases showed normal growth in the presence of stressors that adversely affect growth of bacillithiol-deficient strains, such as paraquat and CdCl2. Thus, the STL bacillithiol transferases represent a new group of proteins that play currently unknown, but potentially significant roles in bacillithiol-dependent reactions. We conclude that these enzymes are highly divergent, perhaps to cope with an equally diverse array of endogenous or exogenous toxic metabolites and oxidants. PMID:29451913

  14. Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis

    PubMed Central

    Wang, Zihao; Udeshi, Namrata D.; Slawson, Chad; Compton, Philip D.; Sakabe, Kaoru; Cheung, Win D.; Shabanowitz, Jeffrey; Hunt, Donald F.; Hart, Gerald W.

    2010-01-01

    Like phosphorylation, the addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways, but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division. PMID:20068230

  15. Regulation of Dauer formation by O-GlcNAcylation in Caenorhabditis elegans.

    PubMed

    Lee, Jeeyong; Kim, Kwang-Youl; Lee, Jihyun; Paik, Young-Ki

    2010-01-29

    Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone daumone, ogt-1 showed suppression of dauer formation, whereas oga-1 exhibited enhancement of dauer formation. Consistent with these findings, treatment of wild-type N2 worms with low concentrations of daumone and the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) enhanced dauer formation, which was dependent on intact O-GlcNAcylation metabolism. We also found that the treatment of daumone enhanced O-GlcNAcylation in vivo. Seven proteins, identified by coupled two-dimensional electrophoresis/liquid chromatography-mass spectroscopy (LC-MS) analysis, were differentially expressed in oga-1(ok1207) worms compared with wild-type N2 worms. The identities of these proteins suggest that O- GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. Using O-GlcNAc labeling with fluorescent dye combined with two-dimensional electrophoresis/LC-MS analysis, we also identified five proteins that were differentially O-GlcNAcylated during dauer formation. Analysis of these candidate O-GlcNAcylated proteins suggests that O-GlcNAcylation may regulate cytoskeleton modifications and protein turnover during dauer formation.

  16. Aniline exposure associated with up-regulated transcriptional responses of three glutathione S-transferase Delta genes in Drosophila melanogaster.

    PubMed

    Chan, Wen-Chiao; Chien, Yi-Chih; Chien, Cheng-I

    2015-03-01

    Complex transcriptional profile of glutathione S-transferase Delta cluster genes occurred in the developmental process of the fruit fly Drosophila melanogaster. The purpose of this project was to quantify the expression levels of Gst Delta class genes altered by aniline exposure and to understand the relationship between aniline dosages and the variation of Gst Delta genes expressed in D. melanogaster. Using RT-PCR expression assays, the expression patterns of the transcript mRNAs of the glutathione S-transferase Delta genes were revealed and their expression levels were measured at eggs, larvae, pupae and adults. The adult stage was selected for further dose-response assays. After analysis, the results indicated that three Gst Delta genes (Gst D2, Gst D5 and Gst D6) were found to show a peak of up-regulated transcriptional response at 6-8h of exposure of aniline. Furthermore, the dose-response relationship of their induction levels within the dose regiments (from 1.2 to 2.0 μl/tube) had been measured. The expression patterns and annotations of these genes were discussed in the context. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Purification and properties of an O-acetyl-transferase from Escherichia coli that can O-acetylate polysialic acid sequences

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

    Higa, H.; Varki, A.

    1986-05-01

    Certain strains of bacteria synthesize an outer polysialic acid (K1) capsule. Some strains of K1/sup +/ E.coli are also capable of adding O-acetyl-esters to the exocyclic hydroxyl groups of the sialic acid residues. Both the capsule and the O-acetyl modification have been correlated with differences in antigenicity and pathogenicity. The authors have developed an assay for an O-acetyl-transferase in E.coli that transfers O-(/sup 3/H)acetyl groups from (/sup 3/H)acetyl-Coenzyme A to colominic acid (fragments of the polysialic acid capsule). Using this assay, the enzyme was solubilized, and purified approx. 600-fold using a single affinity chromatography step with Procion Red-A Agarose. Themore » enzyme also binds to Coenzyme A Sepharose, and can be eluted with high salt or Coenzyme A. The partially purified enzyme has a pH optimum of 7.0 - 7.5, is unaffected by divalent cations, is inhibited by high salt concentrations, is inhibited by Coenzyme A (50% inhibition at 100 ..mu..M), and shows an apparent Km for colominic acid of 3.7 mM (sialic acid concentration). This enzyme could be involved in the O-acetyl +/- form variation seen in some strains of K1/sup +/ E.coli.« less

  18. Regulation of chlorogenic acid biosynthesis by hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase in Lonicera japonica.

    PubMed

    Zhang, Jingru; Wu, Minlin; Li, Weidong; Bai, Genben

    2017-12-01

    For many centuries, Lonicera japonica has been used as an effective herb for the treatment of inflammation and swelling because of the presence of bioactive components such as chlorogenic acid (CGA). To clarify the relationship between L. japonica hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) gene expression and CGA content, an HQT eukaryotic expression system was constructed using Gateway cloning. L. japonica callus transformed with HQT was obtained using Agrobacterium tumefaciens-mediated transformation. We found a positive correlation between CGA content, determined by High-Performance Liquid Chromatography (HPLC), and the expression of HQT, analyzed by semi-quantitative RT-PCR. This study demonstrates that the HQT gene positively regulates CGA synthesis and lays the foundation for further study into enhancing efficacious components of medicinal plants. Copyright © 2017. Published by Elsevier Masson SAS.

  19. Functional O-GlcNAc modifications: Implications in molecular regulation and pathophysiology

    PubMed Central

    Wells, Lance

    2016-01-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification of intracellular proteins. The dynamic and inducible cycling of the modification is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in response to UDP-GlcNAc levels in the hexosamine biosynthetic pathway (HBP). Due to its reliance on glucose flux and substrate availability, a major focus in the field has been on how O-GlcNAc contributes to metabolic disease. For years this post-translational modification has been known to modify thousands of proteins implicated in various disorders, but direct functional connections have until recently remained elusive. New research is beginning to reveal the specific mechanisms through which O-GlcNAc influences cell dynamics and disease pathology including clear examples of O-GlcNAc modification at a specific site on a given protein altering its biological functions. The following review intends to focus primarily on studies in the last half decade linking O-GlcNAc modification of proteins with chromatin-directed gene regulation, developmental processes, and several metabolically related disorders including Alzheimer’s, heart disease and cancer. These studies illustrate the emerging importance of this post-translational modification in biological processes and multiple pathophysiologies. PMID:24524620

  20. γ-Glutamyl transferase 7 is a novel regulator of glioblastoma growth.

    PubMed

    Bui, Timothy T; Nitta, Ryan T; Kahn, Suzana A; Razavi, Seyed-Mostafa; Agarwal, Maya; Aujla, Parvir; Gholamin, Sharareh; Recht, Lawrence; Li, Gordon

    2015-04-07

    Glioblastoma (GBM) is the most malignant primary brain tumor in adults, with a median survival time of one and a half years. Traditional treatments, including radiation, chemotherapy, and surgery, are not curative, making it imperative to find more effective treatments for this lethal disease. γ-Glutamyl transferase (GGT) is a family of enzymes that was shown to control crucial redox-sensitive functions and to regulate the balance between proliferation and apoptosis. GGT7 is a novel GGT family member that is highly expressed in brain and was previously shown to have decreased expression in gliomas. Since other members of the GGT family were found to be altered in a variety of cancers, we hypothesized that GGT7 could regulate GBM growth and formation. To determine if GGT7 is involved in GBM tumorigenesis, we modulated GGT7 expression in two GBM cell lines (U87-MG and U138) and monitored changes in tumorigenicity in vitro and in vivo. We demonstrated for the first time that GBM patients with low GGT7 expression had a worse prognosis and that 87% (7/8) of primary GBM tissue samples showed a 2-fold decrease in GGT7 expression compared to normal brain samples. Exogenous expression of GGT7 resulted in a 2- to 3-fold reduction in proliferation and anchorage-independent growth under minimal growth conditions (1% serum). Decreasing GGT7 expression using either short interfering RNA or short hairpin RNA consistently increased proliferation 1.5- to 2-fold. In addition, intracranial injections of U87-MG cells with reduced GGT7 expression increased tumor growth in mice approximately 2-fold, and decreased mouse survival. To elucidate the mechanism by which GGT7 regulates GBM growth, we analyzed reactive oxygen species (ROS) levels in GBM cells with modulated GGT7 expression. We found that enhanced GGT7 expression reduced ROS levels by 11-33%. Our study demonstrates that GGT7 is a novel player in GBM growth and that GGT7 can play a critical role in tumorigenesis by regulating

  1. Hibiscus cannabinus feruloyl-coa:monolignol transferase

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

    Wilkerson, Curtis; Ralph, John; Withers, Saunia

    The invention relates to isolated nucleic acids encoding a feruloyl-CoA:monolignol transferase and feruloyl-CoA:monolignol transferase enzymes. The isolated nucleic acids and/or the enzymes enable incorporation of monolignol ferulates into the lignin of plants, where such monolignol ferulates include, for example, p-coumaryl ferulate, coniferyl ferulate, and/or sinapyl ferulate. The invention also includes methods and plants that include nucleic acids encoding a feruloyl-CoA:monolignol transferase enzyme and/or feruloyl-CoA:monolignol transferase enzymes.

  2. O-GlcNAcylation and oxidation of proteins: is signalling in the cardiovascular system becoming sweeter?

    PubMed Central

    Lima, Victor V.; Spitler, Kathryn; Choi, Hyehun; Webb, R. Clinton; Tostes, Rita C.

    2012-01-01

    O-GlcNAcylation is an unusual form of protein glycosylation, where a single-sugar [GlcNAc (N-acetylglucosamine)] is added (via β-attachment) to the hydroxyl moiety of serine and threonine residues of nuclear and cytoplasmic proteins. A complex and extensive interplay exists between O-GlcNAcylation and phosphorylation. Many phosphorylation sites are also known glycosylation sites, and this reciprocal occupancy may produce different activities or alter the stability in a target protein. The interplay between these two post-translational modifications is not always reciprocal, as some proteins can be concomitantly phosphorylated and O-GlcNAcylated, and the adjacent phosphorylation or O-GlcNAcylation can regulate the addition of either moiety. Increased cardiovascular production of ROS (reactive oxygen species), termed oxidative stress, has been consistently reported in various chronic diseases and in conditions where O-GlcNAcylation has been implicated as a contributing mechanism for the associated organ injury/protection (for example, diabetes, Alzheimer's disease, arterial hypertension, aging and ischaemia). In the present review, we will briefly comment on general aspects of O-GlcNAcylation and provide an overview of what has been reported for this post-translational modification in the cardiovascular system. We will then specifically address whether signalling molecules involved in redox signalling can be modified by O-GlcNAc (O-linked GlcNAc) and will discuss the critical interplay between O-GlcNAcylation and ROS generation. Experimental evidence indicates that the interactions between O-GlcNAcylation and oxidation of proteins are important not only for cell regulation in physiological conditions, but also under pathological states where the interplay may become dysfunctional and thereby exacerbate cellular injury. PMID:22757958

  3. Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human δ-opioid receptor turnover at the plasma membrane.

    PubMed

    Lackman, Jarkko J; Goth, Christoffer K; Halim, Adnan; Vakhrushev, Sergey Y; Clausen, Henrik; Petäjä-Repo, Ulla E

    2018-01-01

    G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors

  4. O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry.

    PubMed

    Borodkin, Vladimir S; Rafie, Karim; Selvan, Nithya; Aristotelous, Tonia; Navratilova, Iva; Ferenbach, Andrew T; van Aalten, Daan M F

    2018-05-18

    The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer's and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.

  5. Enzymatic Glycosylation by Transferases

    NASA Astrophysics Data System (ADS)

    Blixt, Ola; Razi, Nahid

    Glycosyltransferases are important biological catalysts in cellular systems generating complex cell surface glycans involved in adhesion and signaling processes. Recent advances in glycoscience have increased the demands to access significant amount of glycans representing the glycome. Glycosyltransferases are now playing a key role for in vitro synthesis of oligosaccharides and the bacterial genome are increasingly utilized for cloning and over expression of active transferases in glycosylation reactions. This chapter highlights the recent progress towards preparative synthesis of oligosaccharides representing terminal sequences of glycoproteins and glycolipids using recombinant transferases. Transferases are also being explored in the context of solid-phase synthesis, immobilized on resins and over expression in vivo by engineered bacteria.

  6. Inhibition of O-linked N-acetylglucosamine transferase activity in PC12 cells - A molecular mechanism of organophosphate flame retardants developmental neurotoxicity.

    PubMed

    Gu, Yuxin; Yang, Yu; Wan, Bin; Li, Minjie; Guo, Liang-Hong

    2018-06-01

    Organophosphate flame retardants (OPFRs), as alternatives of brominated flame retardants, can cause neurodevelopmental effects similar to organophosphate pesticides. However, the molecular mechanisms underlying the toxicity remain elusive. O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) regulates numerous neural processes through the O-GlcNAcylation modification of nuclear and cytoplasmic proteins. In this study, we aimed to investigate the molecular mechanisms accounting for the developmental neurotoxicity of OPFRs by identifying potential targets of OPFRs and the attendant effects. Twelve OPFRs were evaluated for inhibition of OGT activity using an electrochemical biosensor. Their potency differed with substituent groups. The alkyl group substituted OPFRs had no inhibitory effect. Instead, the six OPFRs substituted with aromatic or chlorinated alkyl groups inhibited OGT activity significantly, with tri-m-cresyl phosphate (TCrP) being the strongest. The six OPFRs (0-100 μM exposure) also inhibited OGT activity in PC12 cells and decreased protein O-GlcNAcylation level. Inhibition of OGT by OPFRs might be involved in the subsequent toxic effects, including intracellular reactive oxygen species (ROS), calcium level, as well as cell proliferation and autophagy. Molecular docking of the OGT/OPFR complexes provided rationales for the difference in their structure-dependent inhibition potency. Our findings may provide a new biological target of OPFRs in their neurotoxicological actions, which might be a major molecular mechanism of OPFRs developmental neurotoxicity. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis.

    PubMed

    Rivera-Millot, Alex; Lesne, Elodie; Solans, Luis; Coutte, Loic; Bertrand-Michel, Justine; Froguel, Philippe; Dhennin, Véronique; Hot, David; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

    2017-01-01

    The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen.

  8. Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis

    PubMed Central

    Rivera-Millot, Alex; Lesne, Elodie; Solans, Luis; Coutte, Loic; Bertrand-Michel, Justine; Froguel, Philippe; Dhennin, Véronique; Hot, David; Locht, Camille; Antoine, Rudy

    2017-01-01

    The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen. PMID:28493897

  9. Feruloyl-CoA:monolignol transferase

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

    Wilkerson, Curtis; Ralph, John; Withers, Saunia

    The invention relates to nucleic acids encoding a feruloyl-CoA:monolignol transferase and the feruloyl-CoA:monolignol transferase enzyme that enables incorporation of monolignol ferulates, for example, including p-coumaryl ferulate, coniferyl ferulate, and sinapyl ferulate, into the lignin of plants.

  10. Recombinant fungal lectin as a new tool to investigate O-GlcNAcylation processes.

    PubMed

    Machon, Oriane; Baldini, Steffi F; Ribeiro, João P; Steenackers, Agata; Varrot, Annabelle; Lefebvre, Tony; Imberty, Anne

    2017-01-01

    Glycosylation is a group of post-translational modifications that displays a large variety of structures and are implicated in a plethora of biological processes. Therefore, studying glycosylation requires different technical approaches and reliable tools, lectins being part of them. Here, we describe the use of the recombinant mushroom lectin PVL to discriminate O-GlcNAcylation, a modification consisting in the attachment of a single N-acetylglucosamine residue to proteins confined within the cytosolic, nuclear and mitochondrial compartments. Recombinant PVL (Psathyrella velutina lectin) (rPVL) displays significantly stronger affinity for GlcNAc over Neu5Ac residues as verified by thermal shift assays and surface plasmon resonance experiments, being therefore an excellent alternative to WGA (wheat germ agglutinin). Labeling of rPVL with biotin or HRP (horseradish peroxidase) allows its useful and efficient utilization by western blot. The staining of whole cell lysates with  labeled-rPVL was dramatically decreased in response to O-GlcNAc transferase knockdown and seen to increase after pharmacological blockade of O-GlcNAcase. Also, HRP-rPVL seemed to be more sensitive than the anti-O-GlcNAc antibody RL2. Thus, rPVL is a potent new tool to selectively detect O-GlcNAcylated proteins. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. The Importance of Sulfate Adenylyl Transferase in S and O Fractionation by Sulfate Reducing Bacteria

    NASA Astrophysics Data System (ADS)

    Smith, D. A.; Johnston, D. T.; Bradley, A. S.

    2016-12-01

    Microbial sulfate reduction (MSR) is critical to the oxidation of organic matter in modern and ancient oceans, and plays an important role in regulating the redox state of the Earth's surface. The sulfur and oxygen isotopic composition of seawater sulfate and of sulfate minerals reflect the biogeochemical processes that cycle sulfur, of which MSR is among the most important. MSR is a multi-enzymatic reaction network that partitions the isotopes of sulfur and oxygen as a consequence of both the flux of sulfate through this biochemical network and the fractionation imposed by each individual enzyme. MSR affects the δ18O of residual, extracellular sulfate mainly by the equilibration of the MSR intermediate sulfite with extracellular water (Antler et al., 2013 GCA, Wankel et al., 2013 Geobiol). A series of oxidative and exchange reactions catalyzed by APS reductase (APSr), sulfate adenylyl transferase (Sat), and sulfate transporters promote the conversion of water-equilibrated intracellular sulfite to extracellular sulfate. The flux of sulfoxy anions via these proteins will be, at least in part, dependent on the activity of these enzymes. To test this, we examined sulfur and oxygen isotope fractionation in genetically engineered mutants of the sulfate reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH). In these mutants, the activity of Sat has been artificially increased by perturbing the (i) transcriptional repressor Rex and (ii) its binding site upstream of the gene encoding Sat (Christensen et al., 2015 J. Bacteriol). It was predicted that this would minimize the back reaction of Sat, enhance the intracellular pool of APS, and minimize the equilibration between sulfite and adenosine monophosphate (AMP). Both mutants, along with the wild type DvH were grown in batch culture made with water enriched in 18O. Samples were collected throughout batch growth, and we report the evolution of the S and O isotopic composition of sulfate, and of the S isotopic

  12. Increased O-GlcNAcylation of Endothelial Nitric Oxide Synthase Compromises the Anti-contractile Properties of Perivascular Adipose Tissue in Metabolic Syndrome.

    PubMed

    da Costa, Rafael M; da Silva, Josiane F; Alves, Juliano V; Dias, Thiago B; Rassi, Diane M; Garcia, Luis V; Lobato, Núbia de Souza; Tostes, Rita C

    2018-01-01

    Under physiological conditions, the perivascular adipose tissue (PVAT) negatively modulates vascular contractility. This property is lost in experimental and human obesity and in the metabolic syndrome, indicating that changes in PVAT function may contribute to vascular dysfunction associated with increased body weight and hyperglycemia. The O -linked β-N-acetylglucosamine ( O -GlcNAc) modification of proteins ( O -GlcNAcylation) is a unique posttranslational process that integrates glucose metabolism with intracellular protein activity. Increased flux of glucose through the hexosamine biosynthetic pathway and the consequent increase in tissue-specific O -GlcNAc modification of proteins have been linked to multiple facets of vascular dysfunction in diabetes and other pathological conditions. We hypothesized that chronic consumption of glucose, a condition that progresses to metabolic syndrome, leads to increased O -GlcNAc modification of proteins in the PVAT, decreasing its anti-contractile effects. Therefore, the current study was devised to determine whether a high-sugar diet increases O -GlcNAcylation in the PVAT and how increased O -GlcNAc interferes with PVAT vasorelaxant function. To assess molecular mechanisms by which O -GlcNAc contributes to PVAT dysfunction, thoracic aortas surrounded by PVAT were isolated from Wistar rats fed either a control or high sugar diet, for 10 and 12 weeks. Rats chronically fed a high sugar diet exhibited metabolic syndrome features, increased O -GlcNAcylated-proteins in the PVAT and loss of PVAT anti-contractile effect. PVAT from high sugar diet-fed rats for 12 weeks exhibited decreased NO formation, reduced expression of endothelial nitric oxide synthase (eNOS) and increased O -GlcNAcylation of eNOS. High sugar diet also decreased OGA activity and increased superoxide anion generation in the PVAT. Visceral adipose tissue samples from hyperglycemic patients showed increased levels of O -GlcNAc-modified proteins, increased ROS

  13. Swim-exercised mice show a decreased level of protein O-GlcNAcylation and expression of O-GlcNAc transferase in heart.

    PubMed

    Belke, Darrell D

    2011-07-01

    Swim-training exercise in mice leads to cardiac remodeling associated with an improvement in contractile function. Protein O-linked N-acetylglucosamine (O-GlcNAcylation) is a posttranslational modification of serine and threonine residues capable of altering protein-protein interactions affecting gene transcription, cell signaling pathways, and general cell physiology. Increased levels of protein O-GlcNAcylation in the heart have been associated with pathological conditions such as diabetes, ischemia, and hypertrophic heart failure. In contrast, the impact of physiological exercise on protein O-GlcNAcylation in the heart is currently unknown. Swim-training exercise in mice was associated with the development of a physiological hypertrophy characterized by an improvement in contractile function relative to sedentary mice. General protein O-GlcNAcylation was significantly decreased in swim-exercised mice. This effect was mirrored in the level of O-GlcNAcylation of individual proteins such as SP1. The decrease in protein O-GlcNAcylation was associated with a decrease in the expression of O-GlcNAc transferase (OGT) and glutamine-fructose amidotransferase (GFAT) 2 mRNA. O-GlcNAcase (OGA) activity was actually lower in swim-trained than sedentary hearts, suggesting that it did not contribute to the decreased protein O-GlcNAcylation. Thus it appears that exercise-induced physiological hypertrophy is associated with a decrease in protein O-GlcNAcylation, which could potentially contribute to changes in gene expression and other physiological changes associated with exercise.

  14. Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach.

    PubMed

    Gahete, Manuel D; Córdoba-Chacón, Jose; Salvatori, Roberto; Castaño, Justo P; Kineman, Rhonda D; Luque, Raul M

    2010-04-12

    Ghrelin acts as an endocrine link connecting physiological processes regulating food intake, body composition, growth, and energy balance. Ghrelin is the only peptide known to undergo octanoylation. The enzyme mediating this process, ghrelin O-acyltransferase (GOAT), is expressed in the gastrointestinal tract (GI; primary source of circulating ghrelin) as well as other tissues. The present study demonstrates that stomach GOAT mRNA levels correlate with circulating acylated-ghrelin levels in fasted and diet-induced obese mice. In addition, GOAT was found to be expressed in both the pituitary and hypothalamus (two target tissues of ghrelin's actions), and regulated in response to metabolic status. Using primary pituitary cell cultures as a model system to study the regulation of GOAT expression, we found that acylated-ghrelin, but not desacyl-ghrelin, increased GOAT expression. In addition, growth-hormone-releasing hormone (GHRH) and leptin increased, while somatostatin (SST) decreased GOAT expression. The physiologic relevance of these later results is supported by the observation that pituitary GOAT expression in mice lacking GHRH, SST and leptin showed opposite changes to those observed after in vitro treatment with the corresponding peptides. Therefore, it seems plausible that these hormones directly contribute to the regulation of pituitary GOAT. Interestingly, in all the models studied, pituitary GOAT expression paralleled changes in the expression of a dominant spliced-variant of ghrelin (In2-ghrelin) and therefore this transcript may be a primary substrate for pituitary GOAT. Collectively, these observations support the notion that the GI tract is not the only source of acylated-ghrelin, but in fact locally produced des-acylated-ghrelin could be converted to acylated-ghrelin within target tissues by locally active GOAT, to mediate its tissue-specific effects.

  15. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

    NASA Astrophysics Data System (ADS)

    Lira-Navarrete, Erandi; de Las Rivas, Matilde; Compañón, Ismael; Pallarés, María Carmen; Kong, Yun; Iglesias-Fernández, Javier; Bernardes, Gonçalo J. L.; Peregrina, Jesús M.; Rovira, Carme; Bernadó, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon

    2015-05-01

    Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.

  16. Glutathione S - transferases class Pi and Mi and their significance in oncology.

    PubMed

    Marchewka, Zofia; Piwowar, Agnieszka; Ruzik, Sylwia; Długosz, Anna

    2017-06-19

    In this article the current data, which shows that glutathione S-transferases (GST) class Pi and Mi are interesting and promising biomarkers in acute and chronic inflammatory processes as well as in the oncology, were presented based on the review of the latest experimental and clinical studies. The article shows their characteristics, functions and participation (direct - GST Pi, indirect - GST Mi) in the regulation of signaling pathways of JNK kinases, which are involved in cell differentiation. Overexpression of glutathione S-transferases class Pi and Mi in many cancer cells plays a key role in cancer treatment, making them resistant to chemotherapy. GST isoenzymes are involved in the metabolism of various types of xenobiotics and endogenous substrates, so their altered expression in cancer tissues as well as in serum and urine could be an important potential marker of the cancer and an indicator of oxidative stress. The study shows the role of glutathione S-transferases in redox homeostasis of tumor cells and in the mechanism of resistance to anticancer drugs.

  17. Functional significance of O-GlcNAc modification in regulating neuronal properties.

    PubMed

    Hwang, Hongik; Rhim, Hyewhon

    2018-03-01

    Post-translational modifications (PTMs) covalently modify proteins and diversify protein functions. Along with protein phosphorylation, another common PTM is the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine and/or threonine residues. O-GlcNAc modification is similar to phosphorylation in that it occurs to serine and threonine residues and cycles on and off with a similar time scale. However, a striking difference is that the addition and removal of the O-GlcNAc moiety on all substrates are mediated by the two enzymes regardless of proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. O-GlcNAcylation can interact or potentially compete with phosphorylation on serine and threonine residues, and thus serves as an important molecular mechanism to modulate protein functions and activation. However, it has been challenging to address the role of O-GlcNAc modification in regulating protein functions at the molecular level due to the lack of convenient tools to determine the sites and degrees of O-GlcNAcylation. Studies in this field have only begun to expand significantly thanks to the recent advances in detection and manipulation methods such as quantitative proteomics and highly selective small-molecule inhibitors for OGT and OGA. Interestingly, multiple brain regions, especially hippocampus, express high levels of both OGT and OGA, and a number of neuron-specific proteins have been reported to undergo O-GlcNAcylation. This review aims to discuss the recent updates concerning the impacts of O-GlcNAc modification on neuronal functions at multiple levels ranging from intrinsic neuronal properties to synaptic plasticity and animal behaviors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

    PubMed

    Oh, Hye Ji; Moon, Hye Yun; Cheon, Seon Ah; Hahn, Yoonsoo; Kang, Hyun Ah

    2016-10-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

  19. Glutathione S-transferase 4 is a putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium discoideum.

    PubMed

    Kuwayama, Hidekazu; Kikuchi, Haruhisa; Oshima, Yoshiteru; Kubohara, Yuzuru

    2016-12-01

    In the development of the cellular slime mold Dictyostelium discoideum , two chlorinated compounds, the differentiation-inducing factors DIF-1 and DIF-2, play important roles in the regulation of both cell differentiation and chemotactic cell movement. However, the receptors of DIFs and the components of DIF signaling systems have not previously been elucidated. To identify the receptors for DIF-1 and DIF-2, we here performed DIF-conjugated affinity gel chromatography and liquid chromatography-tandem mass spectrometry and identified the glutathione S-transferase GST4 as a major DIF-binding protein. Knockout and overexpression mutants of gst4 ( gst4 - and gst4 OE , respectively) formed fruiting bodies, but the fruiting bodies of gst4 - cells were smaller than those of wild-type Ax2 cells, and those of gst4 OE cells were larger than those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell formation by DIFs in the gst4 mutants were similar to those of Ax2 cells. These results suggest that GST4 is a DIF-binding protein that regulates the sizes of cell aggregates and fruiting bodies in D. discoideum .

  20. Novel role for glutathione S-transferase pi. Regulator of protein S-Glutathionylation following oxidative and nitrosative stress.

    PubMed

    Townsend, Danyelle M; Manevich, Yefim; He, Lin; Hutchens, Steven; Pazoles, Christopher J; Tew, Kenneth D

    2009-01-02

    Glutathione S-transferase Pi (GSTpi) is a marker protein in many cancers and high levels are linked to drug resistance, even when the selecting drug is not a substrate. S-Glutathionylation of proteins is critical to cellular stress response, but characteristics of the forward reaction are not known. Our results show that GSTpi potentiates S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo. Mutational analysis indicated that the catalytic activity of GST is required. GSTpi is itself redox-regulated. S-Glutathionylation on Cys47 and Cys101 autoregulates GSTpi, breaks ligand binding interactions with c-Jun NH2-terminal kinase (JNK), and causes GSTpi multimer formation, all critical to stress response. Catalysis of S-glutathionylation at low pK cysteines in proteins is a novel property for GSTpi and may be a cause for its abundance in tumors and cells resistant to a range of mechanistically unrelated anticancer drugs.

  1. N-Glycan Modification in Aspergillus Species▿

    PubMed Central

    Kainz, Elke; Gallmetzer, Andreas; Hatzl, Christian; Nett, Juergen H.; Li, Huijuan; Schinko, Thorsten; Pachlinger, Robert; Berger, Harald; Reyes-Dominguez, Yazmid; Bernreiter, Andreas; Gerngross, Tillmann; Wildt, Stefan; Strauss, Joseph

    2008-01-01

    The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi. PMID:18083888

  2. Pleiotropic Functions of Glutathione S-Transferase P

    PubMed Central

    Zhang, Jie; Grek, Christina; Ye, Zhi-Wei; Manevich, Yefim; Tew, Kenneth D.; Townsend, Danyelle M.

    2016-01-01

    Glutathione S-transferase P (GSTP) is one member of the GST superfamily that is prevalently expressed in mammals. Known to possess catalytic activity through deprotonating glutathione allowing formation of thioether bonds with electrophilic substrates, more recent discoveries have broadened our understanding of the biological roles of this protein. In addition to catalytic detoxification, other properties so far ascribed to GSTP include chaperone functions, regulation of nitric oxide pathways, regulation of a variety of kinase signaling pathways, and participation in the forward reaction of protein S-glutathionylation. The expression of GSTP has been linked with cancer and other human pathologies and more recently even with drug addiction. With respect to human health, polymorphic variants of GSTP may determine individual susceptibility to oxidative stress and/or be critical in the design and development of drugs that have used redox pathways as a discovery platform. PMID:24974181

  3. Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity[S

    PubMed Central

    Bindesbøll, Christian; Fan, Qiong; Nørgaard, Rikke C.; MacPherson, Laura; Ruan, Hai-Bin; Wu, Jing; Pedersen, Thomas Å.; Steffensen, Knut R.; Yang, Xiaoyong; Matthews, Jason; Mandrup, Susanne; Nebb, Hilde I.; Grønning-Wang, Line M.

    2015-01-01

    Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/β+/+ and LXRα/β−/− mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPβ. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity. PMID:25724563

  4. Conditions Inducing Excessive O-GlcNAcylation Inhibit BMP2-Induced Osteogenic Differentiation of C2C12 Cells.

    PubMed

    Gu, Hanna; Song, Mina; Boonanantanasarn, Kanitsak; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

    2018-01-09

    Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O -linked-β- N -acetylglucosamine glycosylation ( O -GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O -GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N -acetylglucosamine concentrations or by O -GlcNAc transferase (OGT) overexpression. The effect of O -GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N -acetylglucosamine increased O -GlcNAcylation of Runx2 and the total levels of O -GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O -GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing β- N -acetylglucosaminidase. Our findings suggest that excessive protein O -GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.

  5. Glutathione S-transferase class mu regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries

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

    Bhattacharya, Poulomi; Madden, Jill A.; Sen, Nivedita

    2013-02-15

    4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied.more » Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2–8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P < 0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P < 0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P < 0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P < 0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1. - Highlights: ► GSTM protein increases in response to ovarian VCD exposure. ► VCD increases Ask1 mRNA at the onset of follicle loss. ► Ovarian GSTM binds more ASK1 protein during VCD-induced ovotoxicity. ► PI3K regulates ovarian GSTM protein.« less

  6. Down-regulation of glutatione S-transferase α 4 (hGSTA4) in the muscle of thermally injured patients is indicative of susceptibility to bacterial infection

    PubMed Central

    Apidianakis, Yiorgos; Que, Yok-Ai; Xu, Weihong; Tegos, George P.; Zimniak, Piotr; Hamblin, Michael R.; Tompkins, Ronald G.; Xiao, Wenzhong; Rahme, Laurence G.

    2012-01-01

    Patients with severe burns are highly susceptible to bacterial infection. While immunosuppression facilitates infection, the contribution of soft tissues to infection beyond providing a portal for bacterial entry remains unclear. We showed previously that glutathione S-transferase S1 (gstS1), an enzyme with conjugating activity against the lipid peroxidation byproduct 4-hydroxynonenal (4HNE), is important for resistance against wound infection in Drosophila muscle. The importance of the mammalian functional counterpart of GstS1 in the context of wounds and infection has not been investigated. Here we demonstrate that the presence of a burn wound dramatically affects expression of both human (hGSTA4) and mouse (mGsta4) 4HNE scavengers. hGSTA4 is down-regulated significantly within 1 wk of thermal burn injury in the muscle and fat tissues of patients from the large-scale collaborative Inflammation and the Host Response to Injury multicentered study. Similarly, mGsta4, the murine GST with the highest catalytic efficiency for 4HNE, is down-regulated to approximately half of normal levels in mouse muscle immediately postburn. Consequently, 4HNE protein adducts are increased 4- to 5-fold in mouse muscle postburn. Using an open wound infection model, we show that deletion of mGsta4 renders mice more susceptible to infection with the prevalent wound pathogen Pseudomonas aeruginosa, while muscle hGSTA4 expression negatively correlates with burn wound infection episodes per patient. Our data suggest that hGSTA4 down-regulation and the concomitant increase in 4HNE adducts in human muscle are indicative of susceptibility to infection in individuals with severely thermal injuries.—Apidianakis, Y., Que, Y.-A., Xu, W., Tegos, G. P., Zimniak, P., Hamblin, M. R., Tompkins, R. G., Xiao, W., Rahme, L. G. Down-regulation of glutatione S-transferase α 4 (hGSTA4) in the muscle of thermally injured patients is indicative of susceptibility to bacterial infection. PMID:22038048

  7. Protein O-GlcNAcylation: emerging mechanisms and functions

    PubMed Central

    Yang, Xiaoyong; Qian, Kevin

    2017-01-01

    O-GlcNAcylation—the attachment of O-linked N-acetylglucosamine (O-GlcNAc) moieties to cytoplasmic, nuclear and mitochondrial proteins—is a post-translational modification that regulates fundamental cellular processes in metazoans. A single pair of enzymes—O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA)—controls the dynamic cycling of this post-translational modification in a nutrient- and stress-responsive manner. Recent years have seen remarkable advances in our understanding of O-GlcNAcylation at levels ranging from structural and molecular biology to cell signalling and gene regulation to physiology and disease. Emerging from these recent developments are new mechanisms and functions of O-GlcNAcylation that enable us to begin constructing a unified conceptual framework through which to understand the significance of this modification in cellular and organismal physiology. PMID:28488703

  8. Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity.

    PubMed

    Shi, Hao; Munk, Alexander; Nielsen, Thomas S; Daughtry, Morgan R; Larsson, Louise; Li, Shize; Høyer, Kasper F; Geisler, Hannah W; Sulek, Karolina; Kjøbsted, Rasmus; Fisher, Taylor; Andersen, Marianne M; Shen, Zhengxing; Hansen, Ulrik K; England, Eric M; Cheng, Zhiyong; Højlund, Kurt; Wojtaszewski, Jørgen F P; Yang, Xiaoyong; Hulver, Matthew W; Helm, Richard F; Treebak, Jonas T; Gerrard, David E

    2018-05-01

    Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle. We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2). Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

  9. Effect of mannosamine on the formation of lipid-linked oligosaccharides and glycoproteins in canine kidney cells

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

    Pan, Y.T.; Elbein, A.D.

    1985-11-01

    Madin-Darby canine kidney (MDCK) cells normally form lipid-linked oligosaccharides having mostly the Glc3Man9GlcNAc2 oligosaccharide. However, when MDCK cells are incubated in 1 to 10 mM mannosamine and labeled with (2-/sup 3/H)mannose, the major oligosaccharides associated with the dolichol were Man5GlcNAc2 and Man6GlcNAc2 structures. Since both of these oligosaccharides were susceptible to digestion by endo-beta-N-acetylglucosaminidase H, the Man5GlcNAc2 must be different in structure than the Man5GlcNAc2 usually found as a biosynthetic intermediate in the lipid-linked oligosaccharides. Since pulse chase studies indicated that the lesion was in biosynthesis, it appears that mannosamine inhibits the in vivo formation of lipid-linked oligosaccharides perhaps bymore » inhibiting the alpha-1,2-mannosyl transferases. Although the lipid-linked oligosaccharides produced in the presence of mannosamine were smaller in size than those of control cells and did not contain glucose, the oligosaccharides were still transferred in vivo to protein. Furthermore, the oligosaccharide portions of the glycoproteins were still processed as shown by the fact that the glycopeptides were of the complex and hybrid types and were labeled with (/sup 3/H)mannose or (/sup 3/H)galactose.« less

  10. Downregulation of glutathione S-transferase pi in asthma contributes to enhanced oxidative stress.

    PubMed

    Schroer, Kathy T; Gibson, Aaron M; Sivaprasad, Umasundari; Bass, Stacey A; Ericksen, Mark B; Wills-Karp, Marsha; Lecras, Tim; Fitzpatrick, Anne M; Brown, Lou Ann S; Stringer, Keith F; Hershey, Gurjit K Khurana

    2011-09-01

    Glutathione S-transferase pi (GSTPi) is the predominant redox regulator in the lung. Although evidence implicates an important role for GSTPi in asthma, the mechanism for this has remained elusive. We sought to determine how GSTPi is regulated in asthma and to elucidate its role in maintaining redox homeostasis. We elucidated the regulation of GSTPi in children with asthma and used murine models of asthma to determine the role of GSTPi in redox homeostasis. Our findings demonstrate that GSTPi transcript levels are markedly downregulated in allergen- and IL-13-treated murine models of asthma through signal transducer and activator of transcription 6-dependent and independent pathways. Nuclear factor erythroid 2-related factor 2 was also downregulated in these models. The decrease in GSTPi expression was associated with decreased total glutathione S-transferase activity in the lungs of mice. Examination of cystine intermediates uncovered a functional role for GSTPi in regulating cysteine oxidation, whereby GSTPi-deficient mice exhibited increased oxidative stress (increase in percentage cystine) compared with wild-type mice after allergen challenge. GSTPi expression was similarly downregulated in children with asthma. These data collectively suggest that downregulation of GSTPi after allergen challenge might contribute to the asthma phenotype because of disruption of redox homeostasis and increased oxidative stress. Furthermore, GSTPi might be an important therapeutic target for asthma, and evaluation of GSTPi expression might prove beneficial in identifying patients who would benefit from therapy targeting this pathway. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  11. 21 CFR 862.1535 - Ornithine carbamyl transferase test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Test Systems § 862.1535 Ornithine carbamyl transferase test system. (a) Identification. An ornithine carbamyl transferase test system is a device intended to measure the activity of the enzyme ornithine... and treatment of liver diseases, such as infectious hepatitis, acute cholecystitis (inflammation of...

  12. Elevated O-GlcNAcylation promotes gastric cancer cells proliferation by modulating cell cycle related proteins and ERK 1/2 signaling.

    PubMed

    Jiang, Mingzuo; Qiu, Zhaoyan; Zhang, Song; Fan, Xing; Cai, Xiqiang; Xu, Bing; Li, Xiaowei; Zhou, Jinfeng; Zhang, Xiangyuan; Chu, Yi; Wang, Weijie; Liang, Jie; Horvath, Tamas; Yang, Xiaoyong; Wu, Kaichun; Nie, Yongzhan; Fan, Daiming

    2016-09-20

    O-GlcNAc transferase (OGT) is the only enzyme in mammals that catalyzes the attachment of β-D-N-acetylglucosamine (GlcNAc) to serine or threonine residues of target proteins. Hyper-O-GlcNAcylation is becoming increasingly realized as a general feature of cancer and contributes to rapid proliferation of cancer cells. In this study, we demonstrated that O-GlcNAc and OGT levels were increased in all six gastric cancer (GC) cell lines as compared with immortal gastric epithelial cells. Downregulation of the O-GlcNAcylation level by silencing OGT inhibited cell viability and growth rate via the cdk-2, cyclin D1 and ERK 1/2 pathways. In vivo xenograft assays also demonstrated that the hyper-O-GlcNAc level markedly promoted the proliferation of tumors. Moreover, compared with noncancerous tissues, the O-GlcNAcylation level was increased in cancerous tissues. GC patients with higher levels of O-GlcNAcylation exhibited large tumor sizes (≥5 cm), deep tumor invasion (T3 and T4), high AJCC stages (stage III and IV), more lymph node metastases and lower overall survival. Notably, the phosphorylation level of ERK 1/2 was increased progressively with the increase of O-GlcNAcylation in both SGC 7901 and AGS cells. Consistently, human GC tissue arrays also revealed that ERK 1/2 signaling was positively correlated to O-GlcNAcylation (r = 0.348; P = 0.015). Taken together, here we reported that hyper-O-GlcNAcylation significantly promotes GC cells proliferation by modulating cell cycle related proteins and ERK 1/2 signaling, suggesting that inhibition of OGT may be a potential novel therapeutic target of GC.

  13. O-GlcNAcylation of eIF2α regulates the phospho-eIF2α-mediated ER stress response.

    PubMed

    Jang, Insook; Kim, Han Byeol; Seo, Hojoong; Kim, Jin Young; Choi, Hyeonjin; Yoo, Jong Shin; Kim, Jae-woo; Cho, Jin Won

    2015-08-01

    O-GlcNAcylation is highly involved in cellular stress responses including the endoplasmic reticulum (ER) stress response. For example, glucosamine-induced flux through the hexosamine biosynthetic pathway can promote ER stress and ER stress inducers can change the total cellular level of O-GlcNAcylation. However, it is largely unknown which component(s) of the unfolded protein response (UPR) is directly regulated by O-GlcNAcylation. In this study, eukaryotic translation initiation factor 2α (eIF2α), a major branch of the UPR, was O-GlcNAcylated at Ser 219, Thr 239, and Thr 241. Upon ER stress, eIF2α is phosphorylated at Ser 51 by phosphorylated PKR-like ER kinase and this inhibits global translation initiation, except for that of specific mRNAs, including activating transcription factor 4, that induce stress-responsive genes such as C/EBP homologous protein (CHOP). Hyper-O-GlcNAcylation induced by O-GlcNAcase inhibitor (thiamet-G) treatment or O-GlcNAc transferase (OGT) overexpression hindered phosphorylation of eIF2α at Ser 51. The level of O-GlcNAcylation of eIF2α was changed by dithiothreitol treatment dependent on its phosphorylation at Ser 51. Point mutation of the O-GlcNAcylation sites of eIF2α increased its phosphorylation at Ser 51 and CHOP expression and resulted in increased apoptosis upon ER stress. These results suggest that O-GlcNAcylation of eIF2α affects its phosphorylation at Ser 51 and influences CHOP-mediated cell death. This O-GlcNAcylation of eIF2α was reproduced in thiamet-G-injected mouse liver. In conclusion, proper regulation of O-GlcNAcylation and phosphorylation of eIF2α is important to maintain cellular homeostasis upon ER stress. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. The potato suberin feruloyl transferase FHT which accumulates in the phellogen is induced by wounding and regulated by abscisic and salicylic acids

    PubMed Central

    Soler, Marçal; Molinas, Marisa; Figueras, Mercè

    2013-01-01

    The present study provides new insights on the role of the potato (Solanum tuberosum) suberin feruloyl transferase FHT in native and wound tissues, leading to conclusions about hitherto unknown properties of the phellogen. In agreement with the enzymatic role of FHT, it is shown that its transcriptional activation and protein accumulation are specific to tissues that undergo suberization such as the root boundary layers of the exodermis and the endodermis, along with the tuber periderm. Remarkably, FHT expression and protein accumulation within the periderm is restricted to the phellogen derivative cells with phellem identity. FHT levels in the periderm are at their peak near harvest during periderm maturation, with the phellogen becoming meristematically inactive and declining thereafter. However, periderm FHT levels remain high for several months after harvest, suggesting that the inactive phellogen retains the capacity to synthesize ferulate esters. Tissue wounding induces FHT expression and the protein accumulates from the first stages of the healing process onwards. FHT is up-regulated by abscisic acid and down-regulated by salicylic acid, emphasizing the complex regulation of suberin synthesis and wound healing. These findings open up new prospects important for the clarification of the suberization process and yield important information with regard to the skin quality of potatoes. PMID:23918964

  15. A model to environmental monitoring based on glutathione-S-transferase activity and branchial lesions in catfish

    NASA Astrophysics Data System (ADS)

    Neta, Raimunda Nonata Fortes Carvalho; Torres, Audalio Rebelo

    2017-11-01

    In this work, we validate the glutathione-S-transferase and branchial lesions as biomarkers in catfish Sciades herzbergii to obtain a predictive model of the environmental impact effects in a harbor of Brazil. The catfish were sampled from a port known to be contaminated with heavy metals and organic compounds and from a natural reserve in São Marcos Bay, Maranhão. Two biomarkers, hepatic glutathione S-transferase (GST) activity and branchial lesions were analyzed. The values for GST activity were modeled with the occurrence of branchial lesions by fitting a third order polynomial. Results from the mathematical model indicate that GST activity has a strong polynomial relationship with the occurrence of branchial lesions in both the wet and the dry seasons, but only at the polluted port site. Our mathematic model indicates that when the GST ceases to act, serious branchial lesions are observed in the catfish of the contaminated port area.

  16. 21 CFR 862.1535 - Ornithine carbamyl transferase test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Test Systems § 862.1535 Ornithine carbamyl transferase test system. (a) Identification. An ornithine...

  17. 21 CFR 862.1535 - Ornithine carbamyl transferase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Test Systems § 862.1535 Ornithine carbamyl transferase test system. (a) Identification. An ornithine...

  18. Polymorphisms of glutathione S-transferase Mu 1, glutathione S-transferase theta 1 and glutathione S-transferase Pi 1 genes in Hodgkin's lymphoma susceptibility and progression.

    PubMed

    Lourenço, Gustavo J; Néri, Iramaia A; Sforni, Vitor C S; Kameo, Rodolfo; Lorand-Metze, Irene; Lima, Carmen S P

    2009-06-01

    We tested in this study whether the polymorphisms of the glutathione S-transferase Mu1 (GSTM1), glutathione S-transferase Theta 1 (GSTT1) and glutathione S-transferase Pi 1 (GSTP1), involved in metabolism of chemical agents, cell proliferation and cell survival, alter the risk for Hodgkin lymphoma (HL). Genomic DNA from 110 consecutive patients with HL and 226 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. Similar frequencies of the GSTM1 and GSTT1 genotypes were seen in patients and controls. In contrast, the frequency of the GSTP1 wild genotype (59.1%versus 36.3%, P = 0.004) was higher in patients than in controls. Individuals with the wild genotype had a 2.68 (95%CI: 1.38-5.21)-fold increased risk for the disease than others. An excess of the GSTP1 wild genotype was also observed in patients with tumors of stages III + IV when compared with those with tumors of stages I + II (39.1%versus 20.0%, P = 0.03). These results suggest that the wild allele of the GSTP1 gene is linked to an increased risk and high aggressiveness of the HL in our cases but they should be confirmed by further studies with larger cohorts of patients and controls.

  19. Stressful life events and catechol-O-methyl-transferase (COMT) gene in bipolar disorder.

    PubMed

    Hosang, Georgina M; Fisher, Helen L; Cohen-Woods, Sarah; McGuffin, Peter; Farmer, Anne E

    2017-05-01

    A small body of research suggests that gene-environment interactions play an important role in the development of bipolar disorder. The aim of the present study is to contribute to this work by exploring the relationship between stressful life events and the catechol-O-methyl-transferase (COMT) Val 158 Met polymorphism in bipolar disorder. Four hundred eighty-two bipolar cases and 205 psychiatrically healthy controls completed the List of Threatening Experiences Questionnaire. Bipolar cases reported the events experienced 6 months before their worst depressive and manic episodes; controls reported those events experienced 6 months prior to their interview. The genotypic information for the COMT Val 158 Met variant (rs4680) was extracted from GWAS analysis of the sample. The impact of stressful life events was moderated by the COMT genotype for the worst depressive episode using a Val dominant model (adjusted risk difference = 0.09, 95% confidence intervals = 0.003-0.18, P = .04). For the worst manic episodes no significant interactions between COMT and stressful life events were detected. This is the first study to explore the relationship between stressful life events and the COMT Val 158 Met polymorphism focusing solely on bipolar disorder. The results of this study highlight the importance of the interplay between genetic and environmental factors for bipolar depression. © 2017 Wiley Periodicals, Inc.

  20. Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

    PubMed Central

    Kapuria, Vaibhav; Röhrig, Ute F.; Bhuiyan, Tanja; Borodkin, Vladimir S.; van Aalten, Daan M.F.; Zoete, Vincent; Herr, Winship

    2016-01-01

    In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc), O-linked-GlcNAc transferase (OGT) catalyzes Ser/Thr O-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase–protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase–protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT. PMID:27056667

  1. Skp1 prolyl 4-hydroxylase of dictyostelium mediates glycosylation-independent and -dependent responses to O2 without affecting Skp1 stability.

    PubMed

    Zhang, Dongmei; van der Wel, Hanke; Johnson, Jennifer M; West, Christopher M

    2012-01-13

    Cytoplasmic prolyl 4-hydroxylases (PHDs) have a primary role in O(2) sensing in animals via modification of the transcriptional factor subunit HIFα, resulting in its polyubiquitination by the E3(VHL)ubiquitin (Ub) ligase and degradation in the 26 S proteasome. Previously thought to be restricted to animals, a homolog (P4H1) of HIFα-type PHDs is expressed in the social amoeba Dictyostelium where it also exhibits characteristics of an O(2) sensor for development. Dictyostelium lacks HIFα, and P4H1 modifies a different protein, Skp1, an adaptor of the SCF class of E3-Ub ligases related to the E3(VHL)Ub ligase that targets animal HIFα. Normally, the HO-Skp1 product of the P4H1 reaction is capped by a GlcNAc sugar that can be subsequently extended to a pentasaccharide by novel glycosyltransferases. To analyze the role of glycosylation, the Skp1 GlcNAc-transferase locus gnt1 was modified with a missense mutation to block catalysis or a stop codon to truncate the protein. Despite the accumulation of the hydroxylated form of Skp1, Skp1 was not destabilized based on metabolic labeling. However, hydroxylation alone allowed for partial correction of the high O(2) requirement of P4H1-null cells, therefore revealing both glycosylation-independent and glycosylation-dependent roles for hydroxylation. Genetic complementation of the latter function required an enzymatically active form of Gnt1. Because the effect of the gnt1 deficiency depended on P4H1, and Skp1 was the only protein labeled when the GlcNAc-transferase was restored to mutant extracts, Skp1 apparently mediates the cellular functions of both P4H1 and Gnt1. Although Skp1 stability itself is not affected by hydroxylation, its modification may affect the stability of targets of Skp1-dependent Ub ligases.

  2. Glutathione S-transferase pi expression regulates the Nrf2-dependent response to hormetic diselenides.

    PubMed

    Bartolini, D; Commodi, J; Piroddi, M; Incipini, L; Sancineto, L; Santi, C; Galli, F

    2015-11-01

    Glutathione S-transferase pi (GSTP), a phase II gene downstream of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant-responsive element (ARE)/electrophile response element (EpRE) transcription pathway, plays a key role in both the signaling and detoxification response to Se-organic compounds with thiol peroxidase activity. We here investigated the role of GSTP on the Nrf2 activation response of cells challenged with a new class of diselenides derived from the basic structure of diphenyl diselenide [(PhSe)2]. These diselenides, and particularly 2,2'-diselenyl dibenzoic acid (DSBA), behave as mild thiol peroxidases leading to a moderate generation of H2O2 and NOx, and signaling of stress-activated and survival-promoting MAPKs, which ultimately control the mitochondrial pathway of apoptosis. Used in murine embryonic fibroblasts (MEFs) and HepG2 human hepatocarcinoma cells to produce submaximal conditions of stress, the diselenide compounds stimulated Nrf2 nuclear translocation and then the transcription of the same Nrf2 gene as well as of GSTP and other phase II genes. This resulted in a higher degree of protection against H2O2 cytotoxicity (hormetic effect). Diselenide toxicity increased in GSTP knockout MEFs by a higher generation of NOx and stress activated protein kinase (SAPK)/JNK activation. A lowered hormetic potential of these cells was observed in association with an abnormal expression and nuclear translocation of Nrf2 protein. Immunoprecipitation and affinity purification experiments revealed the existence of an Nrf2/GSTP complex in MEFs and HepG2 cells. Covalent oligomers of GSTP subunits were observed in DSBA-treated HepG2 cells. In conclusion, GSTP gene expression influences the Nrf2-dependent response to hormetic diselenides. Mechanistic interpretation for this GSTP-dependent effect may include a direct and redox-sensitive interaction of GSTP with Nrf2 protein. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Abiotic stress and phytohormones affect enzymic activity of 1-O-(indole-3-acetyl)-β-d-glucose: myo-inositol indoleacetyl transferase from rice (Oryza sativa).

    PubMed

    Ciarkowska, Anna; Ostrowski, Maciej; Jakubowska, Anna

    2016-10-20

    Indole-3-acetic acid (IAA) conjugation is a part of mechanism regulating free auxin concentration. 1-O-(indole-3-acetyl)-β-d-glucose: myo-inositol indoleacetyl transferase (IAInos synthase) is an enzyme involved in IAA-ester conjugates biosynthesis. Biotic and abiotic stress conditions can modulate auxin conjugates formation in plants. In this study, we investigated effect of plant hormones (IAA, ABA, SA and 2,4-D) and abiotic stress (drought and salt stress: 150mM NaCl and 300mM NaCl) on expression level and catalytic activity of rice IAInos synthase. Enzymic activity assay indicated that all tested phytohormones affected activity of IAInos synthase, but only ABA had inhibiting effect, while IAA, SA and 2,4-D activated the enzyme. Drought and salt stress induced with lower NaCl concentration resulted in decreased activity of IAInos synthase, but 300mM NaCl had no effect on the enzyme. Despite observed differences in enzymic activities, no changes of expression level, tested by semiquantitative RT-PCR and Western blot, were detected. Based on our results it has been supposed that plant hormones and stress conditions affect IAInos synthase activity on posttranslational level. Copyright © 2016 Elsevier GmbH. All rights reserved.

  4. Dishevelled3 is a novel arginine methyl transferase substrate.

    PubMed

    Bikkavilli, Rama Kamesh; Avasarala, Sreedevi; Vanscoyk, Michelle; Sechler, Marybeth; Kelley, Nicole; Malbon, Craig C; Winn, Robert A

    2012-01-01

    Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient mutant of Dishevelled, R271K, displayed spontaneous membrane localization and robust activation of Wnt signaling; suggesting that differential methylation of Dishevelled plays an important role in Wnt signaling. Thus arginine methylation is shown to be an important switch in regulation of Dishevelled function and Wnt signaling.

  5. ROLE OF STEROID HORMONES AND DECIDUAL INDUCTION IN THE REGULATION OF ADENOSINE DIPHOSPHORIBOSYL TRANSFERASE ACTIVITY IN RAT ENDOMETRIUM

    EPA Science Inventory

    To assess the effect of ovarian steroid hormones on enzyme activity, adenosine diphosphoribosyl transferase (ADPRT) was measured in endometrial nuclei isolated on estrus and on d 4 from rats ovariectomized on estrus (d 0) and treated d 0-3 with (a) vehicle, (b) 1 ug estrone/d (E)...

  6. Retinal O-linked N-acetylglucosamine protein modifications: implications for postnatal retinal vascularization and the pathogenesis of diabetic retinopathy

    PubMed Central

    Sieg, Kelsey M.; Shallow, Keegan D.; Sorenson, Christine M.; Sheibani, Nader

    2013-01-01

    Purpose Hyperglycemia activates several metabolic pathways, including the hexosamine biosynthetic pathway. Uridine diphosphate N-acetylglucosamine (GlcNAc) is the product of the hexosamine biosynthetic pathway and the substrate for O-linked GlcNAc (O-GlcNAc) modification. This modification affects a wide range of proteins by altering their activity, cellular localization, and/or protein interactions. However, the role O-GlcNAcylation may play in normal postnatal retinal vascular development and in the ocular complications of diabetes, including diabetic retinopathy, requires further investigation. Methods The total levels of O-GlcNAc-modified proteins were evaluated by western blot analysis of lysates prepared from retinas obtained at different days during postnatal retinal vascularization and oxygen-induced ischemic retinopathy. Similar experiments were performed with retinal lysate prepared from diabetic Ins2Akita/+ mice with different durations of diabetes and retinal vascular cells cultured under various glucose conditions. The localization of O-GlcNAc-modified proteins in the retinal vasculature was confirmed by immunofluorescence staining. The impact of altered O-GlcNAcylation on the migration of retinal vascular cells was determined using scratch wound and transwell migration assays. Results We detected an increase in protein O-GlcNAcylation during mouse postnatal retinal vascularization and aging, in part through the regulation of the enzymes that control this modification. The study of the diabetic Ins2Akita/+ mouse retina showed an increase in the O-GlcNAc modification of retinal proteins. We also observed an increase in retinal O-GlcNAcylated protein levels during the neovascularization phase of oxygen-induced ischemic retinopathy. Our fluorescence microscopy data confirmed that the alterations in retinal O-GlcNAcylation are similarly represented in the retinal vasculature and in retinal pericytes and endothelial cells. Particularly, the migration of

  7. Heterologous expression and characterization of processing α-glucosidase I from Aspergillus brasiliensis ATCC 9642.

    PubMed

    Miyazaki, Takatsugu; Matsumoto, Yuji; Matsuda, Kana; Kurakata, Yuma; Matsuo, Ichiro; Ito, Yukishige; Nishikawa, Atsushi; Tonozuka, Takashi

    2011-12-01

    A gene for processing α-glucosidase I from a filamentous fungus, Aspergillus brasiliensis (formerly called Aspergillus niger) ATCC 9642 was cloned and fused to a glutathione S-transferase tag. The active construct with the highest production level was a truncation mutant deleting the first 16 residues of the hydrophobic N-terminal domain. This fusion enzyme hydrolyzed pyridylaminated (PA-) oligosaccharides Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA and the products were identified as Glc(2)Man(9)GlcNAc(2)-PA and Glc(2)Man(4)-PA, respectively. Saturation curves were obtained for both Glc(3)Man(9)GlcNAc(2)-PA and Glc(3)Man(4)-PA, and the K (m) values for both substrates were estimated in the micromolar range. When 1 μM Glc(3)Man(4)-PA was used as a substrate, the inhibitors kojibiose and 1-deoxynojirimycin had similar effects on the enzyme; at 20 μM concentration, both inhibitors reduced activity by 50%. © Springer Science+Business Media, LLC 2011

  8. Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

    PubMed

    Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

    2005-01-01

    The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.

  9. Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

    PubMed Central

    Danielson, U H; Esterbauer, H; Mannervik, B

    1987-01-01

    The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism. PMID:3426557

  10. The level of circulating octanoate does not predict ghrelin O-acyl transferase (GOAT)-mediated acylation of ghrelin during fasting.

    PubMed

    Nass, Ralf; Nikolayev, Alexander; Liu, Jianhua; Pezzoli, Suzan S; Farhy, Leon S; Patrie, James; Gaylinn, Bruce D; Heiman, Mark; Thorner, Michael O

    2015-01-01

    Acyl-ghrelin is a 28-amino acid peptide released from the stomach. Ghrelin O-acyl transferase (GOAT) attaches an 8-carbon medium-chain fatty acid (MCFA) (octanoate) to serine 3 of ghrelin. This acylation is necessary for the activity of ghrelin. Animal data suggest that MCFAs provide substrate for GOAT and an increase in nutritional octanoate increases acyl-ghrelin. To address the question of the source of substrate for acylation, we studied whether the decline in ghrelin acylation during fasting is associated with a decline in circulating MCFAs. Eight healthy young men (aged 18-28 years, body mass index range, 20.6-26.2 kg/m(2)) had blood drawn every 10 minutes for acyl- and desacyl-ghrelin and every hour for free fatty acids (FFAs) during the last 24 hours of a 61.5-hour fast and during a fed day. FFAs were measured by a highly sensitive liquid chromatography-mass spectroscopy method. Acyl- and desacyl-ghrelin were measured in an in-house assay; the results were published previously. Ghrelin acylation was assessed by the ratio of acyl-ghrelin to total ghrelin. With the exception of MCFAs C8 and C10, all other FFAs, the MCFAs (C6 and C12), and the long-chain fatty acids (C14-C18) significantly increased with fasting (P < .05). There was no significant association between the fold change in ghrelin acylation and circulating FFAs. These results suggest that changes in circulating MCFAs are not linked to the decline in ghrelin acylation during fasting and support the hypothesis that acylation of ghrelin depends at least partially on the availability of gastroluminal MCFAs or the regulation of GOAT activity.

  11. Identification of O-linked β-d-N-acetylglucosamine-Modified Proteins from Arabidopsis

    PubMed Central

    Xu, Shou-Ling; Chalkley, Robert J.; Wang, Zhi-Yong; Burlingame, Alma L.

    2013-01-01

    The posttranslational modification of proteins with O-linked β-d-N-acetylglucosamine (O-GlcNAc) on serine and threonine residues occurs in all animals and plants. This modification is dynamic and ubiquitous, and regulates many cellular processes, including transcription, signaling and cytokinesis and is associated with several diseases. Cycling of O-GlcNAc is tightly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Plants have two OGTs, SPINDLY (SPY) and SECRET AGENT (SEC); disruption of both causes embryo lethality. Despite O-GlcNAc modification of proteins being discovered more than 20-years ago, identification and mapping of protein GlcNAcylation is still a challenging task. Here we describe the use of lectin affinity chromatography combined with electron transfer dissociation mass spectrometry to enrich and to detect O-GlcNAc modified peptides from Arabidopsis. PMID:22576084

  12. Partial purification and characterization of a mannosyl transferase involved in O -linked mannosylation of glycoproteins in Candida albicans.

    PubMed

    Arroyo-Flores, Blanca L; Calvo-Méndez, Carlos; Flores-Carreón, Arturo; López-Romero, Everardo

    2004-04-01

    Incubation of a mixed membrane fraction of C. albicans with the nonionic detergents Nonidet P-40 or Lubrol solubilized a fraction that catalyzed the transfer of mannose either from endogenously generated or exogenously added dolichol-P-[14C]Man onto endogenous protein acceptors. The protein mannosyl transferase solubilized with Nonidet P-40 was partially purified by a single step of preparative nondenaturing electrophoresis and some of its properties were investigated. Although transfer activity occurred in the absence of exogenous mannose acceptors and thus depended on acceptor proteins isolated along with the enzyme, addition of the protein fraction obtained after chemical de-mannosylation of glycoproteins synthesized in vitro stimulated mannoprotein labeling in a concentration-dependent manner. Other de-mannosylated glycoproteins, such as yeast invertase or glycoproteins extracted from C. albicans, failed to increase the amount of labeled mannoproteins. Mannosyl transfer activity was not influenced by common metal ions such as Mg(2+), Mn(2+) and Ca(2+), but it was stimulated up to 3-fold by EDTA. Common phosphoglycerides such as phosphatidylglycerol and, to a lower extent, phosphatidylinositol and phosphatidylcholine enhanced transfer activity. Interestingly, coupled transfer activity between dolichol phosphate mannose synthase, i.e., the enzyme responsible for Dol-P-Man synthesis, and protein mannosyl transferase could be reconstituted in vitro from the partially purified transferases, indicating that this process can occur in the absence of cell membranes.

  13. Structure of a Novel O-Linked N-Acetyl-d-glucosamine (O-GlcNAc) Transferase, GtfA, Reveals Insights into the Glycosylation of Pneumococcal Serine-rich Repeat Adhesins*

    PubMed Central

    Shi, Wei-Wei; Jiang, Yong-Liang; Zhu, Fan; Yang, Yi-Hu; Shao, Qiu-Yan; Yang, Hong-Bo; Ren, Yan-Min; Wu, Hui; Chen, Yuxing; Zhou, Cong-Zhao

    2014-01-01

    Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a β-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-α-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides. PMID:24936067

  14. Targeting Protein O-GlcNAc Modifications In Breast Cancer

    DTIC Science & Technology

    2010-09-30

    O-GlcNAcation and elevated expression of O-GlcNAc transferase (OGT), the enzyme catalyzing addition of O-GlcNAc to proteins. Reduction of O...regulatory switch mechanism analogous to phosphorylation (28). Cytosolic and nuclear enzymes dynamically catalyze addition (O-GlcNAc transferase or OGT) and...levels, through pharmacological inhibition or genetic knock-down of enzymes that add or remove O-GlcNAc, can inhibit ErbB2-mediated oncogenic

  15. Spectrofluorimetric assay method for glutathione and glutathione transferase using monobromobimane.

    PubMed

    Yakubu, S I; Yakasai, I A; Musa, A

    2011-06-01

    The primary role of glutathione transferase is to defend an organism from toxicities through catalyzing the reaction of glutathione (GSH) with potentially toxic compounds or metabolites to their chemically and biologically inert conjugates. The objective of the study was to develop a simple and sensitive spectrofluorimetric assay method for glutathione transferase using monobromobimane (MBB), a non fluorescent compound with electrophilic site. MBB slowly reacted with glutathione to form fluorescent glutathione conjugate and that the reaction was catalysed by glutathione transferase. Both non-enzymatic and enzymatic reaction products of MBB, in presence of GSH in phosphate buffer (pH 6.5), were measured by following increase of fluorescence at wavelength of 475nm. For validation of the assay method, the kinetic parameters such as the apparent Michaelis-Mente constants and maximum rates of conjugate formation as well as the specific activity of rat hepatic glutathione transferase were determined. The method was found to be sensitive, thus, applied to measure glutathione contents of crude preparation of rat hepatic cytosol fraction.

  16. Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits.

    PubMed Central

    Singh, S V; Partridge, C A; Awasthi, Y C

    1984-01-01

    Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya' is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa') of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya' subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively. Images Fig. 1. Fig. 5. PMID:6433888

  17. The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

    PubMed Central

    Diccianni, M B; Imagawa, M; Muramatsu, M

    1992-01-01

    Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis. Images PMID:1408831

  18. The dyad palindromic glutathione transferase P enhancer binds multiple factors including AP1.

    PubMed

    Diccianni, M B; Imagawa, M; Muramatsu, M

    1992-10-11

    Glutathione Transferase P (GST-P) gene expression is dominantly regulated by an upstream enhancer (GPEI) consisting of a dyad of palindromically oriented imperfect TPA (12-O-tetradecanoyl-phorbol-13-acetate)-responsive elements (TRE). GPEI is active in AP1-lacking F9 cells as well in AP1-containing HeLa cells. Despite GPEI's similarity to a TRE, c-jun co-transfection has only a minimal effect on transactivation. Antisense c-jun and c-fos co-transfection experiments further demonstrate the lack of a role for AP1 in GPEI mediated trans-activation in F9 cells, although endogenously present AP1 can influence GPEI in HeLa cells. Co-transfection of delta fosB with c-jun, which forms an inactive c-Jun/delta FosB heterodimer that binds TRE sequences, inhibits GPEI-mediated transcription in AP1-lacking F9 cells as well as AP1-containing HeLa cells. These data suggest novel factor(s) other than AP1 are influencing GPEI. Binding studies reveal multiple nucleoproteins bind to GPEI. These factors are likely responsible for the high level of GPEI-mediated transcription observed in the absence of AP1 and during hepatocarcinogenesis.

  19. Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains.

    PubMed

    Li, Shan; Zhang, Li; Yao, Qing; Li, Lin; Dong, Na; Rong, Jie; Gao, Wenqing; Ding, Xiaojun; Sun, Liming; Chen, Xing; Chen, She; Shao, Feng

    2013-09-12

    The tumour necrosis factor (TNF) family is crucial for immune homeostasis, cell death and inflammation. These cytokines are recognized by members of the TNF receptor (TNFR) family of death receptors, including TNFR1 and TNFR2, and FAS and TNF-related apoptosis-inducing ligand (TRAIL) receptors. Death receptor signalling requires death-domain-mediated homotypic/heterotypic interactions between the receptor and its downstream adaptors, including TNFR1-associated death domain protein (TRADD) and FAS-associated death domain protein (FADD). Here we discover that death domains in several proteins, including TRADD, FADD, RIPK1 and TNFR1, were directly inactivated by NleB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to inhibit host nuclear factor-κB (NF-κB) signalling. NleB contained an unprecedented N-acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (Arg 235 in the TRADD death domain). NleB GlcNAcylation (the addition of GlcNAc onto a protein side chain) of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signalling in EPEC-infected cells, including NF-κB signalling, apoptosis and necroptosis. Type-III-delivered NleB also blocked FAS ligand and TRAIL-induced cell death by preventing formation of a FADD-mediated death-inducing signalling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification.

  20. S-Nitrosation destabilizes glutathione transferase P1-1.

    PubMed

    Balchin, David; Stoychev, Stoyan H; Dirr, Heini W

    2013-12-23

    Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.

  1. Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration

    PubMed Central

    Pajaud, J; Ribault, C; Ben Mosbah, I; Rauch, C; Henderson, C; Bellaud, P; Aninat, C; Loyer, P; Morel, F; Corlu, A

    2015-01-01

    Glutathione transferases (GST) are phase II enzymes catalyzing the detoxification of endogenous noxious compounds and xenobiotics. They also regulate phosphorylation activities of MAPKinases in a catalytic-independent manner. Previous studies have demonstrated the regulation of JNK-dependent pathway by GSTP1/2. Considering the crucial role of JNK in the early steps of the hepatocyte cell cycle, we sought to determine whether GSTP1/2 were essential for hepatocyte proliferation following partial hepatectomy (PH). Using a conventional double knockout mouse model for the Gstp1 and Gstp2 genes, we found that the lack of GSTP1/P2 reduced the rate of DNA replication and mitotic index during the first wave of hepatocyte proliferation. The lowered proliferation was associated with the decrease in TNFalpha and IL-6 plasma concentrations, reduced hepatic HGF expression and delayed and/or altered activation of STAT3, JNK and ERK1/2 signaling pathways. In addition, the expression and/or activation of cell cycle regulators such as Cyclin D1, CDK4, E2F1 and MCM7 was postponed demonstrating that the absence of GSTP1/2 delayed the entry into and progression through the G1 phase of the cell cycle and impaired the synchrony of proliferation in hepatocytes following PH. Furthermore, while JNK and its downstream targets c-Jun and ATF2 were activated during the early steps of the liver regeneration in wild-type animals, the constitutively active JNK found in the quiescent liver of Gstp1/2 knockout mice underwent a decrease in its activity after PH. Transient induction of antioxidant enzymes and nitric oxide synthase were also delayed or repressed during the regenerative response. Altogether our results demonstrate that GSTP1/2 are a critical regulators of hepatocyte proliferation in the initial phases of liver regeneration. PMID:25590808

  2. Studies on the synthesis and processing of the asparagine-linked carbohydrate units of glycoproteins.

    PubMed

    Spiro, R G; Spiro, M J

    1982-12-24

    It has become apparent in recent years from the work of a number of laboratories that the N-glycosylation of both membrane and secretory glycoproteins is effected by the transfer en bloc to nascent polypeptides of a glucose-containing oligosaccharide (Glc3Man9GlcNAc2) from a dolichyl pyrophosphoryl carrier; this is followed by a series of modifying reactions to yield the mature polymannose and complex asparagine-linked carbohydrate units. The enzymic steps involved in the assembly of the precursor oligosaccharide, its transfer to protein and its subsequent processing represent potential sites for the regulation of glycoprotein synthesis. Studies performed in our laboratory have dealt primarily with thyroid slices and particulate enzymes with special regard to the role of glucose in these events. Thyroglobulin, the major secretory glycoprotein of this tissue, has well defined complex and polymannose saccharide units, and indeed the most complete form of the latter (Man9GlcNAc2) has the same structure as the lipid-linked oligosaccharide without the glucose. Our studies indicate that effective N-glycosylation requires a complete glucose chain (Glc3) and that the glucose sequence is assembled from dolichol-P-glucose in a stepwise manner through the concerted action of at least two transferases in a fashion complementary to the subsequent excision of this sugar by glucosidases. Pulse-chase studies indicate that, after the transfer to protein, the removal of all three glucose residues as well as of the first mannose takes place in the endoplasmic reticulum and three additional mannoses are excised in the Golgi complex, because in the presence of an inhibitor of intracellular transport, carbonyl cyanide m-chlorophenylhydrazone (CCCP), there is a pronounced accumulation of protein-linked Man8GlcNAc2. Studies with metabolic inhibitors (CCCP, antimycin, N2) indicate that, under conditions of energy depletion, glucosylation of oligosaccharide-lipid is selectively impaired

  3. You are what you eat: O-linked N-acetylglucosamine in disease, development and epigenetics.

    PubMed

    Olivier-Van Stichelen, Stéphanie; Hanover, John A

    2015-07-01

    The O-linked N-acetylglucosamine (O-GlcNAc) modification is both responsive to nutrient availability and capable of altering intracellular cellular signalling. We summarize data defining a role for O-GlcNAcylation in metabolic homeostasis and epigenetic regulation of development in the intrauterine environment. O-GlcNAc transferase (OGT) catalyzes nutrient-driven O-GlcNAc addition and is subject to random X-inactivation. OGT plays key roles in growth factor signalling, stem cell biology, epigenetics and possibly imprinting. The O-GlcNAcase, which removes O-GlcNAc, is subject to tight regulation by higher order chromatin structure. O-GlcNAc cycling plays an important role in the intrauterine environment wherein OGT expression is an important biomarker of placental stress. Regulation of O-GlcNAc cycling by X-inactivation, epigenetic regulation and nutrient-driven processes makes it an ideal candidate for a nutrient-dependent epigenetic regulator of human disease. In addition, O-GlcNAc cycling influences chromatin modifiers critical to the regulation and timing of normal development including the polycomb repression complex and the ten-eleven translocation proteins mediating DNA methyl cytosine demethylation. The pathway also impacts the hypothalamic-pituitary-adrenal axis critical to intrauterine programming influencing disease susceptibility in later life.

  4. Hybrid molecule from O2-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid: a glutathione S-transferase π-activated nitric oxide prodrug with selective anti-human hepatocellular carcinoma activity and improved stability.

    PubMed

    Fu, Junjie; Liu, Ling; Huang, Zhangjian; Lai, Yisheng; Ji, Hui; Peng, Sixun; Tian, Jide; Zhang, Yihua

    2013-06-13

    A series of hybrids from O(2)-(2,4-dinitrophenyl)diazeniumdiolate and oleanolic acid (OA) were designed, synthesized, and biologically evaluated as novel nitric oxide (NO)-releasing prodrugs that could be activated by glutathione S-transferase π (GSTπ) overexpressed in a number of cancer cells. It was discovered that the most active compound, 21, released high levels of NO selectively in HCC cells but not in the normal cells and exhibited potent antiproliferative activity in vitro as well as remarkable tumor-retarding effects in vivo. Compared with the reported GSTπ-activated prodrugs JS-K and PABA/NO, 21 exhibited remarkably improved stability in the absence of GSTπ. Importantly, the decomposition of 21 occurred in the presence of GSTπ and was much more effective than in glutathione S-transferase α. Additionally, 21 induced apoptosis in HepG2 cells by arresting the cell cycle at the G2/M phase, activating both the mitochondrion-mediated pathway and the MAPK pathway and enhancing the intracellular production of ROS.

  5. The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A carbohydrate in Streptococcus pyogenes.

    PubMed

    Rush, Jeffrey S; Edgar, Rebecca J; Deng, Pan; Chen, Jing; Zhu, Haining; van Sorge, Nina M; Morris, Andrew J; Korotkov, Konstantin V; Korotkova, Natalia

    2017-11-24

    In many Lactobacillales species ( i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes , synthesizes a key antigenic surface polymer, the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N -acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry, and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphoryl-undecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltransferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

    PubMed

    Matsumoto, M; Imagawa, M; Aoki, Y

    2000-07-01

    Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.

  7. Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

    PubMed Central

    Matsumoto, M; Imagawa, M; Aoki, Y

    2000-01-01

    Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression. PMID:10861232

  8. Activation of Nrf2 is required for up-regulation of the π class of glutathione S-transferase in rat primary hepatocytes with L-methionine starvation.

    PubMed

    Lin, Ai-Hsuan; Chen, Haw-Wen; Liu, Cheng-Tze; Tsai, Chia-Wen; Lii, Chong-Kuei

    2012-07-04

    Numerous genes expression is regulated in response to amino acid shortage, which helps organisms adapt to amino acid limitation. The expression of the π class of glutathione (GSH) S-transferase (GSTP), a highly inducible phase II detoxification enzyme, is regulated mainly by activates activating protein 1 (AP-1) binding to the enhancer I of GSTP (GPEI). Here we show the critical role of nuclear factor erythroid-2-related factor 2 (Nrf2) in up-regulating GSTP gene transcription. Primary rat hepatocytes were cultured in a methionine-restricted medium, and immunoblotting and RT-PCR analyses showed that methionine restriction time-dependently increased GSTP protein and mRNA expression over a 48 h period. Nrf2 translocation to the nucleus, nuclear proteins binding to GPEI, and antioxidant response element (ARE) luciferase reporter activity were increased by methionine restriction as well as by l-buthionine sulfoximine (BSO), a GSH synthesis inhibitor. Transfection with Nrf2 siRNA knocked down Nrf2 expression and reversed the methionine-induced GSTP expression and GPEI binding activity. Chromatin immunoprecipitation assay confirmed the binding of Nrf2 to the GPEI. Phosphorylation of extracellular signal-regulated kinase 2 (ERK2) was increased in methionine-restricted and BSO-treated cells. ERK2 siRNA abolished methionine restriction-induced Nrf2 nuclear translocation, GPEI binding activity, ARE-luciferase reporter activity, and GSTP expression. Our results suggest that the up-regulation of GSTP gene transcription in response to methionine restriction likely occurs via the ERK-Nrf2-GPEI signaling pathway.

  9. Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms*

    PubMed Central

    Nakajima, Kazuki; Ito, Emi; Ohtsubo, Kazuaki; Shirato, Ken; Takamiya, Rina; Kitazume, Shinobu; Angata, Takashi; Taniguchi, Naoyuki

    2013-01-01

    Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using 13C6-glucose and 13C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with 13C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a 13C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using 13C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism. PMID:23720760

  10. Functional Characterization of Lpt3 and Lpt6, the Inner-Core Lipooligosaccharide Phosphoethanolamine Transferases from Neisseria meningitidis▿

    PubMed Central

    Wenzel, Cory Q.; St. Michael, Frank; Stupak, Jacek; Li, Jianjun; Cox, Andrew D.; Richards, James C.

    2010-01-01

    The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His6 and Lpt6-His6 were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro. PMID:19854897

  11. Effect of a glutathione S-transferase inhibitor on oxidative stress and ischemia-reperfusion-induced apoptotic signalling of cultured cardiomyocytes

    PubMed Central

    Röth, E; Marczin, N; Balatonyi, B; Ghosh, S; Kovács, V; Alotti, N; Borsiczky, B; Gasz, B

    2011-01-01

    Oxidative stress and ischemia-reperfusion (I/R) injury are crucial in the pathogenesis of cardiovascular diseases. The antioxidant glutathione S-transferase (GST) is responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. The main objective of the present study was to examine the effect of GST inhibition (with the administration of ethacrynic acid [EA]) on the viability and apoptosis of cardiomyocytes when these cells are exposed to various stress components of I/R and mitogen-activated protein kinase (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase [ERK]) inhibitors. The primary culture of neonatal rat cardiomyocytes was divided into six experimental groups: control group of cells (group 1), cells exposed to H2O2 (group 2), I/R (group 3), I/R and EA (group 4), H2O2 coupled with EA (group 5), and EA alone (group 6). The viability of cardiomyocytes was determined using a colorimetric MTT assay. The apoptosis ratio was evaluated via fluorescein isothiocyanate-labelled annexin V and propidium iodide staining. c-Jun N-terminal kinase, p38, Akt/protein kinase B and ERK/p42-p44 transcription factors were monitored with flow cytometry. c-Jun N-terminal kinase activation increased due to GST inhibition during I/R. EA administration led to a significant increase in p38 activation following both H2O2 treatment and I/R. ERK phosphorylation increased when GST was exposed to I/R. A pronounced decrease in Akt phosphorylation was observed when cells were cotreated with EA and H2O2. GST plays an important role as a regulator of mitogen-activated protein kinase pathways in I/R injury. PMID:22065940

  12. Effects of brefeldin A on oligosaccharide processing. Evidence for decreased branching of complex-type glycans and increased formation of hybrid-type glycans.

    PubMed

    Chawla, D; Hughes, R C

    1991-10-01

    Brefeldin A (BFA), a drug that induces redistribution of Golgi-apparatus proteins into the endoplasmic reticulum, was used to determine the role of subcellular compartmentalization in the processing of asparagine-linked oligosaccharides. Baby-hamster kidney cells were pulse-labelled with [3H]mannose for 30-60 min and chased for up to several hours in the presence or in the absence of BFA or labelled continuously for several hours with and without the drug. Cellular glycoproteins were digested to glycopeptides with Pronase and either fractionated into glycan classes by lectin affinity chromatography or digested further by endoglycosidase H and endoglycosidase D. Released oligosaccharides obtained in the latter procedure were then separated from each other and from endoglycosidase-resistant glycopeptides by paper chromatography. The results show that BFA induces a very fast processing of protein-linked Glc3Man9GlcNAc2 oligosaccharide down to man5GlcNAc2 and conversion into complex-type and hybrid-type glycans. The major difference between untreated and BFA-treated cells is a large increase in bi-antennary and hybrid-type glycans in the latter cells. These results indicate that galactosylation of a mono-antennary GlcNAcMan5GlcNAc2 hybrid blocks subsequent action by mannosidase II and N-acetylglucosaminyl transferase II, producing galactosylated hybrid-type glycans. Similarly, galactosylation of the product of N-acetylglucosaminyltransferases I and II, i.e. a Man3GlcNAc2 core substituted with GlcNAc beta 1----2 on both alpha 1----3- and alpha 1----6-linked mannose residues, blocks branching N-acetylglucosaminyltransferases IV and V, thereby causing an increase in bi-antennary glycans and a decrease in tri- and tetra-antennary glycans.

  13. [Identification of differential proteins of serum in the patients suffering from coal-burning arsenism].

    PubMed

    Han, Bing; Yang, Qin; Luo, Xin-hua; He, Xiao-fei; Wu, Jun; Cheng, Ming-liang

    2009-06-02

    To compare and analyze the differential expression of proteins between coal-burning arsenism serum and normal human serum and identify the proteins related with arseniasis caused by coal-burning. Serum samples were collected from 6 normal subjects and 6 patients suffering from coal-burning arsenism. 2-DE was performed to separate serum proteins. After silver staining, the differential expression of proteins was analyzed and then identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). There were an average of 779 +/- 35 spots and 865 +/- 30 spots on 2-DE matching of two groups and the matching rate was 90.1% between two groups. From these two groups, 60 different protein spots were identified. Up-regulated expression was observed in 25 proteins and down-regulated expression in 35 proteins in the patient serum group. Among which 35 with differential expression above three times were singled out and MALDI-TOF-MS analysis was carried out on them. Thirteen proteins were identified, including keratin 10, apolipoprotein A-V, transferrin, alpha-1-antitrypsin, human zinc-alpha-2-glycoprotein, mitogen-activated protein kinase 3, vacuolar protein sorting 33A, O-linked GlcNAc transferase and etc. Up-regulated expression was observed in 5 proteins and down-regulated expression in 8 proteins in the patient serum group. The well-resolved and reproducible 2-DE serum patterns of patients suffering from coal-burning arsenism were established and some differentially expressed proteins characterized. These data will be used to screen the biomarker and to further study arseniasis caused by coal-burning.

  14. Immediate effects of a single exercise bout on protein O-GlcNAcylation and chromatin regulation of cardiac hypertrophy

    PubMed Central

    Medford, Heidi M.; Porter, Karen

    2013-01-01

    Cardiac hypertrophy induced by pathological stimuli is regulated by a complex formed by the repressor element 1-silencing transcription factor (REST) and its corepressor mSin3A. We previously reported that hypertrophic signaling is blunted by O-linked attachment of β-N-acetylglucosamine (O-GlcNAc) to proteins. Regular exercise induces a physiological hypertrophic phenotype in the heart that is associated with decreased O-GlcNAc levels, but a link between O-GlcNAc, the REST complex, and initiation of exercise-induced cardiac hypertrophy is not known. Therefore, mice underwent a single 15- or 30-min bout of moderate- to high-intensity treadmill running, and hearts were harvested immediately and compared with sedentary controls. Cytosolic O-GlcNAc was lower (P < 0.05) following 15 min exercise with no differences in nuclear levels (P > 0.05). There were no differences in cytosolic or nuclear O-GlcNAc levels in hearts after 30 min exercise (P > 0.05). Cellular compartment levels of O-GlcNAc transferase (OGT, the enzyme that removes the O-GlcNAc moiety from proteins), REST, mSin3A, and histone deacetylases (HDACs) 1, 2, 3, 4, and 5 were not changed with exercise. Immunoprecipitation revealed O-GlcNAcylation of OGT and HDACs 1, 2, 4, and 5 that was not changed with acute exercise; however, exercised hearts did exhibit lower interactions between OGT and REST (P < 0.05) but not between OGT and mSin3A. These data suggest that hypertrophic signaling in the heart may be initiated by as little as 15 min of exercise via intracellular changes in protein O-GlcNAcylation distribution and reduced interactions between OGT and the REST chromatin repressor. PMID:23624624

  15. S-Glutathionylation of Keap1: a new role for glutathione S-transferase pi in neuronal protection.

    PubMed

    Carvalho, Andreia Neves; Marques, Carla; Guedes, Rita C; Castro-Caldas, Margarida; Rodrigues, Elsa; van Horssen, Jack; Gama, Maria João

    2016-05-01

    Oxidative stress is a key pathological feature of Parkinson's disease (PD). Glutathione S-transferase pi (GSTP) is a neuroprotective antioxidant enzyme regulated at the transcriptional level by the antioxidant master regulator nuclear factor-erythroid 2-related factor 2 (Nrf2). Here, we show for the first time that upon MPTP-induced oxidative stress, GSTP potentiates S-glutathionylation of Kelch-like ECH-associated protein 1 (Keap1), an endogenous repressor of Nrf2, in vivo. S-glutathionylation of Keap1 leads to Nrf2 activation and subsequently increases expression of GSTP. This positive feedback regulatory loop represents a novel mechanism by which GSTP elicits antioxidant protection in the brain. © 2016 Federation of European Biochemical Societies.

  16. GLUTATHIONE S-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE

    EPA Science Inventory

    GLUTATHIONE s-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE. M K Ross1 and R A Pegram2. 1Curriculum in Toxicology, University of North Carolina at Chapel Hill; 2Experimental Toxicology Division, NHEERL/ORD, United States Environmental Protection Agency, Research Triangl...

  17. Impacts on silkworm larvae midgut proteomics by transgenic Trichoderma strain and analysis of glutathione S-transferase sigma 2 gene essential for anti-stress response of silkworm larvae.

    PubMed

    Li, Yingying; Dou, Kai; Gao, Shigang; Sun, Jianan; Wang, Meng; Fu, Kehe; Yu, Chuanjin; Wu, Qiong; Li, Yaqian; Chen, Jie

    2015-08-03

    Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors

    PubMed Central

    Kosa, Nicolas M.; Foley, Timothy L.; Burkart, Michael D.

    2016-01-01

    Phosphopantetheinyl transferase (E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human phosphopantetheinyl transferase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity. PMID:24192555

  19. Regulation of asparagine-linked oligosaccharide processing. Oligosaccharide processing in Aedes albopictus mosquito cells.

    PubMed

    Hsieh, P; Robbins, P W

    1984-02-25

    We have examined the synthesis and processing of asparagine-linked oligosaccharides from Aedes albopictus C6/36 mosquito cells. These cells synthesized a glucose-containing lipid-linked oligosaccharide with properties identical to that of Glc3Man9GlcNAc2-PP-dolichol. Results of brief pulse label experiments with [3H]mannose were consistent with the transfer of Glc3Man9GlcNAc2 to protein followed by the rapid removal of glucose residues. Pulse-chase experiments established that further processing of oligosaccharides in C6/36 cells resulted in the removal of up to six alpha-linked mannose residues yielding Man3GlcNAc2 whose structure is identical to that of the trimannosyl "core" of N-linked oligosaccharides of vertebrate cells and yeast. Complex-type oligosaccharides were not observed in C6/36 cells. When Sindbis virus was grown in mosquito cells, Man3GlcNAc2 glycans were preferentially located at the two glycosylation sites which were previously shown to have complex glycans in virus grown in vertebrate cells. These Man3GlcNAc2 structures are the most extensively processed oligosaccharides in A. albopictus, and as such, are analogous to the complex glycans of vertebrate cells. We suggest that determinants of oligosaccharide processing which reside in the polypeptide are universally recognized despite evolutionary divergence of the oligosaccharide-processing pathway between insects and vertebrates.

  20. Analysis of the specificity of sialyltransferases toward mucin core 2, globo, and related structures. identification of the sialylation sequence and the effects of sulfate, fucose, methyl, and fluoro substituents of the carbohydrate chain in the biosynthesis of selectin and siglec ligands, and novel sialylation by cloned alpha2,3(O)sialyltransferase.

    PubMed

    Chandrasekaran, E V; Xue, Jun; Xia, Jie; Chawda, Ram; Piskorz, Conrad; Locke, Robert D; Neelamegham, Sriram; Matta, Khushi L

    2005-11-29

    Sialic acids are key determinants in many carbohydrates involved in biological recognition. We studied the acceptor specificities of three cloned sialyltransferases (STs) [alpha2,3(N)ST, alpha2,3(O)ST, and alpha2,6(N)ST] and another alpha2,3(O)ST present in prostate cancer cell LNCaP toward mucin core 2 tetrasaccharide [Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn] and Globo [Galbeta1,3GalNAcbeta1,3Galalpha-O-Me] structures containing sialyl, fucosyl, sulfo, methyl, or fluoro substituents by identifying the products by electrospray ionization tandem mass spectral analysis and other biochemical methods. The Globo precursor was an efficient acceptor for both alpha2,3(N)ST and alpha2,3(O)ST, whereas only alpha2,3(O)ST used its deoxy analogue (d-Fucbeta1,3GalNAcbeta1,3-Gal-alpha-O-Me); 2-O-MeGalbeta1,3GlcNAc and 4-OMeGalbeta1,4GlcNAc were specific acceptors for alpha2,3(N)ST. Other major findings of this study include: (i) alpha2,3 sialylation of beta1,3Gal in mucin core 2 can proceed even after alpha1,3 fucosylation of beta1,6-linked LacNAc. (ii) Sialylation of beta1,3Gal must precede the sialylation of beta1,4Gal for favorable biosynthesis of mucin core 2 compounds. (iii) alpha2,3 sialylation of the 6-O-sulfoLacNAc moiety in mucin core 2 (e.g., GlyCAM-1) is facilitated when beta1,3Gal has already been alpha2,3 sialylated. (iv) alpha2,6(N)ST was absolutely specific for the beta1,4Gal in mucin core 2. Either alpha1,3 fucosylation or 6-O-sulfation of the GlcNAc moiety reduced the activity. Sialylation of beta1,3Gal in addition to 6-O-sulfation of GlcNAc moiety abolished the activity. (v) Prior alpha2,3 sialylation or 3-O-sulfation of beta1,3Gal would not affect alpha2,6 sialylation of Galbeta1,4GlcNAc of mucin core 2. (vi) A 3- or 4-fluoro substituent in beta1,4Gal resulted in poor acceptors for the cloned alpha2,6(N)ST and alpha2,3(N)ST, whereas 4-fluoro- or 4-OMe-Galbeta1,3GalNAcalpha was a good acceptor for cloned alpha2,3(O)ST. (vii) 4-O-Methylation of beta1

  1. Molecular Cloning of Adenosinediphosphoribosyl Transferase.

    DTIC Science & Technology

    1987-09-08

    nature of the blocking group is unknown, except its identity with pyroglutamic acid was ruled out by its insensitivity to pyroglutaminase (not shown...AdenosinediphosphoribOSyl Transferase (ADPRT) is: 1) the complete amino acid sequence of this large protein is best determined -from the DNA !equence of the gene, 2...enzyme (I), determination of its peptide structure (II) and application of synthetic DNA probes (III) derived from amino acid sequences, resulting in the

  2. Brassinosteroids play a critical role in the regulation of pesticide metabolism in crop plants

    PubMed Central

    Zhou, Yanhong; Xia, Xiaojian; Yu, Gaobo; Wang, Jitao; Wu, Jingxue; Wang, Mengmeng; Yang, Youxin; Shi, Kai; Yu, Yunlong; Chen, Zhixiang; Gan, Jay; Yu, Jingquan

    2015-01-01

    Pesticide residues in agricultural produce pose a threat to human health worldwide. Although the detoxification mechanisms for xenobiotics have been extensively studied in mammalian cells, information about the regulation network in plants remains elusive. Here we show that brassinosteroids (BRs), a class of natural plant hormones, decreased residues of common organophosphorus, organochlorine and carbamate pesticides by 30–70% on tomato, rice, tea, broccoli, cucumber, strawberry, and other plants when treated externally. Genome-wide microarray analysis showed that fungicide chlorothalonil (CHT) and BR co-upregulated 301 genes, including a set of detoxifying genes encoding cytochrome P450, oxidoreductase, hydrolase and transferase in tomato plants. The level of BRs was closely related to the respiratory burst oxidase 1 (RBOH1)-encoded NADPH oxides-dependent H2O2 production, glutathione biosynthesis and the redox homeostasis, and the activity of glutathione S-transferase (GST). Gene silencing treatments showed that BRs decreased pesticide residues in plants likely by promoting their metabolism through a signaling pathway involving BRs-induced H2O2 production and cellular redox change. Our study provided a novel approach for minimizing pesticide residues in crops by exploiting plants' own detoxification mechanisms. PMID:25761674

  3. O-GlcNAcase Expression is Sensitive to Changes in O-GlcNAc Homeostasis.

    PubMed

    Zhang, Zhen; Tan, Ee Phie; VandenHull, Nicole J; Peterson, Kenneth R; Slawson, Chad

    2014-01-01

    O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification involving an attachment of a single β-N-acetylglucosamine moiety to serine or threonine residues in nuclear and cytoplasmic proteins. Cellular O-GlcNAc levels are regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the modification, respectively. The levels of O-GlcNAc can rapidly change in response to fluctuations in the extracellular environment; however, O-GlcNAcylation returns to a baseline level quickly after stimulus removal. This process termed O-GlcNAc homeostasis appears to be critical to the regulation of many cellular functions including cell cycle progress, stress response, and gene transcription. Disruptions in O-GlcNAc homeostasis are proposed to lead to the development of diseases, such as cancer, diabetes, and Alzheimer's disease. O-GlcNAc homeostasis is correlated with the expression of OGT and OGA. We reason that alterations in O-GlcNAc levels affect OGA and OGT transcription. We treated several human cell lines with Thiamet-G (TMG, an OGA inhibitor) to increase overall O-GlcNAc levels resulting in decreased OGT protein expression and increased OGA protein expression. OGT transcript levels slightly declined with TMG treatment, but OGA transcript levels were significantly increased. Pretreating cells with protein translation inhibitor cycloheximide did not stabilize OGT or OGA protein expression in the presence of TMG; nor did TMG stabilize OGT and OGA mRNA levels when cells were treated with RNA transcription inhibitor actinomycin D. Finally, we performed RNA Polymerase II chromatin immunoprecipitation at the OGA promoter and found that RNA Pol II occupancy at the transcription start site was lower after prolonged TMG treatment. Together, these data suggest that OGA transcription was sensitive to changes in O-GlcNAc homeostasis and was potentially regulated by O-GlcNAc.

  4. O-GlcNAcase Expression is Sensitive to Changes in O-GlcNAc Homeostasis

    PubMed Central

    Zhang, Zhen; Tan, Ee Phie; VandenHull, Nicole J.; Peterson, Kenneth R.; Slawson, Chad

    2014-01-01

    O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification involving an attachment of a single β-N-acetylglucosamine moiety to serine or threonine residues in nuclear and cytoplasmic proteins. Cellular O-GlcNAc levels are regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the modification, respectively. The levels of O-GlcNAc can rapidly change in response to fluctuations in the extracellular environment; however, O-GlcNAcylation returns to a baseline level quickly after stimulus removal. This process termed O-GlcNAc homeostasis appears to be critical to the regulation of many cellular functions including cell cycle progress, stress response, and gene transcription. Disruptions in O-GlcNAc homeostasis are proposed to lead to the development of diseases, such as cancer, diabetes, and Alzheimer’s disease. O-GlcNAc homeostasis is correlated with the expression of OGT and OGA. We reason that alterations in O-GlcNAc levels affect OGA and OGT transcription. We treated several human cell lines with Thiamet-G (TMG, an OGA inhibitor) to increase overall O-GlcNAc levels resulting in decreased OGT protein expression and increased OGA protein expression. OGT transcript levels slightly declined with TMG treatment, but OGA transcript levels were significantly increased. Pretreating cells with protein translation inhibitor cycloheximide did not stabilize OGT or OGA protein expression in the presence of TMG; nor did TMG stabilize OGT and OGA mRNA levels when cells were treated with RNA transcription inhibitor actinomycin D. Finally, we performed RNA Polymerase II chromatin immunoprecipitation at the OGA promoter and found that RNA Pol II occupancy at the transcription start site was lower after prolonged TMG treatment. Together, these data suggest that OGA transcription was sensitive to changes in O-GlcNAc homeostasis and was potentially regulated by O-GlcNAc. PMID:25520704

  5. Quantification of butyryl CoA:acetate CoA-transferase genes reveals different butyrate production capacity in individuals according to diet and age.

    PubMed

    Hippe, Berit; Zwielehner, Jutta; Liszt, Kathrin; Lassl, Cornelia; Unger, Frank; Haslberger, Alexander G

    2011-03-01

    The gastrointestinal microbiota produces short-chain fatty acids, especially butyrate, which affect colonic health, immune function and epigenetic regulation. To assess the effects of nutrition and aging on the production of butyrate, the butyryl-CoA:acetate CoA-transferase gene and population shifts of Clostridium clusters lV and XlVa, the main butyrate producers, were analysed. Faecal samples of young healthy omnivores (24 ± 2.5 years), vegetarians (26 ± 5 years) and elderly (86 ± 8 years) omnivores were evaluated. Diet and lifestyle were assessed in questionnaire-based interviews. The elderly had significantly fewer copies of the butyryl-CoA:acetate CoA-transferase gene than young omnivores (P=0.014), while vegetarians showed the highest number of copies (P=0.048). The thermal denaturation of the butyryl-CoA:acetate CoA-transferase gene variant melting curve related to Roseburia/Eubacterium rectale spp. was significantly more variable in the vegetarians than in the elderly. The Clostridium cluster XIVa was more abundant in vegetarians (P=0.049) and in omnivores (P<0.01) than in the elderly group. Gastrointestinal microbiota of the elderly is characterized by decreased butyrate production capacity, reflecting increased risk of degenerative diseases. These results suggest that the butyryl-CoA:acetate CoA-transferase gene is a valuable marker for gastrointestinal microbiota function. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. O-GlcNAc cycling in the developing, adult and geriatric brain.

    PubMed

    Lagerlöf, Olof

    2018-06-01

    Hundreds of proteins in the nervous system are modified by the monosaccharide O-GlcNAc. A single protein is often O-GlcNAcylated on several amino acids and the modification of a single site can play a crucial role for the function of the protein. Despite its complexity, only two enzymes add and remove O-GlcNAc from proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Global and local regulation of these enzymes make it possible for O-GlcNAc to coordinate multiple cellular functions at the same time as regulating specific pathways independently from each other. If O-GlcNAcylation is disrupted, metabolic disorder or intellectual disability may ensue, depending on what neurons are affected. O-GlcNAc's promise as a clinical target for developing drugs against neurodegenerative diseases has been recognized for many years. Recent literature puts O-GlcNAc in the forefront among mechanisms that can help us better understand how neuronal circuits integrate diverse incoming stimuli such as fluctuations in nutrient supply, metabolic hormones, neuronal activity and cellular stress. Here the functions of O-GlcNAc in the nervous system are reviewed.

  7. Biosynthesis of 2-Hydroxyacid-Containing Polyhydroxyalkanoates by Employing butyryl-CoA Transferases in Metabolically Engineered Escherichia coli.

    PubMed

    David, Yokimiko; Joo, Jeong Chan; Yang, Jung Eun; Oh, Young Hoon; Lee, Sang Yup; Park, Si Jae

    2017-11-01

    The authors previously reported the production of polyhydroxyalkanoates (PHAs) containing 2-hydroxyacid monomers by expressing evolved Pseudomonas sp. 6-19 PHA synthase and Clostridium propionicum propionyl-CoA transferase in engineered microorganisms. Here, the authors examined four butyryl-CoA transferases from Roseburia sp., Eubacterium hallii, Faecalibacterium prausnitzii, and Anaerostipes caccae as potential CoA-transferases to support synthesis of polymers having 2HA monomer. In vitro activity analyses of the four butyryl-CoA transferases suggested that each butyryl-CoA transferase has different activities towards 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB), and lactate (LA). When Escherichia coli XL1-Blue expressing Pseudomonas sp. 6-19 PhaC1437 along with one butyryl-CoA transferase is cultured in chemically defined MR medium containing 20 g L -1 of glucose, 2 g L -1 of sodium 3-hydroxybutyrate, and various concentrations of sodium 2-hydroxybutyrate, PHAs consisting of 3HB, 2HB, and LA are produced. The monomer composition of PHAs agreed well with the substrate specificities of butyryl-CoA transferases from E. hallii, F. prausnitzii, and A. caccae, but not Roseburia sp. When E. coli XL1-Blue expressing PhaC1437 and E. hallii butyryl-CoA transferase is cultured in MR medium containing 20 g L -1 of glucose and 2 g L -1 of sodium 2-hydroxybutyrate, P(65.7 mol% 2HB-co-34.3 mol% LA) is produced with the highest PHA content of 30 wt%. Butyryl-CoA transferases also supported the production of P(3HB-co-2HB-co-LA) from glucose as the sole carbon source in E. coli XL1-Blue strains when one of these bct genes is expressed with phaC1437, cimA3.7, leuBCD, panE, and phaAB genes. Butyryl-CoA transferases characterized in this study can be used for engineering of microorganisms that produce PHAs containing novel 2-hydroxyacid monomers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. O-GlcNAcylation enhances anaplastic thyroid carcinoma malignancy.

    PubMed

    Cheng, Y U; Li, Honglun; Li, Jianlin; Li, Jisheng; Gao, Yan; Liu, Baodong

    2016-07-01

    O-linked N -acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation), a dynamic post-translational modification of nuclear and cytoplasmic proteins, may have a critical role in the regulation of biological cell processes and human cancer. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Accumulating evidence suggests that O-GlcNAcylation is involved in a variety of types of human cancer. However, the exact role of O-GlcNAcylation in tumor pathogenesis or progression remains to be established. Computed tomography scans of patients with anaplastic thyroid carcinoma (ATC) reveal a rapid growth rate and invasion. The present study demonstrated that O-GlcNAcylation accelerates the progression of ATC. The global O-GlcNAc level of intracellular proteins was increased by overexpression of OGT or downregulation of OGA activity with the specific inhibitor Thiamet-G. By contrast, the global O-GlcNAc level was decreased by silencing of OGT. MTT assay indicated that O-GlcNAcylation significantly promotes cell proliferation. Furthermore, O-GlcNAcylation enhanced cellular biological functions, such as colony formation ability, migration and invasion, of ATC cells in vitro . The findings of the present study suggest that O-GlcNAcylation is associated with malignant properties of thyroid cancer, and may be a potential target for the diagnosis and treatment of thyroid cancer.

  9. Helicobacter pylori β1,3-N-acetylglucosaminyltransferase for versatile synthesis of type 1 and type 2 poly-LacNAcs on N-linked, O-linked and I-antigen glycans

    PubMed Central

    Peng, Wenjie; Pranskevich, Jennifer; Nycholat, Corwin; Gilbert, Michel; Wakarchuk, Warren; Paulson, James C; Razi, Nahid

    2012-01-01

    Poly-N-acetyllactosamine extensions on N- and O-linked glycans are increasingly recognized as biologically important structural features, but access to these structures has not been widely available. Here, we report a detailed substrate specificity and catalytic efficiency of the bacterial β3-N-acetylglucosaminyltransferase (β3GlcNAcT) from Helicobacter pylori that can be adapted to the synthesis of a rich diversity of glycans with poly-LacNAc extensions. This glycosyltransferase has surprisingly broad acceptor specificity toward type-1, -2, -3 and -4 galactoside motifs on both linear and branched glycans, found commonly on N-linked, O-linked and I-antigen glycans. This finding enables the production of complex ligands for glycan-binding studies. Although the enzyme shows preferential activity for type 2 (Galβ1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in β1-3 linkage to type-1 (Galβ1-3GlcNAc) or type-3/4 (Galβ1-3GalNAcα/β) sequences. Thus, by alternating the use of the H. pylori β3GlcNAcT with galactosyltransferases that make the β1-4 or β1-3 linkages, various N-linked, O-linked and I-antigen acceptors could be elongated with type-2 and type-1 LacNAc repeats. Finally, one-pot incubation of di-LacNAc biantennary N-glycopeptide with the β3GlcNAcT and GalT-1 in the presence of uridine diphosphate (UDP)-GlcNAc and UDP-Gal, yielded products with 15 additional LacNAc units on the precursor, which was seen as a series of sequential ion peaks representing alternative additions of GlcNAc and Gal residues, on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Overall, our data demonstrate a broader substrate specificity for the H. pylori β3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans. PMID:22786570

  10. Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

    PubMed

    Wu, A M; Lin, S R; Chin, L K; Chow, L P; Lin, J Y

    1992-09-25

    The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.

  11. Identification of GIG1, a GlcNAc-Induced Gene in Candida albicans Needed for Normal Sensitivity to the Chitin Synthase Inhibitor Nikkomycin Z▿§

    PubMed Central

    Gunasekera, Angelo; Alvarez, Francisco J.; Douglas, Lois M.; Wang, Hong X.; Rosebrock, Adam P.; Konopka, James B.

    2010-01-01

    The amino sugar N-acetylglucosamine (GlcNAc) is known to be an important structural component of cells from bacteria to humans, but its roles in cell signaling are less well understood. GlcNAc induces two pathways in the human fungal pathogen Candida albicans. One activates cyclic AMP (cAMP) signaling, which stimulates the formation of hyphal cells and the expression of virulence genes, and the other pathway induces genes needed to catabolize GlcNAc. Microarray analysis of gene expression was carried out under four different conditions in order to characterize the transcriptional changes induced by GlcNAc. The most highly induced genes include those that encode a GlcNAc transporter (NGT1) and the GlcNAc catabolic enzymes (HXK1, DAC1, and NAG1). GlcNAc also activated most of the genes whose expression is increased when cells are triggered with other stimuli to form hyphae. Surprisingly, GlcNAc also induced a subset of genes that are regulated by galactose (GAL1, GAL7, and GAL10), which may be due to cross talk between signaling pathways. A novel GlcNAc-induced gene, GIG1, which is not essential for GlcNAc catabolism or the induction of hyphae, was identified. However, a Gig1-green fluorescent protein (GFP) fusion protein was specifically induced by GlcNAc, and not by other sugars. Gig1-GFP localized to the cytoplasm, where GlcNAc metabolism occurs. Significantly, a gig1Δ mutant displayed increased resistance to nikkomycin Z, which inhibits chitin synthase from converting UDP-GlcNAc into cell wall chitin. Gig1 is highly conserved in fungi, especially those that contain GlcNAc catabolic genes. These results implicate Gig1 in GlcNAc metabolism. PMID:20675577

  12. Role of UDP-N-Acetylglucosamine (GlcNAc) and O-GlcNAcylation of Hyaluronan Synthase 2 in the Control of Chondroitin Sulfate and Hyaluronan Synthesis*

    PubMed Central

    Vigetti, Davide; Deleonibus, Sara; Moretto, Paola; Karousou, Eugenia; Viola, Manuela; Bartolini, Barbara; Hascall, Vincent C.; Tammi, Markku; De Luca, Giancarlo; Passi, Alberto

    2012-01-01

    Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1–3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t½ >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies. PMID:22887999

  13. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

    PubMed Central

    Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick; Joshi, Hiren Jitendra; Petersen, Bent Larsen; Vakhrushev, Sergey Y.; Strahl, Sabine; Clausen, Henrik

    2015-01-01

    Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing. PMID:26644575

  14. A little sugar goes a long way: The cell biology of O-GlcNAc

    PubMed Central

    2015-01-01

    Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate–GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport. PMID:25825515

  15. Cloning and expression of clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

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

    Cary, J.W.; Petersen, D.J.; Bennett, G.N.

    1990-06-01

    Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase (butyrate-acetoacetate CoA-transferase) (EC 2.8.3.9)) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The genes encoding the two subunits of this enzyme have been cloned and subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defectmore » in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M{sub r} of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E.coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.« less

  16. The mitochondrial O-linked N-acetylglucosamine transferase (mOGT) in the diabetic patient could be the initial trigger to develop Alzheimer disease.

    PubMed

    Lozano, Liliana; Lara-Lemus, Roberto; Zenteno, Edgar; Alvarado-Vásquez, Noé

    2014-10-01

    Diabetes mellitus (DM) is considered a risk factor for the development of Alzheimer disease (AD); however, how DM favors evolution of AD is still insufficiently understood. Hyperglycemia in DM is associated to an increase in mitochondrial reactive oxygen species (ROS) generation, as well as damage of hippocampal cells, reflected by changes in morphological and mitochondrial functionality. Similar mitochondrial damage has been observed when amyloid beta (Aβ) accumulates in the brain of AD patients. In DM, the excess of glucose in the brain induces higher activity of the hexosamine biosynthesis pathway (HBP), it synthesizes UDP-N-acetylglucosamine (UDP-GlcNAc), which is used by O-linked N-acetylglucosamine transferase (OGT) to catalyze O-GlcNAcylation of numerous proteins. Although O-GlcNAcylation plays an important role in maintaining structure and cellular functionality, chronic activity of this pathway has been associated with insulin resistance and hyperglycemia-induced glucose toxicity. Three different forms of OGT are known: nucleocytoplasmic (ncOGT), short (sOGT), and mitochondrial (mOGT). Previous reports showed that overexpression of ncOGT is not toxic to the cell; in contrast, overexpression of mOGT is associated with cellular apoptosis. In this work, we suggest that hyperglycemia in the diabetic patient could induce greater expression and activity of mOGT, modifying the structure and functionality of mitochondria in hippocampal cells, accelerating neuronal damage, and favoring the start of AD. In consequence, mOGT activity could be a key point for AD development in patients with DM. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Isoenzyme-specific up-regulation of glutathione transferase and aldo-keto reductase mRNA expression by dietary quercetin in rat liver.

    PubMed

    Odbayar, Tseye-Oidov; Kimura, Toshinori; Tsushida, Tojiro; Ide, Takashi

    2009-05-01

    The impact of quercetin on the mRNA expression of hepatic enzymes involved in drug metabolism was evaluated with a DNA microarray and real-time PCR. Male Sprague-Dawley rats were fed an experimental diet containing either 0, 2.5, 5, 10, or 20 g/kg of quercetin for 15 days. The DNA microarray analysis of the gene expression profile in pooled RNA samples from rats fed diets containing 0, 5, and 20 g/kg of quercetin revealed genes of some isoenzymes of glutathione transferase (Gst) and aldo-keto reductase (Akr) to be activated by this flavonoid. Real-time PCR conducted with RNA samples from individual rats fed varying amounts of quercetin together with the microarray analysis showed that quercetin caused marked dose-dependent increases in the mRNA expression of Gsta3, Gstp1, and Gstt3. Some moderate increases were also noted in the mRNA expression of isoenzymes belonging to the Gstm class. Quercetin also dose-dependently increased the mRNA expression of Akr1b8 and Akr7a3. However, it did not affect the parameters of the other Gst and Akr isoenzymes. It is apparent that quercetin increases the mRNA expression of Gst and Akr involved in drug metabolism in an isoenzyme-specific manner. Inasmuch as Gst and Akr isoenzymes up-regulated in their gene expression are involved in the prevention and attenuation of cancer development, this consequence may account for the chemopreventive propensity of quercetin.

  18. Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine.

    PubMed Central

    Heese-Peck, A; Cole, R N; Borkhsenious, O N; Hart, G W; Raikhel, N V

    1995-01-01

    Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized. As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc). Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs. Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays. Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues. Most of these appear to be bound to proteins via a hydroxyl group. This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs. PMID:8589629

  19. Alteration of O-GlcNAcylation affects serine phosphorylation and regulates gene expression and activity of pyruvate kinase M2 in colorectal cancer cells.

    PubMed

    Chaiyawat, Parunya; Chokchaichamnankit, Daranee; Lirdprapamongkol, Kriengsak; Srisomsap, Chantragan; Svasti, Jisnuson; Champattanachai, Voraratt

    2015-10-01

    O-GlcNAcylation is a dynamic post-translational modification that has extensive crosstalk with phosphorylation either at the same or adjacent sites of various proteins. We have previously reported that O-GlcNAcylation level was increased in primary breast and colorectal cancer, but the interplay of the two modifications remains unclear. Therefore, we explored crosstalk of the modifications by RNA interference against O-GlcNAc transferase (OGT) in colorectal cancer cells. Two-dimensional immunoblotting and mass spectrometric analysis showed that the levels of O-GlcNAc and serine phosphorylation of many proteins including serine hydroxymethyltransferase, cytokeratin-8, pyruvate kinase M2 (PKM2), heterogeneous nuclear ribonucleoprotein L, and lamin-B1, were reduced in siOGT cells compared to siScramble cells. In HT29 cells, immunoprecipitated PKM2 revealed decreased O-GlcNAc and serine phosphorylation levels after siOGT knockdown, but increased levels after treatment with Thiamet-G, an inhibitor of O-GlcNAcase (OGA). In addition, when global O-GlcNAcylation was enhanced by treating cells with Thiamet-G, PKM2 expression level was upregulated, but PKM2-specific activity was decreased. On the other hand, in OGT knockdown cells, PKM2 expression level was downregulated, but PKM2-specific activity was increased. Moreover, the metastatic colorectal cancer cells, SW620, had more O-GlcNAc-PKM2 and showed lower PKM2-specific activity compared to the non-metastatic colorectal cancer SW480 cells. These results suggested roles of O-GlcNAcylation in modulating serine phosphorylation, as well as in regulating PKM2 activity and expression. Interfering levels of O-GlcNAcylation of PKM2 may be a novel target in controlling cancer metabolism and tumorigenesis of colorectal cancer.

  20. Detection of Glutathione by Glutathione-S-Transferase-Nanoconjugate Ensemble Electrochemical Device.

    PubMed

    Barman, Ujjwol; Mukhopadhyay, Gargi; Goswami, Namami; Ghosh, Siddhartha Sankar; Paily, Roy P

    2017-06-01

    This paper reports a novel electrochemical method for detection of Glutathione (GSH) using Glutathione-S-Transferase (GST) - ZnO composite nanoparticles to investigate the prospects of the method for detection of cancer at an early stage. The purified GST enzyme was bound with ZnO nanoparticles by electrostatic interactions and the nanocomposite was dropcast on a silicon dioxide wafer. The GST functionalized deposited layer was then used as a chemiresistive channel to detect conjugation reaction between GSH and 1-Chloro-2, 4-Dinitrobenzene (CDNB). The zeta potential values of the ZnO nanoparticles and the GST were found to be 13.4 mV and-6.21 mV, respectively. Around 73.8% binding was observed between the enzyme and ZnO nanoparticles. I - V analysis of the chemiresistive channel showed an increase in conductivity of the channel due to conjugation reaction between GSH and CDNB as compared with that of GSH or CDNB alone. I - V characterization of the GST functionalized layer was performed at various concentrations of GSH and a sensitivity and limit of detection of 5.68 nA/ [Formula: see text] and 41.9 nM were obtained, respectively. Thus from I - V analysis of the chemiresistivechannel, the detectionand quantification of GSH could be obtained. The kinetic parameters of both GST and nanoconjugate of ZnO nanoparticles andGSTwere determinedwith respect to its substrates, GSH and CDNB, using Michaelis-Mentenmodel. This novel approach of detection of GSH bymeans of ZnO nanoparticle and GST enzyme composite can be further analyzed for in vitro experiments, which will lead us to a new and efficient way of detecting certain types of cancers at an early stage.

  1. Reaction of rat liver glutathione S-transferases and bacterial dichloromethane dehalogenase with dihalomethanes.

    PubMed

    Blocki, F A; Logan, M S; Baoli, C; Wackett, L P

    1994-03-25

    Dichloromethane dehalogenase from Methylophilus sp. DM11 is a glutathione S-transferase homolog that is specifically active with dihalomethane substrates. This bacterial enzyme and rat liver glutathione S-transferases were purified to investigate their relative reactivity with CH2Cl2 and related substrates. Rat liver alpha class glutathione transferases were inactive and mu class enzymes showed low activity (7-23 nmol/min/mg of protein) with CH2Cl2. theta class glutathione transferase 5-5 from rat liver and Methylophilus sp. dichloromethane dehalogenase showed specific activities of > or = 1 mumol/min/mg of protein. Apparent Kcat/Km were determined to be 3.3 x 10(4) and 6.0 x 10(4) L M-1 S-1 for the two enzymes, respectively. Dideutero-dichloromethane was processed to dideutereo-formaldehyde, consistent with a nucleophilic halide displacement mechanism. The possibility of a GSCH2X reaction intermediate (GS, glutathione; X, halide) was probed using CH2ClF to generate a more stable halomethylglutathione species (GSCH2F). The reaction of CH2ClF with dichloromethane dehalogenase produced a kinetically identifiable intermediate that decomposed to formaldehyde at a similar rate to synthetic HOCH2CH2SCH2F. 19F-NMR revealed the transient formation of an intermediate identified as GSCH2F by its chemical shift, its triplet resonance, and H-F coupling constant consistent with a fluoromethylthioether. Its decomposition was matched by a stoichiometric formation of fluoride. These studies indicated that the bacterial dichloromethane dehalogenase directs a nucleophilic attack of glutathione on CH2Cl2 to produce a halomethylthioether intermediate. This focuses attention on the mechanism used by theta class glutathione transferases to generate a halomethylthioeter from relatively unreactive dihalomethanes.

  2. Predicting the Retention Behavior of Specific O-Linked Glycopeptides.

    PubMed

    Badgett, Majors J; Boyes, Barry; Orlando, Ron

    2017-09-01

    O -Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze O -glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s). In this paper, HILIC is used to separate peptides with O - N -acetylgalactosamine ( O -GalNAc), O - N -acetylglucosamine ( O -GlcNAc), and O -fucose additions from their native forms, and coefficients representing the extent of hydrophilicity were derived using linear regression analysis as a means to predict the retention times of peptides with these modifications.

  3. Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

    PubMed Central

    Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

    2014-01-01

    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

  4. The Drosophila protein palmitoylome: Characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases

    PubMed Central

    Bannan, Barbra A.; Van Etten, Jamie; Kohler, John A.; Tsoi, Yui; Hansen, Nicole M.; Sigmon, Stacey; Fowler, Elizabeth; Buff, Haley; Williams, Tiffany S.; Ault, Jeffrey G.; Glaser, Robert L.; Korey, Christopher A.

    2010-01-01

    Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization, RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627, and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male-specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2), and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation. PMID:18719403

  5. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... of the enzyme galactose-1-phosphate uridyl transferase in erythrocytes (red blood cells... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II. ...

  6. The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation.

    PubMed Central

    Van Der Heide, Lars P; Hoekman, Marco F M; Smidt, Marten P

    2004-01-01

    FoxO (forkhead box O; forkhead members of the O class) are transcription factors that function under the control of insulin/insulin-like signalling. FoxO factors have been associated with a multitude of biological processes, including cell-cycle, cell death, DNA repair, metabolism and protection from oxidative stress. Central to the regulation of FoxO factors is a shuttling system, which confines FoxO factors to either the nucleus or the cytosol. Shuttling of FoxO requires protein phosphorylation within several domains, and association with 14-3-3 proteins and the nuclear transport machinery. Description of the FoxO-shuttling mechanism contributes to the understanding of FoxO function in relation to signalling and gene regulation. PMID:15005655

  7. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a) Identification...

  8. 21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a) Identification...

  9. 21 CFR 862.1535 - Ornithine carbamyl transferase test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  10. Intracellular APP Domain Regulates Serine-Palmitoyl-CoA Transferase Expression and Is Affected in Alzheimer's Disease

    PubMed Central

    Grimm, Marcus O. W.; Grösgen, Sven; Rothhaar, Tatjana L.; Burg, Verena K.; Hundsdörfer, Benjamin; Haupenthal, Viola J.; Friess, Petra; Müller, Ulrike; Fassbender, Klaus; Riemenschneider, Matthias; Grimm, Heike S.; Hartmann, Tobias

    2011-01-01

    Lipids play an important role as risk or protective factors in Alzheimer's disease (AD), a disease biochemically characterized by the accumulation of amyloid beta peptides (Aβ), released by proteolytic processing of the amyloid precursor protein (APP). Changes in sphingolipid metabolism have been associated to the development of AD. The key enzyme in sphingolipid de novo synthesis is serine-palmitoyl-CoA transferase (SPT). In the present study we identified a new physiological function of APP in sphingolipid synthesis. The APP intracellular domain (AICD) was found to decrease the expression of the SPT subunit SPTLC2, the catalytic subunit of the SPT heterodimer, resulting in that decreased SPT activity. AICD function was dependent on Fe65 and SPTLC2 levels are increased in APP knock-in mice missing a functional AICD domain. SPTLC2 levels are also increased in familial and sporadic AD postmortem brains, suggesting that SPT is involved in AD pathology. PMID:21660213

  11. Structural Basis of Specific Recognition of Non-Reducing Terminal N-Acetylglucosamine by an Agrocybe aegerita Lectin

    PubMed Central

    Ren, Xiao-Ming; Li, De-Feng; Jiang, Shuai; Lan, Xian-Qing; Hu, Yonglin; Sun, Hui; Wang, Da-Cheng

    2015-01-01

    O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently. PMID:26114302

  12. Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons.

    PubMed

    Tan, Elaine Y M; Law, Janice W S; Wang, Chi-Hwa; Lee, Alan Y W

    2007-12-01

    Neurons in post-traumatized mammalian central nervous system show only limited degree of regeneration, which can be attributed to the presence of neurite outgrowth inhibitors in damaged myelin and glial scar, and to the apoptosis of severed central neurons and glial cells during secondary Wallerian degeneration. RhoA GTPase has been implicated as the common denominator in these counter-regeneration events, which shows significant and persistent up-regulation for weeks in injured spinal cord and cerebral infarct after stroke. While the exoenzyme C3 transferase is a potent RhoA inhibitor, its extremely low efficiency of cell entry and degradation in vivo has restricted the therapeutic value. This study aims to circumvent these problems by developing a membrane-permeating form of C3 transferase and a biopolymer-based microsphere depot system for sustainable controlled release of the protein. A membrane-permeating form of C3 transferase was developed by fusing a Tat (trans-activating transcription factor) transduction domain of human immunodeficiency virus to its amino terminal using standard molecular cloning techniques. After confirming efficient cell entry into epithelial and neuroblastoma cells, the resulting recombinant protein TAT-C3 was encapsulated in biocompatible polymer poly(D,L -lactide-co-glycolide) in the form of microspheres by a water-in-oil-in-water (W/O/W) emulsion method. By blending capped and uncapped form of the polymer at different ratios, TAT-C3 protein release profile was modified to suit the expression pattern of endogenous RhoA during CNS injuries. Bioactivity of TAT-C3 released from microspheres was assessed by RhoA ribosylation assay. In contrast to wild-type C3 transferase, the modified TAT-C3 protein was found to efficiently enter NIH3T3 and N1E-115 neuroblastoma cells as early as 6 hours of incubation. The fusion of TAT sequence to C3 transferase imposed no appreciable effects on its biological activity in promoting neurite outgrowth

  13. Glutathione S-transferases and UDP-glycosyltransferases Are Involved in Response to Aluminum Stress in Flax

    PubMed Central

    Dmitriev, Alexey A.; Krasnov, George S.; Rozhmina, Tatiana A.; Kishlyan, Natalya V.; Zyablitsin, Alexander V.; Sadritdinova, Asiya F.; Snezhkina, Anastasiya V.; Fedorova, Maria S.; Yurkevich, Olga Y.; Muravenko, Olga V.; Bolsheva, Nadezhda L.; Kudryavtseva, Anna V.; Melnikova, Nataliya V.

    2016-01-01

    About 30% of the world's ice-free land area is occupied by acid soils. In soils with pH below 5, aluminum (Al) releases to the soil solution, and becomes highly toxic for plants. Therefore, breeding of varieties that are resistant to Al is needed. Flax (Linum usitatissimum L.) is grown worldwide for fiber and seed production. Al toxicity in acid soils is a serious problem for flax cultivation. However, very little is known about mechanisms of flax resistance to Al and the genetics of this resistance. In the present work, we sequenced 16 transcriptomes of flax cultivars resistant (Hermes and TMP1919) and sensitive (Lira and Orshanskiy) to Al, which were exposed to control conditions and aluminum treatment for 4, 12, and 24 h. In total, 44.9–63.3 million paired-end 100-nucleotide reads were generated for each sequencing library. Based on the obtained high-throughput sequencing data, genes with differential expression under aluminum exposure were revealed in flax. The majority of the top 50 up-regulated genes were involved in transmembrane transport and transporter activity in both the Al-resistant and Al-sensitive cultivars. However, genes encoding proteins with glutathione transferase and UDP-glycosyltransferase activity were in the top 50 up-regulated genes only in the flax cultivars resistant to aluminum. For qPCR analysis in extended sampling, two UDP-glycosyltransferases (UGTs), and three glutathione S-transferases (GSTs) were selected. The general trend of alterations in the expression of the examined genes was the up-regulation under Al stress, especially after 4 h of Al exposure. Moreover, in the flax cultivars resistant to aluminum, the increase in expression was more pronounced than that in the sensitive cultivars. We speculate that the defense against the Al toxicity via GST antioxidant activity is the probable mechanism of the response of flax plants to aluminum stress. We also suggest that UGTs could be involved in cell wall modification and protection

  14. Homogentisate solanesyl transferase (HST) cDNA’s in maize

    USDA-ARS?s Scientific Manuscript database

    Maize white seedling 3 (w3) has served as a model albino-seedling mutant since its discovery in 1923. We show that the w3 phenotype is caused by disruptions in homogentisate solanesyl transferase (HST), an enzyme that catalyzes the committed step in plastoquinone-9 (PQ9) biosynthesis. This reaction ...

  15. Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

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

    Fang, Ti; Li, De-Feng; Zhou, Ning-Yi, E-mail: n.zhou@pentium.whiov.ac.cn

    2011-07-08

    Highlights: {yields} Application of site-directed mutagenesis to probe the active site residues of glutathione-dependent maleylpyruvate isomerase. {yields} Two conserved residues, Arg8 and Arg176, in zeta class glutathione S-transferases are critical for maleylpyruvate orientation and enolization. {yields} Arg109, found exclusively in NagL, participates in k{sub cat} regulation. {yields} The T11A mutant exhibited a significantly decreased K{sub m} value for glutathione with little impact on maleylpyruvate kinetics. {yields} The Thr11 residue appears to have significance in the evolution of glutathione S-transferase classes. -- Abstract: The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerizationmore » of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k{sub cat} regulation. Surprisingly, the T11A mutant

  16. Reversal of hypermethylation and reactivation of glutathione S-transferase pi 1 gene by curcumin in breast cancer cell line.

    PubMed

    Kumar, Umesh; Sharma, Ujjawal; Rathi, Garima

    2017-02-01

    One of the mechanisms for epigenetic silencing of tumor suppressor genes is hypermethylation of cytosine residue at CpG islands at their promoter region that contributes to malignant progression of tumor. Therefore, activation of tumor suppressor genes that have been silenced by promoter methylation is considered to be very attractive molecular target for cancer therapy. Epigenetic silencing of glutathione S-transferase pi 1, a tumor suppressor gene, is involved in various types of cancers including breast cancer. Epigenetic silencing of tumor suppressor genes can be reversed by several molecules including natural compounds such as polyphenols that can act as a hypomethylating agent. Curcumin has been found to specifically target various tumor suppressor genes and alter their expression. To check the effect of curcumin on the methylation pattern of glutathione S-transferase pi 1 gene in MCF-7 breast cancer cell line in dose-dependent manner. To check the reversal of methylation pattern of hypermethylated glutathione S-transferase pi 1, MCF-7 breast cancer cell line was treated with different concentrations of curcumin for different time periods. DNA and proteins of treated and untreated cell lines were isolated, and methylation status of the promoter region of glutathione S-transferase pi 1 was analyzed using methylation-specific polymerase chain reaction assay, and expression of this gene was analyzed by immunoblotting using specific antibodies against glutathione S-transferase pi 1. A very low and a nontoxic concentration (10 µM) of curcumin treatment was able to reverse the hypermethylation and led to reactivation of glutathione S-transferase pi 1 protein expression in MCF-7 cells after 72 h of treatment, although the IC 50 value of curcumin was found to be at 20 µM. However, curcumin less than 3 µM of curcumin could not alter the promoter methylation pattern of glutathione S-transferase pi 1. Treatment of breast cancer MCF-7 cells with curcumin

  17. O-GlcNAcase deficiency suppresses skeletal myogenesis and insulin sensitivity in mice through the modulation of mitochondrial homeostasis.

    PubMed

    Wang, Xun; Feng, Zhihui; Wang, Xueqiang; Yang, Liang; Han, Shujun; Cao, Ke; Xu, Jie; Zhao, Lin; Zhang, Yong; Liu, Jiankang

    2016-06-01

    O-GlcNAcylation is implicated in modulating mitochondrial function, which is closely involved in regulating muscle metabolism. The presence of O-GlcNAcase (OGA), the enzyme involved in the removal of O-GlcNAc, in mitochondria was recently confirmed in rats. In the present study, we investigated the regulation of myogenesis and muscle insulin sensitivity to OGA in mice, with a focus on mitochondria. C57BL/6J mice fed a high-fat diet for 4 months were used to observe mitochondrial density, activity and O-GlcNAcylation in muscle. Small interfering RNA and overexpression vectors were used to modulate protein content in vitro. High-fat feeding decreased the OGA level and largely increased mitochondrial O-GlcNAcylation in mouse skeletal muscle that was accompanied by decreased levels of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), decreased mitochondrial density and disrupted mitochondrial complex activities. Knockdown of OGA in C2C12 myoblasts promoted PGC-1α degradation, resulting in the suppression of mitochondrial biogenesis and myogenesis, whereas neither knockdown of O-GlcNAc transferase nor overexpression of OGA had significant effects on myogenesis. Mitochondrial dysfunction as evidenced by decreased ATP content and increased reactive oxygen species production, and increased lipid and protein oxidation was observed in both myoblasts and myotubes after OGA knockdown. Meanwhile, elevated O-GlcNAcylation through either OGA knockdown or treatment with the OGA inhibitor PUGNAc and the O-GlcNAc transferase substrate D-GlcNAc suppressed myotube insulin signalling transduction and glucose uptake. OGA overexpression had no significant effect on insulin sensitivity but sufficiently improved the insulin resistance induced by D-GlcNAc treatment. These data suggest that OGA can modulate mitochondrial density via PGC-1α and mitochondrial function via protein O-GlcNAcylation. In this manner, OGA appears to play a key role in myogenesis and the

  18. FoxO6 regulates memory consolidation and synaptic function

    PubMed Central

    Salih, Dervis A.M.; Rashid, Asim J.; Colas, Damien; de la Torre-Ubieta, Luis; Zhu, Ruo P.; Morgan, Alexander A.; Santo, Evan E.; Ucar, Duygu; Devarajan, Keerthana; Cole, Christina J.; Madison, Daniel V.; Shamloo, Mehrdad; Butte, Atul J.; Bonni, Azad; Josselyn, Sheena A.; Brunet, Anne

    2012-01-01

    The FoxO family of transcription factors is known to slow aging downstream from the insulin/IGF (insulin-like growth factor) signaling pathway. The most recently discovered FoxO isoform in mammals, FoxO6, is highly enriched in the adult hippocampus. However, the importance of FoxO factors in cognition is largely unknown. Here we generated mice lacking FoxO6 and found that these mice display normal learning but impaired memory consolidation in contextual fear conditioning and novel object recognition. Using stereotactic injection of viruses into the hippocampus of adult wild-type mice, we found that FoxO6 activity in the adult hippocampus is required for memory consolidation. Genome-wide approaches revealed that FoxO6 regulates a program of genes involved in synaptic function upon learning in the hippocampus. Consistently, FoxO6 deficiency results in decreased dendritic spine density in hippocampal neurons in vitro and in vivo. Thus, FoxO6 may promote memory consolidation by regulating a program coordinating neuronal connectivity in the hippocampus, which could have important implications for physiological and pathological age-dependent decline in memory. PMID:23222102

  19. Poly-N-acetylglucosamine mediates biofilm formation and antibiotic resistance in Actinobacillus pleuropneumoniae

    PubMed Central

    Izano, Era A.; Sadovskaya, Irina; Vinogradov, Evgeny; Mulks, Martha H.; Velliyagounder, Kabilan; Ragunath, Chandran; Kher, William B.; Ramasubbu, Narayanan; Jabbouri, Saïd; Perry, Malcolm B.; Kaplan, Jeffrey B.

    2007-01-01

    Most field isolates of the swine pathogen Actinobacillus pleuropneumoniae form tenacious biofilms on abiotic surfaces in vitro. We purified matrix polysaccharides from biofilms produced by A. pleuropneumoniae field isolates IA1 and IA5 (serotypes 1 and 5, respectively), and determined their chemical structures by using NMR spectroscopy. Both strains produced matrix polysaccharides consisting of linear chains of N-acetyl-D-glucosamine (GlcNAc) residues in β(1,6) linkage (poly-β-1,6-GlcNAc or PGA). A small percentage of the GlcNAc residues in each polysaccharide were N-deacetylated. These structures were nearly identical to those of biofilm matrix polysaccharides produced by Escherichia coli, Staphylococcus aureus and S. epidermidis. PCR analyses indicated that a gene encoding the PGA-specific glycoside transferase enzyme PgaC was present on the chromosome of 15 out of 15 A. pleuropneumoniae reference strains (serotypes 1-12) and 76 out of 77 A. pleuropneumoniae field isolates (serotypes 1, 5 and 7). A pgaC mutant of strain IA5 failed to form biofilms in vitro, as did wild-type strains IA1 and IA5 when grown in broth supplemented with the PGA-hydrolyzing enzyme dispersin B. Treatment of IA5 biofilms with dispersin B rendered them more sensitive to killing by ampicillin. Our findings suggest that PGA functions as a major biofilm adhesin in A. pleuropneumoniae. Biofilm formation may have relevance to the colonization and pathogenesis of A. pleuropneumoniae in pigs. PMID:17412552

  20. Electrospun Microfiber Scaffolds with Anti-Inflammatory Tributanoylated N-Acetyl-d-Glucosamine Promote Cartilage Regeneration

    PubMed Central

    Kim, Chaekyu; Shores, Lucas; Guo, Qiongyu; Aly, Ahmed; Jeon, Ok Hee; Kim, Do Hun; Bernstein, Nicholas; Bhattacharya, Rahul; Chae, Jemin Jeremy; Yarema, Kevin J.

    2016-01-01

    Tissue-engineering strategies offer promising tools for repairing cartilage damage; however, these strategies suffer from limitations under pathological conditions. As a model disease for these types of nonideal systems, the inflammatory environment in an osteoarthritic (OA) joint limits the efficacy of engineered therapeutics by disrupting joint homeostasis and reducing its capacity for regeneration. In this work, we investigated a sugar-based drug candidate, a tributanoylated N-acetyl-d-glucosamine analogue, called 3,4,6-O-Bu3GlcNAc, that is known to reduce nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in osteoarthritis. 3,4,6-O-Bu3GlcNAc not only inhibited NFκB signaling but also exerted chondrogenic and anti-inflammatory effects on chondrocytes isolated from patients with osteoarthritis. 3,4,6-O-Bu3GlcNAc also increased the expression of extracellular matrix proteins and induced cartilage tissue production in three-dimensional in vitro hydrogel culture systems. To translate these chondrogenic and anti-inflammatory properties to tissue regeneration in osteoarthritis, we implanted 3,4,6-O-Bu3GlcNAc-loaded poly(lactic-co-glycolic acid) microfiber scaffolds into rats. The drug-laden scaffolds were biocompatible, and when seeded with human OA chondrocytes, similarly promoted cartilage tissue formation. 3,4,6-O-Bu3GlcNAc combined with the appropriate structural environment could be a promising therapeutic approach for osteoarthritis. PMID:27019285

  1. Demystifying O-GlcNAcylation: hints from peptide substrates.

    PubMed

    Shi, Jie; Ruijtenbeek, Rob; Pieters, Roland J

    2018-03-22

    O-GlcNAcylation, analogous to phosphorylation, is an essential post-translational modification of proteins at Ser/Thr residues with a single β-N-acetylglucosamine moiety. This dynamic protein modification regulates many fundamental cellular processes and its deregulation has been linked to chronic diseases such as cancer, diabetes and neurodegenerative disorders. Reversible attachment and removal of O-GlcNAc is governed only by O-GlcNAc transferase and O-GlcNAcase, respectively. Peptide substrates, derived from natural O-GlcNAcylation targets, function in the catalytic cores of these two enzymes by maintaining interactions between enzyme and substrate, which makes them ideal models for the study of O-GlcNAcylation and deglycosylation. These peptides provide valuable tools for a deeper understanding of O-GlcNAc processing enzymes. By taking advantage of peptide chemistry, recent progress in the study of activity and regulatory mechanisms of these two enzymes has advanced our understanding of their fundamental specificities as well as their potential as therapeutic targets. Hence, this review summarizes the recent achievements on this modification studied at the peptide level, focusing on enzyme activity, enzyme specificity, direct function, site-specific antibodies and peptide substrate-inspired inhibitors.

  2. [Regulation of terpene metabolism]. [Mentha piperita, Mentha spicata

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

    Croteau, R.

    1989-01-01

    Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

  3. Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase.

    PubMed

    Murphy, Jesse R; Mullins, Elwood A; Kappock, T Joseph

    2016-01-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.

  4. Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase

    PubMed Central

    Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

    2016-01-01

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. PMID:27242998

  5. Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

    DOE PAGES

    Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

    2016-05-23

    Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. Here in this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes andmore » orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. Finally, the ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.« less

  6. Structural and functional analysis of an enhancer GPEI having a phorbol 12-O-tetradecanoate 13-acetate responsive element-like sequence found in the rat glutathione transferase P gene.

    PubMed

    Okuda, A; Imagawa, M; Maeda, Y; Sakai, M; Muramatsu, M

    1989-10-05

    We have recently identified a typical enhancer, termed GPEI, located about 2.5 kilobases upstream from the transcription initiation site of the rat glutathione transferase P gene. Analyses of 5' and 3' deletion mutants revealed that the cis-acting sequence of GPEI contained the phorbol 12-O-tetradecanoate 13-acetate responsive element (TRE)-like sequence in it. For the maximal activity, however, GPEI required an adjacent upstream sequence of about 19 base pairs in addition to the TRE-like sequence. With the DNA binding gel-shift assay, we could detect protein(s) that specifically binds to the TRE-like sequence of GPEI fragment, which was possibly c-jun.c-fos complex or a similar protein complex. The sequence immediately upstream of the TRE-like sequence did not have any activity by itself, but augmented the latter activity by about 5-fold.

  7. A QUANTITATIVE MODEL FOR XENOBIOTIC METABOLIZING ENZYME (XME) INDUCTION REGULATED BY THE PREGNANE X RECEPTOR (PXR)

    EPA Science Inventory

    The nuclear receptor, PXR, is an integral part of the regulation of hepatic metabolism. It has been shown to regulate specific CYPs (phase I drug-metabolizing enzymes) as well as certain phase II drug metabolism activities, including UDP-glucuronosyl transferase (UGT), sulfotran...

  8. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

  9. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

  10. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

  11. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

  12. 21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

  13. Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase: an Enzyme of the Anaerobic Toluene Catabolic Pathway in Denitrifying Bacteria†

    PubMed Central

    Leutwein, Christina; Heider, Johann

    2001-01-01

    Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (R)-benzylsuccinate as first intermediate, which is further metabolized via β-oxidation to benzoyl-coenzyme A (CoA) and succinyl-CoA. A specific succinyl-CoA:(R)-benzylsuccinate CoA-transferase activating (R)-benzylsuccinate to the CoA-thioester was purified and characterized from Thauera aromatica. The enzyme is fully reversible and forms exclusively the 2-(R)-benzylsuccinyl-CoA isomer. Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (R)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an α2β2 conformation. Identity of the subunits with the products of the previously identified toluene-induced bbsEF genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (E)-cinnamoyl-CoA:(R)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases. As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates. PMID:11418570

  14. O-GlcNAcylation of histone deacetylases 1 in hepatocellular carcinoma promotes cancer progression.

    PubMed

    Zhu, Guizhou; Tao, Tao; Zhang, Dongmei; Liu, Xiaojuan; Qiu, Huiyuan; Han, LiJian; Xu, Zhiwei; Xiao, Ying; Cheng, Chun; Shen, Aiguo

    2016-08-01

    Hepatocellular carcinoma (HCC) is a malignant tumor originating in the liver. Previous studies have indicated that O-GlcNAc transferase (OGT) and histone deacetylase-1 (HDAC1) play important roles in the pathogenesis of HCC. In the present study, we investigated the physical link between OGT and HDAC1. The O-GlcNAcylation of HDAC1 is overexpressed in HCC. We found that HDAC1 has two major sites of O-GlcNAcylation in its histone deacetylase domain. HDAC1 O-GlcNAcylation increases the activated phosphorylation of HDAC1, which enhances its enzyme activity. HDAC1 O-GlcNAc mutants promote the p21 transcription regulation through affecting the acetylation levels of histones from chromosome, and then influence the proliferation of HCC cells. We also found that mutants of O-GlcNAcylation site of HDAC1 affect invasion and migration of HepG2 cells. E-cadherin level is highly up-regulated in HDAC1 O-GlcNAc mutant-treated liver cancer cells, which inhibit the occurrence and development of HCC. Our findings suggest that OGT promotes the O-GlcNAc modification of HDAC1in the development of HCC. Therefore, inhibiting O-GlcNAcylation of HDAC1 may repress the progression of HCC. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  15. Matrin 3 co-immunoprecipitates with the heat shock proteins glucose-regulated protein 78 (GRP78), GRP75 and glutathione S-transferase π isoform 2 (GSTπ2) in thymoma cells.

    PubMed

    Osman, Ahmed M; van Loveren, Henk

    2014-06-01

    Here, we report evidence that matrin 3 (MATR3), a highly conserved inner nuclear matrix phosphoprotein, whose function is largely unknown, interacts specifically with the heat shock proteins glucose-regulated protein 78 (GRP78), GRP75 and glutathione S-transferase π isoform 2 (GSTπ2). Using immunoprecipitation experiments of lysates obtained from control and tributyltin oxide (TBTO)-treated thymoma cell line (EL4), we identified MATR3 and its partners by MS/MS analysis and confirmed by immunoblot. We also show that MATR3 undergoes degradation as reported before and that this cleavage process, which is inhibited by the broad-spectrum caspase inhibitor, z-VAD-FMK, is more marked in TBTO-treated cells. Further, we found that the heat shock protein glucose-regulated protein 78 was downregulated in the TBTO-treated cells. The GRP78 protein is known to protect cells from apoptosis by complexing with procaspase 7 thereby preventing caspase activation cascade. By immunoblot analysis, we found that the levels of procaspases-3 and -7 were lower in TBTO-treated cells; in contrast, the level of p20, the active form of caspase 3, was relatively higher in the treated cells compared to that of control cells. We propose that the TBTO-mediated downregulation of GRP78 triggers the caspase cascade pathway leading to MATR3 degradation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  16. Ascorbate Peroxidase and Catalase Activities and Their Genetic Regulation in Plants Subjected to Drought and Salinity Stresses.

    PubMed

    Sofo, Adriano; Scopa, Antonio; Nuzzaci, Maria; Vitti, Antonella

    2015-06-12

    Hydrogen peroxide (H2O2), an important relatively stable non-radical reactive oxygen species (ROS) is produced by normal aerobic metabolism in plants. At low concentrations, H2O2 acts as a signal molecule involved in the regulation of specific biological/physiological processes (photosynthetic functions, cell cycle, growth and development, plant responses to biotic and abiotic stresses). Oxidative stress and eventual cell death in plants can be caused by excess H2O2 accumulation. Since stress factors provoke enhanced production of H2O2 in plants, severe damage to biomolecules can be possible due to elevated and non-metabolized cellular H2O2. Plants are endowed with H2O2-metabolizing enzymes such as catalases (CAT), ascorbate peroxidases (APX), some peroxiredoxins, glutathione/thioredoxin peroxidases, and glutathione sulfo-transferases. However, the most notably distinguished enzymes are CAT and APX since the former mainly occurs in peroxisomes and does not require a reductant for catalyzing a dismutation reaction. In particular, APX has a higher affinity for H2O2 and reduces it to H2O in chloroplasts, cytosol, mitochondria and peroxisomes, as well as in the apoplastic space, utilizing ascorbate as specific electron donor. Based on recent reports, this review highlights the role of H2O2 in plants experiencing water deficit and salinity and synthesizes major outcomes of studies on CAT and APX activity and genetic regulation in drought- and salt-stressed plants.

  17. Ascorbate Peroxidase and Catalase Activities and Their Genetic Regulation in Plants Subjected to Drought and Salinity Stresses

    PubMed Central

    Sofo, Adriano; Scopa, Antonio; Nuzzaci, Maria; Vitti, Antonella

    2015-01-01

    Hydrogen peroxide (H2O2), an important relatively stable non-radical reactive oxygen species (ROS) is produced by normal aerobic metabolism in plants. At low concentrations, H2O2 acts as a signal molecule involved in the regulation of specific biological/physiological processes (photosynthetic functions, cell cycle, growth and development, plant responses to biotic and abiotic stresses). Oxidative stress and eventual cell death in plants can be caused by excess H2O2 accumulation. Since stress factors provoke enhanced production of H2O2 in plants, severe damage to biomolecules can be possible due to elevated and non-metabolized cellular H2O2. Plants are endowed with H2O2-metabolizing enzymes such as catalases (CAT), ascorbate peroxidases (APX), some peroxiredoxins, glutathione/thioredoxin peroxidases, and glutathione sulfo-transferases. However, the most notably distinguished enzymes are CAT and APX since the former mainly occurs in peroxisomes and does not require a reductant for catalyzing a dismutation reaction. In particular, APX has a higher affinity for H2O2 and reduces it to H2O in chloroplasts, cytosol, mitochondria and peroxisomes, as well as in the apoplastic space, utilizing ascorbate as specific electron donor. Based on recent reports, this review highlights the role of H2O2 in plants experiencing water deficit and salinity and synthesizes major outcomes of studies on CAT and APX activity and genetic regulation in drought- and salt-stressed plants. PMID:26075872

  18. Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

    PubMed

    Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

    2002-08-16

    A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

  19. O-GlcNAcylation: A New Cancer Hallmark?

    PubMed

    Fardini, Yann; Dehennaut, Vanessa; Lefebvre, Tony; Issad, Tarik

    2013-01-01

    O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression.

  20. O-GlcNAcylation: A New Cancer Hallmark?

    PubMed Central

    Fardini, Yann; Dehennaut, Vanessa; Lefebvre, Tony; Issad, Tarik

    2013-01-01

    O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression. PMID:23964270

  1. Purification and Biochemical Characterization of Glutathione S-Transferase from Down Syndrome and Normal Children Erythrocytes: A Comparative Study

    ERIC Educational Resources Information Center

    Hamed, Ragaa R.; Maharem, Tahany M.; Abdel-Meguid, Nagwa; Sabry, Gilane M.; Abdalla, Abdel-Monem; Guneidy, Rasha A.

    2011-01-01

    Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was…

  2. Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice

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

    Conklin, Daniel J., E-mail: dj.conklin@louisville.edu; Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40292; Haberzettl, Petra

    2015-06-01

    High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100–300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK·MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null thanmore » WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein–acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1–5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10–20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity. - Graphical abstract: Cyclophosphamide (CY) treatment results in P450-mediated metabolic formation of phosphoramide mustard and acrolein (3-propenal). Acrolein is either metabolized

  3. O-GlcNAc cycling: Emerging Roles in Development and Epigenetics

    PubMed Central

    Love, Dona C.; Krause, Michael W.; Hanover, John A.

    2010-01-01

    The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan. Surprisingly, from flies to man, the O-GlcNAcase (OGA, MGEA5) gene is embedded within the NK cluster, the most evolutionarily ancient of three homeobox gene clusters regulated by PcG repression. PcG repression also plays a key role in maintaining stem cell identity, recruiting the DNA methyltransferase machinery for imprinting, and in X-chromosome inactivation. Intriguingly, the Ogt gene resides near the Xist locus in vertebrates and is subject to regulation by PcG-dependent X-inactivation. OGT is also an enzymatic component of the human dosage compensation complex. These ‘evo-devo’ relationships linking O-GlcNAc cycling to higher order chromatin structure provide insights into how nutrient availability may influence the epigenetic regulation of gene expression. O-GlcNAc cycling at promoters and PcG repression represent concrete mechanisms by which nutritional information may be transmitted across generations in the intra-uterine environment. Thus, the nutrient-sensing hexosamine signaling pathway may be a key contributor to the metabolic deregulation resulting from prenatal exposure to famine, or the ‘vicious cycle’ observed in children of mothers with type-2 diabetes and metabolic disease. PMID:20488252

  4. The NEXT-A (N-terminal EXtension with Transferase and ARS) reaction.

    PubMed

    Taki, Masumi; Kuroiwa, Hiroyuki; Sisido, Masahiko

    2009-01-01

    L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein. A nonnatural amino acid is once charged onto an E. coli tRNA(Phe) by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides alphaA294G mutation on the ARS, alphaT251A, betaG318W, or betaA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.

  5. In vitro synthesis of intermediates involved in the assembly of enterobacterial common antigen (ECA)

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

    Barr, K.; Wolski, S.; Kroto, J.

    1986-05-01

    ECA is a cell surface antigen found in all bacteria belonging to the family Enterobacteriaceae. The serological specificity of ECA is determined by a linear heteropolysaccharide comprised of trisaccharide repeat units; the component sugars are N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-mannosaminuronic acid (ManNAcUA), and 4-acetamido-D-fucose (Fuc4NAc). In vivo studies have suggested that GlcNAc-pyrophosphorylundecaprenol (GlcNAc-PP-lipid) is an intermediate in ECA synthesis. More recently, they have demonstrated UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase activity in cell envelope preparations of E. coli. Radioactivity from UDP-(/sup 3/H)Glc-NAc was incorporated into endogenous lipid acceptor, and the labeled product was characterized as GlcNAc-PP-lipid (lipid I). Transferase activity was inhibited by tunicamycin andmore » UMP, but it was unaffected by UDP. The reaction was reversible, and the synthesis of UDP-(/sup 3/H)GlcNAc from UMP and (/sup 3/H)GlcNAc-PP-lipid was also sensitive to tunicamycin. The simultaneous addition of UDP-(/sup 14/C)ManNAcUA and UDP-(/sup 3/H)GlcNAc to cell envelope preparations resulted in the synthesis of a more polar lipid (lipid II) that contained both labeled sugars in equimolar amounts. Synthesis of lipid II was dependent on prior synthesis of lipid I. Accordingly, (/sup 3/H)GlcNAc-PP-lipid that had been synthesized in vivo served as an acceptor in vitro of ManNAcUA residues from UDP-ManNAcUA. Lipid II has been tentatively identified as ManNAcUA-GlcNAc-pyrophosphorylundecaprenol.« less

  6. Ketone isosteres of 2-N-acetamidosugars as substrates for metabolic cell surface engineering

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

    Hang, Howard C.; Bertozzi, Carolyn R.

    2000-08-22

    Novel chemical reactivity can be engendered on cell surfaces by the metabolic incorporation of unnatural sugars into cell surface glycoconjuagtes. 2-N-Acetamido sugars such as GalNAc and GlcNAc are abundant components of cell surface glycoconjugates, and hence attractive targets for metabolic cell surface engineering. Here we report (1) the synthesis of isosteric analogs bearing a ketone group in place of the N-acetamido group, and (2) evaluation of their metabolic incorporation into mammalian cell surface glycans. A ketone isostere of GalNAc was metabolized by CHO cells through the salvage pathway and delivered to O-linked glycoproteins on the cell surface. Its residence atmore » the core position of O-linked glycans is suggested by studies with a-benzyl GalNAc, an inhibitor of O-linked oligosaccharide extension. A mutant CHO cell line lacking endogenous UDP-GalNAc demonstrated enhanced metabolism of the GalNAc analog, suggesting that competition with native intermediates might limits enzymatic transformation in mammalian cells. A ketone isostere of GlcNAc could not be detected on CHO or human cell surfaces after incubation. Thus, the enzymes in the GlcNAc salvage pathway might be less permissive of unnatural substrates than those comprising the GalNAc salvage pathway. Alternatively, high levels of endogenous GlcNAc derivatives might compete with the ketone isostere and prevent its incorporation into oligosaccharides.« less

  7. O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis

    PubMed Central

    Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping; Matsumoto, Peter A.; Dawdy, Andrew; Barnhill, Benjamin; Oldenhof, Harriëtte; Hartweck, Lynn M.; Maitra, Sushmit; Thomas, Stephen G.; Cockrell, Shelley; Boyce, Michael; Shabanowitz, Jeffrey; Hunt, Donald F.; Olszewski, Neil E.; Sun, Tai-ping

    2016-01-01

    The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein–protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors—PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)—that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development. PMID:26773002

  8. O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis.

    PubMed

    Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping; Matsumoto, Peter A; Dawdy, Andrew; Barnhill, Benjamin; Oldenhof, Harriëtte; Hartweck, Lynn M; Maitra, Sushmit; Thomas, Stephen G; Cockrell, Shelley; Boyce, Michael; Shabanowitz, Jeffrey; Hunt, Donald F; Olszewski, Neil E; Sun, Tai-Ping

    2016-01-15

    The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development. © 2016 Zentella et al.; Published by Cold Spring Harbor Laboratory Press.

  9. Association of manganese superoxide dismutase and glutathione S-transferases genotypes with myocardial infarction in patients with type 2 diabetes mellitus.

    PubMed

    Kariž, Stojan; Nikolajević Starčević, Jovana; Petrovič, Daniel

    2012-10-01

    In the present study we investigated the association between genetic polymorphisms with functional effects on redox regulation: Val16Ala of manganese superoxide dismutase (MnSOD), polymorphic deletions of glutathione S-transferases M1 (GSTM1) and T1 (GSTT1) and Ile105Val of glutathione S-transferase P1 (GSTP1) and myocardial infarction (MI) in a group of patients with type 2 diabetes mellitus. The study population consisted of 463 Caucasian subjects with type 2 diabetes mellitus of more than 10 years' duration: 206 patients with MI and 257 patients with no history of coronary artery disease (CAD). Genotypes were determined by polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP) and with multiplex PCR. The genotype distributions of tested single nucleotide polymorphisms did not show significant difference between cases and controls. After adjustment for age, gender, smoking, BMI, duration of diabetes and lipid parameters carriers of GSTM1/GSTT1-null haplotype showed an increased risk for MI (OR=3.22, 95% CI 1.37-5.04, p=0.03). The GSTM1/GSTT1 haplotype might be a genetic risk factor for MI in patients with type 2 diabetes mellitus. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  10. Differential expression of two glutathione S-transferases identified from the American dog tick, Dermacentor variabilis.

    PubMed

    Dreher-Lesnick, S M; Mulenga, A; Simser, J A; Azad, A F

    2006-08-01

    Reciprocal signalling and gene expression play a cardinal role during pathogen-host molecular interactions and are prerequisite to the maintenance of balanced homeostasis. Gene expression repertoire changes during rickettsial infection and glutathione-S-transferases (GSTs) were among the genes found up-regulated in Rickettsia-infected Dermacentor variabilis. GSTs are well known to play an important part in cellular stress responses in the host. We have cloned two full-length GSTs from D. variabilis (DvGST1 and DvGST2). Comparison of these two DvGST molecules with those of other species indicate that DvGST1 is related to the mammalian class theta and insect class delta GSTs, while DvGST2 does not seem to fall in the same family. Northern blotting analyses revealed differential expression patterns, where DvGST1 and DvGST2 transcripts are found in the tick gut, with DvGST2 transcripts also present in the ovaries. Both DvGST transcripts are up-regulated upon tick feeding. Challenge of fed adult ticks with Escherichia coli injection showed decreased transcript amounts compared with ticks injected with phosphate-buffered saline (sham) and naïve ticks.

  11. O-GlcNAcomic Profiling Identifies Widespread O-Linked β-N-Acetylglucosamine Modification (O-GlcNAcylation) in Oxidative Phosphorylation System Regulating Cardiac Mitochondrial Function*♦

    PubMed Central

    Ma, Junfeng; Liu, Ting; Wei, An-Chi; Banerjee, Partha; O'Rourke, Brian; Hart, Gerald W.

    2015-01-01

    Dynamic cycling of O-linked β-N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic proteins serves as a nutrient sensor to regulate numerous biological processes. However, mitochondrial protein O-GlcNAcylation and its effects on function are largely unexplored. In this study, we performed a comparative analysis of the proteome and O-GlcNAcome of cardiac mitochondria from rats acutely (12 h) treated without or with thiamet-G (TMG), a potent and specific inhibitor of O-GlcNAcase. We then determined the functional consequences in mitochondria isolated from the two groups. O-GlcNAcomic profiling finds that over 88 mitochondrial proteins are O-GlcNAcylated, with the oxidative phosphorylation system as a major target. Moreover, in comparison with controls, cardiac mitochondria from TMG-treated rats did not exhibit altered protein abundance but showed overall elevated O-GlcNAcylation of many proteins. However, O-GlcNAc was unexpectedly down-regulated at certain sites of specific proteins. Concomitantly, TMG treatment resulted in significantly increased mitochondrial oxygen consumption rates, ATP production rates, and enhanced threshold for permeability transition pore opening by Ca2+. Our data reveal widespread and dynamic mitochondrial protein O-GlcNAcylation, serving as a regulator to their function. PMID:26446791

  12. Survey of O-GlcNAc level variations in Xenopus laevis from oogenesis to early development.

    PubMed

    Dehennaut, Vanessa; Lefebvre, Tony; Leroy, Yves; Vilain, Jean-Pierre; Michalski, Jean-Claude; Bodart, Jean-François

    2009-04-01

    Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I-V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.

  13. FBXW10 is negatively regulated in transcription and expression level by protein O-GlcNAcylation.

    PubMed

    Feng, Zhou; Hui, Yan; Ling, Li; Xiaoyan, Liu; Yuqiu, Wang; Peng, Wang; Lianwen, Zhang

    2013-08-23

    Intricate cross-talks exist among multiple post-translational modifications that play critical roles in various cellular events, such as the control of gene expression and regulation of protein function. Here, the cross-talk between O-GlcNAcylation and ubiquitination was investigated in HEK293T cells. By PCR array, 84 ubiquitination-related genes were explored in transcription level in response to the elevation of total protein O-GlcNAcylation due to over-expression of OGT, inhibition of OGA or GlcN treatment. Varied genes were transcriptionally regulated by using different method. But FBXW10, an F-box protein targeting specific proteins for ubiquitination, could be negatively regulated in all ways, suggesting its regulation by protein O-GlcNAcylation. By RT-PCR and Western blot analysis, it was found that FBXW10 could be sharply down-regulated in mRNA and protein level in GlcN-treated cells in a time-dependent way, in line with the enhancement of protein O-GlcNAcylation. It was also found that endogenous FBXW10 was modified by O-GlcNAc in HEK293T cells, implying O-GlcNAcylation might regulate FBXW10 in multiple levels. These findings indicate that O-GlcNAcylation is involved in the regulation of ubiquitination-related genes, and help us understand the cross-talk between O-GlcNAcylation and ubiquitination. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  15. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  16. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  17. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  18. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  19. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  20. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  1. 21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  2. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  3. 21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

  4. Nutrient-driven O-GlcNAc in proteostasis and neurodegeneration.

    PubMed

    Akan, Ilhan; Olivier-Van Stichelen, Stephanie; Bond, Michelle R; Hanover, John A

    2018-01-01

    Proteostasis is essential in the mammalian brain where post-mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient-sensitive nucleocytoplasmic post-translational modification O-linked N-acetylglucosamine (O-GlcNAc) regulates protein homeostasis. The O-GlcNAc modification is highly abundant in the mammalian brain and has been linked to proteopathies, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. C. elegans, Drosophila, and mouse models harboring O-GlcNAc transferase- and O-GlcNAcase-knockout alleles have helped define the role O-GlcNAc plays in development as well as age-associated neurodegenerative disease. These enzymes add and remove the single monosaccharide from protein serine and threonine residues, respectively. Blocking O-GlcNAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. Findings in C. elegans and Drosophila model systems indicate that the dynamic turnover of O-GlcNAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment cell fate decisions. In addition, pathways of autophagy and proteasomal degradation depend on a transcriptional network that is also reliant on O-GlcNAc cycling. Like the quality control system in the endoplasmic reticulum which uses a 'mannose timer' to monitor protein folding, we propose that cytoplasmic proteostasis relies on an 'O-GlcNAc timer' to help regulate the lifetime and fate of nuclear and cytoplasmic proteins. O-GlcNAc-dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Brain-selective knockout mouse models will be an important tool for understanding the role of O-GlcNAc in the

  5. Elevated O-GlcNAcylation promotes colonic inflammation and tumorigenesis by modulating NF-κB signaling.

    PubMed

    Yang, Yong Ryoul; Kim, Dae Hyun; Seo, Young-Kyo; Park, Dohyun; Jang, Hyun-Jun; Choi, Soo Youn; Lee, Yong Hwa; Lee, Gyun Hui; Nakajima, Kazuki; Taniguchi, Naoyuki; Kim, Jung-Min; Choi, Eun-Jeong; Moon, Hyo Youl; Kim, Il Shin; Choi, Jang Hyun; Lee, Ho; Ryu, Sung Ho; Cocco, Lucio; Suh, Pann-Ghill

    2015-05-20

    O-GlcNAcylation is a reversible post-translational modification. O-GlcNAc addition and removal is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. More recent evidence indicates that regulation of O-GlcNAcylation is important for inflammatory diseases and tumorigenesis. In this study, we revealed that O-GlcNAcylation was increased in the colonic tissues of dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced colitis-associated cancer (CAC) animal models. Moreover, the O-GlcNAcylation level was elevated in human CAC tissues compared with matched normal counterparts. To investigate the functional role of O-GlcNAcylation in colitis, we used OGA heterozygote mice, which have an increased level of O-GlcNAcylation. OGA(+/-) mice have higher susceptibility to DSS-induced colitis than OGA(+/+) mice. OGA(+/-) mice exhibited a higher incidence of colon tumors than OGA(+/+) mice. In molecular studies, elevated O-GlcNAc levels were shown to enhance the activation of NF-κB signaling through increasing the binding of RelA/p65 to its target promoters. We also found that Thr-322 and Thr352 in the p65-O-GlcNAcylation sites are critical for p65 promoter binding. These results suggest that the elevated O-GlcNAcylation level in colonic tissues contributes to the development of colitis and CAC by disrupting regulation of NF-κB-dependent transcriptional activity.

  6. Electron transfer dissociation (ETD): The mass spectrometric breakthrough essential for O-GlcNAc protein site assignments – A study of the O-GlcNAcylated protein Host Cell Factor C1

    PubMed Central

    Myers, Samuel A.; Daou, Salima; Affar, El Bachir; Burlingame, AL

    2014-01-01

    The development of electron-based, unimolecular dissociation mass spectrometric methods, i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile post-translational modification (PTM) site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine) signaling has especially advanced with the advent of ETD mass spectrometry. Only within the last five years have proteomic-scale experiments utilizing ETD allowed the assignment of hundreds of O-GlcNAc sites within cells and subcellular structures. Our ability to identify and unambiguously assign the site of O-GlcNAc modifications using ETD is rapidly increasing our understanding of this regulatory glycosylation and its potential interaction with other PTMs. Here, we discuss the advantages of using ETD, complimented with collisional-activation mass spectrometry (CID/CAD), in a study of O-GlcNAc modified peptides of the extensively O-GlcNAcylated protein Host Cell Factor C1 (HCF-1). HCF-1 is a transcriptional co-regulator, forms a stable complex with O-GlcNAc transferase and is involved in control of cell cycle progression. ETD, along with higher energy collisional dissociation (HCD) mass spectrometry, was employed to assign the PTMs of the HCF-1 protein isolated from HEK293T cells. These include nineteen sites of O-GlcNAcylation, two sites of phosphorylation and two sites bearing dimethylarginine, and showcase the residue-specific, PTM complexity of this regulator of cell proliferation. PMID:23335398

  7. A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.

    PubMed

    Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean

    2005-04-01

    Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

  8. Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

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

    Torres, Rodrigo; Lan, Benson; Latif, Yama

    2014-04-01

    The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NOmore » levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully

  9. Cyanidin-3-O-β-glucoside regulates fatty acid metabolism via an AMP-activated protein kinase-dependent signaling pathway in human HepG2 cells

    PubMed Central

    2012-01-01

    Background Hepatic metabolic derangements are key components in the development of fatty liver disease. AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and carnitine palmitoyl transferase 1 (CPT-1) pathway. In this study, cyanidin-3-O-β-glucoside (Cy-3-g), a typical anthocyanin pigment was used to examine its effects on AMPK activation and fatty acid metabolism in human HepG2 hepatocytes. Results Anthocyanin Cy-3-g increased cellular AMPK activity in a calmodulin kinase kinase dependent manner. Furthermore, Cy-3-g substantially induced AMPK downstream target ACC phosphorylation and inactivation, and then decreased malonyl CoA contents, leading to stimulation of CPT-1 expression and significant increase of fatty acid oxidation in HepG2 cells. These effects of Cy-3-g are largely abolished by pharmacological and genetic inhibition of AMPK. Conclusion This study demonstrates that Cy-3-g regulates hepatic lipid homeostasis via an AMPK-dependent signaling pathway. Targeting AMPK activation by anthocyanin may represent a promising approach for the prevention and treatment of obesity-related nonalcoholic fatty liver disease. PMID:22243683

  10. Microsomal Glutathione Transferase 1 Protects Against Toxicity Induced by Silica Nanoparticles but Not by Zinc Oxide Nanoparticles

    PubMed Central

    2012-01-01

    Microsomal glutathione transferase 1 (MGST1) is an antioxidant enzyme located predominantly in the mitochondrial outer membrane and endoplasmic reticulum and has been shown to protect cells from lipid peroxidation induced by a variety of cytostatic drugs and pro-oxidant stimuli. We hypothesized that MGST1 may also protect against nanomaterial-induced cytotoxicity through a specific effect on lipid peroxidation. We evaluated the induction of cytotoxicity and oxidative stress by TiO2, CeO2, SiO2, and ZnO in the human MCF-7 cell line with or without overexpression of MGST1. SiO2 and ZnO nanoparticles caused dose- and time-dependent toxicity, whereas no obvious cytotoxic effects were induced by nanoparticles of TiO2 and CeO2. We also noted pronounced cytotoxicity for three out of four additional SiO2 nanoparticles tested. Overexpression of MGST1 reversed the cytotoxicity of the main SiO2 nanoparticles tested and for one of the supplementary SiO2 nanoparticles but did not protect cells against ZnO-induced cytotoxic effects. The data point toward a role of lipid peroxidation in SiO2 nanoparticle-induced cell death. For ZnO nanoparticles, rapid dissolution was observed, and the subsequent interaction of Zn2+ with cellular targets is likely to contribute to the cytotoxic effects. A direct inhibition of MGST1 by Zn2+ could provide a possible explanation for the lack of protection against ZnO nanoparticles in this model. Our data also showed that SiO2 nanoparticle-induced cytotoxicity is mitigated in the presence of serum, potentially through masking of reactive surface groups by serum proteins, whereas ZnO nanoparticles were cytotoxic both in the presence and in the absence of serum. PMID:22303956

  11. Crosstalk between phosphorylation and O-GlcNAcylation: friend or foe.

    PubMed

    van der Laarse, Saar A M; Leney, Aneika C; Heck, Albert J R

    2018-05-02

    A wide variety of protein post-translational modifications (PTMs) decorate cellular proteins, regulating their structure, interactions and ultimately their function. The density of co-occurring PTMs on proteins can be very high, where multiple PTMs can positively or negatively influence each other's actions, termed PTM crosstalk. In this review, we highlight recent progress in the area of PTM crosstalk, whereby we focus on crosstalk between protein phosphorylation and O-GlcNAcylation. These two PTMs largely target identical (i.e., Ser and Thr) amino acids in proteins. Phosphorylation/O-GlcNAcylation crosstalk comes in many flavors, for instance by competition for the same site/residue (reciprocal crosstalk), as well as by modifications influencing each other in proximity or even distal on the protein sequence. PTM crosstalk is observed on the writers of these modifications (i.e., kinases and O-GlcNAc transferase), on the erasers (i.e., phosphatases and O-GlcNAcase), and on the readers and the substrates. We describe examples of all these different flavors of crosstalk, and additionally the methods that are emerging to better investigate in particular phosphorylation/O-GlcNAcylation crosstalk. © 2018 Federation of European Biochemical Societies.

  12. Genomic organization of plant aminopropyl transferases.

    PubMed

    Rodríguez-Kessler, Margarita; Delgado-Sánchez, Pablo; Rodríguez-Kessler, Gabriela Theresia; Moriguchi, Takaya; Jiménez-Bremont, Juan Francisco

    2010-07-01

    Aminopropyl transferases like spermidine synthase (SPDS; EC 2.5.1.16), spermine synthase and thermospermine synthase (SPMS, tSPMS; EC 2.5.1.22) belong to a class of widely distributed enzymes that use decarboxylated S-adenosylmethionine as an aminopropyl donor and putrescine or spermidine as an amino acceptor to form in that order spermidine, spermine or thermospermine. We describe the analysis of plant genomic sequences encoding SPDS, SPMS, tSPMS and PMT (putrescine N-methyltransferase; EC 2.1.1.53). Genome organization (including exon size, gain and loss, as well as intron number, size, loss, retention, placement and phase, and the presence of transposons) of plant aminopropyl transferase genes were compared between the genomic sequences of SPDS, SPMS and tSPMS from Zea mays, Oryza sativa, Malus x domestica, Populus trichocarpa, Arabidopsis thaliana and Physcomitrella patens. In addition, the genomic organization of plant PMT genes, proposed to be derived from SPDS during the evolution of alkaloid metabolism, is illustrated. Herein, a particular conservation and arrangement of exon and intron sequences between plant SPDS, SPMS and PMT genes that clearly differs with that of ACL5 genes, is shown. The possible acquisition of the plant SPMS exon II and, in particular exon XI in the monocot SPMS genes, is a remarkable feature that allows their differentiation from SPDS genes. In accordance with our in silico analysis, functional complementation experiments of the maize ZmSPMS1 enzyme (previously considered to be SPDS) in yeast demonstrated its spermine synthase activity. Another significant aspect is the conservation of intron sequences among SPDS and PMT paralogs. In addition the existence of microsynteny among some SPDS paralogs, especially in P. trichocarpa and A. thaliana, supports duplication events of plant SPDS genes. Based in our analysis, we hypothesize that SPMS genes appeared with the divergence of vascular plants by a processes of gene duplication and the

  13. Characterization of a lambda-cyhalothrin metabolizing glutathione S-transferase CpGSTd1 from Cydia pomonella (L.).

    PubMed

    Liu, Jiyuan; Yang, Xueqing; Zhang, Yalin

    2014-11-01

    In insects, glutathione S-transferases (GSTs) are enzymes involved in detoxification of insecticides. However, few data are available for the codling moth, Cydia pomonella (L.). In this study, we cloned a delta class GST gene CpGSTd1 from C. pomonella. Real-time quantitative PCR shows that CpGSTd1 was up-regulated with aging, and the mRNA level of CpGSTd1 was higher in the fat body and silk glands than in other tissues. The expression level of CpGSTd1 exposure to insecticide suggests that CpGSTd1 is up-regulated after chlorpyrifos-methyl and lambda-cyhalothrin treatments. Both lambda-cyhalothrin and chlorpyrifos-methyl altered GST activity in vivo. The purified CpGSTd1 protein exhibits a high catalytic efficiency with CDNB and was inhibited by lambda-cyhalothrin and chlorpyrifos-methyl in vitro. Metabolism assays indicate that lambda-cyhalothrin was significantly metabolized while chlorpyrifos-methyl was not metabolized by CpGSTd1. Binding free energy analysis suggests that CpGSTd1 binding is tighter with lambda-cyhalothrin than with chlorpyrifos-methyl. Our study suggests that CpGSTd1 plays a key role in the metabolism of insecticides in C. pomonella.

  14. Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola.

    PubMed

    Calmes, Benoit; Morel-Rouhier, Mélanie; Bataillé-Simoneau, Nelly; Gelhaye, Eric; Guillemette, Thomas; Simoneau, Philippe

    2015-06-18

    Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were 'orphans'. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs.

  15. FoxO regulates microtubule dynamics and polarity to promote dendrite branching in Drosophila sensory neurons

    PubMed Central

    Sears, James C.; Broihier, Heather T.

    2016-01-01

    The size and shape of dendrite arbors are defining features of neurons and critical determinants of neuronal function. The molecular mechanisms establishing arborization patterns during development are not well understood, though properly regulated microtubule (MT) dynamics and polarity are essential. We previously found that FoxO regulates axonal MTs, raising the question of whether it also regulates dendritic MTs and morphology. Here we demonstrate that FoxO promotes dendrite branching in all classes of Drosophila dendritic arborization (da) neurons. FoxO is required both for initiating growth of new branches and for maintaining existing branches. To elucidate FoxO function, we characterized MT organization in both foxO null and overexpressing neurons. We find that FoxO directs MT organization and dynamics in dendrites. Moreover, it is both necessary and sufficient for anterograde MT polymerization, which is known to promote dendrite branching. Lastly, FoxO promotes proper larval nociception, indicating a functional consequence of impaired da neuron morphology in foxO mutants. Together, our results indicate that FoxO regulates dendrite structure and function and suggest that FoxO-mediated pathways control MT dynamics and polarity. PMID:27546375

  16. Global deletion of glutathione S-Transferase A4 exacerbates developmental nonalcoholic steatohepatitis

    USDA-ARS?s Scientific Manuscript database

    We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor a (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We us...

  17. Lactobacillus casei Ferments the N-Acetylglucosamine Moiety of Fucosyl-α-1,3-N-Acetylglucosamine and Excretes l-Fucose

    PubMed Central

    Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio

    2012-01-01

    We have previously characterized from Lactobacillus casei BL23 three α-l-fucosidases, AlfA, AlfB, and AlfC, which hydrolyze in vitro natural fucosyl-oligosaccharides. In this work, we have shown that L. casei is able to grow in the presence of fucosyl-α-1,3-N-acetylglucosamine (Fuc-α-1,3-GlcNAc) as a carbon source. Interestingly, L. casei excretes the l-fucose moiety during growth on Fuc-α-1,3-GlcNAc, indicating that only the N-acetylglucosamine moiety is being metabolized. Analysis of the genomic sequence of L. casei BL23 shows that downstream from alfB, which encodes the α-l-fucosidase AlfB, a gene, alfR, that encodes a transcriptional regulator is present. Divergently from alfB, three genes, alfEFG, that encode proteins with homology to the enzyme IIAB (EIIAB), EIIC, and EIID components of a mannose-class phosphoenolpyruvate:sugar phosphotransferase system (PTS) are present. Inactivation of either alfB or alfF abolishes the growth of L. casei on Fuc-α-1,3-GlcNAc. This proves that AlfB is involved in Fuc-α-1,3-GlcNAc metabolism and that the transporter encoded by alfEFG participates in the uptake of this disaccharide. A mutation in the PTS general component enzyme I does not eliminate the utilization of Fuc-α-1,3-GlcNAc, suggesting that the transport via the PTS encoded by alfEFG is not coupled to phosphorylation of the disaccharide. Transcriptional analysis with alfR and ccpA mutants shows that the two gene clusters alfBR and alfEFG are regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor AlfR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This work reports for the first time the characterization of the physiological role of an α-l-fucosidase in lactic acid bacteria and the utilization of Fuc-α-1,3-GlcNAc as a carbon source for bacteria. PMID:22544237

  18. Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines.

    PubMed

    Sawers, L; Ferguson, M J; Ihrig, B R; Young, H C; Chakravarty, P; Wolf, C R; Smith, G

    2014-09-09

    Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT-PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.

  19. Amino Sugars Enhance the Competitiveness of Beneficial Commensals with Streptococcus mutans through Multiple Mechanisms

    PubMed Central

    Farivar, Tanaz; Burne, Robert A.

    2016-01-01

    ABSTRACT Biochemical and genetic aspects of the metabolism of the amino sugars N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) by commensal oral streptococci and the effects of these sugars on interspecies competition with the dental caries pathogen Streptococcus mutans were explored. Multiple S. mutans wild-type isolates displayed long lag phases when transferred from glucose-containing medium to medium with GlcNAc as the primary carbohydrate source, but commensal streptococci did not. Competition in liquid coculture or dual-species biofilms between S. mutans and Streptococcus gordonii showed that S. gordonii was particularly dominant when the primary carbohydrate was GlcN or GlcNAc. Transcriptional and enzymatic assays showed that the catabolic pathway for GlcNAc was less highly induced in S. mutans than in S. gordonii. Exposure to H2O2, which is produced by S. gordonii and antagonizes the growth of S. mutans, led to reduced mRNA levels of nagA and nagB in S. mutans. When the gene for the transcriptional regulatory NagR was deleted in S. gordonii, the strain produced constitutively high levels of nagA (GlcNAc-6-P deacetylase), nagB (GlcN-6-P deaminase), and glmS (GlcN-6-P synthase) mRNA. Similar to NagR of S. mutans (NagRSm), the S. gordonii NagR protein (NagRSg) could bind to consensus binding sites (dre) in the nagA, nagB, and glmS promoter regions of S. gordonii. Notably, NagRSg binding was inhibited by GlcN-6-P, but G-6-P had no effect, unlike for NagRSm. This study expands the understanding of amino sugar metabolism and NagR-dependent gene regulation in streptococci and highlights the potential for therapeutic applications of amino sugars to prevent dental caries. IMPORTANCE Amino sugars are abundant in the biosphere, so the relative efficiency of particular bacteria in a given microbiota to metabolize these sources of carbon and nitrogen might have a profound impact on the ecology of the community. Our investigation reveals that several oral commensal

  20. Amino Sugars Enhance the Competitiveness of Beneficial Commensals with Streptococcus mutans through Multiple Mechanisms.

    PubMed

    Zeng, Lin; Farivar, Tanaz; Burne, Robert A

    2016-06-15

    Biochemical and genetic aspects of the metabolism of the amino sugars N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) by commensal oral streptococci and the effects of these sugars on interspecies competition with the dental caries pathogen Streptococcus mutans were explored. Multiple S. mutans wild-type isolates displayed long lag phases when transferred from glucose-containing medium to medium with GlcNAc as the primary carbohydrate source, but commensal streptococci did not. Competition in liquid coculture or dual-species biofilms between S. mutans and Streptococcus gordonii showed that S. gordonii was particularly dominant when the primary carbohydrate was GlcN or GlcNAc. Transcriptional and enzymatic assays showed that the catabolic pathway for GlcNAc was less highly induced in S. mutans than in S. gordonii Exposure to H2O2, which is produced by S. gordonii and antagonizes the growth of S. mutans, led to reduced mRNA levels of nagA and nagB in S. mutans When the gene for the transcriptional regulatory NagR was deleted in S. gordonii, the strain produced constitutively high levels of nagA (GlcNAc-6-P deacetylase), nagB (GlcN-6-P deaminase), and glmS (GlcN-6-P synthase) mRNA. Similar to NagR of S. mutans (NagRSm), the S. gordonii NagR protein (NagRSg) could bind to consensus binding sites (dre) in the nagA, nagB, and glmS promoter regions of S. gordonii Notably, NagRSg binding was inhibited by GlcN-6-P, but G-6-P had no effect, unlike for NagRSm This study expands the understanding of amino sugar metabolism and NagR-dependent gene regulation in streptococci and highlights the potential for therapeutic applications of amino sugars to prevent dental caries. Amino sugars are abundant in the biosphere, so the relative efficiency of particular bacteria in a given microbiota to metabolize these sources of carbon and nitrogen might have a profound impact on the ecology of the community. Our investigation reveals that several oral commensal bacteria have a much

  1. Inherited glutathione-S-transferase deficiency is a risk factor for pulmonary asbestosis.

    PubMed

    Smith, C M; Kelsey, K T; Wiencke, J K; Leyden, K; Levin, S; Christiani, D C

    1994-09-01

    Pulmonary diseases attributable to asbestos exposure constitute a significant public health burden, yet few studies have investigated potential genetic determinants of susceptibility to asbestos-related diseases. The glutathione-S-transferases are a family of conjugating enzymes that both catalyze the detoxification of a variety of potentially cytotoxic electrophilic agents and act in the generation of sulfadipeptide leukotriene inflammatory mediators. The gene encoding glutathione-S-transferase class mu (GSTM-1) is polymorphic; approximately 50% of Caucasian individuals have a homozygous deletion of this gene and do not produce functional enzyme. Glutathione-S-transferase mu (GST-mu) deficiency has been previously reported to be associated with smoking-induced lung cancer. We conducted a cross-sectional study to examine the prevalence of the homozygous deletion for the GSTM-1 gene in members of the carpentry trade occupationally exposed to asbestos. Members of the United Brotherhood of Carpenters and Joiners of America attending their 1991 National Union conference were invited to participate. Each participant was offered a chest X-ray and was asked to complete a comprehensive questionnaire and have their blood drawn. All radiographs were assessed for the presence of pneumoconiosis in a blinded fashion by a National Institute for Occupational Safety and Health-certified International Labor Office "B" reader. Individual GSTM-1 status was determined using polymerase chain reaction methods. Six hundred fifty-eight workers were studied. Of these, 80 (12.2%) had X-ray abnormalities associated with asbestos exposure. Individuals genetically deficient in GST-mu were significantly more likely to have radiographic evidence of nonmalignant asbestos-related disease than those who were not deficient (chi 2 = 5.0; P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. The identification and characterisation of a functional interaction between arginyl-tRNA-protein transferase and topoisomerase II.

    PubMed

    Barker, Catherine R; Mouchel, Nathalie A P; Jenkins, John R

    2006-04-07

    Topoisomerase II is required for the viability of all eukaryotic cells. It plays important roles in DNA replication, recombination, chromosome segregation, and the maintenance of the nuclear scaffold. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between arginyl-tRNA-protein transferase (Ate1) and the N-terminal domain of the S. cerevisiae topoisomerase II, including the potential site of interaction. Ate1 is a component of the N-end rule protein degradation pathway which targets proteins for degradation. We also propose a previously unidentified role for Ate1 in modulating the level of topoisomerase II through the cell cycle.

  3. Glutathione S-Transferases Interact with AMP-Activated Protein Kinase: Evidence for S-Glutathionylation and Activation In Vitro

    PubMed Central

    Polge, Cécile; Ramirez, Sacnicte; Michelland, Sylvie; Sève, Michel; Vertommen, Didier; Rider, Mark; Lentze, Nicolas; Auerbach, Daniel; Schlattner, Uwe

    2013-01-01

    AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions. PMID:23741294

  4. GRASP55 Senses Glucose Deprivation through O-GlcNAcylation to Promote Autophagosome-Lysosome Fusion.

    PubMed

    Zhang, Xiaoyan; Wang, Leibin; Lak, Behnam; Li, Jie; Jokitalo, Eija; Wang, Yanzhuang

    2018-04-23

    The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. GLUTATHIONE S-TRANSFERASE THETA 1-1-DEPENDENT METABOLISM OF THE DISINFECTION BYPRODUCT BROMODICHLOROMETHANE

    EPA Science Inventory

    ABSTRACT
    Bromodichloromethane (BDCM), a prevalent drinking water disinfection by-product, was previously shown to be mutagenic in Salmonella expressing glutathione S-transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the...

  6. [Regulation of terpene metabolism]. Annual progress report, March 15, 1988--March 14, 1989

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

    Croteau, R.

    1989-12-31

    Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

  7. DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1

    EPA Science Inventory


    DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1. R A Pegram1 and M K Ross2. 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC; 1Pharmacokinetics Branch, NHEERL, ORD, United States Environmental Protection Ag...

  8. Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii: synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases.

    PubMed

    Ruiz-Baca, Estela; Villagómez-Castro, Julio C; Leal-Morales, Carlos A; Sabanero-López, Myrna; Flores-Carreón, Arturo; López-Romero, Everardo

    2005-01-01

    A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.

  9. Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines

    PubMed Central

    Sawers, L; Ferguson, M J; Ihrig, B R; Young, H C; Chakravarty, P; Wolf, C R; Smith, G

    2014-01-01

    Background: Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. Methods: Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT–PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. Results: Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. Conclusions: Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients. PMID:25010864

  10. Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling

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

    Singhal, Sharad S., E-mail: ssinghal@coh.org; Singh, Sharda P.; Singhal, Preeti

    2015-12-15

    4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxidesmore » and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes — higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. - Highlights: • GSTs are the major

  11. Synthesis and regulation of chlorogenic acid in potato: Rerouting phenylpropanoid flux in HQT silenced lines

    USDA-ARS?s Scientific Manuscript database

    Chlorogenic acid (CGA) is the major phenolic sink in potato tubers and can constitute over 90% of total phenylpropanoids. The regulation of CGA biosynthesis in potato and the role of the CGA biosynthetic gene hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase (HQT) was characterized. A sucros...

  12. O-Linked N-Acetylglucosaminylation of Sp1 Inhibits the Human Immunodeficiency Virus Type 1 Promoter▿

    PubMed Central

    Jochmann, Ramona; Thurau, Mathias; Jung, Susan; Hofmann, Christian; Naschberger, Elisabeth; Kremmer, Elisabeth; Harrer, Thomas; Miller, Matthew; Schaft, Niels; Stürzl, Michael

    2009-01-01

    Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5′ long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5′ LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-d-glucosamine (O-GlcNAc), we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4+ T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS. PMID:19193796

  13. The role of endoxyloglucan transferase in the organization of plant cell walls.

    PubMed

    Nishitani, K

    1997-01-01

    The plant cell wall plays a central role in morphogenesis as well as responsiveness to environmental signals. Xyloglucans are the principal component of the plant cell wall matrix and serve as cross-links between cellulose microfibrils to form the cellulose-xyloglucan framework. Endoxyloglucan transferase (EXGT), which was isolated and characterized in 1992, is an enzyme that mediates molecular grafting reaction between xyloglucan molecules. Structural studies on cDNAs encoding EXGT and its related proteins have disclosed the ubiquitous presence in the plant kingdom of a large multigene family of xyloglucan-related proteins (XRPs). Each XRP functions as either hydrolase or transferase acting on xyloglucans and is considered to be responsible for rearrangement of the cellulose-xyloglucan framework, the processes essential for the construction, modification, and degradation of plant cell walls. Different XRP genes exhibit potentially different expression profiles with respect to tissue specificity and responsiveness to hormonal and mechanical signals. The molecular approach to individual XRP genes will open a new path for exploring the controlling mechanisms by which the plant cell wall is constructed and reformed during plant growth and development.

  14. "Nutrient-sensing" and self-renewal: O-GlcNAc in a new role.

    PubMed

    Sharma, Nikita S; Saluja, Ashok K; Banerjee, Sulagna

    2018-06-01

    Whether embryonic, hematopoietic or cancer stem cells, this metabolic reprogramming is dependent on the nutrient-status and bioenergetic pathways that is influenced by the micro-environmental niches like hypoxia. Thus, the microenvironment plays a vital role in determining the stem cell fate by inducing metabolic reprogramming. Under the influence of the microenvironment, like hypoxia, the stem cells have increased glucose and glutamine uptake which result in activation of hexosamine biosynthesis pathway (HBP) and increased O-GlcNAc Transferase (OGT). The current review is focused on understanding how HBP, a nutrient-sensing pathway (that leads to increased OGT activity) is instrumental in regulating self-renewal not only in embryonic and hematopoietic stem cells (ESC/HSC) but also in cancer stem cells.

  15. Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Fa, Svetlana; Pogrmic-Majkic, Kristina; Andric, Nebojsa

    2016-01-05

    Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Expression profiling of selected glutathione transferase genes in Zea mays (L.) seedlings infested with cereal aphids.

    PubMed

    Sytykiewicz, Hubert; Chrzanowski, Grzegorz; Czerniewicz, Paweł; Sprawka, Iwona; Łukasik, Iwona; Goławska, Sylwia; Sempruch, Cezary

    2014-01-01

    The purpose of this report was to evaluate the expression patterns of selected glutathione transferase genes (gst1, gst18, gst23 and gst24) in the tissues of two maize (Zea mays L.) varieties (relatively resistant Ambrozja and susceptible Tasty Sweet) that were colonized with oligophagous bird cherry-oat aphid (Rhopalosiphum padi L.) or monophagous grain aphid (Sitobion avenae L.). Simultaneously, insect-triggered generation of superoxide anion radicals (O2•-) in infested Z. mays plants was monitored. Quantified parameters were measured at 1, 2, 4, 8, 24, 48 and 72 h post-initial aphid infestation (hpi) in relation to the non-infested control seedlings. Significant increases in gst transcript amounts were recorded in aphid-stressed plants in comparison to the control seedlings. Maximal enhancement in the expression of the gst genes in aphid-attacked maize plants was found at 8 hpi (gst23) or 24 hpi (gst1, gst18 and gst24) compared to the control. Investigated Z. mays cultivars formed excessive superoxide anion radicals in response to insect treatments, and the highest overproduction of O2•- was noted 4 or 8 h after infestation, depending on the aphid treatment and maize genotype. Importantly, the Ambrozja variety could be characterized as having more profound increments in the levels of gst transcript abundance and O2•- generation in comparison with the Tasty Sweet genotype.

  17. Indigofera suffruticosa Mill extracts up-regulate the expression of the π class of glutathione S-transferase and NAD(P)H: quinone oxidoreductase 1 in rat Clone 9 liver cells.

    PubMed

    Chen, Chun-Chieh; Liu, Chin-San; Li, Chien-Chun; Tsai, Chia-Wen; Yao, Hsien-Tsung; Liu, Te-Chung; Chen, Haw-Wen; Chen, Pei-Yin; Wu, Yu-Ling; Lii, Chong-Kuei; Liu, Kai-Li

    2013-09-01

    Because induction of phase II detoxification enzyme is important for chemoprevention, we study the effects of Indigofera suffruticosa Mill, a medicinal herb, on the expression of π class of glutathione S-transferase (GSTP) and NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Both water and ethanolic extracts of I. suffruticosa significantly increased the expression and enzyme activities of GSTP and NQO1. I. suffruticosa extracts up-regulated GSTP promoter activity and the binding affinity of nuclear factor erythroid 2-related factor 2 (Nrf2) with the GSTP enhancer I oligonucleotide. Moreover, I. suffruticosa extracts increased nuclear Nrf2 accumulation as well as ARE transcriptional activity. The level of phospho-ERK was augmented by I. suffruticosa extracts, and the ERK inhibitor PD98059 abolished the I. suffruticosa extract-induced ERK activation and GSTP and NQO-1 expression. Moreover, I. suffruticosa extracts, especially the ethanolic extract increased the glutathione level in mouse liver and red blood cells as well as Clone 9 liver cells. The efficacy of I. suffruticosa extracts in induction of phase II detoxification enzymes and glutathione content implies that I. suffruticosa could be considered as a potential chemopreventive agent. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Differential substrate behaviours of ethylene oxide and propylene oxide towards human glutathione transferase theta hGSTT1-1.

    PubMed

    Thier, R; Wiebel, F A; Bolt, H M

    1999-11-01

    The transformation of ethylene oxide (EO), propylene oxide (PO) and 1-butylene oxide (1-BuO) by human glutathione transferase theta (hGSTT1-1) was studied comparatively using 'conjugator' (GSTT1 + individuals) erythrocyte lysates. The relative sequence of velocity of enzymic transformation was PO > EO > 1-BuO. The faster transformation of PO compared to EO was corroborated in studies with human and rat GSTT1-1 (hGSTT1-1 and rGSTT1-1, respectively) expressed by Salmonella typhimurium TA1535. This sequence of reactivities of homologous epoxides towards GSTT1-1 contrasts to the sequence observed in homologous alkyl halides (methyl bromide, MBr; ethyl bromide, EtBr; n-propyl bromide, PrBr) where the relative sequence MeBr > EtBr > PrBr is observed. The higher reactivity towards GSTT1-1 of propylene oxide compared to ethylene oxide is consistent with a higher chemical reactivity. This is corroborated by experimental data of acid-catalysed hydrolysis of a number of aliphatic epoxides, including ethylene oxide and propylene oxide and consistent with semi-empirical molecular orbital modelings.

  19. A Tyrosine-Reactive Irreversible Inhibitor for Glutathione S-Transferase Pi (GSTP1)

    PubMed Central

    Crawford, L. A.; Weerapana, E.

    2016-01-01

    Glutathione S-Transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, we report LAS17, a dichlorotriazine-containing compound that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition. PMID:27113843

  20. A tyrosine-reactive irreversible inhibitor for glutathione S-transferase Pi (GSTP1).

    PubMed

    Crawford, L A; Weerapana, E

    2016-05-24

    Glutathione S-transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, we report LAS17, a dichlorotriazine-containing compound that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition.

  1. Agm1/Pgm3-Mediated Sugar Nucleotide Synthesis Is Essential for Hematopoiesis and Development▿

    PubMed Central

    Greig, Kylie T.; Antonchuk, Jennifer; Metcalf, Donald; Morgan, Phillip O.; Krebs, Danielle L.; Zhang, Jian-Guo; Hacking, Douglas F.; Bode, Lars; Robb, Lorraine; Kranz, Christian; de Graaf, Carolyn; Bahlo, Melanie; Nicola, Nicos A.; Nutt, Stephen L.; Freeze, Hudson H.; Alexander, Warren S.; Hilton, Douglas J.; Kile, Benjamin T.

    2007-01-01

    Carbohydrate modification of proteins includes N-linked and O-linked glycosylation, proteoglycan formation, glycosylphosphatidylinositol anchor synthesis, and O-GlcNAc modification. Each of these modifications requires the sugar nucleotide UDP-GlcNAc, which is produced via the hexosamine biosynthesis pathway. A key step in this pathway is the interconversion of GlcNAc-6-phosphate (GlcNAc-6-P) and GlcNAc-1-P, catalyzed by phosphoglucomutase 3 (Pgm3). In this paper, we describe two hypomorphic alleles of mouse Pgm3 and show there are specific physiological consequences of a graded reduction in Pgm3 activity and global UDP-GlcNAc levels. Whereas mice lacking Pgm3 die prior to implantation, animals with less severe reductions in enzyme activity are sterile, exhibit changes in pancreatic architecture, and are anemic, leukopenic, and thrombocytopenic. These phenotypes are accompanied by specific rather than wholesale changes in protein glycosylation, suggesting that while universally required, the functions of certain proteins and, as a consequence, certain cell types are especially sensitive to reductions in Pgm3 activity. PMID:17548465

  2. Characterization of the endomannosidase pathway for the processing of N-linked oligosaccharides in glucosidase II-deficient and parent mouse lymphoma cells.

    PubMed

    Moore, S E; Spiro, R G

    1992-04-25

    Studies on N-linked oligosaccharide processing in the mouse lymphoma glucosidase II-deficient mutant cell line (PHAR2.7) as well as the parent BW5147 cells indicated that the former maintain their capacity to synthesize complex carbohydrate units through the use of the deglucosylation mechanism provided by endomannosidase. The in vivo activity of this enzyme was evident in the mutant cells from their production of substantial amounts of glucosylated mannose saccharides, predominantly Glc2Man; moreover, in the presence of 1-deoxymannojirimycin or kifunensine to prevent processing by mannosidase I, N-linked Man8GlcNAc2 was observed entirely in the form of the characteristic isomer in which the terminal mannose of the alpha 1,3-linked branch is missing (isomer A). In contrast, parent lymphoma cells, as well as HepG2 cells in the presence of 1-deoxymannojirimycin accumulated Man9GlcNAc2 as the primary deglucosylated N-linked oligosaccharide and contained only about 16% of their Man8GlcNAc2 as isomer A. In the presence of the glucosidase inhibitor castanospermine the mutant released Glc3Man instead of Glc2Man, and the parent cells converted their deglucosylation machinery to the endomannosidase route. Despite the mutant's capacity to accommodate a large traffic through this pathway no increase in the in vitro determined endomannosidase activity was evident. The exclusive utilization of endomannosidase by the mutant for the deglucosylation of its predominant N-linked Glc2Man9GlcNAc2 permitted an exploration of the in vivo site of this enzyme's action. Pulse-chase studies utilizing sucrose-D2O density gradient centrifugation indicated that the Glc2Man9GlcNAc2 to Man8GlcNAc2 conversion is a relatively late event that is temporally separated from the endoplasmic reticulum-situated processing of Glc3Man9GlcNAc2 to Glc2Man9GlcNAc2 and in contrast to the latter takes place in the Golgi compartment.

  3. Crystal structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product.

    PubMed

    Hadfield, A T; Harvey, D J; Archer, D B; MacKenzie, D A; Jeenes, D J; Radford, S E; Lowe, G; Dobson, C M; Johnson, L N

    1994-11-11

    The crystal structure of a mutant hen egg white lysozyme, in which the key catalytic residue aspartic acid 52 has been changed to a serine residue (D52S HEWL), has been determined and refined to a crystallographic R value of 0.173 for all data F > 0 between 8 and 1.9 A resolution. The D52S HEWL structure is very similar to the native HEWL structure (r.m.s. deviation of main-chain atoms 0.20 A). Small shifts that result from the change in hydrogen bonding pattern on substitution of Asp by Ser were observed in the loop between beta-strands in the region of residues 46 to 49. D52S HEWL exhibits less than 1% activity against the bacterial cell wall substrate. Cocrystallisation experiments with the hexasaccharide substrate beta(1-4) polymer of N-acetyl-D-glucosamine (GlcNAc6) resulted in crystals between 5 days and 14 days after the initial mixing of enzyme and substrate. Analysis by laser absorption mass spectrometry of the oligosaccharides present after incubation with native and D52S HEWL under conditions similar to those used for crystal growth showed that after 14 days with native HEWL complete catalysis to GlcNAc3. GlcNAc2 and GlcNac had occurred but with D52S HEWL only partial catalysis to the major products GlcNAc4 and GlcNAc2 had occurred and at least 50% of the GlcNAc6 remained intact. X-ray analysis of the D52S-oligosaccharide complex crystals showed that they contained the product GlcNAc4. The structure of the D52S HEWL-GlcNAc4 complex has been determined and refined to an R value of 0.160 for data between 8 and 2 A resolution. GlcNAc4 occupies sites A to D in the active site cleft. Careful refinement and examination of 2Fo-Fc electron density maps showed that the sugar in site D has the sofa conformation, a conformation previously observed with the HEWL complex with tetra-N-acetylglucosamine lactone transition state analogue, the HEWL complex with the cell wall trisaccharide and the phage T4 lysozyme complex with a cell wall product. The semi-axial C(5)-C(6

  4. FoxO1 transcriptional activities in VEGF expression and beyond: a key regulator in functional angiogenesis?

    PubMed

    Ren, Bin

    2018-04-24

    FoxO1 has emerged as an important regulator of angiogenesis. Recent work published in this Journal shows that FoxO1 regulates VEGF expression in keratinocytes and is required for angiogenesis in wound healing. Since FoxO1 also regulates CD36 transcription, and endothelial cell differentiation and vascular maturation, this transcription factor may be essential for the formation of functional vascular networks via coupling the regulation of CD36 in vascular endothelial cells under physiological and pathological conditions. Although many outstanding questions remain to be answered, the mechanisms by which FoxO1 regulates VEGF in keratinocytes provide insight into the development of functional angiogenesis and further our understanding of vascular biology. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  5. Enhancement of endoplasmic reticulum (ER) degradation of misfolded Null Hong Kong alpha1-antitrypsin by human ER mannosidase I.

    PubMed

    Hosokawa, Nobuko; Tremblay, Linda O; You, Zhipeng; Herscovics, Annette; Wada, Ikuo; Nagata, Kazuhiro

    2003-07-11

    Misfolded glycoproteins synthesized in the endoplasmic reticulum (ER) are degraded by cytoplasmic proteasomes, a mechanism known as ERAD (ER-associated degradation). In the present study, we demonstrate that ERAD of the misfolded genetic variant-null Hong Kong alpha1-antitrypsin is enhanced by overexpression of the ER processing alpha1,2-mannosidase (ER ManI) in HEK 293 cells, indicating the importance of ER ManI in glycoprotein quality control. We showed previously that EDEM, an enzymatically inactive mannosidase homolog, interacts with misfolded alpha1-antitrypsin and accelerates its degradation (Hosokawa, N., Wada, I., Hasegawa, K., Yorihuzi, T., Tremblay, L. O., Herscovics, A., and Nagata, K. (2001) EMBO Rep. 2, 415-422). Herein we demonstrate a combined effect of ER ManI and EDEM on ERAD of misfolded alpha1-antitrypsin. We also show that misfolded alpha1-antitrypsin NHK contains labeled Glc1Man9GlcNAc and Man5-9GlcNAc released by endo-beta-N-acetylglucosaminidase H in pulse-chase experiments with [2-3H]mannose. Overexpression of ER ManI greatly increases the formation of Man8GlcNAc, induces the formation of Glc1Man8GlcNAc and increases trimming to Man5-7GlcNAc. We propose a model whereby the misfolded glycoprotein interacts with ER ManI and with EDEM, before being recognized by downstream ERAD components. This detailed characterization of oligosaccharides associated with a misfolded glycoprotein raises the possibility that the carbohydrate recognition determinant triggering ERAD may not be restricted to Man8GlcNAc2 isomer B as previous studies have suggested.

  6. O-GlcNAc: a novel regulator of immunometabolism.

    PubMed

    Machacek, Miranda; Slawson, Chad; Fields, Patrick E

    2018-06-01

    The rapidly expanding field of immunometabolism focuses on how metabolism controls the function of immune cells. CD4 + T cells are essential for the adaptive immune response leading to the eradication of specific pathogens. However, when T cells are inappropriately over-active, they can drive autoimmunity, allergic disease, and chronic inflammation. The mechanisms by which metabolic changes influence function in CD4 + T cells are not fully understood. The post-translational protein modification, O-GlcNAc (O-linked β-N-acetylglucosamine), dynamically cycles on and off of intracellular proteins as cells respond to their environment and flux through metabolic pathways changes. As the rate of O-GlcNAc cycling fluctuates, protein function, stability, and/or localization can be affected. Thus, O-GlcNAc is critically poised at the nexus of cellular metabolism and function. This review highlights the intra- and extracellular metabolic factors that influence CD4 + T cell activation and differentiation and how O-GlcNAc regulates these processes. We also propose areas of future research that may illuminate O-GlcNAc's role in the plasticity and pathogenicity of CD4 + T cells and uncover new potential therapeutic targets.

  7. Genetic analysis of the roles of agaA, agaI, and agaS genes in the N-acetyl-D-galactosamine and D-galactosamine catabolic pathways in Escherichia coli strains O157:H7 and C

    PubMed Central

    2013-01-01

    Background The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C. Results Knockout mutants in agaA, agaI, and agaS were constructed to test their roles in Aga and Gam utilization. Knockout mutants in the N-acetylglucosamine (GlcNAc) pathway genes nagA and nagB coding for GlcNAc-6-P deacetylase and glucosamine-6-P deaminase/isomerase, respectively, and double knockout mutants ΔagaA ΔnagA and ∆agaI ∆nagB were also constructed to investigate if there is any interplay of these enzymes between the Aga/Gam and the GlcNAc pathways. It is shown that Aga utilization was unaffected in ΔagaA mutants but ΔagaA ΔnagA mutants were blocked in Aga and GlcNAc utilization. E. coli C ΔnagA could not grow on GlcNAc but could grow when the aga/gam regulon was constitutively expressed. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA resulted in growth on both Aga and GlcNAc. It was also found that ΔagaI, ΔnagB, and ∆agaI ΔnagB mutants were unaffected in utilization of Aga and Gam. Importantly, ΔagaS mutants were blocked in Aga and Gam utilization. Expression analysis of relevant genes in these strains with different genetic backgrounds by real time RT-PCR supported these observations. Conclusions Aga utilization was not affected in ΔagaA mutants because nagA was expressed and substituted for agaA. Complementation of ΔagaA ΔnagA mutants with either agaA or nagA also showed that both agaA and nagA can substitute for each other. The ∆agaI, ∆nagB, and ∆agaI ∆nagB mutants were

  8. C. elegans EAK-3 inhibits dauer arrest via nonautonomous regulation of nuclear DAF-16/FoxO activity

    PubMed Central

    Zhang, Yanmei; Xu, Jinling; Puscau, Cristina; Kim, Yongsoon; Wang, Xi; Alam, Hena; Hu, Patrick J.

    2008-01-01

    SUMMARY Insulin regulates development, metabolism, and lifespan via a conserved PI3K/Akt pathway that promotes cytoplasmic sequestration of FoxO transcription factors. The regulation of nuclear FoxO is poorly understood. In the nematode Caenorhabditis elegans, insulin-like signaling functions in larvae to inhibit dauer arrest and acts during adulthood to regulate lifespan. In a screen for genes that modulate C. elegans insulin-like signaling, we identified eak-3, which encodes a novel protein that is specifically expressed in the two endocrine XXX cells. The dauer arrest phenotype of eak-3 mutants is fully suppressed by mutations in daf-16/FoxO, which encodes the major target of C. elegans insulin-like signaling, and daf-12, which encodes a nuclear receptor regulated by steroid hormones known as dafachronic acids. eak-3 mutation does not affect DAF-16/FoxO subcellular localization but enhances expression of the direct DAF-16/FoxO target sod-3 in a daf-16/FoxO- and daf-12-dependent manner. eak-3 mutants have normal lifespans, suggesting that EAK-3 decouples insulin-like regulation of development and longevity. We propose that EAK-3 activity in the XXX cells promotes the synthesis and/or secretion of a hormone that acts in parallel to AKT-1 to inhibit the expression of DAF-16/FoxO target genes. Similar hormonal pathways may regulate FoxO target gene expression in mammals. PMID:18241854

  9. Drug resistance in epithelial ovarian cancer: P-glycoprotein and glutation S-transferase. Can they play an important role in detecting response to platinum-based chemotherapy as a first-line therapy.

    PubMed

    Simşek, T; Ozbilim, G; Gülkesen, H; Kaya, H; Sargin, F; Karaveli, S

    2001-01-01

    Drug resistance is important for the treatment of ovarian cancer. P-glycoprotein and glutation S-transferase as resistance markers play an important role in the effectivity of chemotherapeutical agents. The role of P-glycoprotein and glutation S-transferase in the treatment of epithelial ovarian cancer is not well understood. We investigated the relation between P-glycoprotein and glutation S-transferase level for response to platinum-based chemotherapy in epithelial ovarian cancer. We reviewed 30 cases diagnosed as epithelial ovarian cancer and treated with platinum-based chemotherapy in the Department of Obstetrics and Gynecology, Akdeniz University School of Medicine. The material was attained from initial parafin-embeded blocks stained for P-glycoprotein and glutation S-transferase. The cases that were diagnosed and treated before attending our clinic were not enrolled in the study. Mean age was 58.2 (25-70) and mean gravida 4.1 (0-10). Twenty-four patients (80%) were glutation S-transferase positive. Three cases (10%) out of 30 had positive reaction for P-glycoprotein. No difference was revealed regarding chemotherapy response rate among the cases showing glutation S-transferase positivity and P-glycoprotein negativity. Detection of glutation S-transferase and P-glycoprotein levels in epithelial ovarian cancer tissue is not important for response to platinum-based chemotherapy as a first line.

  10. Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes.

    PubMed

    Erales, Jenny; Marchand, Virginie; Panthu, Baptiste; Gillot, Sandra; Belin, Stéphane; Ghayad, Sandra E; Garcia, Maxime; Laforêts, Florian; Marcel, Virginie; Baudin-Baillieu, Agnès; Bertin, Pierre; Couté, Yohann; Adrait, Annie; Meyer, Mélanie; Therizols, Gabriel; Yusupov, Marat; Namy, Olivier; Ohlmann, Théophile; Motorin, Yuri; Catez, Frédéric; Diaz, Jean-Jacques

    2017-12-05

    Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

  11. FoxO1 in dopaminergic neurons regulates energy homeostasis and targets tyrosine hydroxylase

    PubMed Central

    Doan, Khanh V.; Kinyua, Ann W.; Yang, Dong Joo; Ko, Chang Mann; Moh, Sang Hyun; Shong, Ko Eun; Kim, Hail; Park, Sang-Kyu; Kim, Dong-Hoon; Kim, Inki; Paik, Ji-Hye; DePinho, Ronald A.; Yoon, Seul Gi; Kim, Il Yong; Seong, Je Kyung; Choi, Yun-Hee; Kim, Ki Woo

    2016-01-01

    Dopaminergic (DA) neurons are involved in the integration of neuronal and hormonal signals to regulate food consumption and energy balance. Forkhead transcriptional factor O1 (FoxO1) in the hypothalamus plays a crucial role in mediation of leptin and insulin function. However, the homoeostatic role of FoxO1 in DA system has not been investigated. Here we report that FoxO1 is highly expressed in DA neurons and mice lacking FoxO1 specifically in the DA neurons (FoxO1 KODAT) show markedly increased energy expenditure and interscapular brown adipose tissue (iBAT) thermogenesis accompanied by reduced fat mass and improved glucose/insulin homoeostasis. Moreover, FoxO1 KODAT mice exhibit an increased sucrose preference in concomitance with higher dopamine and norepinephrine levels. Finally, we found that FoxO1 directly targets and negatively regulates tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of the catecholamine synthesis, delineating a mechanism for the KO phenotypes. Collectively, these results suggest that FoxO1 in DA neurons is an important transcriptional factor that directs the coordinated control of energy balance, thermogenesis and glucose homoeostasis. PMID:27681312

  12. O-GlcNAcylation of Orphan Nuclear Receptor Estrogen-Related Receptor γ Promotes Hepatic Gluconeogenesis.

    PubMed

    Misra, Jagannath; Kim, Don-Kyu; Jung, Yoon Seok; Kim, Han Byeol; Kim, Yong-Hoon; Yoo, Eun-Kyung; Kim, Byung Gyu; Kim, Sunghoon; Lee, In-Kyu; Harris, Robert A; Kim, Jeong-Sun; Lee, Chul-Ho; Cho, Jin Won; Choi, Hueng-Sik

    2016-10-01

    Estrogen-related receptor γ (ERRγ) is a major positive regulator of hepatic gluconeogenesis. Its transcriptional activity is suppressed by phosphorylation signaled by insulin in the fed state, but whether posttranslational modification alters its gluconeogenic activity in the fasted state is not known. Metabolically active hepatocytes direct a small amount of glucose into the hexosamine biosynthetic pathway, leading to protein O-GlcNAcylation. In this study, we demonstrate that ERRγ is O-GlcNAcylated by O-GlcNAc transferase in the fasted state. This stabilizes the protein by inhibiting proteasome-mediated protein degradation, increasing ERRγ recruitment to gluconeogenic gene promoters. Mass spectrometry identifies two serine residues (S317, S319) present in the ERRγ ligand-binding domain that are O-GlcNAcylated. Mutation of these residues destabilizes ERRγ protein and blocks the ability of ERRγ to induce gluconeogenesis in vivo. The impact of this pathway on gluconeogenesis in vivo was confirmed by the observation that decreasing the amount of O-GlcNAcylated ERRγ by overexpressing the deglycosylating enzyme O-GlcNAcase decreases ERRγ-dependent glucose production in fasted mice. We conclude that O-GlcNAcylation of ERRγ serves as a major signal to promote hepatic gluconeogenesis. © 2016 by the American Diabetes Association.

  13. O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.

    PubMed

    Makino, Ayako; Dai, Anzhi; Han, Ying; Youssef, Katia D; Wang, Weihua; Donthamsetty, Reshma; Scott, Brian T; Wang, Hong; Dillmann, Wolfgang H

    2015-11-01

    Cardiovascular disease is the primary cause of morbidity and mortality in diabetes, and endothelial dysfunction is commonly seen in these patients. Increased O-linked N-acetylglucosamine (O-GlcNAc) protein modification is one of the central pathogenic features of diabetes. Modification of proteins by O-GlcNAc (O-GlcNAcylation) is regulated by two key enzymes: β-N-acetylglucosaminidase [O-GlcNAcase (OGA)], which catalyzes the reduction of protein O-GlcNAcylation, and O-GlcNAc transferase (OGT), which induces O-GlcNAcylation. However, it is not known whether reducing O-GlcNAcylation can improve endothelial dysfunction in diabetes. To examine the effect of endothelium-specific OGA overexpression on protein O-GlcNAcylation and coronary endothelial function in diabetic mice, we generated tetracycline-inducible, endothelium-specific OGA transgenic mice, and induced OGA by doxycycline administration in streptozotocin-induced type 1 diabetic mice. OGA protein expression was significantly decreased in mouse coronary endothelial cells (MCECs) isolated from diabetic mice compared with control MCECs, whereas OGT protein level was markedly increased. The level of protein O-GlcNAcylation was increased in diabetic compared with control mice, and OGA overexpression significantly decreased the level of protein O-GlcNAcylation in MCECs from diabetic mice. Capillary density in the left ventricle and endothelium-dependent relaxation in coronary arteries were significantly decreased in diabetes, while OGA overexpression increased capillary density to the control level and restored endothelium-dependent relaxation without changing endothelium-independent relaxation. We found that connexin 40 could be the potential target of O-GlcNAcylation that regulates the endothelial functions in diabetes. These data suggest that OGA overexpression in endothelial cells improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes. Copyright © 2015 the

  14. β(1,3)-Glucanosyl-Transferase Activity Is Essential for Cell Wall Integrity and Viability of Schizosaccharomyces pombe

    PubMed Central

    de Medina-Redondo, María; Arnáiz-Pita, Yolanda; Clavaud, Cécile; Fontaine, Thierry; del Rey, Francisco; Latgé, Jean Paul; Vázquez de Aldana, Carlos R.

    2010-01-01

    Background The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of β-glucans. The β(1,3)-glucan synthase complex synthesizes linear β(1,3)-glucans, which remain unorganized until they are cross-linked to other β(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding β(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed. Methodology/Principal Findings Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display β(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast. Conclusions/Significance We conclude that β(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth. PMID:21124977

  15. Bisubstrate Kinetics of Glutathione S-Transferase: A Colorimetric Experiment for the Introductory Biochemistry Laboratory

    ERIC Educational Resources Information Center

    Stefanidis, Lazaros; Scinto, Krystal V.; Strada, Monica I.; Alper, Benjamin J.

    2018-01-01

    Most biochemical transformations involve more than one substrate. Bisubstrate enzymes catalyze multiple chemical reactions in living systems and include members of the transferase, oxidoreductase, and ligase enzyme classes. Working knowledge of bisubstrate enzyme kinetic models is thus of clear importance to the practicing biochemist. However,…

  16. New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

    PubMed

    Anumula, Kalyan Rao

    2012-07-01

    Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

  17. Identification of Glutathione S-Transferase Pi as a Protein Involved in Parkinson Disease Progression

    PubMed Central

    Shi, Min; Bradner, Joshua; Bammler, Theo K.; Eaton, David L.; Zhang, JianPeng; Ye, ZuCheng; Wilson, Angela M.; Montine, Thomas J.; Pan, Catherine; Zhang, Jing

    2009-01-01

    Parkinson disease (PD) typically affects the cortical regions during the later stages of disease, with neuronal loss, gliosis, and formation of diffuse cortical Lewy bodies in a significant portion of patients with dementia. To identify novel proteins involved in PD progression, we prepared synaptosomal fractions from the frontal cortices of pathologically verified PD patients at different stages along with age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique. Approximately 100 proteins displayed significant differences in their relative abundances between PD patients at various stages and controls; three of these proteins were validated using independent techniques. One of the confirmed proteins, glutathione S-transferase Pi, was further investigated in cellular models of PD, demonstrating that its level was intimately associated with several critical cellular processes that are directly related to neurodegeneration in PD. These results have, for the first time, suggested that the levels of glutathione S-transferase Pi may play an important role in modulating the progression of PD. PMID:19498008

  18. The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism.

    PubMed

    Thom, R; Dixon, D P; Edwards, R; Cole, D J; Lapthorn, A J

    2001-05-18

    The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed. Copyright 2001 Academic Press.

  19. Expression Profiling of Selected Glutathione Transferase Genes in Zea mays (L.) Seedlings Infested with Cereal Aphids

    PubMed Central

    Sytykiewicz, Hubert; Chrzanowski, Grzegorz; Czerniewicz, Paweł; Sprawka, Iwona; Łukasik, Iwona; Goławska, Sylwia; Sempruch, Cezary

    2014-01-01

    The purpose of this report was to evaluate the expression patterns of selected glutathione transferase genes (gst1, gst18, gst23 and gst24) in the tissues of two maize (Zea mays L.) varieties (relatively resistant Ambrozja and susceptible Tasty Sweet) that were colonized with oligophagous bird cherry-oat aphid (Rhopalosiphum padi L.) or monophagous grain aphid (Sitobion avenae L.). Simultaneously, insect-triggered generation of superoxide anion radicals (O2 •−) in infested Z. mays plants was monitored. Quantified parameters were measured at 1, 2, 4, 8, 24, 48 and 72 h post-initial aphid infestation (hpi) in relation to the non-infested control seedlings. Significant increases in gst transcript amounts were recorded in aphid-stressed plants in comparison to the control seedlings. Maximal enhancement in the expression of the gst genes in aphid-attacked maize plants was found at 8 hpi (gst23) or 24 hpi (gst1, gst18 and gst24) compared to the control. Investigated Z. mays cultivars formed excessive superoxide anion radicals in response to insect treatments, and the highest overproduction of O2 •− was noted 4 or 8 h after infestation, depending on the aphid treatment and maize genotype. Importantly, the Ambrozja variety could be characterized as having more profound increments in the levels of gst transcript abundance and O2 •− generation in comparison with the Tasty Sweet genotype. PMID:25365518

  20. Inorganic Arsenic Induces NRF2-Regulated Antioxidant Defenses in Both Cerebral Cortex and Hippocampus in Vivo.

    PubMed

    Zhang, Yang; Duan, Xiaoxu; Li, Jinlong; Zhao, Shuo; Li, Wei; Zhao, Lu; Li, Wei; Nie, Huifang; Sun, Guifang; Li, Bing

    2016-08-01

    Inorganic arsenic is reported to induce the reactive oxygen species-mediated oxidative stress, which is supposed to be one of the main mechanisms of arsenic-related neurological diseases. Nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of antioxidant defense systems, up-regulates the expression of target genes to fight against oxidative damages caused by harmful substances, including metals. In the present study, mice were used as a model to investigate the oxidative stress levels and the expressions of NRF2-regulated antioxidant substances in both cerebral cortex and hippocampus with 5, 10 and 20 mg/kg NaAsO2 exposure intra-gastrically. Our results showed that acute NaAsO2 treatment resulted in decreased total anti-oxidative capacity (T-AOC) and increased maleic dialdehyde production in the nervous system. We also detected rapidly elevation of NRF2 protein levels by enhancement of Nrf2 transcription, especially at 20 mg/kg NaAsO2 exposure group. In the meantime, mRNA and protein levels of Nrf2 encoding antioxidant enzymes heme oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) were consistently elevated time- and dose-dependently both in the cerebral cortex and hippocampus. Taken together, the presence study demonstrated the activation of NRF2 pathway, an early antioxidant defensive response, in both cerebral cortex and hippocampus upon inorganic arsenic (iAs) exposure in vivo. A better knowledge on the roles of NRF2 pathway in maintaining cellular redox homeostasis would be helpful for the strategies on improvement of neurotoxicity related to this metalloid.

  1. Imidazopyridine and Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase.

    PubMed

    Genin, Michael J; Gonzalez Valcarcel, Isabel C; Holloway, William G; Lamar, Jason; Mosior, Marian; Hawkins, Eric; Estridge, Thomas; Weidner, Jeffrey; Seng, Thomas; Yurek, David; Adams, Lisa A; Weller, Jennifer; Reynolds, Vincent L; Brozinick, Joseph T

    2016-06-23

    To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats.

  2. Expanding the substrate scope of chitooligosaccharide oxidase from Fusarium graminearum by structure-inspired mutagenesis.

    PubMed

    Ferrari, Alessandro R; Lee, Misun; Fraaije, Marco W

    2015-06-01

    Chitooligosaccharide oxidase from Fusarium graminearum (ChitO) oxidizes N-acetyl-D-glucosamine (GlcNAc) and its oligomers with high efficiency at the C1-hydroxyl moiety while it shows poor or no activity with other carbohydrates. By sequence and structural comparison with other known carbohydrate oxidases (glucooligosaccharide oxidase from Acremonium strictum and lactose oxidase from Microdochium nivale) eleven mutants were designed to redirect the catalytic scope of ChitO for improved oxidation of lactose, cellobiose and maltose. The catalytic properties of the most interesting mutants were further improved by combining single mutations. This has resulted in the creation of a set of ChitO variants that display totally different substrate tolerances. One ChitO variant shows a dramatic improvement in catalytic efficiency towards oxidation of glucose, cellobiose, lactose, and maltose. We also describe a ChitO variant with the highest catalytic efficiency in GlcNAc oxidation so far reported in the literature. © 2015 Wiley Periodicals, Inc.

  3. Glutathione-S-transferase profiles in the emerald ash borer, Agrilus planipennis.

    PubMed

    Rajarapu, Swapna Priya; Mittapalli, Omprakash

    2013-05-01

    The emerald ash borer, Agrilus planipennis Fairmaire is a recently discovered invasive insect pest of ash, Fraxinus spp. in North America. Glutathione-S-transferases (GST) are a multifunctional superfamily of enzymes which function in conjugating toxic compounds to less toxic and excretable forms. In this study, we report the molecular characterization and expression patterns of different classes of GST genes in different tissues and developmental stages plus their specific activity. Multiple sequence alignment of all six A. planipennis GSTs (ApGST-E1, ApGST-E2, ApGST-E3, ApGST-O1, ApGST-S1 and ApGST-μ1) revealed conserved features of insect GSTs and a phylogenetic analysis grouped the GSTs within the epsilon, sigma, omega and microsomal classes of GSTs. Real time quantitative PCR was used to study field collected samples. In larval tissues high mRNA levels for ApGST-E1, ApGST-E3 and ApGST-O1 were obtained in the midgut and Malpighian tubules. On the other hand, ApGST-E2 and ApGST-S1 showed high mRNA levels in fat body and ApGST-μ1 showed constitutive levels in all the tissues assayed. During development, mRNA levels for ApGST-E2 were observed to be the highest in feeding instars, ApGST-S1 in prepupal instars; while the others showed constitutive patterns in all the developmental stages examined. At the enzyme level, total GST activity was similar in all the tissues and developmental stages assayed. Results obtained suggest that A. planipennis is potentially primed with GST-driven detoxification to metabolize ash allelochemicals. To our knowledge this study represents the first report of GSTs in A. planipennis and also in the family of wood boring beetles. Copyright © 2013 Elsevier Inc. All rights reserved.

  4. O-Linked β-N-Acetylglucosaminylation (O-GlcNAcylation) in Primary and Metastatic Colorectal Cancer Clones and Effect of N-Acetyl-β-d-glucosaminidase Silencing on Cell Phenotype and Transcriptome*

    PubMed Central

    Yehezkel, Galit; Cohen, Liz; Kliger, Adi; Manor, Esther; Khalaila, Isam

    2012-01-01

    O-Linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a regulatory post-translational modification occurring on the serine or threonine residues of nucleocytoplasmic proteins. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), which are responsible for O-GlcNAc addition and removal, respectively. Although O-GlcNAcylation was found to play a significant role in several pathologies such as type II diabetes and neurodegenerative diseases, the role of O-GlcNAcylation in the etiology and progression of cancer remains vague. Here, we followed O-GlcNAcylation and its catalytic machinery in metastatic clones of human colorectal cancer and the effect of OGA knockdown on cellular phenotype and on the transcriptome. The colorectal cancer SW620 metastatic clone exhibited increased O-GlcNAcylation and decreased OGA expression compared with its primary clone, SW480. O-GlcNAcylation elevation in SW620 cells, through RNA interference of OGA, resulted in phenotypic alterations that included acquisition of a fibroblast-like morphology, which coincides with epithelial metastatic progression and growth retardation. Microarray analysis revealed that OGA silencing altered the expression of about 1300 genes, mostly involved in cell movement and growth, and specifically affected metabolic pathways of lipids and carbohydrates. These findings support the involvement of O-GlcNAcylation in various aspects of tumor cell physiology and suggest that this modification may serve as a link between metabolic changes and cancer. PMID:22730328

  5. Nuclear glutathione S-transferase pi prevents apoptosis by reducing the oxidative stress-induced formation of exocyclic DNA products.

    PubMed

    Kamada, Kensaku; Goto, Shinji; Okunaga, Tomohiro; Ihara, Yoshito; Tsuji, Kentaro; Kawai, Yoshichika; Uchida, Koji; Osawa, Toshihiko; Matsuo, Takayuki; Nagata, Izumi; Kondo, Takahito

    2004-12-01

    We previously found that nuclear glutathione S-transferase pi (GSTpi) accumulates in cancer cells resistant to anticancer drugs, suggesting that it has a role in the acquisition of resistance to anticancer drugs. In the present study, the effect of oxidative stress on the nuclear translocation of GSTpi and its role in the protection of DNA from damage were investigated. In human colonic cancer HCT8 cells, the hydrogen peroxide (H(2)O(2))-induced increase in nuclear condensation, the population of sub-G(1) peak, and the number of TUNEL-positive cells were observed in cells pretreated with edible mushroom lectin, an inhibitor of the nuclear transport of GSTpi. The DNA damage and the formation of lipid peroxide were dependent on the dose of H(2)O(2) and the incubation time. Immunological analysis showed that H(2)O(2) induced the nuclear accumulation of GSTpi but not of glutathione peroxidase. Formation of the 7-(2-oxo-hepyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct by the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with 2'-deoxyguanosine was inhibited by GSTpi in the presence of glutathione. The conjugation product of 4-oxo-2-nonenal, a lipid aldehyde of 13-HPODE, with GSH in the presence of GSTpi, was identified by LS/MS. These results suggested that nuclear GSTpi prevents H(2)O(2)-induced DNA damage by scavenging the formation of lipid-peroxide-modified DNA.

  6. FoxO is a critical regulator of stem cell maintenance in immortal Hydra.

    PubMed

    Boehm, Anna-Marei; Khalturin, Konstantin; Anton-Erxleben, Friederike; Hemmrich, Georg; Klostermeier, Ulrich C; Lopez-Quintero, Javier A; Oberg, Hans-Heinrich; Puchert, Malte; Rosenstiel, Philip; Wittlieb, Jörg; Bosch, Thomas C G

    2012-11-27

    Hydra's unlimited life span has long attracted attention from natural scientists. The reason for that phenomenon is the indefinite self-renewal capacity of its stem cells. The underlying molecular mechanisms have yet to be explored. Here, by comparing the transcriptomes of Hydra's stem cells followed by functional analysis using transgenic polyps, we identified the transcription factor forkhead box O (FoxO) as one of the critical drivers of this continuous self-renewal. foxO overexpression increased interstitial stem cell and progenitor cell proliferation and activated stem cell genes in terminally differentiated somatic cells. foxO down-regulation led to an increase in the number of terminally differentiated cells, resulting in a drastically reduced population growth rate. In addition, it caused down-regulation of stem cell genes and antimicrobial peptide (AMP) expression. These findings contribute to a molecular understanding of Hydra's immortality, indicate an evolutionarily conserved role of FoxO in controlling longevity from Hydra to humans, and have implications for understanding cellular aging.

  7. Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization

    USDA-ARS?s Scientific Manuscript database

    The extensive similarities between helminth proteins and allergens are thought to contribute to helminth-driven allergic sensitization. We investigated the molecular and structural similarities between Bla g 5, a major glutathione-S transferase (GST) allergen of cockroaches, and the GST of Wucherer...

  8. OsWS1 involved in cuticular wax biosynthesis is regulated by osa-miR1848.

    PubMed

    Xia, Kuaifei; Ou, Xiaojin; Gao, Chunzhi; Tang, Huadan; Jia, Yongxia; Deng, Rufang; Xu, Xinlan; Zhang, Mingyong

    2015-12-01

    Cuticular wax forms a hydrophobic layer covering aerial plant organs and acting as a protective barrier against biotic and abiotic stresses. Compared with well-known wax biosynthetic pathway, molecular regulation of wax biosynthesis is less known. Here, we show that rice OsWS1, a member of the membrane-bound O-acyl transferase gene family, involved in wax biosynthesis and was regulated by an osa-miR1848. OsWS1-tagged green fluorescent protein localized to the endoplasmic reticulum (ER). Compared with wild-type rice, OsWS1 overexpression plants displayed a 3% increase in total wax, especially a 35% increase in very long-chain fatty acids, denser wax papillae around the stoma, more cuticular wax crystals formed on leaf and stem surfaces, pollen coats were thicker and more seedlings survived after water-deficit treatment. In contrast, OsWS1-RNAi and osa-miR1848 overexpression plants exhibited opposing changes. Gene expression analysis showed that overexpression of osa-miR1848 down-regulated OsWS1 transcripts; furthermore, expression profiles of OsWS1 and osa-miR1848 were inversely correlated in the leaf, panicle and stem, and upon water-deficit treatment. These results suggest that OsWS1 is regulated by osa-miR1848 and participates in cuticular wax formation. © 2015 John Wiley & Sons Ltd.

  9. Glutathione S-transferase M1 and glutathione S-transferase T1 genotype in chronic pancreatitis: a meta-analysis.

    PubMed

    Zhong, Yanjun; Zou, Runmei; Cao, Jie; Peng, Mou

    2015-02-01

    A meta-analysis to determine the association between chronic pancreatitis and glutathione-S transferase (GST) mu 1 (GSTM1) and theta 1 (GSTT1) deletions. Case-control studies concerning the relationship between chronic pancreatitis and GSTM1 or GSTT1 deletions were identified (up to October 2013). Meta-analyses of the association between GSTM1 and GSTT1 genotype and chronic pancreatitis or alcoholic chronic pancreatitis (ACP) were performed. Seven studies were included in the meta-analysis (650 patients/1382 controls for GSTM1 and 536 patients/1304 controls for GSTT1). There were no significant relationships between GSTM1/GSTT1 and chronic pancreatitis or GSTT1 and ACP. There was a significant association between GSTM1 null genotype and ACP (odds ratio 1.16, 95% confidence intervals 1.03, 1.30). The GSTM1 null genotype was significantly associated with ACP risk. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  10. The antitumour activity of alkylating agents is not correlated with the levels of glutathione, glutathione transferase and O6-alkylguanine-DNA-alkyltransferase of human tumour xenografts. EORTC SPG and PAMM Groups.

    PubMed

    D'Incalci, M; Bonfanti, M; Pifferi, A; Mascellani, E; Tagliabue, G; Berger, D; Fiebig, H H

    1998-10-01

    Twenty-three human xenografts, including five colon, five gastric, nine lung (three small cell lung cancer) and four breast carcinomas, were investigated for their sensitivity to nitrosoureas, dacarbazine (DTIC), cyclophosphamide (CTX) and cisplatin (DDP). In 12 cases, at least one of the drugs produced complete or partial remission, in 2, a minor regression was observed and in the other 9, treatment was ineffective. The level of sensitivity to each drug, using a score from 1 to 5, was correlated to three biochemical parameters reported to be involved in resistance to alkylating agents: glutathione (GSH), glutathione transferase (GST) and O6-alkylguanine-DNA-alkyltransferase (AGT). A wide variability was found in these parameters in the xenografts investigated. No correlation was found between any of the three parameters and sensitivity to the drugs used or between sensitivity to one drug and to any of the other drugs tested. These results illustrate the complexity of the question of resistance to alkylating agents and indicate that, at least in xenografts, the biochemical parameters examined are not predictive of response to alkylating agents.

  11. The Role of Stress-Induced O-GlcNAc Protein Modification in the Regulation of Membrane Transport

    PubMed Central

    Fisi, Viktória; Miseta, Attila

    2017-01-01

    O-linked N-acetylglucosamine (O-GlcNAc) is a posttranslational modification that is increasingly recognized as a signal transduction mechanism. Unlike other glycans, O-GlcNAc is a highly dynamic and reversible process that involves the addition and removal of a single N-acetylglucosamine molecule to Ser/Thr residues of proteins. UDP-GlcNAc—the direct substrate for O-GlcNAc modification—is controlled by the rate of cellular metabolism, and thus O-GlcNAc is dependent on substrate availability. Serving as a feedback mechanism, O-GlcNAc influences the regulation of insulin signaling and glucose transport. Besides nutrient sensing, O-GlcNAc was also implicated in the regulation of various physiological and pathophysiological processes. Due to improvements of mass spectrometry techniques, more than one thousand proteins were detected to carry the O-GlcNAc moiety; many of them are known to participate in the regulation of metabolites, ions, or protein transport across biological membranes. Recent studies also indicated that O-GlcNAc is involved in stress adaptation; overwhelming evidences suggest that O-GlcNAc levels increase upon stress. O-GlcNAc elevation is generally considered to be beneficial during stress, although the exact nature of its protective effect is not understood. In this review, we summarize the current data regarding the oxidative stress-related changes of O-GlcNAc levels and discuss the implications related to membrane trafficking. PMID:29456783

  12. Glutathione S-transferase Pi mediates proliferation of androgen-independent prostate cancer cells

    PubMed Central

    Hokaiwado, Naomi; Takeshita, Fumitaka; Naiki-Ito, Aya; Asamoto, Makoto; Ochiya, Takahiro; Shirai, Tomoyuki

    2008-01-01

    Prostate cancers generally acquire an androgen-independent growth capacity with progression, resulting in resistance to antiandrogen therapy. Therefore, identification of the genes regulated through this process may be important for understanding the mechanisms of prostate carcinogenesis. We here utilized androgen-dependent/independent transplantable tumors, newly established with the ‘transgenic rat adenocarcinoma in prostate’ (TRAP) model, to analyze their gene expression using microarrays. Among the overexpressed genes in androgen-independent prostate cancers compared with the androgen-dependent tumors, glutathione S-transferase pi (GST-pi) was included. In line with this, human prostate cancer cell lines PC3 and DU145 (androgen independent) had higher expression of GST-pi compared with LNCaP (androgen dependent) as determined by semiquantitative reverse transcription–polymerase chain reaction analysis. To investigate the roles of GST-pi expression in androgen-independent human prostate cancers, GST-pi was knocked down by a small interfering RNA (siRNA), resulting in significant decrease of the proliferation rate in the androgen-independent PC3 cell line. In vivo, administration of GST-pi siRNA–atelocollagen complex decreased GST-pi protein expression, resulting in enhanced numbers of TdT mediated dUTP-biotin nick-end labering (TUNEL)-positive apoptotic cells. These findings suggest that GST-pi might play important roles in proliferation of androgen-independent human prostate cancer cells. PMID:18413363

  13. Regulation of Protein Degradation by O-GlcNAcylation: Crosstalk with Ubiquitination*

    PubMed Central

    Ruan, Hai-Bin; Nie, Yongzhan; Yang, Xiaoyong

    2013-01-01

    The post-translational modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates essential cellular processes such as signal transduction, transcription, translation, and protein degradation. Misfolded, damaged, and unwanted proteins are tagged with a chain of ubiquitin moieties for degradation by the proteasome, which is critical for cellular homeostasis. In this review, we summarize the current knowledge of the interplay between O-GlcNAcylation and ubiquitination in the control of protein degradation. Understanding the mechanisms of action of O-GlcNAcylation in the ubiquitin-proteosome system shall facilitate the development of therapeutics for human diseases such as cancer, metabolic syndrome, and neurodegenerative diseases. PMID:23824911

  14. The glutathione-S-transferase Mu 1 null genotype modulates ozone-induced airway inflammation in humans*

    EPA Science Inventory

    Background: The Glutathione-S-Transferase Mu 1 null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. Howev...

  15. Climate-mediated changes in marine ecosystem regulation during El Niño.

    PubMed

    Lindegren, Martin; Checkley, David M; Koslow, Julian A; Goericke, Ralf; Ohman, Mark D

    2018-02-01

    The degree to which ecosystems are regulated through bottom-up, top-down, or direct physical processes represents a long-standing issue in ecology, with important consequences for resource management and conservation. In marine ecosystems, the role of bottom-up and top-down forcing has been shown to vary over spatio-temporal scales, often linked to highly variable and heterogeneously distributed environmental conditions. Ecosystem dynamics in the Northeast Pacific have been suggested to be predominately bottom-up regulated. However, it remains unknown to what extent top-down regulation occurs, or whether the relative importance of bottom-up and top-down forcing may shift in response to climate change. In this study, we investigate the effects and relative importance of bottom-up, top-down, and physical forcing during changing climate conditions on ecosystem regulation in the Southern California Current System (SCCS) using a generalized food web model. This statistical approach is based on nonlinear threshold models and a long-term data set (~60 years) covering multiple trophic levels from phytoplankton to predatory fish. We found bottom-up control to be the primary mode of ecosystem regulation. However, our results also demonstrate an alternative mode of regulation represented by interacting bottom-up and top-down forcing, analogous to wasp-waist dynamics, but occurring across multiple trophic levels and only during periods of reduced bottom-up forcing (i.e., weak upwelling, low nutrient concentrations, and primary production). The shifts in ecosystem regulation are caused by changes in ocean-atmosphere forcing and triggered by highly variable climate conditions associated with El Niño. Furthermore, we show that biota respond differently to major El Niño events during positive or negative phases of the Pacific Decadal Oscillation (PDO), as well as highlight potential concerns for marine and fisheries management by demonstrating increased sensitivity of pelagic

  16. G₂/M cell cycle arrest by an N-acetyl-D-glucosamine specific lectin from Psathyrella asperospora.

    PubMed

    Rouf, Razina; Stephens, Alexandre S; Spaan, Lina; Arndt, Nadia X; Day, Christopher J; May, Tom W; Tiralongo, Evelin; Tiralongo, Joe

    2014-01-01

    A new N-acetyl-D-glucosamine (GlcNAc) specific lectin was identified and purified from the fruiting body of the Australian indigenous mushroom Psathyrella asperospora. The functional lectin, named PAL, showed hemagglutination activity against neuraminidase treated rabbit and human blood types A, B and O, and exhibited high binding specificity towards GlcNAc, as well as mucin and fetuin, but not against asialofetuin. PAL purified to homogeneity by a combination of ammonium sulfate precipitation, chitin affinity chromatography and size exclusion chromatography, was monomeric with a molecular mass of 41.8 kDa, was stable at temperatures up to 55 °C and between pH 6-10, and did not require divalent cations for optimal activity. De novo sequencing of PAL using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc recognizing lectins from Psathyrella velutina (PVL) and Agrocybe aegerita (AAL-II) in both the carbohydrate binding and calcium binding sites. Significantly, PAL was also found to exert a potent anti-proliferative effect on HT29 cells (IC50 0.48 μM) that was approximately 3-fold greater than that observed on VERO cells; a difference found to be due to the differential expression of cell surface GlcNAc on HT29 and VERO cells. Further characterization of this activity using propidium iodine staining revealed that PAL induced cell cycle arrest at G2/M phase in a manner dependent on its ability to bind GlcNAc.

  17. Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.

    PubMed

    Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David

    2010-03-23

    NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

  18. Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors.

    PubMed

    Balcik-Ercin, Pelin; Cetin, Metin; Yalim-Camci, Irem; Odabas, Gorkem; Tokay, Nurettin; Sayan, A Emre; Yagci, Tamer

    2018-03-07

    Protein Atlas dataset and, again, observed an inverse correlation in all analyzed tumor types, except malignant melanoma. In contrast to a generally negative or weak ZEB2 expression, we found that most tumor tissues exhibited a strong or moderate GALNT3 expression. Our observation that ZEB2 negatively regulates a GalNAc-transferase (GALNT3) that is involved in O-glycosylation adds another layer of complexity to the role of ZEB2 in cancer progression and metastasis. Proteins glycosylated by GALNT3 may be exploited as novel diagnostics and/or therapeutic targets.

  19. Exploring the transferase activity of Ffase from Schwanniomyces occidentalis, a β-fructofuranosidase showing high fructosyl-acceptor promiscuity.

    PubMed

    Piedrabuena, David; Míguez, Noa; Poveda, Ana; Plou, Francisco J; Fernández-Lobato, María

    2016-10-01

    The β-fructofuranosidase from the yeast Schwanniomyces occidentalis (Ffase) produces the prebiotic sugars 6-kestose and 1-kestose by transfructosylation of sucrose, which makes it of biotechnological interest. In this study, the hydrolase and transferase activity of this enzyme was kinetically characterized and its potential to synthesize new fructosylated products explored. A total of 40 hydroxylated compounds were used as potential fructosyl-acceptor alternatives to sucrose. Only 17 of them, including some monosaccharides, disaccharides, and oligosaccharides as well as alditols and glycosides were fructosylated. The best alternative acceptors were the alditols. The major transfer product of the reaction including mannitol was purified and characterized as 1-O-β-D-fructofuranosyl-D-mannitol, whose maximum concentration reached 44 g/L, representing about 7.3 % of total compounds in the mixture and 89 % of all products generated by transfructosylation. The reactions including erythritol produced 35 g/L of an isomer mixture comprising 1- and 4-O-β-D-fructofuranosyl-D-erythritol. In addition, Ffase produced 24 g/L of the disaccharide blastose by direct fructosylation of glucose, which makes it the first enzyme characterized from yeast showing this ability. Thus, novel fructosylated compounds with potential applications in food and pharmaceutical industries can be obtained due to the Ffase fructosyl-acceptor promiscuity.

  20. Human glutathione S-transferase P1-1 functions as an estrogen receptor α signaling modulator

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

    Liu, Xiyuan; An, Byoung Ha; Kim, Min Jung

    2014-09-26

    Highlights: • GSTP induces the classical ERα signaling event. • The functional GSTP is a prerequisite for GSTP-induced ERα transcription activity. • The expression of RIP140, a transcription cofactor, was inhibited by GSTP protein. • We propose the novel non-enzymatic role of GSTP. - Abstract: Estrogen receptor α (ERα) plays a crucial role in estrogen-mediated signaling pathways and exerts its action as a nuclear transcription factor. Binding of the ligand-activated ERα to the estrogen response element (ERE) is a central part of ERα-associated signal transduction pathways and its aberrant modulation is associated with many disease conditions. Human glutathione S-transferase P1-1more » (GSTP) functions as an enzyme in conjugation reactions in drug metabolism and as a regulator of kinase signaling pathways. It is overexpressed in tumors following chemotherapy and has been associated with a poor prognosis in breast cancer. In this study, a novel regulatory function of GSTP has been proposed in which GSTP modulates ERE-mediated ERα signaling events. Ectopic expression of GSTP was able to induce the ERα and ERE-mediated transcriptional activities in ERα-positive but GSTP-negative MCF7 human breast cancer cells. This inductive effect of GSTP on the ERE-transcription activity was diminished when the cells express a mutated form of the enzyme or are treated with a GSTP-specific chemical inhibitor. It was found that GSTP inhibited the expression of the receptor interacting protein 140 (RIP140), a negative regulator of ERα transcription, at both mRNA and protein levels. Our study suggests a novel non-enzymatic role of GSTP which plays a significant role in regulating the classical ERα signaling pathways via modification of transcription cofactors such as RIP140.« less

  1. Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication.

    PubMed

    Nakamura, Shoko; Horie, Masayuki; Daidoji, Tomo; Honda, Tomoyuki; Yasugi, Mayo; Kuno, Atsushi; Komori, Toshihisa; Okuzaki, Daisuke; Narimatsu, Hisashi; Nakaya, Takaaki; Tomonaga, Keizo

    2016-02-15

    Influenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained from galnt3-knockout mice. Interestingly, IAV-infected galnt3-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells. Viral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However, the details of how

  2. Influenza A Virus-Induced Expression of a GalNAc Transferase, GALNT3, via MicroRNAs Is Required for Enhanced Viral Replication

    PubMed Central

    Nakamura, Shoko; Horie, Masayuki; Daidoji, Tomo; Honda, Tomoyuki; Yasugi, Mayo; Kuno, Atsushi; Komori, Toshihisa; Okuzaki, Daisuke; Narimatsu, Hisashi; Nakaya, Takaaki

    2015-01-01

    ABSTRACT Influenza A virus (IAV) affects the upper and lower respiratory tracts and rapidly induces the expression of mucins, which are common O-glycosylated proteins, on the epithelial surfaces of the respiratory tract. Although mucin production is associated with the inhibition of virus transmission as well as characteristic clinical symptoms, little is known regarding how mucins are produced on the surfaces of respiratory epithelial cells and how they affect IAV replication. In this study, we found that two microRNAs (miRNAs), miR-17-3p and miR-221, which target GalNAc transferase 3 (GALNT3) mRNA, are rapidly downregulated in human alveolar basal epithelial cells during the early stage of IAV infection. We demonstrated that the expression of GALNT3 mRNA is upregulated in an IAV replication-dependent fashion and leads to mucin production in bronchial epithelial cells. A lectin microarray analysis revealed that the stable expression of GALNT3 by human alveolar basal epithelial cells induces mucin-type O-glycosylation modifications similar to those present in IAV-infected cells, suggesting that GALNT3 promotes mucin-type O-linked glycosylation in IAV-infected cells. Notably, analyses using short interfering RNAs and miRNA mimics showed that GALNT3 knockdown significantly reduces IAV replication. Furthermore, IAV replication was markedly decreased in embryonic fibroblast cells obtained from galnt3-knockout mice. Interestingly, IAV-infected galnt3-knockout mice exhibited high mortality and severe pathological alterations in the lungs compared to those of wild-type mice. Our results demonstrate not only the molecular mechanism underlying rapid mucin production during IAV infection but also the contribution of O-linked glycosylation to the replication and propagation of IAV in lung cells. IMPORTANCE Viral infections that affect the upper or lower respiratory tracts, such as IAV, rapidly induce mucin production on the epithelial surfaces of respiratory cells. However

  3. The N-end rule pathway and regulation by proteolysis

    PubMed Central

    Varshavsky, Alexander

    2011-01-01

    The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. The main determinant of an N-degron is a destabilizing N-terminal residue of a protein. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N-end rule and the Arg/N-end rule pathways. The Ac/N-end rule pathway targets proteins containing Nα-terminally acetylated (Nt-acetylated) residues. The Arg/N-end rule pathway recognizes unacetylated N-terminal residues and involves N-terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt-acetylated. Thus, most proteins harbor a specific degradation signal, termed AcN-degron, from the moment of their birth. Specific N-end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N-degrons include methionine-aminopeptidases, caspases, calpains, Nt-acetylases, Nt-amidases, arginyl-transferases, and leucyl-transferases. Regulated degradation of specific proteins by the N-end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation, and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights. PMID:21633985

  4. Up-regulation of CYP1A1 and phase II enzymes by 5-ring isomeric polycyclic aromatic hydrocarbons in precision-cut rat hepatic slices: Importance of molecular shape.

    PubMed

    Pushparajah, Daphnee; Lewis, Dfv; Ioannides, Costas

    2017-04-01

    The objectives of the present study were two-fold: (a) to evaluate the role of molecular shape on the interaction of polycyclic aromatic hydrocarbons (PAHs) with the Ah receptor and CYP1A1 upregulation, and (b) to evaluate the potential of PAHs to induce epoxide hydrolase and glutathione S-transferase, two major enzymes involved in their metabolism. In order to achieve these objectives, precision-cut rat liver slices were incubated with a range of concentrations of seven 5-ring isomeric PAHs, namely benzo[c]chrysene, benzo[b]chrysene, benzo[g]chrysene, dibenzo[a,j]anthracene, dibenzo[a,c]anthracene, picene and pentacene, for 24h. All compounds, with the exception of pentacene, elevated the O-deethylation of ethoxyresorufin, an activity associated with CYP1A1; induction of this enzyme by the various PAHs correlated with their avidity for the Ah receptor. None of the PAHs studied increased epoxide hydrolase activity, monitored using benzo[a]pyrene 4,5-oxide. Of the seven PAHs, only benzo[g]chrysene elevated glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole as substrates. No relationship could be established between length or length/width and interaction with the Ah receptor and CYP1A1 up-regulation indicating that other structural or electronic factors are likely to be more important. Finally, 5-ring PAHs are poor inducers of the epoxide hydrolase and glutathione S-transferase enzyme systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

    PubMed Central

    Rosenwald, A G; Stanley, P; Krag, S S

    1989-01-01

    A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Stanley, Mol. Cell. Biol. 6:1268-1275, 1986). In this paper we provide evidence that cellular carbohydrates from Lec9 cells also contain an increased proportion of beta-1,6-branched carbohydrates, although they do not possess significantly increased activity of the beta-1,6 branching enzyme (GlcNAc-transferase V). Biosynthetic labeling experiments show that a substantial degree of underglycosylation occurs in Lec9 cells and that this affects several classes of glycoproteins. Lec9 cells synthesize ca. 40-fold less Glc3Man9GlcNAc2-P-P-lipid and ca. 2-fold less Man5GlcNAc2-P-P-lipid than parental cells do. In addition, Lec9 cells possess ca. fivefold less protein-bound oligosaccharide intermediates, and one major species is resistant to release by endo-beta-N-acetylglucosaminidase H (endo H). Membranes of Lec9 cells exhibit normal mannosylphosphoryldolichol synthase, glucosylphosphoryldolichol synthase, and N-acetylglucosaminylphosphate transferase activities in the presence of exogenous dolichyl phosphate. However, in the absence of exogenous dolichyl phosphate, mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities are reduced in membranes of Lec9 cells, indicating that membranes of Lec9 cells are deficient in lipid phosphate. This was confirmed by analysis of lipids labeled by [3H]mevalonate, which showed that Lec9 cells have less lipid phosphate than parental CHO cells. Mechanisms by which a defect in the synthesis of dolichol

  6. Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

    PubMed

    Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

    2016-07-01

    We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

  7. Identification of a negative regulator of gibberellin action, HvSPY, in barley.

    PubMed Central

    Robertson, M; Swain, S M; Chandler, P M; Olszewski, N E

    1998-01-01

    To broaden our understanding of the molecular mechanisms of gibberellin (GA) action, we isolated a spindly clone (HvSPY) from barley cultivar Himalaya and tested whether the HvSPY protein would modulate GA action in barley aleurone. The HvSPY cDNA showed high sequence identity to Arabidopsis SPY along its entire length, and the barley protein functionally complemented the spy-3 mutation. HvSPY and SPY proteins showed sequence relatedness with animal O-linked N-acetylglucosamine transferases (OGTs), suggesting that they may also have OGT activity. HvSPY has a locus distinct from that of Sln, a mutation that causes the constitutive GA responses of slender barley, which phenotypically resembles Arabidopsis spy mutants. The possibility that the HvSPY gene encodes a negative regulator of GA action was tested by expressing HvSPY in a barley aleurone transient assay system. HvSPY coexpression largely abolished GA3-induced activity of an alpha-amylase promoter. Surprisingly, HvSPY coexpression increased reporter gene activity from an abscisic acid (ABA)-inducible gene promoter (dehydrin), even in the absence of exogenous ABA. These results show that HvSPY modulates the transcriptional activities of two hormonally regulated promoters: negatively for a GA-induced promoter and positively for an ABA-induced promoter. PMID:9634587

  8. Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

    PubMed

    Axarli, Irine; Muleta, Abdi W; Chronopoulou, Evangelia G; Papageorgiou, Anastassios C; Labrou, Nikolaos E

    2017-01-01

    Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher k cat /K m and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Šand 2.3Šresolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. RNA interference knockdown of DNA methyl-transferase 3 affects gene alternative splicing in the honey bee

    PubMed Central

    Li-Byarlay, Hongmei; Li, Yang; Stroud, Hume; Feng, Suhua; Newman, Thomas C.; Kaneda, Megan; Hou, Kirk K.; Worley, Kim C.; Elsik, Christine G.; Wickline, Samuel A.; Jacobsen, Steven E.; Ma, Jian; Robinson, Gene E.

    2013-01-01

    Studies of DNA methylation from fungi, plants, and animals indicate that gene body methylation is ancient and highly conserved in eukaryotic genomes, but its role has not been clearly defined. It has been postulated that regulation of alternative splicing of transcripts was an original function of DNA methylation, but a direct experimental test of the effect of methylation on alternative slicing at the whole genome level has never been performed. To do this, we developed a unique method to administer RNA interference (RNAi) in a high-throughput and noninvasive manner and then used it to knock down the expression of DNA methyl-transferase 3 (dnmt3), which is required for de novo DNA methylation. We chose the honey bee (Apis mellifera) for this test because it has recently emerged as an important model organism for studying the effects of DNA methylation on development and social behavior, and DNA methylation in honey bees is predominantly on gene bodies. Here we show that dnmt3 RNAi decreased global genomic methylation level as expected and in addition caused widespread and diverse changes in alternative splicing in fat tissue. Four different types of splicing events were affected by dnmt3 gene knockdown, and change in two types, exon skipping and intron retention, was directly related to decreased methylation. These results demonstrate that one function of gene body DNA methylation is to regulate alternative splicing. PMID:23852726

  10. Preliminary X-ray crystallographic analysis of glutathione transferase zeta 1 (GSTZ1a-1a)

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

    Boone, Christopher D.; Zhong, Guo; Smeltz, Marci

    2014-01-21

    Crystals of glutathione transferase zeta 1 were grown and shown to diffract X-rays to 3.1 Å resolution. They belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, β = 69.9, γ = 73.4°.

  11. Glycan gimmickry by parasitic helminths: a strategy for modulating the host immune response?

    PubMed

    van Die, Irma; Cummings, Richard D

    2010-01-01

    Parasitic helminths (worms) co-evolved with vertebrate immune systems to enable long-term survival of worms in infected hosts. Among their survival strategies, worms use their glycans within glycoproteins and glycolipids, which are abundant on helminth surfaces and in their excretory/ secretory products, to regulate and suppress host immune responses. Many helminths express unusual and antigenic (nonhost-like) glycans, including those containing polyfucose, tyvelose, terminal GalNAc, phosphorylcholine, methyl groups, and sugars in unusual linkages. In addition, some glycan antigens are expressed that share structural features with those in their intermediate and vertebrate hosts (host-like glycans), including Le(X) (Galbeta1-4[Fucalpha1-3]GlcNAc-), LDNF (GalNAcbeta1-4[Fucalpha1-3]GlcNAc-), LDN (GalNAcbeta1-4GlcNAc-), and Tn (GalNAcalpha1-O-Thr/Ser) antigens. The expression of host-like glycan determinants is remarkable and suggests that helminths may gain advantages by synthesizing such glycans. The expression of host-like glycans by parasites previously led to the concept of "molecular mimicry," in which molecules are either derived from the pathogen or acquired from the host to evade recognition by the host immune system. However, recent discoveries into the potential of host glycan-binding proteins (GBPs), such as C-type lectin receptors and galectins, to functionally interact with various host-like helminth glycans provide new insights. Host GBPs through their interactions with worm-derived glycans participate in shaping innate and adaptive immune responses upon infection. We thus propose an alternative concept termed "glycan gimmickry," which is defined as an active strategy of parasites to use their glycans to target GBPs within the host to promote their survival.

  12. Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners

    PubMed Central

    Yu, Seok-Ho; Boyce, Michael; Wands, Amberlyn M.; Bond, Michelle R.; Bertozzi, Carolyn R.; Kohler, Jennifer J.

    2012-01-01

    O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation. PMID:22411826

  13. Luminal flow regulates NO and O2− along the nephron

    PubMed Central

    Cabral, Pablo D.

    2011-01-01

    Urinary flow is not constant but in fact highly variable, altering the mechanical forces (shear stress, stretch, and pressure) exerted on the epithelial cells of the nephron as well as solute delivery. Nitric oxide (NO) and superoxide (O2−) play important roles in various processes within the kidney. Reductions in NO and increases in O2− lead to abnormal NaCl and water absorption and hypertension. In the last few years, luminal flow has been shown to be a regulator of NO and O2− production along the nephron. Increases in luminal flow enhance fluid, Na, and bicarbonate transport in the proximal tubule. However, we know of no reports directly addressing flow regulation of NO and O2− in this segment. In the thick ascending limb, flow-stimulated NO and O2− formation has been extensively studied. Luminal flow stimulates NO production by nitric oxide synthase type 3 and its translocation to the apical membrane in medullary thick ascending limbs. These effects are mediated by flow-induced shear stress. In contrast, flow-induced stretch and NaCl delivery stimulate O2− production by NADPH oxidase in this segment. The interaction between flow-induced NO and O2− is complex and involves more than one simply scavenging the other. Flow-induced NO prevents flow from increasing O2− production via cGMP-dependent protein kinase in thick ascending limbs. In macula densa cells, shear stress increases NO production and this requires that the primary cilia be intact. The role of luminal flow in NO and O2− production in the distal tubule is not known. In cultured inner medullary collecting duct cells, shear stress enhances nitrite accumulation, a measure of NO production. Although much progress has been made on this subject in the last few years, there are still many unanswered questions. PMID:21345976

  14. Glycosylation and processing of high-mannose oligosaccharides of thyroid-stimulating hormone subunits: comparison to nonsecretory cell glycoproteins.

    PubMed

    Ronin, C; Stannard, B S; Rosenbloom, I L; Magner, J A; Weintraub, B D

    1984-09-25

    Thyroid-stimulating hormone (TSH) subunit glycosylation was compared to that of total cell glycoproteins in mouse thyrotropic tumors. Lipid-linked oligosaccharides, total cell glycoproteins, and TSH subunits were labeled with either [3H]mannose, [3H]galactose, or [3H]glucose in pulse and pulse-chase experiments. The various oligosaccharides were isolated respectively by lipid extraction and mild acid hydrolysis, by selective immunoprecipitation, or by acid precipitation followed by trypsin and endoglycosidase H treatment. The nature of the oligosaccharides was assessed by their migration in paper chromatography, their relative incorporation of different precursors, and also their resistance to alpha-mannosidase. At 60 min, lipid-linked oligosaccharides were found to be composed of Glc3-2Man9GlcNAc2, Man9-8GlcNAc2, and Man5GlcNAc2. At 10 or 60 min of labeling, total cell proteins contained Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2, Glc1Man8GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2. The largest oligosaccharide, Glc3Man9GlcNAc2, had an unusually long half-life of about 2 h. In contrast, no Glc3Man9GlcNAc2 was found either on TSH + alpha subunits or on free beta subunits isolated either by immunoprecipitation or by sodium dodecyl sulfate gel electrophoresis. Instead, primarily Man9GlcNAc2 was found after a 10-min pulse both on TSH + alpha subunits and on beta subunits. When the pulse was followed by a chase up to 2 h, there was a progressive increase in Man8GlcNAc2 in higher amounts on TSH + alpha-subunit carbohydrate chains than on beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. N-acetylglucosamine, the building block of chitin, inhibits growth of Neurospora crassa.

    PubMed

    Gaderer, Romana; Seidl-Seiboth, Verena; de Vries, Ronald P; Seiboth, Bernhard; Kappel, Lisa

    2017-10-01

    N-acetylglucosamine (GlcNAc) is the monomer of the polysaccharide chitin, an essential structural component of the fungal cell wall and the arthropod exoskeleton. We recently showed that the genes encoding the enzymes for GlcNAc catabolism are clustered in several ascomycetes. In the present study we tested these fungi for growth on GlcNAc and chitin. All fungi, containing the GlcNAc gene cluster, could grow on GlcNAc with the exception of four independent Neurospora crassa wild-type isolates, which were however able to grow on chitin. GlcNAc even inhibited their growth in the presence of other carbon sources. Genes involved in GlcNAc catabolism were strongly upregulated in the presence of GlcNAc, but during growth on chitin their expression was not increased. Deletion of hxk-3 (encoding the first catabolic enzyme, GlcNAc-hexokinase) and ngt-1 (encoding the GlcNAc transporter) improved growth of N. crassa on GlcNAc in the presence of glycerol. A crucial step in GlcNAc catabolism is enzymatic conversion from glucosamine-6-phosphate to fructose-6-phosphate, catalyzed by the glucosamine-6-phosphate deaminase, DAM-1. To assess, if DAM-1 is compromised in N. crassa, the orthologue from Trichoderma reesei, Trdam1, was expressed in N. crassa. Trdam1 expression partially alleviated the negative effects of GlcNAc in the presence of a second carbon source, but did not fully restore growth on GlcNAc. Our results indicate that the GlcNAc-catabolism pathway is bypassed during growth of N. crassa on chitin by use of an alternative pathway, emphasizing the different strategies that have evolved in the fungal kingdom for chitin utilization. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Glutathione S-transferase pi mediates MPTP-induced c-Jun N-terminal kinase activation in the nigrostriatal pathway.

    PubMed

    Castro-Caldas, Margarida; Carvalho, Andreia Neves; Rodrigues, Elsa; Henderson, Colin; Wolf, C Roland; Gama, Maria João

    2012-06-01

    Parkinson's disease (PD) is a progressive movement disorder resulting from the death of dopaminergic neurons in the substantia nigra. Neurotoxin-based models of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) recapitulate the neurological features of the disease, triggering a cascade of deleterious events through the activation of the c-Jun N-terminal kinase (JNK). The molecular mechanisms underlying the regulation of JNK activity under cellular stress conditions involve the activation of several upstream kinases along with the fine-tuning of different endogenous JNK repressors. Glutathione S-transferase pi (GSTP), a phase II detoxifying enzyme, has been shown to inhibit JNK-activated signaling by protein-protein interactions, preventing c-Jun phosphorylation and the subsequent trigger of the cell death cascade. Here, we use C57BL/6 wild-type and GSTP knockout mice treated with MPTP to evaluate the regulation of JNK signaling by GSTP in both the substantia nigra and the striatum. The results presented herein show that GSTP knockout mice are more susceptible to the neurotoxic effects of MPTP than their wild-type counterparts. Indeed, the administration of MPTP induces a progressive demise of nigral dopaminergic neurons together with the degeneration of striatal fibers at an earlier time-point in the GSTP knockout mice when compared to the wild-type mice. Also, MPTP treatment leads to increased p-JNK levels and JNK catalytic activity in both wild-type and GSTP knockout mice midbrain and striatum. Moreover, our results demonstrate that in vivo GSTP acts as an endogenous regulator of the MPTP-induced cellular stress response by controlling JNK activity through protein-protein interactions.

  17. O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis.

    PubMed

    Zeng, Qinghua; Zhao, Rui-Xun; Chen, Jianfeng; Li, Yining; Li, Xiang-Dong; Liu, Xiao-Long; Zhang, Wei-Ming; Quan, Cheng-Shi; Wang, Yi-Shu; Zhai, Ying-Xian; Wang, Jian-Wei; Youssef, Mariam; Cui, Rutao; Liang, Jiyong; Genovese, Nicholas; Chow, Louise T; Li, Yu-Lin; Xu, Zhi-Xiang

    2016-08-16

    High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18-transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc.

  18. O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis

    PubMed Central

    Zeng, Qinghua; Zhao, Rui-Xun; Chen, Jianfeng; Li, Yining; Li, Xiang-Dong; Liu, Xiao-Long; Zhang, Wei-Ming; Quan, Cheng-Shi; Wang, Yi-Shu; Zhai, Ying-Xian; Wang, Jian-Wei; Youssef, Mariam; Cui, Rutao; Liang, Jiyong; Genovese, Nicholas; Chow, Louise T.; Li, Yu-Lin; Xu, Zhi-Xiang

    2016-01-01

    High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18–transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc. PMID:27482104

  19. Modulation of ethoxyresorufin O-deethylase and glutathione S-transferase activities in Nile tilapia (Oreochromis niloticus) by polycyclic aromatic hydrocarbons containing two to four rings: implications in biomonitoring aquatic pollution.

    PubMed

    Pathiratne, Asoka; Hemachandra, Chamini K

    2010-08-01

    Despite ubiquity of polycyclic aromatic hydrocarbons (PAHs) in the tropical environments, little information is available concerning responses of tropical fish to PAHs and associated toxicity. In the present study, effects of five PAHs containing two to four aromatic rings on hepatic CYP1A dependent ethoxyresorufin O-deethylase (EROD), glutathione S-transferase (GST) and serum sorbitol dehydrogenase (SDH) activities in Nile tilapia, a potential fish species for biomonitoring pollution in tropical waters, were evaluated. Results showed that EROD activities were induced by the PAHs containing four aromatic rings (pyrene and chrysene) in a dose dependent manner. However PAHs with two to three aromatic rings (naphthalene, phenanthrene and fluoranthene) caused no effect or inhibition of EROD activities depending on the dose and the duration. Fluoranthene was the most potent inhibitor. SDH results demonstrated that high doses of fluoranthene induced hepatic damage. GST activity was induced by the lowest dose of phenanthrene, fluoranthene and chrysene but high doses had no effect. The results indicate that induction of EROD enzyme in Nile tilapia is a useful biomarker of exposure to PAHs such as pyrene and chrysene. However EROD inhibiting PAHs such as fluoranthene in the natural environment may modulate the EROD inducing potential of other PAHs thereby influencing PAH exposure assessments.

  20. Characterization of spermidine hydroxycinnamoyl transferases from eggplant (Solanum melongena L.) and its wild relative Solanum richardii Dunal

    USDA-ARS?s Scientific Manuscript database

    Eggplant produces a variety of hydroxycinnamic acid amides (HCAAs) that play an important role in plant development and adaptation to environmental changes. However, the HCAA pathway remains largely uncharacterized in Solanaceae. In this study, a spermidine hydroxycinnamoyl transferase (SHT) from eg...

  1. Branched-chain amino acid catabolism is a conserved regulator of physiological ageing.

    PubMed

    Mansfeld, Johannes; Urban, Nadine; Priebe, Steffen; Groth, Marco; Frahm, Christiane; Hartmann, Nils; Gebauer, Juliane; Ravichandran, Meenakshi; Dommaschk, Anne; Schmeisser, Sebastian; Kuhlow, Doreen; Monajembashi, Shamci; Bremer-Streck, Sibylle; Hemmerich, Peter; Kiehntopf, Michael; Zamboni, Nicola; Englert, Christoph; Guthke, Reinhard; Kaleta, Christoph; Platzer, Matthias; Sühnel, Jürgen; Witte, Otto W; Zarse, Kim; Ristow, Michael

    2015-12-01

    Ageing has been defined as a global decline in physiological function depending on both environmental and genetic factors. Here we identify gene transcripts that are similarly regulated during physiological ageing in nematodes, zebrafish and mice. We observe the strongest extension of lifespan when impairing expression of the branched-chain amino acid transferase-1 (bcat-1) gene in C. elegans, which leads to excessive levels of branched-chain amino acids (BCAAs). We further show that BCAAs reduce a LET-363/mTOR-dependent neuro-endocrine signal, which we identify as DAF-7/TGFβ, and that impacts lifespan depending on its related receptors, DAF-1 and DAF-4, as well as ultimately on DAF-16/FoxO and HSF-1 in a cell-non-autonomous manner. The transcription factor HLH-15 controls and epistatically synergizes with BCAT-1 to modulate physiological ageing. Lastly and consistent with previous findings in rodents, nutritional supplementation of BCAAs extends nematodal lifespan. Taken together, BCAAs act as periphery-derived metabokines that induce a central neuro-endocrine response, culminating in extended healthspan.

  2. Branched-chain amino acid catabolism is a conserved regulator of physiological ageing

    PubMed Central

    Mansfeld, Johannes; Urban, Nadine; Priebe, Steffen; Groth, Marco; Frahm, Christiane; Hartmann, Nils; Gebauer, Juliane; Ravichandran, Meenakshi; Dommaschk, Anne; Schmeisser, Sebastian; Kuhlow, Doreen; Monajembashi, Shamci; Bremer-Streck, Sibylle; Hemmerich, Peter; Kiehntopf, Michael; Zamboni, Nicola; Englert, Christoph; Guthke, Reinhard; Kaleta, Christoph; Platzer, Matthias; Sühnel, Jürgen; Witte, Otto W.; Zarse, Kim; Ristow, Michael

    2015-01-01

    Ageing has been defined as a global decline in physiological function depending on both environmental and genetic factors. Here we identify gene transcripts that are similarly regulated during physiological ageing in nematodes, zebrafish and mice. We observe the strongest extension of lifespan when impairing expression of the branched-chain amino acid transferase-1 (bcat-1) gene in C. elegans, which leads to excessive levels of branched-chain amino acids (BCAAs). We further show that BCAAs reduce a LET-363/mTOR-dependent neuro-endocrine signal, which we identify as DAF-7/TGFβ, and that impacts lifespan depending on its related receptors, DAF-1 and DAF-4, as well as ultimately on DAF-16/FoxO and HSF-1 in a cell-non-autonomous manner. The transcription factor HLH-15 controls and epistatically synergizes with BCAT-1 to modulate physiological ageing. Lastly and consistent with previous findings in rodents, nutritional supplementation of BCAAs extends nematodal lifespan. Taken together, BCAAs act as periphery-derived metabokines that induce a central neuro-endocrine response, culminating in extended healthspan. PMID:26620638

  3. Structural studies on molecular interactions between camel peptidoglycan recognition protein, CPGRP-S, and peptidoglycan moieties N-acetylglucosamine and N-acetylmuramic acid.

    PubMed

    Sharma, Pradeep; Yamini, Shavait; Dube, Divya; Singh, Amar; Sinha, Mau; Dey, Sharmistha; Mitra, Dipendra K; Kaur, Punit; Sharma, Sujata; Singh, Tej P

    2012-06-22

    Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

  4. Glutathione Transferase U13 Functions in Pathogen-Triggered Glucosinolate Metabolism.

    PubMed

    Piślewska-Bednarek, Mariola; Nakano, Ryohei Thomas; Hiruma, Kei; Pastorczyk, Marta; Sanchez-Vallet, Andrea; Singkaravanit-Ogawa, Suthitar; Ciesiołka, Danuta; Takano, Yoshitaka; Molina, Antonio; Schulze-Lefert, Paul; Bednarek, Paweł

    2018-01-01

    Glutathione (GSH) and indole glucosinolates (IGs) exert key functions in the immune system of the model plant Arabidopsis ( Arabidopsis thaliana ). Appropriate GSH levels are important for execution of both pre- and postinvasive disease resistance mechanisms to invasive pathogens, whereas an intact PENETRATION2 (PEN2)-pathway for IG metabolism is essential for preinvasive resistance in this species. Earlier indirect evidence suggested that the latter pathway involves conjugation of GSH with unstable products of IG metabolism and further processing of the resulting adducts to biologically active molecules. Here we describe the identification of Glutathione- S -Transferase class-tau member 13 (GSTU13) as an indispensable component of the PEN2 immune pathway for IG metabolism. gstu13 mutant plants are defective in the pathogen-triggered biosynthesis of end products of the PEN2 pathway, including 4-O-β-d-glucosyl-indol-3-yl formamide, indole-3-ylmethyl amine, and raphanusamic acid. In line with this metabolic defect, lack of functional GSTU13 results in enhanced disease susceptibility toward several fungal pathogens including Erysiphe pisi , Colletotrichum gloeosporioides , and Plectosphaerella cucumerina Seedlings of gstu13 plants fail also to deposit the (1,3)-β-glucan cell wall polymer, callose, after recognition of the bacterial flg22 epitope. We show that GSTU13 mediates specifically the role of GSH in IG metabolism without noticeable impact on other immune functions of this tripeptide. We postulate that GSTU13 connects GSH with the pathogen-triggered PEN2 pathway for IG metabolism to deliver metabolites that may have numerous functions in the innate immune system of Arabidopsis. © 2018 American Society of Plant Biologists. All Rights Reserved.

  5. Structure elucidation of the O-specific polysaccharide by NMR spectroscopy and selective cleavage and genetic characterization of the O-antigen of Escherichia albertii O5.

    PubMed

    Naumenko, Olesya I; Zheng, Han; Wang, Jianping; Senchenkova, Sof'ya N; Wang, Hong; Shashkov, Alexander S; Chizhov, Alexander O; Li, Qun; Knirel, Yuriy A; Xiong, Yanwen

    2018-03-02

    The O-specific polysaccharide (O-antigen) was obtained by mild acid degradation of the lipopolysaccharide of Escherichia albertii O5 (strain T150248) and studied by sugar analysis, selective cleavages of glycosidic linkages, and 1D and 2D 1 H and 13 C NMR spectroscopy. Partial solvolysis with anh (anhydrous) CF 3 CO 2 H and hydrolysis with 0.05 M CF 3 CO 2 H cleaved predominantly the glycosidic linkage of β-GalpNAc or β-Galf, respectively, whereas the linkages of α-GlcpNAc and β-Galp were stable. Mixtures of the corresponding tri- and tetra-saccharides thus obtained were studied by NMR spectroscopy and high-resolution ESI MS. The following new structure was established for the tetrasaccharide repeat (O-unit) of the O-polysaccharide: →4)-α-d-GlcpNAc-(1 → 4)-β-d-Galp6Ac-(1 → 6)-β-d-Galf-(1 → 3)-β-d-GalpNAc-(1→where the degree of O-acetylation of d-Galp is ∼70%. The O-polysaccharide studied has a β-d-Galp-(1 → 6)-β-d-Galf-(1 → 3)-β-d-GalpNAc trisaccharide fragment in common with the O-polysaccharides of E. albertii O7, Escherichia coli O124 and O164, and Shigella dysenteriae type 3 studied earlier. The orf5-7 in the O-antigen gene cluster of E. albertii O5 are 47%, 78%, and 75% identical on the amino acid level to genes for predicted enzymes of E. albertii O7, including Galp-transferase wfeS, UDP-d-Galp mutase glf, and Galf-transferase wfeT, respectively, which are putatively involved with the synthesis of the shared trisaccharide fragment of the O-polysaccharides. The occurrence upstream of the O-antigen gene cluster of a 4-epimerase gene gnu for conversion of undecaprenyl diphosphate-linked d-GlcNAc (UndPP-d-GlcNAc) into UndPP-d-GalNAc indicates that d-GalNAc is the first monosaccharide of the O-unit, and hence the O-units are interlinked in the O-polysaccharide of E. albertii O5 by the β-d-GalpNAc-(1 → 4)-α-d-GlcpNAc linkage. Copyright © 2017. Published by Elsevier Ltd.

  6. N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity.

    PubMed

    Lee, Sang-Min; Booe, Jason M; Gingell, Joseph J; Sjoelund, Virginie; Hay, Debbie L; Pioszak, Augen A

    2017-07-05

    The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for the treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-Glycan-free CTR ECD produced in Escherichia coli exhibited ∼10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N-glycans, or in HEK293S GnTI - cells, which yield core N-glycans (Man 5 GlcNAc 2 ). PNGase F-catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ∼10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N → Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

  7. Polymorphisms of Glutathione S-transferases Omega-1 among ethnic populations in China

    PubMed Central

    Fu, Songbo; Wu, Jie; Chen, Feng; Sun, Dianjun; Fu, Songbin

    2008-01-01

    Background Glutathione S-transferases (GSTs) is a genetic factor for many diseases and exhibits great diversities among various populations. We assessed association of the genotypes of Glutathione S-transferases Omega-1 (GSTO1) A140D with ethnicity in China. Results Peripheral blood samples were obtained from 1314 individuals from 14 ethnic groups. Polymorphisms of GSTO1 A140D were measured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Logistic regression was employed to adjustment for regional factor. The frequency of GSTO1 140A allele was 15.49% in the total 14 ethnic populations. Compared to Han ethnic group, two ethnic populations were more likely to have AA or CA genotype [odds ratio (OR): 1.77, 95% confidence interval (95% CI): 1.05–2.98 for Uygur and OR: 1.78, 95% CI: 1.18–2.69 for Hui]. However, there were no statistically significant differences across 14 ethnic groups when region factor was adjusted. In Han ethnicity, region was significantly associated with AA or CA genotype. Han individuals who resided in North-west of China were more likely to have these genotypes than those in South of China (OR: 1.63, 95% CI: 1.21–2.20). Conclusion The prevalence of the GSTO1 140A varied significantly among different regional populations in China, which showed that geography played a more important role in the population differentiation for this allele than the ethnicity/race. PMID:18400112

  8. Suppressive role of OGT-mediated O-GlcNAcylation of BAP1 in retinoic acid signaling.

    PubMed

    Moon, Seungtae; Lee, Yong-Kyu; Lee, Sang-Wang; Um, Soo-Jong

    2017-10-07

    BRCA1-associated protein 1 (BAP1) has been implicated in diverse biological functions, including tumor suppression. However, its regulation via glycosylation and its role in embryonic stem (ES) cells are poorly defined. BAP1 was recently reported to interact with O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT). Here, we confirmed the physical interaction and investigated its functional significance. The O-GlcNAcylation of BAP1, which requires OGT, was examined in vivo and in vitro, and was proven using alloxan, an OGT inhibitor. OGT promoted the BAP1-induced repression of retinoic acid (RA)-induced RA receptor (RAR) activation. The repressive activity of BAP1 was relieved by alloxan but exacerbated by PUGNAc, an O-GlcNAcase (OGA) inhibitor. Finally, we addressed the role of O-GlcNAcylation in the RA-induced differentiation of murine ES cells. Alkaline phosphatase staining revealed the cooperation of RA and alloxan for impairing the pluripotency of ES cells. This cooperation was also observed by measuring the size of embryonic bodies and the expression of Sox2, a pluripotency marker. Overall, our data suggest that OGT-mediated O-GlcNAcylation of BAP1 prefers the maintenance of pluripotency, whereas its inhibition facilitates RA-induced differentiation in ES cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Reciprocal relationship between alpha1,2 mannosidase processing and reglucosylation in the rough endoplasmic reticulum of Man-P-Dol deficient cells.

    PubMed

    Duvet, S; Chirat, F; Mir, A M; Verbert, A; Dubuisson, J; Cacan, R

    2000-02-01

    The study of the glycosylation pathway of a mannosylphosphoryldolichol-deficient CHO mutant cell line (B3F7) reveals that truncated Glc(0-3)Man5GlcNAc2 oligosaccharides are transferred onto nascent proteins. Pulse-chase experiments indicate that these newly synthesized glycoproteins are retained in intracellular compartments and converted to Man4GlcNAc2 species. In this paper, we demonstrate that the alpha1,2 mannosidase, which is involved in the processing of Man5GlcNAc2 into Man4GlcNAc2, is located in the rough endoplasmic reticulum. The enzyme was shown to be inhibited by kifunensine and deoxymannojirimycin, indicating that it is a class I mannosidase. In addition, Man4GlcNAc2 species were produced at the expense of Glc1Man5GlcNAc2 species. Thus, the trimming of Man5GlcNAc2 to Man4GlcNAc2, which is catalyzed by this mannosidase, could be involved in the control of the glucose-dependent folding pathway.

  10. The insect repellent DEET (N,N-diethyl-3-methylbenzamide) increases the synthesis of glutathione S-transferase in cultured mosquito cells.

    PubMed

    Hellestad, Vanessa J; Witthuhn, Bruce A; Fallon, Ann M

    2011-04-01

    DEET (N,N-diethyl-3-methylbenzamide) is the active ingredient used in many commonly used insect repellents, but its mode of action remains poorly understood. Efforts to identify properties that could lead to the development of more effective active ingredients have distinguished among DEET's repellent, deterrent, and insecticidal activities. We used an Aedes albopictus mosquito cell line to evaluate DEET's toxicological properties in the absence of sensory input mediated by the olfactory system. When cells were treated with DEET and labeled with [(35)S]methionine/cysteine, a single 25-kDa protein was induced, relative to other proteins, on SDS-polyacrylamide gels. The 25-kDa band from DEET-treated cells was enriched in peptides corresponding to glutathione S-transferase D10 and/or theta in the Aedes aegypti genome. Consistent with the increased expression of the labeled protein, DEET-treated cells had increased glutathione S-transferase activity, and the radiolabeled band bound to Sepharose 4B containing reduced glutathione. By analyzing partial tryptic digests, we established that DEET induces the homolog of A. aegypti glutathione S-transferase, class theta, corresponding to protein XP_001658009.1 in the NCBI database. This specific effect of DEET at the subcellular level suggests that DEET induces physiological responses that extend beyond recognition by the peripheral olfactory system.

  11. FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression.

    PubMed

    Gopal, Keshav; Saleme, Bruno; Al Batran, Rami; Aburasayn, Hanin; Eshreif, Amina; Ho, Kim L; Ma, Wayne K; Almutairi, Malak; Eaton, Farah; Gandhi, Manoj; Park, Edwards A; Sutendra, Gopinath; Ussher, John R

    2017-09-01

    Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation and a critical regulator of metabolic flexibility during the fasting to feeding transition. PDH is regulated via both PDH kinases (PDHK) and PDH phosphatases, which phosphorylate/inactivate and dephosphorylate/activate PDH, respectively. Our goal was to determine whether the transcription factor forkhead box O1 (FoxO1) regulates PDH activity and glucose oxidation in the heart via increasing the expression of Pdk4 , the gene encoding PDHK4. To address this question, we differentiated H9c2 myoblasts into cardiac myocytes and modulated FoxO1 activity, after which Pdk4 /PDHK4 expression and PDH phosphorylation/activity were assessed. We assessed binding of FoxO1 to the Pdk4 promoter in cardiac myocytes in conjunction with measuring the role of FoxO1 on glucose oxidation in the isolated working heart. Both pharmacological (1 µM AS1842856) and genetic (siRNA mediated) inhibition of FoxO1 decreased Pdk4 /PDHK4 expression and subsequent PDH phosphorylation in H9c2 cardiac myocytes, whereas 10 µM dexamethasone-induced Pdk4 /PDHK4 expression was abolished via pretreatment with 1 µM AS1842856. Furthermore, transfection of H9c2 cardiac myocytes with a vector expressing FoxO1 increased luciferase activity driven by a Pdk4 promoter construct containing the FoxO1 DNA-binding element region, but not in a Pdk4 promoter construct lacking this region. Finally, AS1842856 treatment in fasted mice enhanced glucose oxidation rates during aerobic isolated working heart perfusions. Taken together, FoxO1 directly regulates Pdk4 transcription in the heart, thereby controlling PDH activity and subsequent glucose oxidation rates. NEW & NOTEWORTHY Although studies have shown an association between FoxO1 activity and pyruvate dehydrogenase kinase 4 expression, our study demonstrated that pyruvate dehydrogenase kinase 4 is a direct transcriptional target of FoxO1 (but not FoxO3/FoxO4) in the heart. Furthermore, we

  12. Neuroantibodies (NAB) in African-American Children: Associations with Gender, Glutathione-S-Transferase (GST)Pi Polymorphisms (SNP) and Heavy Metals

    EPA Science Inventory

    CONTACT (NAME ONLY): Hassan El-Fawal Abstract Details PRESENTATION TYPE: Platform or Poster CURRENT CATEGORY: Neurodegenerative Disease | Biomarkers | Neurotoxicity, Metals KEYWORDS: Autoantibodies, Glutathione-S-Transferase, DATE/TIME LAST MODIFIED: DATE/TIME SUBMITTED: Abs...

  13. FoxO1 regulates apoptosis induced by asbestos in the MT-2 human T-cell line.

    PubMed

    Matsuzaki, Hidenori; Lee, Suni; Maeda, Megumi; Kumagai-Takei, Naoko; Nishimura, Yasumitsu; Otsuki, Takemi

    2016-09-01

    Asbestos is known to cause malignant mesothelioma and lung cancer. Recent studies implicate tumor immunity in the development of various tumors, including malignant mesothelioma. In order to establish an in vitro T-cell model to clarify the effects of long-term exposure of asbestos on tumor immunity, in this study, human T-cell line MT-2 cells were cultured with asbestos for longer than 8 months and the resultant cells (MT-2Rst) were assessed for the expression of forkhead transcription factor FoxO1. Gene expression analysis revealed that the amount of FoxO1 mRNA decreased after long-term exposure of the MT-2 cells to asbestos. In accordance with this reduction in FoxO1, pro-apoptotic Foxo1 target genes Puma, Fas ligand and Bim were also seen to be down-regulated in MT-2Rst cells. Furthermore, shRNA-mediated knock-down of FoxO1 reduced the number of apoptotic parental MT-2 cells after treatment with asbestos. On the other hand, over-expression of FoxO1 did not affect asbestos-induced apoptosis in MT-2Rst cells. These results suggested that FoxO1 played an important role in regulating asbestos-induced apoptosis and confirmed the presence of multiple pathways regulating resistance to asbestos in MT-2Rst cells.

  14. Global Analysis of Apicomplexan Protein S-Acyl Transferases Reveals an Enzyme Essential for Invasion

    PubMed Central

    Frénal, Karine; Tay, Chwen L; Mueller, Christina; Bushell, Ellen S; Jia, Yonggen; Graindorge, Arnault; Billker, Oliver; Rayner, Julian C; Soldati-Favre, Dominique

    2013-01-01

    The advent of techniques to study palmitoylation on a whole proteome scale has revealed that it is an important reversible modification that plays a role in regulating multiple biological processes. Palmitoylation can control the affinity of a protein for lipid membranes, which allows it to impact protein trafficking, stability, folding, signalling and interactions. The publication of the palmitome of the schizont stage of Plasmodium falciparum implicated a role for palmitoylation in host cell invasion, protein export and organelle biogenesis. However, nothing is known so far about the repertoire of protein S-acyl transferases (PATs) that catalyse this modification in Apicomplexa. We undertook a comprehensive analysis of the repertoire of Asp-His-His-Cys cysteine-rich domain (DHHC-CRD) PAT family in Toxoplasma gondii and Plasmodium berghei by assessing their localization and essentiality. Unlike functional redundancies reported in other eukaryotes, some apicomplexan-specific DHHCs are essential for parasite growth, and several are targeted to organelles unique to this phylum. Of particular interest is DHHC7, which localizes to rhoptry organelles in all parasites tested, including the major human pathogen P. falciparum. TgDHHC7 interferes with the localization of the rhoptry palmitoylated protein TgARO and affects the apical positioning of the rhoptry organelles. This PAT has a major impact on T. gondii host cell invasion, but not on the parasite’s ability to egress. PMID:23638681

  15. Glutathione Transferase from Trichoderma virens Enhances Cadmium Tolerance without Enhancing Its Accumulation in Transgenic Nicotiana tabacum

    PubMed Central

    Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan

    2011-01-01

    Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and

  16. Metabolomic Analysis of Blood Plasma after Oral Administration of N-acetyl-d-Glucosamine in Dogs

    PubMed Central

    Osaki, Tomohiro; Kurozumi, Seiji; Sato, Kimihiko; Terashi, Taro; Azuma, Kazuo; Murahata, Yusuke; Tsuka, Takeshi; Ito, Norihiko; Imagawa, Tomohiro; Minami, Saburo; Okamoto, Yoshiharu

    2015-01-01

    N-acetyl-d-glucosamine (GlcNAc) is a monosaccharide that polymerizes linearly through (1,4)-β-linkages. GlcNAc is the monomeric unit of the polymer chitin. GlcNAc is a basic component of hyaluronic acid and keratin sulfate found on the cell surface. The aim of this study was to examine amino acid metabolism after oral GlcNAc administration in dogs. Results showed that plasma levels of ectoine were significantly higher after oral administration of GlcNAc than prior to administration (p < 0.001). To our knowledge, there have been no reports of increased ectoine concentrations in the plasma. The mechanism by which GlcNAc administration leads to increased ectoine plasma concentration remains unclear; future studies are required to clarify this mechanism. PMID:26262626

  17. Potentiation of angiogenic switch in capillary endothelial cells by cAMP: A cross-talk between up-regulated LLO biosynthesis and the HSP-70 expression.

    PubMed

    Martínez, Juan A; Tavárez, José J; Oliveira, Caroline M; Banerjee, Dipak K

    2006-05-01

    During tumor growth and invasion, the endothelial cells from a relatively quiescent endothelium start proliferating. The exact mechanism of switching to a new angiogenic phenotype is currently unknown. We have examined the role of intracellular cAMP in this process. When a non-transformed capillary endothelial cell line was treated with 2 mM 8Br-cAMP, cell proliferation was enhanced by approximately 70%. Cellular morphology indicated enhanced mitosis after 32-40 h with almost one-half of the cell population in the S phase. Bcl-2 expression and caspase-3, -8, and -9 activity remained unaffected. A significant increase in the Glc(3)Man(9)GlcNAc(2)-PP-Dol biosynthesis and turnover, Factor VIIIC N-glycosylation, and cell surface expression of N-glycans was observed in cells treated with 8Br-cAMP. Dol-P-Man synthase activity in the endoplasmic reticulum membranes also increased. A 1.4-1.6-fold increase in HSP-70 and HSP-90 expression was also observed in 8Br-cAMP treated cells. On the other hand, the expression of GRP-78/Bip was 2.3-fold higher compared to that of GRP-94 in control cells, but after 8Br-cAMP treatment for 32 h, it was reduced by 3-fold. GRP-78/Bip expression in untreated cells was 1.2-1.5-fold higher when compared with HSP-70 and HSP-90, whereas that of the GRP-94 was 1.5-1.8-fold lower. After 8Br-cAMP treatment, GRP-78/Bip expression was reduced 4.5-4.8-fold, but the GRP-94 was reduced by 1.5-1.6-fold only. Upon comparison, a 2.9-fold down-regulation of GRP-78/Bip was observed compared to GRP-94. We, therefore, conclude that a high level of Glc(3)Man(9)GlcNAc(2)-PP-Dol, resulting from 8Br-cAMP stimulation up-regulated HSP-70 expression and down-regulated that of the GRP-78/Bip, maintained adequate protein folding, and reduced endoplasmic reticulum stress. As a result capillary endothelial cell proliferation was induced.

  18. Regulation of RAW 264.7 macrophages behavior on anodic TiO2 nanotubular arrays

    NASA Astrophysics Data System (ADS)

    Yao, Shenglian; Feng, Xujia; Li, Wenhao; Wang, Lu-Ning; Wang, Xiumei

    2017-12-01

    Titanium (Ti) implants with TiO2 nanotubular arrays on the surface could regulate cells adhesion, proliferation and differentiation to determine the bone integration. Additionally, the regulation of immune cells could improve osteogenesis or lead in appropriate immune reaction. Thus, we evaluate the behavior of RAW264.7 macrophages on TiO2 nanotubular arrays with a wide range diameter (from 20 to 120 nm) fabricated by an electrochemical anodization process. In this work, the proliferation, cell viability and cytokine/chemokine secretion were evaluated by CCK-8, live/dead staining and ELISA, respectively. SEM and confocal microscopy were used to observe the adhesion morphology. Results showed that the small size nanotube surface was benefit for the macrophages adhesion and proliferation, while larger size surface could reduce the inflammatory response. These findings contribute to the design of immune-regulating Ti implants surface that supports successful implantation.

  19. Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation.

    PubMed

    Robles-Martinez, Leobarda; Mendez, Tavis L; Apodaca, Jennifer; Das, Siddhartha

    2017-01-01

    The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2-3 fold higher than of GlcCer. Although both activities follow Michaelis-Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Gene Regulation by Retinoid Receptors in Human Mammary Epithelial Cells

    DTIC Science & Technology

    2002-10-01

    ceptors for collagen and fibronectin in human fibrosarcoma cells possessing Xia, Y-, S.G. Gil, and W.G. Carter. 1996. Anchorage mediated by integrin...simultaneously block all TdT terminal deoxynucleotidyl transferase pRB retinoblastoma protein wild-type RAR isoforms (-o, -P, and -- ) (Damm et CAT ...P0 pM treated with 0, 0,1, 1.0, or 10 jIM ATRA for 24 hours and cell lysates were assayed for CAT reporter activity. pCMV-GH was used as a transfection

  1. N-Acetylglucosamine: Production and Applications

    PubMed Central

    Chen, Jeen-Kuan; Shen, Chia-Rui; Liu, Chao-Lin

    2010-01-01

    N-Acetylglucosamine (GlcNAc) is a monosaccharide that usually polymerizes linearly through (1,4)-β-linkages. GlcNAc is the monomeric unit of the polymer chitin, the second most abundant carbohydrate after cellulose. In addition to serving as a component of this homogeneous polysaccharide, GlcNAc is also a basic component of hyaluronic acid and keratin sulfate on the cell surface. In this review, we discuss the industrial production of GlcNAc, using chitin as a substrate, by chemical, enzymatic and biotransformation methods. Also, newly developed methods to obtain GlcNAc using glucose as a substrate in genetically modified microorganisms are introduced. Moreover, GlcNAc has generated interest not only as an underutilized resource but also as a new functional material with high potential in various fields. Here we also take a closer look at the current applications of GlcNAc, and several new and cutting edge approaches in this fascinating area are thoroughly discussed. PMID:20948902

  2. The O-antigen structure of bacterium Comamonas aquatica CJG.

    PubMed

    Wang, Xiqian; Kondakova, Anna N; Zhu, Yutong; Knirel, Yuriy A; Han, Aidong

    2017-11-01

    Genus Comamonas is a group of bacteria that are able to degrade a variety of environmental waste. Comamonas aquatica CJG (C. aquatica) in this genus is able to absorb low-density lipoprotein but not high-density lipoprotein of human serum. Using 1 H and 13 C NMR spectroscopy, we found that the O-polysaccharide (O-antigen) of this bacterium is comprised of a disaccharide repeat (O-unit) of d-glucose and 2-O-acetyl-l-rhamnose, which is shared by Serratia marcescens O6. The O-antigen gene cluster of C. aquatica, which is located between coaX and tnp4 genes, contains rhamnose synthesis genes, glycosyl and acetyl transferase genes, and ATP-binding cassette transporter genes, and therefore is consistent with the O-antigen structure determined here.

  3. A methionine-free diet associated with nitrosourea treatment down-regulates methylguanine-DNA methyl transferase activity in patients with metastatic cancer.

    PubMed

    Thivat, Emilie; Durando, Xavier; Demidem, Aïcha; Farges, Marie-Chantal; Rapp, Maryse; Cellarier, Eric; Guenin, Samuel; D'Incan, Michel; Vasson, Marie-Paule; Chollet, Philippe

    2007-01-01

    Methionine (MET) depletion used in association with chemotherapy improves the therapeutic index in animal models. This potentiating effect may be due to tumor cell sensitization to chloroethylnitrosoureas through their MET dependency and the down-regulation of O6- methylguanine-DNA methyltransferase (MGMT). Our purpose was to evaluate the impact of the association of a dietary MET restriction with nitrosourea treatment on MGMT activity in peripheral blood mononuclear cells (PBMCs). Six patients with metastatic cancer (melanoma and glioma) received 4 cycles of a MET-free diet with cystemustine (60 mg/m2). MGMT activity in PBMCs decreased by an average of 13% from 553+/-90 fnol/mg before the diet to 413+/-59 fmol/mg after the diet + chemotherapy period (p=0.029). The decrease of MGMT activity was not affected by the duration of the MET-free diet period but seems to be correlated to the plasma MET depletion induced by the MET-free diet.

  4. Arabidopsis F-box protein containing a Nictaba-related lectin domain interacts with N-acetyllactosamine structures.

    PubMed

    Stefanowicz, Karolina; Lannoo, Nausicaä; Proost, Paul; Van Damme, Els J M

    2012-01-01

    The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N- and O-glycans containing N-acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly-N-acetyllactosamine ([Galβ1-4GlcNAc]n) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides.

  5. Protection against oxidative DNA damage and stress in human prostate by glutathione S-transferase P1

    PubMed Central

    Kanwal, Rajnee; Pandey, Mitali; Bhaskaran, Natarajan; MacLennan, Gregory T; Fu, Pingfu; Ponsky, Lee E; Gupta, Sanjay

    2014-01-01

    The pi-class glutathione S-transferase (GSTP1) actively protect cells from carcinogens and electrophilic compounds. Loss of GSTP1 expression via promoter hypermethylation is the most common epigenetic alteration observed in human prostate cancer. Silencing of GSTP1 can increase generation of reactive oxygen species (ROS) and DNA damage in cells. In this study we investigated whether loss of GSTP1 contributes to increased DNA damage that may predispose men to a higher risk of prostate cancer. We found significantly elevated (103%; P<0.0001) levels of 8-oxo-2′-deoxogunosine (8-OHdG), an oxidative DNA damage marker, in adenocarcinomas, compared to benign counterparts, which positively correlated (r=0.2) with loss of GSTP1 activity (34%; P<0.0001). Silencing of GSTP1 using siRNA approach in normal human prostate epithelial RWPE1 cells caused increased intracellular production of ROS and higher susceptibility of cells to H2O2-mediated oxidative stress. Additionally, human prostate carcinoma LNCaP cells, which contain a silenced GSTP1 gene, were genetically modified to constitutively express high levels of GSTP1. Induction of GSTP1 activity lowered endogenous ROS levels in LNCaP-pLPCX-GSTP1 cells, and when exposed to H2O2, these cells exhibited significantly reduced production of ROS and 8-OHdG levels, compared to vector control LNCaP-pLPCX cells. Furthermore, exposure of LNCaP cells to green tea polyphenols caused re-expression of GSTP1, which protected the cells from H2O2-mediated DNA damage through decreased ROS production compared to non-exposed cells. These results suggest that loss of GSTP1 expression in human prostate cells, a process that increases their susceptibility to oxidative stress-induced DNA damage, may be an important target for primary prevention of prostate cancer. PMID:22833520

  6. Combined glutathione S transferase M1/T1 null genotypes is associated with type 2 diabetes mellitus

    PubMed Central

    POROJAN, MIHAI D.; BALA, CORNELIA; ILIES, ROXANA; CATANA, ANDREEA; POPP, RADU A.; DUMITRASCU, DAN L.

    2015-01-01

    Background Due to new genetic insights, a considerably large number of genes and polymorphic gene variants are screened and linked with the complex pathogenesis of type 2 diabetes (DM). Our study aimed to investigate the association between the two isoforms of the glutathione S-transferase genes (Glutathione S transferase isoemzyme type M1- GSTM1 and Glutathione S transferase isoemzyme type T1-GSTT1) and the prevalence of DM in the Northern Romanian population. Methods We conducted a cross-sectional, randomized, case-control study evaluating the frequency of GSTM1 and GSTT1 null alleles in patients diagnosed with DM. A total of 106 patients diagnosed with DM and 124 healthy controls were included in the study. GSTM1 and GSTT1 null alleles genotyping was carried out using Multiplex PCR amplification of relevant gene fragments, followed by gel electrophoresis analysis of the resulting amplicons. Results Molecular analysis did not reveal an increased frequency of the null GSTM1 and GSTT1 alleles (mutant genotypes) respectively in the DM group compared to controls (p=0.171, OR=1.444 CI=0.852–2.447; p=0.647, OR=0.854, CI=0.436–1.673). Nevertheless, the combined GSTM1/GSTT1 null genotypes were statistically significantly higher in DM patients compared to control subjects (p=0.0021, OR=0.313, CI=0.149–0.655) Conclusions The main finding of our study is that the combined, double GSTM1/GSTT1 null genotypes are to be considered among the polymorphic genetic risk factors for type 2 DM. PMID:26528065

  7. Uncoupling of acetylation from phosphorylation regulates FoxO1 function independent of its subcellular localization.

    PubMed

    Qiang, Li; Banks, Alexander S; Accili, Domenico

    2010-08-27

    The activity of transcription factor FoxO1 is regulated by phosphorylation-dependent nuclear exclusion and deacetylation-dependent nuclear retention. It is unclear whether and how these two post-translational modifications affect each other. To answer this question, we expressed FoxO1 cDNAs with combined mutations of phosphorylation and acetylation sites in HEK-293 cells and analyzed their subcellular localization patterns. We show that mutations mimicking the acetylated state (KQ series) render FoxO1 more sensitive to Akt-mediated phosphorylation and nuclear exclusion and can reverse the constitutively nuclear localization of phosphorylation-defective FoxO1. Conversely, mutations mimicking the deacetylated state (KR series) promote FoxO1 nuclear retention. Oxidative stress and the Sirt1 activator resveratrol are thought to promote FoxO1 deacetylation and nuclear retention, thus increasing its activity. Accordingly, FoxO1 deacetylation was required for the effect of oxidative stress (induced by H(2)O(2)) to retain FoxO1 in the nucleus. H(2)O(2) also inhibited FoxO1 phosphorylation on Ser-253 and Thr-24, the key insulin-regulated sites, irrespective of its acetylation. In contrast, the effect of resveratrol was independent of FoxO1 acetylation and its phosphorylation on Ser-253 and Thr-24, suggesting that resveratrol acts on FoxO1 in a Sirt1- and Akt-independent manner. The dissociation of deacetylation from dephosphorylation in H(2)O(2)-treated cells indicates that the two modifications can occur independently of each other. It can be envisaged that FoxO1 exists in multiple nuclear forms with distinct activities depending on the balance of acetylation and phosphorylation.

  8. Engineering of N-acetylglucosamine metabolism for improved antibiotic production in Streptomyces coelicolor A3(2) and an unsuspected role of NagA in glucosamine metabolism.

    PubMed

    Świątek, Magdalena A; Urem, Mia; Tenconi, Elodie; Rigali, Sébastien; van Wezel, Gilles P

    2012-01-01

    N-acetylglucosamine (GlcNAc), the monomer of chitin and constituent of bacterial peptidoglycan, is a preferred carbon and nitrogen source for streptomycetes. Recent studies have revealed new functions of GlcNAc in nutrient signaling of bacteria. Exposure to GlcNAc activates development and antibiotic production of Streptomyces coelicolor under poor growth conditions (famine) and blocks these processes under rich conditions (feast). Glucosamine-6-phosphate (GlcN-6P) is a key molecule in this signaling pathway and acts as an allosteric effector of a pleiotropic transcriptional repressor DasR, the regulon of which includes the GlcNAc metabolic enzymes N-actetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase (NagA) and GlcN-6P deaminase (NagB). Intracellular accumulation of GlcNAc-6P and GlcN-6P enhanced production of the pigmented antibiotic actinorhodin. When the nagB mutant was challenged with GlcNAc or GlcN, spontaneous second-site mutations that relieved the toxicity of the accumulated sugar phosphates were obtained. Surprisingly, deletion of nagA also relieved toxicity of GlcN, indicating novel linkage between the GlcN and GlcNAc utilization pathways. The strongly enhanced antibiotic production observed for many suppressor mutants shows the potential of the modulation of GlcNAc and GlcN metabolism as a metabolic engineering tool toward the improvement of antibiotic productivity or even the discovery of novel compounds.

  9. Protective role for ovarian glutathione S-transferase isoform pi during 7,12-dimethylbenz[a]anthracene-induced ovotoxicity

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

    Bhattacharya, Poulomi, E-mail: poulomib@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2012-04-15

    7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all developmental stages. This study investigated a role for the glutathione S-transferase (Gst) isoforms alpha (a), mu (m) and pi (p) and the transcription factors, Ahr and Nrf2, during DMBA-induced ovotoxicity, and their regulation by phosphatidylinositol-3 kinase (PI3K) signaling. Negative regulation of JNK by GSTP during DMBA exposure was also studied. Post-natal day (PND) 4 Fischer 344 rat ovaries were exposed to vehicle control (1% DMSO) ± DMBA (1 μM) or vehicle control (1% DMSO) ± LY294002 (PI3K inhibitor; 20 μM) for 1, 2, 4, or 6 days. Total RNA or protein was isolated,more » followed by RT-PCR or Western blotting to determine mRNA or protein level, respectively. Immunoprecipitation using an anti-GSTP antibody was performed to determine interaction between GSTP and JNK, followed by Western blotting to determine JNK and p-c-Jun protein level. DMBA had no impact on Gsta, Gstm or Nrf2 mRNA level, but increased Gstp mRNA and protein after 2 days. Ahr mRNA and protein increased after 2 and 4 days of DMBA exposure, respectively and DMBA increased NRF2 protein level after 4 days. JNK bound to GSTP was increased during DMBA exposure, with a concomitant decrease in unbound JNK and p-c-Jun. Ahr and Gstp mRNA were decreased (2 days) and increased (4 days) by PI3K inhibition, while Gstm mRNA increased (P < 0.05) after both time points, and there was no effect on Nrf2 mRNA. PI3K inhibition increased AHR, NRF2 and GSTP protein level. These findings support involvement of ovarian GSTP during DMBA exposure, and indicate a regulatory role for the PI3K signaling pathway on ovarian xenobiotic metabolism gene expression. -- Highlights: ► Ovarian GSTP is activated in response to DMBA exposure. ► AhR and Nrf2 transcription factors are up-regulated by DMBA. ► PI3K signaling regulates Ahr, Nrf2 and Gstp expression. ► GSTP negatively regulates ovarian JNK in response to DMBA exposure.« less

  10. Role of O2 in regulating tissue respiration in dog muscle working in situ

    NASA Technical Reports Server (NTRS)

    Hogan, M. C.; Arthur, P. G.; Bebout, D. E.; Hochachka, P. W.; Wagner, P. D.

    1992-01-01

    This study was designed to investigate the role of tissue oxygenation in some of the factors that are thought to regulate muscle respiration and metabolism. Tissue oxygenation was altered by reductions in O2 delivery (muscle blood flow x arterial O2 content), induced by decreases in arterial PO2 (PaO2). O2 uptake (VO2) was measured in isolated in situ canine gastrocnemius at rest and while working at two stimulation intensities (isometric tetanic contractions at 0.5 and 1 contractions/s) on three separate occasions, with only the level of PaO2 (78, 30, and 21 Torr) being different for each occasion. Muscle blood flow was held constant (pump perfusion) at each work intensity for the three different levels of PaO2. Muscle biopsies were obtained at the end of each rest and work period. Muscle VO2 was significantly less (P less than 0.05) at both stimulation intensities for the hypoxemic conditions, whereas [ATP] was reduced only during the highest work intensity during both hypoxemic conditions (31% reduction at 21 Torr PaO2 and 17% at 30 Torr). For each level of PaO2, the relationships between the changes that occurred in VO2 and levels of phosphocreatine, ADP, and ATP/ADP.P(i) as the stimulation intensity was increased were significantly correlated; however, the slopes and intercepts of these lines were significantly different for each PaO2. Thus a greater change in any of the proposed regulators of tissue respiration (e.g., phosphocreatine, ADP) was required to achieve a given VO2 as PaO2 was decreased.(ABSTRACT TRUNCATED AT 250 WORDS).

  11. Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases.

    PubMed

    Foddy, L; Hughes, R C

    1988-08-01

    We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.

  12. NagA-dependent uptake of N-acetyl-glucosamine and N-acetyl-chitin oligosaccharides across the outer membrane of Caulobacter crescentus.

    PubMed

    Eisenbeis, Simone; Lohmiller, Stefanie; Valdebenito, Marianne; Leicht, Stefan; Braun, Volkmar

    2008-08-01

    Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-beta-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a beta-barrel composed of 22 antiparallel beta-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc)(3) and (GlcNAc)(5) requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.

  13. Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro.

    PubMed

    Trombley, Michael P; Post, Deborah M B; Rinker, Sherri D; Reinders, Lorri M; Fortney, Kate R; Zwickl, Beth W; Janowicz, Diane M; Baye, Fitsum M; Katz, Barry P; Spinola, Stanley M; Bauer, Margaret E

    2015-01-01

    Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.

  14. Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation.

    PubMed

    Spiro, R G

    2004-05-01

    Misfolded or incompletely assembled multisubunit glycoproteins undergo endoplasmic reticulum-associated degradation (ERAD) regulated in large measure by their N-linked polymannose oligosaccharides. In this quality control system lectin interaction with Glc(3)Man(9)GlcNAc(2) glycans after trimming with endoplasmic reticulum (ER) alpha-glucosidases and alpha-mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and proteolytic degradation. Monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins take part in the calnexin/calreticulin glucosylation-deglucosylation cycle, while the Man(8)GlcNAc(2) isomer B product of ER mannosidase I interacts with EDEM. Proteasomal degradation requires retrotranslocation into the cytosol through a Sec61 channel and deglycosylation by peptide: N-glycosidase (PNGase); in alternate models both PNGase and proteasomes may be either free in the cytosol or ER membrane-imbedded/attached. Numerous proteins appear to undergo nonproteasomal degradation in which deglycosylation and proteolysis take place in the ER lumen. The released free oligosaccharides (OS) are transported to the cytosol as OS-GlcNAc(2) along with similar components produced by the hydrolytic action of the oligosaccharyltransferase, where they together with OS from the proteasomal pathway are trimmed to Man(5)GlcNAc(1) by the action of cytosolic endo-beta- N-acetylglucosaminidase and alpha-mannosidase before entering the lysosomes. Some misfolded glycoproteins can recycle between the ER, intermediate and Golgi compartments, where they are further processed before ERAD. Moreover, properly folded glycoproteins with mannose-trimmed glycans can be deglucosylated in the Golgi by endomannosidase, thereby releasing calreticulin and permitting formation of complex OS. A number of regulatory controls have been described, including the glucosidase-glucosyltransferase shuttle, which controls the level of Glc(3)Man(9)GlcNAc(2)-P-P-Dol, and the unfolded protein

  15. Effects of imidacloprid on detoxifying enzyme glutathione S-transferase on Folsomia candida (Collembola).

    PubMed

    Sillapawattana, Panwad; Schäffer, Andreas

    2017-04-01

    Chemical analyses of the environment can document contamination by various xenobiotics, but it is also important to understand the effect of pollutants on living organisms. Thus, in the present work, we investigated the effect of the pesticide imidacloprid on the detoxifying enzyme glutathione S-transferase (GST) from Folsomia candida (Collembola), a standard test organism for estimating the effects of pesticides and environmental pollutants on non-target soil arthropods. Test animals were treated with different concentrations of imidacloprid for 48 h. Changes in steady-state levels of GST messenger RNA (mRNA) and GST enzyme activity were investigated. Extracted proteins were separated according to their sizes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved protein bands were detected by silver staining. The size of the glutathione (GSH) pool in Collembola was also determined. A predicted protein sequence of putative GSTs was identified with animals from control group. A 3-fold up-regulation of GST steady-state mRNA levels was detected in the samples treated with 5.0 mg L -1 imidacloprid compared to the control, while a 2.5- and 2.0- fold up-regulation was found in organisms treated with 2.5 and 7.5 mg L -1 imidacloprid, respectively. GST activity increased with increasing imidacloprid amounts from an initial activity of 0.11 μmol min -1  mg -1 protein in the control group up to 0.25 μmol min -1  mg -1 protein in the sample treated with the 5.0 mg L -1 of pesticide. By contrast, the total amount of GSH decreased with increasing imidacloprid concentration. The results suggest that the alteration of GST activity, steady-state level of GST mRNA, and GSH level may be involved in the response of F. candida to the exposure of imidacloprid and can be used as biomarkers to monitor the toxic effects of imidacloprid and other environmental pollutants on Collembola.

  16. Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica.

    PubMed

    Borjian, Farshad; Johnsen, Ulrike; Schönheit, Peter; Berg, Ivan A

    2017-01-01

    Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria ) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to β-methylmalyl-CoA. The cleavage of β-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C 4 -dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica , succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii , and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as

  17. MicroRNA-137 Negatively Regulates H2O2-Induced Cardiomyocyte Apoptosis Through CDC42

    PubMed Central

    Wang, Junnan; Xu, Rihao; Wu, Junduo; Li, Zhibo

    2015-01-01

    Background Oxidative stress, inducing cardiomyocyte apoptosis or myocardial ischemia, is the major denominator of many cardiac diseases. In this study, we intended to explore the regulatory function of microRNA-137 (miR-137) in oxidative stress-induced cardiomyocyte apoptosis. Material/Methods Cardiomyocytes were extracted from newborn C57BL/6 mice and cultured in vitro. Apoptosis was induced by H2O2, and evaluated by TUNEL assay. The effect of cardiomyocyte apoptosis on gene expression of miR-137 was evaluated by qRT-PCR. Lentivirus was used to stably down-regulate miR-137, and the subsequent effects of miR-137 down-regulation on cardiomyocyte apoptosis, its targeted gene CDC42, and caspase pathway were evaluated by TUNEL assay, dual-luciferase reporter assay, and Western blot assay, respectively. Finally, CDC42 was down-regulated by siRNA and its effect on miR-137-mediated cardiomyocyte apoptosis protection was examined. Results H2O2 induced significant apoptosis and up-regulated miR-137 in cardiomyocytes, whereas lentivirus-mediated miR-137 down-regulation protected against apoptosis. CDC42 was the direct target gene of miR-137 and proteins of CDC42, caspase-3, and caspase-9 were all regulated by miR-137 down-regulation in cardiomyocyte apoptosis. SiRNA-mediated CDC42 down-regulation reversed the protection of miR-137 down-regulation against cardiomyocyte apoptosis. Conclusions Our work demonstrated miR-137 and CDC42 are critical regulators in cardiomyocyte apoptosis. It may help to identify the molecular targets to prevent myocardial injury in human patients. PMID:26566162

  18. The recognition of three different epitopes for the H-type 2 human blood group determinant by lectins of Ulex europaeus, Galactia tenuiflora and Psophocarpus tetragonolobus (winged bean).

    PubMed

    Du, M H; Spohr, U; Lemieux, R U

    1994-10-01

    The chemical mapping of the regions of H-type 2 human blood group-related trisaccharide (Fuc alpha (1-2)Gal beta (1-4)GlcNAc beta Me) that are recognized by three different lectins, the so-called epitopes, are reviewed together with an account of how and why oligosaccharides form specific complexes with proteins as presently viewed in this laboratory. The occasion is used to report the synthesis of the various mono-O-methyl derivatives of the above trisaccharide that were used in these investigations. Also, Fuc alpha (1-2)Gal beta (1-4)Xyl beta Me was synthesized in order to examine whether or not the hydroxymethyl group of the GlcNAc residue participates in the binding reaction.

  19. The synthesis of ethacrynic acid thiazole derivatives as glutathione S-transferase pi inhibitors.

    PubMed

    Li, Ting; Liu, Guyue; Li, Hongcai; Yang, Xinmei; Jing, Yongkui; Zhao, Guisen

    2012-04-01

    Glutathione S-transferase pi (GSTpi) is a phase II enzyme which protects cells from death and detoxifies chemotherapeutic agents in cancer cells. Ethacrynic acid (EA) is a weak GSTpi inhibitor. Structure modifications were done to improve the ability of EA to inhibit GSTpi activity. Eighteen EA thiazole derivatives were designed and synthesized. Compounds 9a, 9b and 9c with a replacement of carboxyl group of EA by a heterocyclic thiazole exhibited improvement over EA to inhibit GSTpi activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Inhibition of processing of plant N-linked oligosaccharides by castanospermine.

    PubMed

    Hori, H; Pan, Y T; Molyneux, R J; Elbein, A D

    1984-02-01

    Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that inhibits lysosomal alpha- and beta-glucosidase. It also inhibits processing of influenza viral glycoproteins by inhibiting glucosidase I and leads to altered glycoproteins with Glc3Man7GlcNAc2 structures. Castanospermine was tested as an inhibitor of glycoprotein processing in suspension-cultured soybean cells. Soybean cells were pulse-labeled with [2-3H]mannose and chased for varying periods in unlabeled medium. In normal cells, the initial glycopeptides contained oligosaccharides having Glc3Man9GlcNAc2 to Glc1Man9GlcNAc2 structures and these were trimmed during the chase to Man9GlcNac2 to Man7GlcNAc2 structures. In the presence of castanospermine, no trimming of glucose residues occurred although some mannose residues were apparently still removed. Thus, the major oligosaccharide in the glycopeptides of castanospermine-incubated cells after a 90-min chase was a Glc3Man7GlcNAc2 structure. Smaller amounts of Glc3Man6GlcNAc2 and Glc3Man5GlcNAc2 were also identified. Thus, in plant cells, castanospermine also prevents the removal of the outermost glucose residue.

  1. N-acetylglucosamine increases symptoms and fungal burden in a murine model of oral candidiasis.

    PubMed

    Ishijima, Sanae A; Hayama, Kazumi; Takahashi, Miki; Holmes, Ann R; Cannon, Richard D; Abe, Shigeru

    2012-04-01

    The amino sugar N-acetylglucosamine (GlcNAc) is an in vitro inducer of the hyphal mode of growth of the opportunistic pathogen Candida albicans. The development of hyphae by C. albicans is considered to contribute to the pathogenesis of mucosal oral candidiasis. GlcNAc is also a commonly used nutritional supplement for the self-treatment of conditions such as arthritis. To date, no study has investigated whether ingestion of GlcNAc has an effect on the in vivo growth of C. albicans or the pathogenesis of a C. albicans infection. Using a murine model of oral candidiasis, we have found that administration of GlcNAc, but not glucose, increased oral symptoms of candidiasis and fungal burden. Groups of mice were given GlcNAc in either water or in a viscous carrier, i.e., 1% methylcellulose. There was a dose-dependent relationship between GlcNAc concentration and the severity of oral symptoms. Mice given the highest dose of GlcNAc, 45.2 mM, also showed a significant increase in fungal burden, and increased histological evidence of infection compared to controls given water alone. We propose that ingestion of GlcNAc, as a nutritional supplement, may have an impact on oral health in people susceptible to oral candidiasis.

  2. Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

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

    Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

    Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

  3. BLT1-mediated O-GlcNAcylation is required for NOX2-dependent migration, exocytotic degranulation and IL-8 release of human mast cell induced by Trichomonas vaginalis-secreted LTB4.

    PubMed

    Min, Arim; Lee, Young Ah; Kim, Kyeong Ah; Shin, Myeong Heon

    2018-05-31

    Trichomonas vaginalis is a sexually-transmitted protozoan parasite that causes vaginitis and cervicitis. Although mast cell activation is important for provoking tissue inflammation during infection with parasites, information regarding the signaling mechanisms in mast cell activation and T. vaginalis infection is limited. O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serine and threonine residues that functions as a critical regulator of intracellular signaling, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). We investigated if O-GlcNAcylation was associated with mast cell activation induced by T. vaginalis-derived secretory products (TvSP). Modified TvSP collected from live trichomonads treated with the 5-lipooxygenase inhibitor AA861 inhibited migration of mast cells. This result suggested that mast cell migration was caused by stimulation of T. vaginalis-secreted leukotrienes. Using the BLT1 antagonist U75302 or BLT1 siRNA, we found that migration of mast cells was evoked via LTB 4 receptor (BLT1). Furthermore, TvSP induced protein O-GlcNAcylation and OGT expression in HMC-1 cells, which was prevented by transfection with BLT1 siRNA. TvSP-induced migration, ROS generation, CD63 expression and IL-8 release were significantly suppressed by pretreatmemnt with OGT inhibitor ST045849 or OGT siRNA. These results suggested that BLT1-mediated OGlcNAcylation was important for mast cell activation during trichomoniasis. Copyright © 2018. Published by Elsevier Masson SAS.

  4. Regulation of Phospholipid Synthesis in Escherichia coli by Guanosine Tetraphosphate

    PubMed Central

    Merlie, John P.; Pizer, Lewis I.

    1973-01-01

    Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with 32P-inorganic phosphate and 32P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells. Images PMID:4583220

  5. IL-15 regulates memory CD8+ T cell O-glycan synthesis and affects trafficking

    PubMed Central

    Nolz, Jeffrey C.; Harty, John T.

    2014-01-01

    Memory and naive CD8+ T cells exhibit distinct trafficking patterns. Specifically, memory but not naive CD8+ T cells are recruited to inflamed tissues in an antigen-independent manner. However, the molecular mechanisms that regulate memory CD8+ T cell trafficking are largely unknown. Here, using murine models of infection and T cell transfer, we found that memory but not naive CD8+ T cells dynamically regulate expression of core 2 O-glycans, which interact with P- and E-selectins to modulate trafficking to inflamed tissues. Following infection, antigen-specific effector CD8+ T cells strongly expressed core 2 O-glycans, but this glycosylation pattern was lost by most memory CD8+ T cells. After unrelated infection or inflammatory challenge, memory CD8+ T cells synthesized core 2 O-glycans independently of antigen restimulation. The presence of core 2 O-glycans subsequently directed these cells to inflamed tissue. Memory and naive CD8+ T cells exhibited the opposite pattern of epigenetic modifications at the Gcnt1 locus, which encodes the enzyme that initiates core 2 O-glycan synthesis. The open chromatin configuration in memory CD8+ T cells permitted de novo generation of core 2 O-glycans in a TCR-independent, but IL-15–dependent, manner. Thus, IL-15 stimulation promotes antigen-experienced memory CD8+ T cells to generate core 2 O-glycans, which subsequently localize them to inflamed tissues. These findings suggest that CD8+ memory T cell trafficking potentially can be manipulated to improve host defense and immunotherapy. PMID:24509081

  6. BIOTRANSFORMATION AND GENOTOXICITY OF THE DRINKING WATER DISINFECTION BYPRODUCT BROMODICHLOROMETHANE: DNA BINDING MEDIATED BY GLUTATHIONE TRANSFERASE THETA 1-1

    EPA Science Inventory

    The drinking water disinfection byproduct bromodichloromethane (CHBrCl2) was
    previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione
    transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study
    to inve...

  7. Dimethyl adenosine transferase (KsgA) deficiency in Salmonella Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens

    USDA-ARS?s Scientific Manuscript database

    : Dimethyladenosine transferase (KsgA) performs diverse roles in bacteria including ribosomal maturation, DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that ksgA mutation in Salmonella Enteritidis results in impaired invasiveness i...

  8. Caribbean yellow band disease compromises the activity of catalase and glutathione S-transferase in the reef-building coral Orbicella faveolata exposed to anthracene.

    PubMed

    Montilla, Luis Miguel; Ramos, Ruth; García, Elia; Cróquer, Aldo

    2016-05-03

    Healthy and diseased corals are threatened by different anthropogenic sources, such as pollution, a problem expected to become more severe in the near future. Despite the fact that coastal pollution and coral diseases might represent a serious threat to coral reef health, there is a paucity of controlled experiments showing whether the response of diseased and healthy corals to xenobiotics differs. In this study, we exposed healthy and Caribbean yellow band disease (CYBD)-affected Orbicella faveolata colonies to 3 sublethal concentrations of anthracene to test if enzymatic responses to this hydrocarbon were compromised in CYBD-affected tissues. For this, a 2-factorial fully orthogonal design was used in a controlled laboratory bioassay, using tissue condition (2 levels: apparently healthy and diseased) and pollutant concentration (4 levels: experimental control, 10, 30 and 100 ppb concentration) as fixed factors. A permutation-based ANOVA (PERMANOVA) was used to test the effects of condition and concentration on the specific activity of 3 enzymatic biomarkers: catalase, glutathione S-transferase, and glutathione peroxidase. We found a significant interaction between the concentration of anthracene and the colony condition for catalase (Pseudo-F = 3.84, df = 3, p < 0.05) and glutathione S-transferase (Pseudo-F = 3.29, df = 3, p < 0.05). Moreover, our results indicated that the enzymatic response to anthracene in CYBD-affected tissues was compromised, as the activity of these enzymes decreased 3- to 4-fold compared to healthy tissues. These results suggest that under a potential scenario of increasing hydrocarbon coastal pollution, colonies of O. faveolata affected with CYBD might become more vulnerable to the deleterious effects of chemical pollution.

  9. Intracellular compartmentalization and degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis.

    PubMed

    Moore, S E; Spiro, R G

    1994-04-29

    The intracellular site for the degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis has been studied by permeabilizing the plasma membrane of metabolically radiolabeled HepG2 cells with streptolysin O. This pore-forming agent permitted us to examine the breakdown in both the cytosolic and vesicular compartments of the previously recognized (Anumula, K. R., and Spiro, R. G. (1983) J. Biol. Chem. 258, 15274-15282) polymannose components terminating in a di-N-acetylchitobiose sequence (OS-Glc-NAc2) or a single N-acetylglucosamine residue (OS-Glc-NAc1) residue. Pulse-chase studies indicated that although the OS-GlcNAc2 saccharides were about equally distributed between vesicles and cytosol and rapidly disappeared after reaching the Man8 stage, the OS-GlcNAc1 species were found predominantly in the extravesicular compartment and there underwent a distinctive demannosylation sequence resulting in the formation of a Man5GlcNAc isomer (Man alpha 1-->2Man alpha 1-->2Man alpha 1-->3(Man alpha 1-->6)Man beta 1-->4GlcNAc) which was different from the product of Golgi processing enzymes. Further trimming of this cytosolic limit product required its translocation into a vesicular compartment, believed to be lysosomes, in which Man2-4GlcNAc components appeared as the metabolic chase progressed. The accumulation of Glc1Man5GlcNAc in the cytosol during the chase suggested that glucose interferes with the cytosolic-vesicular transfer and this became even more evident by the pronounced pile-up of extravesicular Glc3Man5GlcNAc when the cells were incubated in the presence of castanospermine. Although the biological significance and mechanism of free polymannose oligosaccharide entry into the cytosol is not yet known, the possibility that it may reflect an endoplasmic reticulum-situated degradative process of glycoproteins merits consideration.

  10. Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits.

    PubMed

    McElduff, A; Watkinson, A; Hedo, J A; Gorden, P

    1986-11-01

    The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the

  11. Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits.

    PubMed Central

    McElduff, A; Watkinson, A; Hedo, J A; Gorden, P

    1986-01-01

    The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the

  12. O-GlcNAc Modification of the runt-Related Transcription Factor 2 (Runx2) Links Osteogenesis and Nutrient Metabolism in Bone Marrow Mesenchymal Stem Cells*

    PubMed Central

    Nagel, Alexis K.; Ball, Lauren E.

    2014-01-01

    Runx2 is the master switch controlling osteoblast differentiation and formation of the mineralized skeleton. The post-translational modification of Runx2 by phosphorylation, ubiquitinylation, and acetylation modulates its activity, stability, and interactions with transcriptional co-regulators and chromatin remodeling proteins downstream of osteogenic signals. Characterization of Runx2 by electron transfer dissociation tandem mass spectrometry revealed sites of O-linked N-acetylglucosamine (O-GlcNAc) modification, a nutrient-responsive post-translational modification that modulates the action of numerous transcriptional effectors. O-GlcNAc modification occurs in close proximity to phosphorylated residues and novel sites of arginine methylation within regions known to regulate Runx2 transactivation. An interaction between Runx2 and the O-GlcNAcylated, O-GlcNAc transferase enzyme was also detected. Pharmacological inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc from Ser/Thr residues, enhanced basal (39.9%) and BMP2/7-induced (43.3%) Runx2 transcriptional activity in MC3T3-E1 pre-osteoblasts. In bone marrow-derived mesenchymal stem cells differentiated for 6 days in osteogenic media, inhibition of OGA resulted in elevated expression (24.3%) and activity (65.8%) of alkaline phosphatase (ALP) an early marker of bone formation and a transcriptional target of Runx2. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells in the presence of BMP2/7 for 8 days culminated in decreased OGA activity (39.0%) and an increase in the abundance of O-GlcNAcylated Runx2, as compared with unstimulated cells. Furthermore, BMP2/7-induced ALP activity was enhanced by 35.6% in bone marrow-derived mesenchymal stem cells differentiated in the presence of the OGA inhibitor, demonstrating that direct or BMP2/7-induced inhibition of OGA is associated with increased ALP activity. Altogether, these findings link O-GlcNAc cycling to the Runx2

  13. 78 FR 23843 - Special Local Regulations; Moss Point Rockin' the Riverfront Festival; Robertson Lake & O'Leary...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-23

    ...-AA08 Special Local Regulations; Moss Point Rockin' the Riverfront Festival; Robertson Lake & O'Leary... Festival high speed boat races. Entry into, transiting or anchoring in this area is prohibited to all... Rockin' the Riverfront Festival; Robertson Lake & O'Leary Lake; Moss Point, MS. (a) Location. The...

  14. What is the Efficiency of ATP Signaling from Erythrocytes to Regulate Distribution of O2 Supply within the Microvasculature?

    PubMed Central

    C.G., Ellis; S., Milkovich; D., Goldman

    2012-01-01

    Erythrocytes appear to be ideal sensors for regulating microvascular O2 supply since they release the potent vasodilator adenosine 5′-triphosphate (ATP) in an O2 saturation dependent manner. Whether erythrocytes play a significant role in regulating O2 supply in the complex environment of diffusional O2 exchange among capillaries, arterioles and venules, depends on the efficiency with which erythrocytes signal the vascular endothelium. If one assumes that the distribution of purinergic receptors is uniform throughout the microvasculature, then the most efficient site for signaling should occur in capillaries, where the erythrocyte membrane is in close proximity to the endothelium. ATP released from erythrocytes would diffuse a short distance to P2y receptors inducing an increase in blood flow possibly the result of endothelial hyperpolarization. We hypothesize that this hyperpolarization varies across the capillary bed dependent upon erythrocyte supply rate and the flux of O2 from these erythrocytes to support O2 metabolism. This would suggest that the capillary bed would be the most effective site for erythrocytes to communicate tissue oxygen needs. Electrically coupled endothelial cells conduct the integrated signal upstream where arterioles adjust vascular resistance, thus enabling ATP released from erythrocytes to regulate the magnitude and distribution of O2 supply to individual capillary networks. PMID:22587367

  15. Transgenic expression of a maize geranyl geranyl transferase gene sequence in maize callus increases resistance to ear rot pathogens

    USDA-ARS?s Scientific Manuscript database

    Determining the genes responsible for pest resistance in maize can allow breeders to develop varieties with lower losses and less contamination with undesirable toxins. A gene sequence coding for a geranyl geranyl transferase-like protein located in a fungal ear rot resistance quantitative trait loc...

  16. TXNIP regulates myocardial fatty acid oxidation via miR-33a signaling.

    PubMed

    Chen, Junqin; Young, Martin E; Chatham, John C; Crossman, David K; Dell'Italia, Louis J; Shalev, Anath

    2016-07-01

    Myocardial fatty acid β-oxidation is critical for the maintenance of energy homeostasis and contractile function in the heart, but its regulation is still not fully understood. While thioredoxin-interacting protein (TXNIP) has recently been implicated in cardiac metabolism and mitochondrial function, its effects on β-oxidation have remained unexplored. Using a new cardiomyocyte-specific TXNIP knockout mouse and working heart perfusion studies, as well as loss- and gain-of-function experiments in rat H9C2 and human AC16 cardiomyocytes, we discovered that TXNIP deficiency promotes myocardial β-oxidation via signaling through a specific microRNA, miR-33a. TXNIP deficiency leads to increased binding of nuclear factor Y (NFYA) to the sterol regulatory element binding protein 2 (SREBP2) promoter, resulting in transcriptional inhibition of SREBP2 and its intronic miR-33a. This allows for increased translation of the miR-33a target genes and β-oxidation-promoting enzymes, carnitine octanoyl transferase (CROT), carnitine palmitoyl transferase 1 (CPT1), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase-β (HADHB), and AMPKα and is associated with an increase in phospho-AMPKα and phosphorylation/inactivation of acetyl-CoA-carboxylase. Thus, we have identified a novel TXNIP-NFYA-SREBP2/miR-33a-AMPKα/CROT/CPT1/HADHB pathway that is conserved in mouse, rat, and human cardiomyocytes and regulates myocardial β-oxidation. Copyright © 2016 the American Physiological Society.

  17. Anoxia-responsive regulation of the FoxO transcription factors in freshwater turtles, Trachemys scripta elegans.

    PubMed

    Krivoruchko, Anastasia; Storey, Kenneth B

    2013-11-01

    The forkhead class O (FoxO) transcription factors are important regulators of multiple aspects of cellular metabolism. We hypothesized that activation of these transcription factors could play crucial roles in low oxygen survival in the anoxia-tolerant turtle, Trachemys scripta elegans. Two FoxOs, FoxO1 and FoxO3, were examined in turtle tissues in response to 5 and 20h of anoxic submergence using techniques of RT-PCR, western immunoblotting and DNA-binding assays to assess activation. Transcript levels of FoxO-responsive genes were also quantified using RT-PCR. FoxO1 was anoxia-responsive in the liver, with increases in transcript levels, protein levels, nuclear levels and DNA-binding of 1.7-4.8fold in response to anoxia. Levels of phosphorylated FoxO1 also decreased to 57% of control values in response to 5h of anoxia, indicating activation. FoxO3 was activated in the heart, kidney and liver in response to anoxia, with nuclear levels increasing by 1.5-3.7fold and DNA-binding activity increasing by 1.3-2.9fold. Transcript levels of two FoxO-target genes, p27kip1 and catalase, also rose by 2.4-2.5fold in the turtle liver under anoxia. The results suggest that the FoxO transcription factors are activated in response to anoxia in T. scripta elegans, potentially contributing to the regulation of stress resistance and metabolic depression. This study provides the first demonstration of activation of FoxOs in a natural model for vertebrate anoxia tolerance, further improving understanding of how tissues can survive without oxygen. © 2013.

  18. Hha Controls Escherichia coli O157:H7 Biofilm Formation by Differential Regulation of Global Transcriptional Regulators FlhDC and CsgD

    PubMed Central

    Bearson, Bradley L.

    2013-01-01

    Although molecular mechanisms promoting adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation are not fully understood. In this study, we demonstrate that biofilm formation in EHEC O157:H7 strain 86-24 is highly repressed compared to that in an isogenic hha mutant. The hha mutant produced large quantities of biofilm compared to the wild-type strain at 30°C and 37°C. Complementation of the hha mutant reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production. While swimming motility and expression of the flagellar gene fliC were significantly reduced, the expression of csgA (encoding curlin of curli fimbriae) and the ability to bind Congo red were significantly enhanced. The expression of both fliC and csgA and the phenotypes of motility and curli production affected by these two genes, respectively, were restored to wild-type levels in the complemented hha mutant. The csgA deletion abolished biofilm formation in the hha mutant and wild-type strain, and csgA complementation restored biofilm formation to these strains, indicating the importance of csgA and curli in biofilm formation. The regulatory effects of Hha on flagellar and curli gene expression appear to occur via the induction and repression of FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant, respectively. In gel shift assays Hha interacted with flhDC and csgD promoters. In conclusion, Hha regulates biofilm formation in EHEC O157:H7 by differential regulation of FlhDC and CsgD, the global regulators of motility and curli production, respectively. PMID:23377937

  19. Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD.

    PubMed

    Sharma, Vijay K; Bearson, Bradley L

    2013-04-01

    Although molecular mechanisms promoting adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation are not fully understood. In this study, we demonstrate that biofilm formation in EHEC O157:H7 strain 86-24 is highly repressed compared to that in an isogenic hha mutant. The hha mutant produced large quantities of biofilm compared to the wild-type strain at 30°C and 37°C. Complementation of the hha mutant reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production. While swimming motility and expression of the flagellar gene fliC were significantly reduced, the expression of csgA (encoding curlin of curli fimbriae) and the ability to bind Congo red were significantly enhanced. The expression of both fliC and csgA and the phenotypes of motility and curli production affected by these two genes, respectively, were restored to wild-type levels in the complemented hha mutant. The csgA deletion abolished biofilm formation in the hha mutant and wild-type strain, and csgA complementation restored biofilm formation to these strains, indicating the importance of csgA and curli in biofilm formation. The regulatory effects of Hha on flagellar and curli gene expression appear to occur via the induction and repression of FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant, respectively. In gel shift assays Hha interacted with flhDC and csgD promoters. In conclusion, Hha regulates biofilm formation in EHEC O157:H7 by differential regulation of FlhDC and CsgD, the global regulators of motility and curli production, respectively.

  20. 78 FR 9866 - Special Local Regulation; Moss Point Rockin' the Riverfront Festival; Robertson Lake & O'Leary...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-12

    ... 1625-AA08 Special Local Regulation; Moss Point Rockin' the Riverfront Festival; Robertson Lake & O... Riverfront Festival high speed boat races. Entry into, transiting or anchoring in this area is prohibited to... Point Rockin' the Riverfront Festival; Robertson Lake & O'Leary Lake; Moss Point, MS. (a) Location. The...

  1. Degradation of Misfolded Endoplasmic Reticulum Glycoproteins in Saccharomyces cerevisiae Is Determined by a Specific Oligosaccharide Structure

    PubMed Central

    Jakob, Claude A.; Burda, Patricie; Roth, Jürgen; Aebi, Markus

    1998-01-01

    In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal α1,2 glucose and the first α1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man8GlcNAc2 oligosaccharide. Reduced rate of degradation was mainly observed for mis- folded CPY bearing Man6GlcNAc2, Man7GlcNAc2 and Man9GlcNAc2 oligosaccharides, whereas Man8GlcNAc2 and, to a lesser extent, Man5GlcNAc2 oligosaccharides supported degradation. These results suggest a role for the Man8GlcNAc2 oligosaccharide in the degradation process. They may indicate the presence of a Man8GlcNAc2-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae. PMID:9732283

  2. Sphingobium sp. SYK-6 LigG involved in lignin degradation is structurally and biochemically related to the glutathione transferase ω class.

    PubMed

    Meux, Edgar; Prosper, Pascalita; Masai, Eiji; Mulliert, Guillermo; Dumarçay, Stéphane; Morel, Mélanie; Didierjean, Claude; Gelhaye, Eric; Favier, Frédérique

    2012-11-16

    SpLigG is one of the three glutathione transferases (GSTs) involved in the process of lignin breakdown in the soil bacterium Sphingobium sp. SYK-6. Sequence comparisons showed that SpLigG and several proteobacteria homologues form an independent cluster within cysteine-containing GSTs. The relationship between SpLigG and other GSTs was investigated. The X-ray structure and biochemical properties of SpLigG indicate that this enzyme belongs to the omega class of glutathione transferases. However, the hydrophilic substrate binding site of SpLigG, together with its known ability to stereoselectively deglutathionylate the physiological substrate α-glutathionyl-β-hydroxypropiovanillone, argues for broadening the definition of the omega class. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Targeted deletion of apoptosis signal-regulating kinase 1 attenuates left ventricular remodeling

    PubMed Central

    Yamaguchi, Osamu; Higuchi, Yoshiharu; Hirotani, Shinichi; Kashiwase, Kazunori; Nakayama, Hiroyuki; Hikoso, Shungo; Takeda, Toshihiro; Watanabe, Tetsuya; Asahi, Michio; Taniike, Masayuki; Matsumura, Yasushi; Tsujimoto, Ikuko; Hongo, Kenichi; Kusakari, Yoichiro; Kurihara, Satoshi; Nishida, Kazuhiko; Ichijo, Hidenori; Hori, Masatsugu; Otsu, Kinya

    2003-01-01

    Left ventricular remodeling that occurs after myocardial infarction (MI) and pressure overload is generally accepted as a determinant of the clinical course of heart failure. The molecular mechanism of this process, however, remains to be elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We used ASK1 knockout mice (ASK-/-) to test the hypothesis that ASK1 is involved in development of left ventricular remodeling. ASK-/- hearts showed no morphological or histological defects. Echocardiography and cardiac catheterization revealed normal global structure and function. Left ventricular structural and functional remodeling were determined 4 weeks after coronary artery ligation or thoracic transverse aortic constriction (TAC). ASK-/- had significantly smaller increases in left ventricular end-diastolic and end-systolic ventricular dimensions and smaller decreases in fractional shortening in both experimental models compared with WT mice. The number of terminal deoxynucleotidyl transferase biotin-dUDP nick end-labeling-positive myocytes after MI or TAC was decreased in ASK-/- compared with that in WT mice. Overexpression of a constitutively active mutant of ASK1 induced apoptosis in isolated rat neonatal cardiomyocytes, whereas neonatal ASK-/- cardiomyocytes were resistant to H2O2-induced apoptosis. An in vitro kinase assay showed increased ASK1 activity in heart after MI or TAC in WT mice. Thus, ASK1 plays an important role in regulating left ventricular remodeling by promoting apoptosis. PMID:14665690

  4. Hyper-O-GlcNAcylation induces cisplatin resistance via regulation of p53 and c-Myc in human lung carcinoma.

    PubMed

    Luanpitpong, Sudjit; Angsutararux, Paweorn; Samart, Parinya; Chanthra, Nawin; Chanvorachote, Pithi; Issaragrisil, Surapol

    2017-09-06

    Aberrant metabolism in hexosamine biosynthetic pathway (HBP) has been observed in several cancers, affecting cellular signaling and tumor progression. However, the role of O-GlcNAcylation, a post-translational modification through HBP flux, in apoptosis remains unclear. Here, we found that hyper-O-GlcNAcylation in lung carcinoma cells by O-GlcNAcase inhibition renders the cells to apoptosis resistance to cisplatin (CDDP). Profiling of various key regulatory proteins revealed an implication of either p53 or c-Myc in the apoptosis regulation by O-GlcNAcylation, independent of p53 status. Using co-immunoprecipitation and correlation analyses, we found that O-GlcNAcylation of p53 under certain cellular contexts, i.e. high p53 activation, promotes its ubiquitin-mediated proteasomal degradation, resulting in a gain of oncogenic and anti-apoptotic functions. By contrast, O-GlcNAcylation of c-Myc inhibits its ubiquitination and subsequent proteasomal degradation. Gene manipulation studies revealed that O-GlcNAcylation of p53/c-Myc is in part a regulator of CDDP-induced apoptosis. Accordingly, we classified CDDP resistance by hyper-O-GlcNAcylation in lung carcinoma cells as either p53 or c-Myc dependence based on their molecular targets. Together, our findings provide novel mechanisms for the regulation of lung cancer cell apoptosis that could be important in understanding clinical drug resistance and suggest O-GlcNAcylation as a potential target for cancer therapy.

  5. Cell cycle-dependent O-GlcNAc modification of tobacco histones and their interaction with the tobacco lectin.

    PubMed

    Delporte, Annelies; De Zaeytijd, Jeroen; De Storme, Nico; Azmi, Abdelkrim; Geelen, Danny; Smagghe, Guy; Guisez, Yves; Van Damme, Els J M

    2014-10-01

    The Nicotiana tabacum agglutinin or Nictaba is a nucleocytoplasmic lectin that is expressed in tobacco after the plants have been exposed to jasmonate treatment or insect herbivory. Nictaba specifically recognizes GlcNAc residues. Recently, it was shown that Nictaba is interacting in vitro with the core histone proteins from calf thymus. Assuming that plant histones - similar to their animal counterparts - undergo O-GlcNAcylation, this interaction presumably occurs through binding of the lectin to the O-GlcNAc modification present on the histones. Hereupon, the question was raised whether this modification also occurs in plants and if it is cell cycle dependent. To this end, histones were purified from tobacco BY-2 suspension cells and the presence of O-GlcNAc modifications was checked. Concomitantly, O-GlcNAcylation of histone proteins was studied. Our data show that similar to animal histones plant histones are modified by O-GlcNAc in a cell cycle-dependent fashion. In addition, the interaction between Nictaba and tobacco histones was confirmed using lectin chromatography and far Western blot analysis. Collectively these findings suggest that Nictaba can act as a modulator of gene transcription through its interaction with core histones. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  6. Function and phylogeny of bacterial butyryl-CoA:acetate transferases and their diversity in the proximal colon of swine

    USDA-ARS?s Scientific Manuscript database

    Studying the host-associated butyrate-producing bacterial community is important because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl-coA:acetate transferase (2.3.8.3) as a the main gene for butyrate production in intestinal ecosystems; h...

  7. Testosterone regulation of Akt/mTORC1/FoxO3a Signaling in Skeletal Muscle

    PubMed Central

    White, James P.; Gao, Song; Puppa, Melissa J.; Sato, Shuichi; Welle, Stephen L.; Carson, James A.

    2012-01-01

    Low endogenous testosterone production, known as hypogonadism is commonly associated with conditions inducing muscle wasting. Akt signaling can control skeletal muscle mass through mTOR regulation of protein synthesis and FoxO regulation of protein degradation, and this pathway has been previously identified as a target of androgen signaling. However, the testosterone sensitivity of Akt/mTOR signaling requires further understanding in order to grasp the significance of varied testosterone levels seen with wasting disease on muscle protein turnover regulation. Therefore, the purpose of this study is to determine the effect of androgen availability on muscle Akt/mTORC1/FoxO3a regulation in skeletal muscle and cultured C2C12 myotubes. C57BL/6 mice were either castrated for 42 days or castrated and treated with the nandrolone decanoate (ND) (6 mg/kg bw/wk). Testosterone loss (TL) significantly decreased volitional grip strength, body weight, and gastrocnemius (GAS) muscle mass, and ND reversed these changes. Related to muscle mass regulation, TL decreased muscle IGF-1 mRNA, the rate of myofibrillar protein synthesis, Akt phosphorylation, and the phosphorylation of Akt targets, GSK3β, PRAS40 and FoxO3a. TL induced expression of FoxO transcriptional targets, MuRF1, atrogin1 and REDD1. Muscle AMPK and raptor phosphorylation, mTOR inhibitors, were not altered by low testosterone. ND restored IGF-1 expression and Akt/mTORC1 signaling while repressing expression of FoxO transcriptional targets. Testosterone (T) sensitivity of Akt/mTORC1 signaling was examined in C2C12 myotubes, and mTOR phosphorylation was induced independent of Akt activation at low T concentrations, while a higher T concentration was required to activate Akt signaling. Interestingly, low concentration T was sufficient to amplify myotube mTOR and Akt signaling after 24h of T withdrawal, demonstrating the potential in cultured myotubes for a T initiated positive feedback mechanism to amplify Akt

  8. Aberrant Epigenetic Alterations of Glutathione-S-Transferase P1 in Age-Related Nuclear Cataract.

    PubMed

    Chen, Jia; Zhou, Jing; Wu, Jian; Zhang, Guowei; Kang, Lihua; Ben, Jindong; Wang, Yong; Qin, Bai; Guan, Huaijin

    2017-03-01

    Oxidative damage of lens tissue contributes to the formation of age-related cataract. Pi-class glutathione-S-transferase (GSTP1) plays a role in the removal of oxidative adducts by transferring them to glutathione. To assess epigenetic regulation of GSTP1 and its potential role in age-related nuclear cataract (ARNC) pathogenesis, we evaluated GSTP1 mRNA expression, methylation, and chromatin modifications in lenses from ARNC patients. The mRNA and protein of lens GSTP1 were assayed by relative quantitative real-time polymerase chain reaction (qRT-PCR) and Western blots. Methylation of the GSTP1 promoter was determined by bisulfite genomic sequencing. Chromatin modification was detected by chromatin immunoprecipitation. DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities were also assayed by enzyme-linked immunosorbent assay (ELISA)-like reaction. To assess the effect of DNA methylation on the mRNA expression of GSTP1, human lens epithelium HLE-B3 cells were treated with the demethylation compound 5-aza-dC, followed by qRT-PCR assay. GSTP1 mRNA and protein levels were significantly reduced in lens epithelium and cortex of ARNC cases versus age-matched controls. The changes corresponded to hypermethylation of the GSTP1 promoter CpG islands. The loss of GSTP1 mRNA and protein and the increased DNA promoter methylation might be correlated with the severity of the ARNC. ARNC lenses also had lower acetylation of histone proteins H3, H4, and lower methylation of H3K4, and higher methylation of H3K9. Histone modifications were not correlated with the severity of the ARNCs. DNMT and HDAC were elevated in lenses from ARNCs compared with controls. Demethylation treatment of HLE-B3 cells with 5-aza-dC enhanced the expression of GSTP1. Epigenetic alteration of GSTP1 regulates its expression in lens epithelial and cortical tissues. These changes likely contribute to the pathogenesis of ARNC.

  9. A phosphoethanolamine transferase specific for the 4'-phosphate residue of Cronobacter sakazakii lipid A.

    PubMed

    Liu, L; Li, Y; Wang, X; Guo, W

    2016-11-01

    Investigate how Cronobacter sakazakii modify their lipid A structure to avoid recognition by the host immune cells. Lipid A modification was observed in C. sakazakii BAA894 grown at pH 5·0 but not pH 7·0. Overexpression of C. sakazakii gene ESA_RS09200 in Escherichia coli W3110 caused a phosphoethanolamine (PEA) modification of lipid A; when ESA_RS09200 was deleted in C. sakazakii BAA894, this lipid A modification disappeared. Lipid A modification was observed in BAA894 grown at pH 5·0 when the 1- phosphate residue of lipid A was removed, but disappeared when the 4'- phosphate residue of lipid A was removed. When ESA_RS16430, the orthologous gene of E. coli pmrA, was deleted in C. sakazakii BAA894, this PEA modification of lipid A was still observed, suggesting that this modification was not regulated by the PmrA-PmrB system. Compared to the wild-type BAA894, ESA_RS09200 deletion mutant showed decreased resistance to cationic antimicrobial peptides (CAMP), increased recognition by TLR4/MD2, decreased ability to invade and persist in mammalian cells. ESA_RS09200 in C. sakazakii BAA894 encodes a PEA transferase that specifically adds a PEA to the 4'-phosphate residue of lipid A, but not regulated by the PmrA-PmrB system. PEA modification of lipid A reduces recognition and killing by the host innate immune system. This study showed that modification of the lipid A moiety of C. sakazakii with PEA increased resistance to CAMP and recognition of the immune response although signalling of TLR4/MD2 cascade, suggesting that the organism could not successfully evade the host innate immune system without the transference of PEA to its lipid A moiety. © 2016 The Society for Applied Microbiology.

  10. Removal of O-GlcNAcylation is important for pig preimplantation development

    PubMed Central

    SHIBUTANI, Mihiro; MORI, Takeshi; MIYANO, Takashi; MIYAKE, Masashi

    2015-01-01

    Glucose has been recognized as an energy source for a long time, but it has recently been suggested that the hexosamine biosynthesis pathway (HBP) and downstream protein O-GlcNAcylation have important functions in mouse preimplantation development. Thus, whether or not O-GlcNAcylation was present and what functions O-GlcNAcylation has in pig preimplantation development were investigated in the present study. The expressions of mRNA of glutaminefructose-6-phosphate aminotransferase (Gfpt), O-GlcNAc transferase (Ogt) and O-GlcNAcase (Oga), which are involved in the HBP and O-GlcNAc cycling, were examined in pig parthenogenetic diploids at each preimplantation developmental stage. Gfpt and Ogt were detected in diploids at all stages. Though Oga was detected at all stages except the 4-cell stage, OGA proteins were detected in diploids from the 2-cell to blastocyst stage. Furthermore, O-GlcNAcylated proteins in MII oocytes and diploids were also detected by immunofluorescence at every stage. Inhibition of OGT by 4.0 mM BADGP did not affect development up to the blastocyst stage, while inhibition of OGA by 300 µM PUGNAc decreased the proportion of diploids beyond the 4-cell stage. Four-cell diploids cultured with PUGNAc until 48 h developed to the blastocyst stage after culture in a PUGNAc-free medium until 144 h after electrostimulation. RNA polymerase II (Pol II) phosphorylation, which indicates the onset of mRNA transcription, was detected in nuclei of diploids in the control group at 48 h but not in the PUGNAc-treated group. These results indicate that HBP and O-GlcNAcylation have important functions in pig preimplantation development and that inhibition of OGA is fatal for development. It is also suggested that OGA inhibition disrupts normal Pol II regulation and may cause a zygotic gene activation error. PMID:26004176

  11. Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells.

    PubMed

    Jones, Jane T; Qian, Xi; van der Velden, Jos L J; Chia, Shi Biao; McMillan, David H; Flemer, Stevenson; Hoffman, Sidra M; Lahue, Karolyn G; Schneider, Robert W; Nolin, James D; Anathy, Vikas; van der Vliet, Albert; Townsend, Danyelle M; Tew, Kenneth D; Janssen-Heininger, Yvonne M W

    2016-08-01

    Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor. Copyright

  12. Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase π

    PubMed Central

    Ralat, Luis A.; Colman, Roberta F.

    2003-01-01

    Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites. PMID:14573868

  13. Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi.

    PubMed

    Ralat, Luis A; Colman, Roberta F

    2003-11-01

    Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.

  14. Hyper-O-GlcNAcylation inhibits the induction of heat shock protein 70 (Hsp 70) by sodium arsenite in HeLa cells.

    PubMed

    Miura, Yuri; Sato, Takatoshi; Sakurai, Yoko; Sakai, Ryo; Hiraoka, Wakako; Endo, Tamao

    2014-01-01

    O-Linked β-N-acetylglucosamine-modification (O-GlcNAcylation) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. However, the role of O-GlcNAcylation in the induction of heat shock proteins (Hsps) by arsenite remains unclear. We used O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc), an inhibitor of O-GlcNAcase, and glucosamine (GlcN), an enhancer of the hexosamine biosynthesis pathway, or O-GlcNAc transferase (OGT) short interfering RNA (siRNA) to enhance or suppress cellular O-GlcNAcylation levels, respectively, in HeLa cells. The exposure to arsenite increased O-GlcNAcylation and Hsp 70 levels in HeLa cells. However, the pre-treatment with PUGNAc or GlcN, which enhanced O-GlcNAcylation levels, decreased the arsenite-induced expression of Hsp 70. The pre-treatment with OGT siRNA, which suppressed O-GlcNAcylation levels, did not affect the induction of Hsp 70. We then examined the effects of O-GlcNAcylation on the nuclear translocation and phosphorylation of heat shock factor 1 (HSF1), and found that neither the nuclear translocation nor phosphorylation of HSF1 was regulated by O-GlcNAcylation. Finally, Hsp 70 mRNA expression was induced by arsenite, whereas the addition of PUGNAc slightly suppressed its induction. These results indicate that O-GlcNAcylation is related to arsenite-induced Hsp 70 expression, and demonstrated that hyper-O-GlcNAcylation inhibited the induction of Hsp 70 via transcriptional factors instead of HSF1.

  15. Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state

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

    Osterman, Ilya A.; Khabibullina, Nelli F.; Komarova, Ekaterina S.

    The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of themore » tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.« less

  16. Increased Expression of a myo-Inositol Methyl Transferase in Mesembryanthemum crystallinum Is Part of a Stress Response Distinct from Crassulacean Acid Metabolism Induction 1

    PubMed Central

    Vernon, Daniel M.; Bohnert, Hans J.

    1992-01-01

    The facultative halophyte Mesembryanthemum crystallinum responds to osmotic stress by switching from C3 photosynthesis to Crassulacean acid metabolism (CAM). This shift to CAM involves the stress-initiated up-regulation of mRNAs encoding CAM enzymes. The capability of the plants to induce a key CAM enzyme, phosphoenolpyruvate carboxylase, is influenced by plant age, and it has been suggested that adaptation to salinity in M. crystallinum may be modulated by a developmental program that controls molecular responses to stress. We have compared the effects of plant age on the expression of two salinity-induced genes: Gpdl, which encodes the photosynthesis-related enzyme glyceraldehyde 3-phosphate dehydrogenase, and Imtl, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imtl mRNA accumulation and the accompanying increase in pinitol in stressed Mesembryanthemum exhibit a pattern of induction distinct from that observed for CAM-related genes. We conclude that the molecular mechanisms that trigger Imtl and pinitol accumulation in response to salt stress in M. crystallinum differ in some respects from those that lead to CAM induction. There may be multiple signals or pathways that regulate inducible components of salinity tolerance in this facultative halophyte. ImagesFigure 1Figure 2 PMID:16669095

  17. Functional Analysis of the N-Acetylglucosamine Metabolic Genes of Streptomyces coelicolor and Role in Control of Development and Antibiotic Production

    PubMed Central

    Świątek, Magdalena A.; Tenconi, Elodie; Rigali, Sébastien

    2012-01-01

    N-Acetylglucosamine, the monomer of chitin, is a favored carbon and nitrogen source for streptomycetes. Its intracellular catabolism requires the combined actions of the N-acetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase NagA and the glucosamine-6-phosphate (GlcN-6P) deaminase/isomerase NagB. GlcNAc acts as a signaling molecule in the DasR-mediated nutrient sensing system, activating development and antibiotic production under poor growth conditions (famine) and blocking these processes under rich conditions (feast). In order to understand how a single nutrient can deliver opposite information according to the nutritional context, we carried out a mutational analysis of the nag metabolic genes nagA, nagB, and nagK. Here we show that the nag genes are part of the DasR regulon in Streptomyces coelicolor, which explains their transcriptional induction by GlcNAc. Most likely as the result of the intracellular accumulation of GlcN-6P, nagB deletion mutants fail to grow in the presence of GlcNAc. This toxicity can be alleviated by the additional deletion of nagA. We recently showed that in S. coelicolor, GlcNAc is internalized as GlcNAc-6P via the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). Considering the relevance of GlcNAc for the control of antibiotic production, improved insight into GlcNAc metabolism in Streptomyces may provide new leads toward biotechnological applications. PMID:22194457

  18. Estrogen induced {beta}-1,4-galactosyltransferase 1 expression regulates proliferation of human breast cancer MCF-7 cells

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

    Choi, Hee-Jung; Division of Applied Medicine, School of Korean Medicine, Pusan National University, Yangsan-city, Gyeongsangnam-do; Chung, Tae-Wook

    2012-10-05

    Highlights: Black-Right-Pointing-Pointer We examined the regulation and biological functions of B4GALT1 expression induced by estrogen. Black-Right-Pointing-Pointer Estrogen-induced B4GALT1 expression through the direct binding of ER-{alpha} to ERE in MCF-7 cells. Black-Right-Pointing-Pointer B4GALT1 expression activates the proliferation of MCF-7 cells via its receptor function. Black-Right-Pointing-Pointer Thus, we suggest B4GALT1 as a molecular target for inhibiting breast cancer proliferation. -- Abstract: Beta 1,4-galactosyltransferase 1 (B4GALT1) synthesizes galactose {beta}-1,4-N-acetylglucosamine (Gal{beta}1-4GlcNAc) groups on N-linked sugar chains of glycoproteins, which play important roles in many biological events, including the proliferation and migration of cancer cells. A previous microarray study reported that this gene is expressedmore » by estrogen treatment in breast cancer. In this study, we examined the regulatory mechanisms and biological functions of estrogen-induced B4GALT1 expression. Our data showed that estrogen-induced expression of B4GALT1 is localized in intracellular compartments and in the plasma membrane. In addition, B4GALT1 has an enzyme activity involved in the production of the Gal{beta}1-4GlcNAc structure. The result from a promoter assay and chromatin immunoprecipitation revealed that 3 different estrogen response elements (EREs) in the B4GALT1 promoter are critical for responsiveness to estrogen. In addition, the estrogen antagonists ICI 182,780 and ER-{alpha}-ERE binding blocker TPBM inhibit the expression of estrogen-induced B4GALT1. However, the inhibition of signal molecules relating to the extra-nuclear pathway, including the G-protein coupled receptors, Ras, and mitogen-activated protein kinases, had no inhibitory effects on B4GALT1 expression. The knock-down of the B4GALT1 gene and the inhibition of membrane B4GALT1 function resulted in the significant inhibition of estrogen-induced proliferation of MCF-7 cells

  19. Protein O-GlcNAcylation: A critical regulator of the cellular response to stress.

    PubMed

    Chatham, John C; Marchase, Richard B

    2010-01-01

    The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of chronic disease processes. However, there is increasing evidence that O-GlcNAc levels are increased in response to stress and that acute augmentation of this response is cytoprotective, at least in the short term. Conversely, a reduction in O-GlcNAc levels appears to be associated with decreased cell survival in response to an acute stress. Here we summarize our current understanding of protein O-GlcNAcylation on the cellular response to stress and in mediating cellular protective mechanisms focusing primarily on the cardiovascular system as an example. We consider the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis and explore the parallels between O-GlcNAc signaling and redox signaling. We also discuss the apparent paradox between the reported adverse effects of increased O-GlcNAcylation with its recently reported role in mediating cell survival mechanisms.

  20. Structures of the Oligosaccharides of the Glycoprotein Coded by Early Region E3 of Adenovirus 2

    PubMed Central

    Kornfeld, Rosalind; Wold, William S. M.

    1981-01-01

    Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-3H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-β-N-acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man9GlcNAc and Man8GlcNAc and small amounts of Man7GlcNAc and Man6GlcNAc. The pulse-chase sample had predominantly Man8GlcNAc and much less Man9GlcNAc, indicating that processing of the Man9GlcNAc to Man8GlcNAc had occurred during the chase period. Thus, Man8GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with α-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man9GlcNAc were identical to those of the lipid-linked Glc3Man9GlcNAc2 donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell-coded glycoprotein. Images PMID:7321093

  1. Structures of the oligosaccharides of the glycoprotein coded by early region E3 of adenovirus 2.

    PubMed

    Kornfeld, R; Wold, W S

    1981-11-01

    Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-(3)H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-beta-N-acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man(9)GlcNAc and Man(8)GlcNAc and small amounts of Man(7)GlcNAc and Man(6)GlcNAc. The pulse-chase sample had predominantly Man(8)GlcNAc and much less Man(9)GlcNAc, indicating that processing of the Man(9)GlcNAc to Man(8)GlcNAc had occurred during the chase period. Thus, Man(8)GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with alpha-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man(9)GlcNAc were identical to those of the lipid-linked Glc(3)Man(9)GlcNAc(2) donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell

  2. EDEM2 initiates mammalian glycoprotein ERAD by catalyzing the first mannose trimming step

    PubMed Central

    Ninagawa, Satoshi; Okada, Tetsuya; Sumitomo, Yoshiki; Kamiya, Yukiko; Kato, Koichi; Horimoto, Satoshi; Ishikawa, Tokiro; Takeda, Shunichi; Sakuma, Tetsushi; Yamamoto, Takashi

    2014-01-01

    Glycoproteins misfolded in the endoplasmic reticulum (ER) are subjected to ER-associated glycoprotein degradation (gpERAD) in which Htm1-mediated mannose trimming from the oligosaccharide Man8GlcNAc2 to Man7GlcNAc2 is the rate-limiting step in yeast. In contrast, the roles of the three Htm1 homologues (EDEM1/2/3) in mammalian gpERAD have remained elusive, with a key controversy being whether EDEMs function as mannosidases or as lectins. We therefore conducted transcription activator-like effector nuclease–mediated gene knockout analysis in human cell line and found that all endogenous EDEMs possess mannosidase activity. Mannose trimming from Man8GlcNAc2 to Man7GlcNAc2 is performed mainly by EDEM3 and to a lesser extent by EDEM1. Most surprisingly, the upstream mannose trimming from Man9GlcNAc2 to Man8GlcNAc2 is conducted mainly by EDEM2, which was previously considered to lack enzymatic activity. Based on the presence of two rate-limiting steps in mammalian gpERAD, we propose that mammalian cells double check gpERAD substrates before destruction by evolving EDEM2, a novel-type Htm1 homologue that catalyzes the first mannose trimming step from Man9GlcNAc2. PMID:25092655

  3. Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity

    PubMed Central

    1984-01-01

    The processing of asparagine-linked oligosaccharides on the alpha- chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N- acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2

  4. Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity.

    PubMed

    Hickman, S; Theodorakis, J L; Greco, J M; Brown, P H

    1984-02-01

    The processing of asparagine-linked oligosaccharides on the alpha-chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N-acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2

  5. Structure and gene cluster of the O-antigen of Escherichia coli O54.

    PubMed

    Naumenko, Olesya I; Guo, Xi; Senchenkova, Sof'ya N; Geng, Peng; Perepelov, Andrei V; Shashkov, Alexander S; Liu, Bin; Knirel, Yuriy A

    2018-06-15

    Mild acid hydrolysis of the lipopolysaccharide of Escherichia coli O54 afforded an O-polysaccharide, which was studied by sugar analysis, solvolysis with anhydrous trifluoroacetic acid, and 1 H and 13 C NMR spectroscopy. Solvolysis cleaved predominantly the linkage of β-d-Ribf and, to a lesser extent, that of β-d-GlcpNAc, whereas the other linkages, including the linkage of α-l-Rhap, were stable under selected conditions (40 °C, 5 h). The following structure of the O-polysaccharide was established: →4)-α-d-GalpA-(1 → 2)-α-l-Rhap-(1 → 2)-β-d-Ribf-(1 → 4)-β-d-Galp-(1 → 3)-β-d-GlcpNAc-(1→ The O-antigen gene cluster of E. coli O54 was analyzed and found to be consistent in general with the O-polysaccharide structure established but there were two exceptions: i) in the cluster, there were genes for phosphoserine phosphatase and serine transferase, which have no apparent role in the O-polysaccharide synthesis, and ii) no ribofuranosyltransferase gene was present in the cluster. Both uncommon features are shared by some other enteric bacteria. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. PABA/NO lead optimization: Improved targeting of cytotoxicity to glutathione S-transferase P1-overexpressing cancer cells.

    PubMed

    Kim, Youseung; Maciag, Anna E; Cao, Zhao; Deschamps, Jeffrey R; Saavedra, Joseph E; Keefer, Larry K; Holland, Ryan J

    2015-08-01

    PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells. Published by Elsevier Ltd.

  7. Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases.

    PubMed

    Hagenbucher, Jan; Stekman, Hilda; Rodriguez-Gil, Alfonso; Kracht, Michael; Schmitz, M Lienhard

    2017-01-01

    The family of seven-in-absentia (SIAH) ubiquitin E3 ligases functions in the control of numerous key signaling pathways. These enzymes belong to the RING (really interesting new gene) group of E3 ligases and mediate the attachment of ubiquitin chains to substrates, which then leads to their proteasomal degradation. Here, we describe a protocol that allows measuring SIAH-mediated ubiquitination and degradation of its client proteins as exemplified by acetyl transferases using simple overexpression experiments. The impact of SIAH expression on the relative amounts of target proteins and their mRNAs can be quantified by Western blotting and quantitative PCR (qPCR) as described here.

  8. Identification of Glutathione S-Transferase (GST) Genes from a Dark Septate Endophytic Fungus (Exophiala pisciphila) and Their Expression Patterns under Varied Metals Stress

    PubMed Central

    Qiao, Qin; Liu, Lei; Wang, Jun-Ling; Cao, Guan-Hua; Li, Tao; Zhao, Zhi-Wei

    2015-01-01

    Glutathione S-transferases (GSTs) compose a family of multifunctional enzymes that play important roles in the detoxification of xenobiotics and the oxidative stress response. In the present study, twenty four GST genes from the transcriptome of a metal-tolerant dark septate endophyte (DSE), Exophiala pisciphila, were identified based on sequence homology, and their responses to various heavy metal exposures were also analyzed. Phylogenetic analysis showed that the 24 GST genes from E. pisciphila (EpGSTs) were divided into eight distinct classes, including seven cytosolic classes and one mitochondrial metaxin 1-like class. Moreover, the variable expression patterns of these EpGSTs were observed under different heavy metal stresses at their effective concentrations for inhibiting growth by 50% (EC50). Lead (Pb) exposure caused the up-regulation of all EpGSTs, while cadmium (Cd), copper (Cu) and zinc (Zn) treatments led to the significant up-regulation of most of the EpGSTs (p < 0.05 to p < 0.001). Furthermore, although heavy metal-specific differences in performance were observed under various heavy metals in Escherichia coli BL21 (DE3) transformed with EpGSTN-31, the over-expression of this gene was able to enhance the heavy metal tolerance of the host cells. These results indicate that E. Pisciphila harbored a diverse of GST genes and the up-regulated EpGSTs are closely related to the heavy metal tolerance of E. pisciphila. The study represents the first investigation of the GST family in E. pisciphila and provides a primary interpretation of heavy metal detoxification for E. pisciphila. PMID:25884726

  9. Mechanosensing regulates virulence in Escherichia coli O157:H7.

    PubMed

    Islam, Md Shahidul; Krachler, Anne Marie

    2016-01-01

    Enterohemorrhagic Escherichia coli O157:H7 is a food-borne pathogen transmitted via the fecal-oral route, and can cause bloody diarrhea and hemolytic uremic syndrome (HUS) in the human host. Although a range of colonization factors, Shiga toxins and a type III secretion system (T3SS) all contribute to disease development, the locus of enterocyte effacement (LEE) encoded T3SS is responsible for the formation of lesions in the intestinal tract. While a variety of chemical cues in the host environment are known to up-regulate LEE expression, we recently demonstrated that changes in physical forces at the site of attachment are required for localized, full induction of the system and thus spatial regulation of virulence in the intestinal tract. Here, we discuss our findings in the light of other recent studies describing mechanosensing of the host and force-dependent induction of virulence mechanisms. We discuss potential mechanisms of mechanosensing and mechanotransduction, and the level of conservation across bacterial species.

  10. 2-acylamido analogues of N-acetylglucosamine prime formation of chitin oligosaccharides by yeast chitin synthase 2

    USDA-ARS?s Scientific Manuscript database

    Chitin, a polymer of beta-1,4-linked N-acetylglucosamine (GlcNAc), is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases (CSs) transfer GlcNAc from UDP-GlcNAc to pre-existing chitin chains in reactions that are typically stimulated by free GlcNAc. The eff...

  11. The E3 Ubiquitin Ligase Adaptor Protein Skp1 Is Glycosylated by an Evolutionarily Conserved Pathway That Regulates Protist Growth and Development.

    PubMed

    Rahman, Kazi; Zhao, Peng; Mandalasi, Msano; van der Wel, Hanke; Wells, Lance; Blader, Ira J; West, Christopher M

    2016-02-26

    Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro-154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by five sugars via the action of three glycosyltransferases, Gnt1, PgtA, and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal, and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi.

    PubMed

    Su, Chang; Lu, Yang; Liu, Haoping

    2016-10-03

    N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription.

  13. N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi

    PubMed Central

    Su, Chang; Lu, Yang; Liu, Haoping

    2016-01-01

    N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription. PMID:27694804

  14. Transfer of free polymannose-type oligosaccharides from the cytosol to lysosomes in cultured human hepatocellular carcinoma HepG2 cells.

    PubMed

    Saint-Pol, A; Bauvy, C; Codogno, P; Moore, S E

    1997-01-13

    Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H-like enzyme and a mannosidase to yield the linear isomer of Man5GlcNAc (Man[alpha 1-2]Man[alpha 1-2]Man[alpha 1-3][Man alpha 1-6]Man[beta 1-4] GlcNAc). Here we have examined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse-chase incubations with D-[2-3H]mannose followed by permeabilization of cells with streptolysin O free oligosaccharides were isolated from the resulting cytosolic and membrane-bound compartments. Control pulse-chase experiments revealed that total cellular free oligosaccharides are lost from HepG2 cells with a half-life of 3-4 h. In contrast use of the vacuolar H+/ATPase inhibitor, concanamycin A, stabilized total cellular free oligosaccharides and enabled us to demonstrate a translocation of partially trimmed oligosaccharides from the cytosol into a membrane-bound compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 microM swainsonine, which provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels which lead to the accumulation of the linear isomer of Man5GlcNAc in the cytosol. Subcellular fractionation studies on Percoll density gradients revealed that the cytosol-generated linear isomer of Man5GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes.

  15. Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

    USDA-ARS?s Scientific Manuscript database

    We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzym...

  16. The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

    PubMed Central

    López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M. Isabel G.

    2013-01-01

    With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097

  17. Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling.

    PubMed

    Singhal, Sharad S; Singh, Sharda P; Singhal, Preeti; Horne, David; Singhal, Jyotsana; Awasthi, Sanjay

    2015-12-15

    4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes - higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. Copyright © 2015 Elsevier Inc. All rights

  18. Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in Butyrate-Producing Bacteria from the Human Large Intestine

    PubMed Central

    Duncan, Sylvia H.; Barcenilla, Adela; Stewart, Colin S.; Pryde, Susan E.; Flint, Harry J.

    2002-01-01

    Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine. PMID:12324374

  19. Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

    PubMed

    Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

    2007-08-01

    INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

  20. A Xanthomonas uridine 5'-monophosphate transferase inhibits plant immune kinases.

    PubMed

    Feng, Feng; Yang, Fan; Rong, Wei; Wu, Xiaogang; Zhang, Jie; Chen, She; He, Chaozu; Zhou, Jian-Min

    2012-04-15

    Plant innate immunity is activated on the detection of pathogen-associated molecular patterns (PAMPs) at the cell surface, or of pathogen effector proteins inside the plant cell. Together, PAMP-triggered immunity and effector-triggered immunity constitute powerful defences against various phytopathogens. Pathogenic bacteria inject a variety of effector proteins into the host cell to assist infection or propagation. A number of effector proteins have been shown to inhibit plant immunity, but the biochemical basis remains unknown for the vast majority of these effectors. Here we show that the Xanthomonas campestris pathovar campestris type III effector AvrAC enhances virulence and inhibits plant immunity by specifically targeting Arabidopsis BIK1 and RIPK, two receptor-like cytoplasmic kinases known to mediate immune signalling. AvrAC is a uridylyl transferase that adds uridine 5'-monophosphate to and conceals conserved phosphorylation sites in the activation loop of BIK1 and RIPK, reducing their kinase activity and consequently inhibiting downstream signalling.

  1. Glutathione S-transferase mediates an ageing response to mitochondrial dysfunction

    PubMed Central

    Dancy, Beverley M.; Brockway, Nicole; Ramadasan-Nair, Renjini; Yang, Yoing; Sedensky, Margaret M.; Morgan, Philip G.

    2016-01-01

    To understand primary mitochondrial disease, we utilized a complex I-deficient Caenorhabditis elegans mutant, gas-1. These animals strongly upregulate the expression of gst-14 (encoding a glutathione S-transferase). Knockdown of gst-14 dramatically extends the lifespan of gas-1 and increases hydroxynonenal (HNE) modified mitochondrial proteins without improving complex I function. We observed no change in reactive oxygen species levels as measured by Mitosox staining, consistent with a potential role of GST-14 in HNE clearance. The upregulation of gst-14 in gas-1 animals is specific to the pharynx. These data suggest that an HNE-mediated response in the pharynx could be beneficial for lifespan extension in the context of complex I dysfunction in C. elegans. Thus, whereas HNE is typically considered damaging, our work is consistent with recent reports of its role in signaling, and that in this case, the signal is pro-longevity in a model of mitochondrial dysfunction. PMID:26704446

  2. Diversity within the O-linked protein glycosylation systems of acinetobacter species.

    PubMed

    Scott, Nichollas E; Kinsella, Rachel L; Edwards, Alistair V G; Larsen, Martin R; Dutta, Sucharita; Saba, Julian; Foster, Leonard J; Feldman, Mario F

    2014-09-01

    The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison of the 26 glycoproteins identified revealed that different A. baumannii strains target similar protein substrates, both in characteristics of the sites of O-glycosylation and protein identity. Surprisingly, glycan micro-heterogeneity was also observed within nearly all isolates examined demonstrating glycan heterogeneity is a widespread phenomena in Acinetobacter O-linked glycosylation. By comparing the 11 main glycoforms and over 20 alternative glycoforms characterized within the 15 strains, trends within the glycan utilized for O-linked glycosylation could be observed. These trends reveal Acinetobacter O-linked glycosylation favors short (three to five residue) glycans with limited branching containing negatively charged sugars such as GlcNAc3NAcA4OAc or legionaminic/pseudaminic acid derivatives. These observations suggest that although highly diverse, the capsule/O-linked glycan biosynthetic pathways generate glycans with similar characteristics across all A. baumannii. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Structure and binding analysis of Polyporus squamosus lectin in complex with the Neu5Acα2-6Galβ1-4GlcNAc human-type influenza receptor

    PubMed Central

    Kadirvelraj, Renuka; Grant, Oliver C; Goldstein, Irwin J; Winter, Harry C; Tateno, Hiroaki; Fadda, Elisa; Woods, Robert J

    2011-01-01

    Glycan chains that terminate in sialic acid (Neu5Ac) are frequently the receptors targeted by pathogens for initial adhesion. Carbohydrate-binding proteins (lectins) with specificity for Neu5Ac are particularly useful in the detection and isolation of sialylated glycoconjugates, such as those associated with pathogen adhesion as well as those characteristic of several diseases including cancer. Structural studies of lectins are essential in order to understand the origin of their specificity, which is particularly important when employing such reagents as diagnostic tools. Here, we report a crystallographic and molecular dynamics (MD) analysis of a lectin from Polyporus squamosus (PSL) that is specific for glycans terminating with the sequence Neu5Acα2-6Galβ. Because of its importance as a histological reagent, the PSL structure was solved (to 1.7 Å) in complex with a trisaccharide, whose sequence (Neu5Acα2-6Galβ1-4GlcNAc) is exploited by influenza A hemagglutinin for viral adhesion to human tissue. The structural data illuminate the origin of the high specificity of PSL for the Neu5Acα2-6Gal sequence. Theoretical binding free energies derived from the MD data confirm the key interactions identified crystallographically and provide additional insight into the relative contributions from each amino acid, as well as estimates of the importance of entropic and enthalpic contributions to binding. PMID:21436237

  4. Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

    PubMed Central

    Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

    2012-01-01

    Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

  5. Expression Patterns of Glutathione Transferase Gene (GstI) in Maize Seedlings Under Juglone-Induced Oxidative Stress

    PubMed Central

    Sytykiewicz, Hubert

    2011-01-01

    Juglone (5-hydroxy-1,4-naphthoquinone) has been identified in organs of many plant species within Juglandaceae family. This secondary metabolite is considered as a highly bioactive substance that functions as direct oxidant stimulating the production of reactive oxygen species (ROS) in acceptor plants. Glutathione transferases (GSTs, E.C.2.5.1.18) represent an important group of cytoprotective enzymes participating in detoxification of xenobiotics and limiting oxidative damages of cellular macromolecules. The purpose of this study was to investigate the impact of tested allelochemical on growth and development of maize (Zea mays L.) seedlings. Furthermore, the effect of juglone-induced oxidative stress on glutathione transferase (GstI) gene expression patterns in maize seedlings was recorded. It was revealed that 4-day juglone treatment significantly stimulated the transcriptional activity of GstI in maize seedlings compared to control plants. By contrast, at the 6th and 8th day of experiments the expression gene responses were slightly lower as compared with non-stressed seedlings. Additionally, the specific gene expression profiles, as well as the inhibition of primary roots and coleoptile elongation were proportional to juglone concentrations. In conclusion, the results provide strong molecular evidence that allelopathic influence of juglone on growth and development of maize seedlings may be relevant with an induction of oxidative stress in acceptor plants. PMID:22174645

  6. Dexamethasone-Induced Skeletal Muscle Atrophy Increases O-GlcNAcylation in C2C12 Cells.

    PubMed

    Massaccesi, Luca; Goi, Giancarlo; Tringali, Cristina; Barassi, Alessandra; Venerando, Bruno; Papini, Nadia

    2016-08-01

    Skeletal muscle atrophy is a well-known adverse effect of chronic treatment with glucocorticoids and it also occurs when stress conditions such as sepsis and cachexia increase the release of endogenous glucocorticoids. Although the mechanisms of action of these hormones have been elucidated, the possible molecular mechanisms causing atrophy are not yet fully understood. The involvement of the O-GlcNAcylation process has recently been reported in disuse atrophy. O-GlcNAcylation, a regulatory post-translational modification of nuclear and cytoplasmic proteins consists in the attachment of O-GlcNAc residues on cell proteins and is regulated by two enzymes: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays a crucial role in many cellular processes and it seems to be related to skeletal muscle physiological function. The aim of this study is to investigate the involvement of O-GlcNAcylation in glucocorticoid-induced atrophy by using an "in vitro" model, achieved by treatment of C2C12 with 10 μM dexamethasone for 48 h. In atrophic condition, we observed that O-GlcNAc levels in cell proteins increased and concomitantly protein phosphorylation on serine and threonine residues decreased. Analysis of OGA expression at mRNA and protein levels showed a reduction in this enzyme in atrophic myotubes, whereas no significant changes of OGT expression were found. Furthermore, inhibition of OGA activity by Thiamet G induced atrophy marker expression. Our current findings suggest that O-GlcNAcylation is involved in dexamethasone-induced atrophy. In particular, we propose that the decrease in OGA content causes an excessive and mostly durable level of O-GlcNAc residues on sarcomeric proteins that might modify their function and stability. J. Cell. Biochem. 117: 1833-1842, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. GCN5L1 modulates cross-talk between mitochondria and cell signaling to regulate FoxO1 stability and gluconeogenesis.

    PubMed

    Wang, Lingdi; Scott, Iain; Zhu, Lu; Wu, Kaiyuan; Han, Kim; Chen, Yong; Gucek, Marjan; Sack, Michael N

    2017-09-12

    The mitochondrial enriched GCN5-like 1 (GCN5L1) protein has been shown to modulate mitochondrial protein acetylation, mitochondrial content and mitochondrial retrograde signaling. Here we show that hepatic GCN5L1 ablation reduces fasting glucose levels and blunts hepatic gluconeogenesis without affecting systemic glucose tolerance. PEPCK and G6Pase transcript levels are downregulated in hepatocytes from GCN5L1 liver specific knockout mice and their upstream regulator, FoxO1 protein levels are decreased via proteasome-dependent degradation and via reactive oxygen species mediated ERK-1/2 phosphorylation. ERK inhibition restores FoxO1, gluconeogenic enzyme expression and glucose production. Reconstitution of mitochondrial-targeted GCN5L1 blunts mitochondrial ROS, ERK activation and increases FoxO1, gluconeogenic enzyme expression and hepatocyte glucose production. We suggest that mitochondrial GCN5L1 modulates post-translational control of FoxO1, regulates gluconeogenesis and controls metabolic pathways via mitochondrial ROS mediated ERK activation. Exploring mechanisms underpinning GCN5L1 mediated ROS signaling may expand our understanding of the role of mitochondria in gluconeogenesis control.Hepatic gluconeogenesis is tightly regulated at transcriptional level and is essential for survival during prolonged fasting. Here Wang et al. show that the mitochondrial enriched GCN5-like 1 protein controls hepatic glucose production by regulating FoxO1 protein levels via proteasome-dependent degradation and, in turn, gluconeogenic gene expression.

  8. The processing of asparagine-linked oligosaccharides in HT-29 cells is a function of their state of enterocytic differentiation. An accumulation of Man9,8-GlcNAc2-Asn species is indicative of an impaired N-glycan trimming in undifferentiated cells.

    PubMed

    Ogier-Denis, E; Codogno, P; Chantret, I; Trugnan, G

    1988-05-05

    Studies on the regulation of the enterocytic differentiation of the human colon cancer cell line HT-29, which is differentiated in the absence (Glc-) but not in the presence of glucose (Glc+), have recently shown that the post-translational processing of sucrase-isomaltase and particularly its glycosylation vary as a function of cell differentiation (Trugnan G., Rousset, M., Chantret, I., Barbat, A., and Zweibaum, A. (1987) J. Cell Biol. 104, 1199-1205). Other studies indicate that in undifferentiated HT-29 Glc+ cells there is an accumulation of UDP-N-acetylhexosamine, which is involved in the glycosylation process (Wice, B. M., Trugnan, G., Pinto, M., Rousset, M., Chevalier, G., Dussaulx, E., Lacroix, B., and Zweibaum, A. (1985) J. Biol. Chem. 260, 139-146). The purpose of the present work is to investigate whether an overall alteration of protein glycosylation is associated with the inability of HT-29 cells to differentiate. At least three alterations are detected: (i) after a 10-min pulse, the incorporation of D-[2-3H]mannose in undifferentiated cells is severely reduced, compared to differentiated cells. (ii) After a 24-h period of labeling with D-[2-3H]mannose, undifferentiated cells accumulate more than 60% of the radioactivity in the high mannose glycopeptides, whereas differentiated HT-29 Glc- cells accumulate only 38%. (iii) The analysis of the high mannose oligosaccharides transferred "en bloc" from the lipid precursor shows that Man9,8-GlcNAc2 species accumulate in undifferentiated cells, whereas no such accumulation can be detected in differentiated cells. This glycosylation pattern is consistent with an impairment of the trimming of high mannose into complex glycans. It is concluded that N-glycan processing is correlated with the state of enterocytic differentiation of HT-29 cells.

  9. Isomerization of 1-O-indol-3-ylacetyl-beta-D-glucose. Enzymatic hydrolysis of 1-O, 4-O, and 6-O-indol-3-ylacetyl-beta-D-glucose and the enzymatic synthesis of indole-3-acetyl glycerol by a hormone metabolizing complex

    NASA Technical Reports Server (NTRS)

    Kowalczyk, S.; Bandurski, R. S.

    1990-01-01

    The first compound in the series of reactions leading to the ester conjugates of indole-3-acetic acid (IAA) in kernels of Zea mays sweet corn is the acyl alkyl acetal, 1-O-indol-3-ylacetyl-beta-D-glucose (1-O-IAGlu). The enzyme catalyzing the synthesis of this compound is UDP-glucose:indol-3-ylacetate glucosyl-transferase (IAGlu synthase). The IAA moiety of the high energy compound 1-O-IAGlu may be enzymatically transferred to myo-inositol or to glycerol or the 1-O-IAGlu may be enzymatically hydrolyzed. Alternatively, nonenzymatic acyl migration may occur to yield the 2-O, 4-O, and 6-O esters of IAA and glucose. The 4-O and 6-O esters may then be enzymatically hydrolyzed to yield free IAA and glucose. This work reports new enzymatic activities, the transfer of IAA from 1-O-IAGlu to glycerol, and the enzyme-catalyzed hydrolysis of 4-O and 6-O-IAGlu. Data is also presented on the rate of non-enzymatic acyl migration of IAA from the 1-O to the 4-O and 6-O positions of glucose. We also report that enzymes catalyzing the synthesis of 1-O-IAGlu and the hydrolysis of 1-O, 4-O, and 6-O-IAGlu fractionate as a hormone metabolizing complex. The association of synthetic and hydrolytic capabilities in enzymes which cofractionate may have physiological significance.

  10. Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells. [Pisum sativum

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

    Camirand, A.; Brummell, D.; MacLachlan, G.

    1987-07-01

    Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-(/sup 14/C) fucose and UDP-(/sup 14/C)xylose to xyloglucan occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with (/sup 3/H)fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.

  11. Transforming growth factor-beta and Forkhead box O transcription factors as cardiac fibroblast regulators.

    PubMed

    Norambuena-Soto, Ignacio; Núñez-Soto, Constanza; Sanhueza-Olivares, Fernanda; Cancino-Arenas, Nicole; Mondaca-Ruff, David; Vivar, Raul; Díaz-Araya, Guillermo; Mellado, Rosemarie; Chiong, Mario

    2017-05-23

    Fibroblasts play several homeostatic roles, including electrical coupling, paracrine signaling and tissue repair after injury. Fibroblasts have low secretory activity. However, in response to injury, they differentiate to myofibroblasts. These cells have an increased extracellular matrix synthesis and secretion, including collagen fibers, providing stiffness to the tissue. In pathological conditions myofibroblasts became resistant to apoptosis, remaining in the tissue, causing excessive extracellular matrix secretion and deposition, which contributes to the progressive tissue remodeling. Therefore, increased myofibroblast content within damaged tissue is a characteristic hallmark of heart, lung, kidney and liver fibrosis. Recently, it was described that cardiac fibroblast to myofibroblast differentiation is triggered by the transforming growth factor β1 (TGF-β1) through a Smad-independent activation of Forkhead box O (FoxO). FoxO proteins are a transcription factor family that includes FoxO1, FoxO3, FoxO4 and FoxO6. In several cells types, they play an important role in cell cycle arrest, oxidative stress resistance, cell survival, energy metabolism, and cell death. Here, we review the role of FoxO family members on the regulation of cardiac fibroblast proliferation and differentiation.

  12. Aralia elata inhibits neurodegeneration by downregulating O-GlcNAcylation of NF-κB in diabetic mice.

    PubMed

    Kim, Seong-Jae; Kim, Min-Jun; Choi, Mee-Young; Kim, Yoon-Sook; Yoo, Ji-Myong; Hong, Eun-Kyung; Ju, Sunmi; Choi, Wan-Sung

    2017-01-01

    To investigate the role of O-GlcNAcylation of nuclear factor-kappa B (NF-κB) in retinal ganglion cell (RGC) death and analysedthe effect of Aralia elata (AE) on neurodegeneration in diabetic mice. C57BL/6mice with streptozotocin-induced diabetes were fed daily with AE extract or control (CTL) diet at the onset of diabetes mellitus (DM). Two months after injection of streptozotocin or saline, the degree of cell death and the expression of O-GlcNAc transferase (OGT), N-acetyl-b-D-glucosaminidase (OGA), O-GlcNAcylated proteins, and O-GlcNAcylation of NF-κB were examined. AE did not affect the metabolic status of diabetic mice. The decrease in the inner retinal thickness ( P <0.001 vs CTL, P <0.01 vs DM) and increases in RGCs with terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling ( P <0.001 vs CTL, P <0.0001 vs DM), glial activation, and active caspase-3 ( P <0.0001 vs CTL, P <0.0001 vs DM) were blocked in diabetic retinas of AE extract-fed mice. Expression levels of protein O-GlcNAcylation and OGT were increased in diabetic retinas ( P <0.0001 vs CTL), and the level of O-GlcNAcylation of the NF-κB p65 subunit was higher in diabetic retinas than in controls ( P <0.0001 vs CTL). AE extract downregulated O-GlcNAcylation of NF-κB and prevented neurodegeneration induced by hyperglycemia ( P <0.0001 vs DM). O-GlcNAcylation of NF-κB is concerned in neuronal degeneration and that AE prevents diabetes-induced RGC apoptosis via downregulation of NF-κB O-GlcNAcylation. Hence, O-GlcNAcylation may be a new object for the treatment of DR, and AE may have therapeutic possibility to prevent diabetes-induced neurodegeneration.

  13. Regulation of Epigenetic Modifiers, Including KDM6B, by Interferon-γ and Interleukin-4 in Human Macrophages.

    PubMed

    Yıldırım-Buharalıoğlu, Gökçe; Bond, Mark; Sala-Newby, Graciela B; Hindmarch, Charles C T; Newby, Andrew C

    2017-01-01

    Interferon-γ (IFN-γ) or interleukin-4 (IL-4) drives widely different transcriptional programs in macrophages. However, how IFN-γ and IL-4 alter expression of histone-modifying enzymes involved in epigenetic regulation and how this affects the resulting phenotypic polarization is incompletely understood. We investigated steady-state messenger RNA levels of 84 histone-modifying enzymes and related regulators in colony-stimulating factor-1 differentiated primary human macrophages using quantitative polymerase chain reaction. IFN-γ or IL-4 treatment for 6-48 h changed 11 mRNAs significantly. IFN-γ increased CIITA, KDM6B, and NCOA1, and IL-4 also increased KDM6B by 6 h. However, either cytokine decreased AURKB, ESCO2, SETD6, SUV39H1, and WHSC1, whereas IFN-γ alone decreased KAT2A, PRMT7, and SMYD3 mRNAs only after 18 h, which coincided with decreased cell proliferation. Rendering macrophages quiescent by growth factor starvation or adenovirus-mediated overexpression of p27 kip1 inhibited expression of AURKB, ESCO2, SUV39H1, and WHSC1, and mRNA levels were restored by overexpressing the S-phase transcription factor E2F1, implying their expression, at least partly, depended on proliferation. However, CIITA, KDM6B, NCOA1, KAT2A, PRMT7, SETD6, and SMYD3 were regulated independently of effects on proliferation. Silencing KDM6B, the only transcriptional activator upregulated by both IFN-γ and IL-4, pharmacologically or with short hairpin RNA, blunted a subset of responses to each cytokine. These findings demonstrate that IFN-γ or IL-4 can regulate the expression of histone acetyl transferases and histone methyl transferases independently of effects on proliferation and that upregulation of the histone demethylase, KDM6B, assists phenotypic polarization by both cytokines.

  14. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2) Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    PubMed

    Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi

    2015-01-01

    Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and

  15. S-glutathionyl-(chloro)hydroquinone reductases: a new class of glutathione transferases functioning as oxidoreductases

    PubMed Central

    Belchik, Sara M.; Xun, Luying

    2011-01-01

    Glutathione transferases (GSTs) are best known for transferring glutathione (GSH) to hydrophobic organic compounds, making the conjugates more soluble. However, the omega-class GSTs of animals and the lambda-class GSTs and dehydroascorbate reductases (DHARs) of plants have little or no activity for GSH transfer. Instead, they catalyze GSH-dependent oxidoreductions. The lambda-class GSTs reduce disulfide bonds, the DHARs reduce the disulfide bonds and dehydroascorbate, and the omega-class GSTs can reduce more substrates, including disulfide bonds, dehydroascorbate, and dimethylarsinate. Glutathionyl-(chloro)hydroquinone reductases (GS-HQRs) are the newest class of GSTs that mainly catalyze oxidoreductions. Besides the activities of the other three classes, GS-HQRs also reduce GS-hydroquinones, including GS-trichloro-p-hydroquinone, GS-dichloro-p-hydroquinone, GS-2-hydroxy-p-hydroquinone, and GS-p-hydroquinone. They are conserved and widely distributed in bacteria, fungi, protozoa, and plants, but not in animals. The four classes are phylogenetically more related to each other than to other GSTs, and they share a Cys-Pro motif at the GSH-binding site. Hydroquinones are metabolic intermediates of certain aromatic compounds. They can be auto-oxidized by O2 to benzoquinones, which spontaneously react with GSH to form GS-hydroquinones via Michael’s addition. GS-HQRs are expected to channel GS-hydroquinones, formed spontaneously or enzymatically, back to hydroquinones. When the released hydroquinones are intermediates of metabolic pathways, GS-HQRs play a maintenance role for the pathways. Further, the common presence of GS-HQRs in plants, green algae, cyanobacteria, and halobacteria suggest a beneficial role in the light-using organisms. PMID:21425927

  16. Can CuO nanoparticles lead to epigenetic regulation of antioxidant enzyme system?

    PubMed

    Chibber, Sandesh; Shanker, Rishi

    2017-01-01

    Copper has been used from ancient time in various applications. Scientists have exploited its means of exposure and consequences to living organisms. The peculiar property of nanomaterials that is a high surface to volume ratio has increased the range of application in products. Copper oxide nanoparticles (CuO NPs) are widely used in industrial applications such as semiconductor devices, gas sensor, batteries, solar energy converter, microelectronics, heat transfer fluids and consumer products. In contrast, acute toxicity of CuO NPs has also been reported. Subsequently, human and environmental health may be at a high risk. Their frequent use can also contaminate ecosystems. Therefore, the toxicity of CuO NPs needs to be thoroughly understood. In this review, we have tried to discuss the recent facts and mechanism that have been explored for CuO NPs-induced toxicity at a cellular, in vivo and ecotoxicological level. Accordingly, the main cause for induction of toxicity by CuO NPs is the generation of reactive oxygen species (ROS) followed by the mitochondrial destruction that leads to apoptosis via the intrinsic pathway or under the condition such as hypoxia cell on exposure to CuO NPs may commit to necrosis. Moreover, CuO NPs also result in activation of MAPK pathways, ERKs and JNK/SAPK thus play an important role in the activation of AP-1. Furthermore, CuO NPs also leads to up-regulation of p53 and caspase three genes. Therefore, careful measures are required to explore omic technology to understand the molecular mechanism of the deleterious effects caused by CuO NPs. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  17. O-GlcNAcylation of NF-κB Promotes Lung Metastasis of Cervical Cancer Cells via Upregulation of CXCR4 Expression.

    PubMed

    Ali, Akhtar; Kim, Sung Hwan; Kim, Min Jun; Choi, Mee Young; Kang, Sang Soo; Cho, Gyeong Jae; Kim, Yoon Sook; Choi, Jun-Young; Choi, Wan Sung

    2017-07-31

    C-X-C chemokine receptor 4 (CXCR4) stimulates cancer metastasis. NF-κB regulates CXCR4 expression in cancer cells, and O-GlcNAc modification of NF-κB promotes its transcriptional activity. Here, we determined whether CXCR4 expression is affected by O-GlcNAcylation of NF-κB in lung metastasis of cervical cancer. We found elevated levels of O-linked-N-actylglucosamine transferase (OGT) and O-GlcNAcylation in cervical cancer cells compared to those in non-malignant epithelial cells and detected increased expression of NF-κB p65 (p65) and CXCR4 in cervical cancer cells. Knockdown of OGT inhibited the O-GlcNAcylation of p65 and decreased CXCR4 expression levels in HeLa cells. Thiamet G treatment increased O-GlcNAcylated p65, which subsequently enhanced CXCR4 expression levels. Inhibition of O-GlcNAcylation by 6-Diazo-5-oxo-L-norleucine (DON) treatment decreased p65 activation, eventually inhibiting CXCR4 expression in HeLa cells. Lung tissues from mice engrafted with OGT-knockdown HeLa cells (shOGT) exhibited lower expression of Ki-67 and HPV E6 and E7 oncogenes compared to lung tissues from mice engrafted with control HeLa cells (shCTL). In addition, lung tissues from mice engrafted with shOGT cells exhibited lower p65 and CXCR4 immunoreactivity compared to tissues from mice engrafted with shCTL cells. Taken together, our data suggest that p65 O-GlcNAcylation promotes lung metastasis of cervical cancer cells by activating CXCR4 expression.

  18. Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1

    PubMed Central

    YANG, LIANG; LIU, REN; MA, HONG-BIN; YING, MING-ZHEN; WANG, YA-JIE

    2015-01-01

    The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 (GSTP1) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G2/M phase arrest in the GSTP1-expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1-expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G2/M phase arrest. PMID:26622693

  19. Radiosensitivity in HeLa cervical cancer cells overexpressing glutathione S-transferase π 1.

    PubMed

    Yang, Liang; Liu, Ren; Ma, Hong-Bin; Ying, Ming-Zhen; Wang, Ya-Jie

    2015-09-01

    The aims of the present study were to investigate the effect of overexpressed exogenous glutathione S-transferase π 1 ( GSTP1 ) gene on the radiosensitivity of the HeLa human cervical cancer cell line and conduct a preliminarily investigation into the underlying mechanisms of the effect. The full-length sequence of human GSTP1 was obtained by performing a polymerase chain reaction (PCR) using primers based on the GenBank sequence of GSTP1. Subsequently, the gene was cloned into a recombinant eukaryotic expression plasmid, and the resulting construct was confirmed by restriction analysis and DNA sequencing. A HeLa cell line that was stably expressing high levels of GSTP1 was obtained through stable transfection of the constructed plasmids using lipofectamine and screening for G418 resistance, as demonstrated by reverse transcription-PCR. Using the transfected HeLa cells, a colony formation assay was conducted to detect the influence of GSTP1 overexpression on the cell radiosensitivity. Furthermore, flow cytometry was used to investigate the effect of GSTP1 overexpression on cell cycle progression, with the protein expression levels of the cell cycle regulating factor cyclin B1 detected using western blot analysis. Colony formation and G 2 /M phase arrest in the GSTP1 -expressing cells were significantly increased compared with the control group (P<0.01). In addition, the expression of cyclin B1 was significantly reduced in the GSTP1 -expressing cells. These results demonstrated that increased expression of GSTP1 inhibits radiosensitivity in HeLa cells. The mechanism underlying this effect may be associated with the ability of the GSTP1 protein to reduce cyclin B1 expression, resulting in significant G 2 /M phase arrest.

  20. Curcumin activates human glutathione S-transferase P1 expression through antioxidant response element.

    PubMed

    Nishinaka, Toru; Ichijo, Yusuke; Ito, Maki; Kimura, Masayoshi; Katsuyama, Masato; Iwata, Kazumi; Miura, Takeshi; Terada, Tomoyuki; Yabe-Nishimura, Chihiro

    2007-05-15

    Curcumin is a plant-derived diferuloylmethane compound extracted from Curcuma longa, possessing antioxidative and anticarcinogenic properties. Antioxidants and oxidative stress are known to induce the expression of certain classes of detoxification enzymes. Since the upregulation of detoxifying enzymes affects the drug metabolism and cell defense system, it is important to understand the gene regulation by such agents. In this study, we demonstrated that curcumin could induce the expression of human glutathione S-transferase P1 (GSTP1). In HepG2 cells treated with 20muM curcumin, the level of GSTP1 mRNA was significantly increased. In luciferase reporter assays, curcumin augmented the promoter activity of a reporter construct carrying 336bp upstream of the 5'-flanking region of the GSTP1 gene. Mutation analyses revealed that the region including antioxidant response element (ARE), which overlaps AP1 in sequence, was essential to the response to curcumin. While the introduction of a wild-type Nrf2 expression construct augmented the promoter activity of the GSTP1 gene, co-expression of a dominant-negative Nrf2 abolished the responsiveness to curcumin. In addition, curcumin activated the expression of the luciferase gene from a reporter construct carrying multiple ARE consensus sequences but not one with multiple AP1 sites. In a gel mobility shift assay with an oligonucleotide with GSTP1 ARE, an increase in the amount of the binding complex was observed in the nuclear extracts of curcumin-treated HepG2 cells. These results suggested that ARE is the primary sequence for the curcumin-induced transactivation of the GSTP1 gene. The induction of GSTP1 may be one of the mechanisms underlying the multiple actions of curcumin.

  1. Potential coordination role between O-GlcNAcylation and epigenetics.

    PubMed

    Wu, Donglu; Cai, Yong; Jin, Jingji

    2017-10-01

    Dynamic changes of the post-translational O-GlcNAc modification (O-GlcNAcylation) are controlled by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the glycoside hydrolase O-GlcNAcase (OGA) in cells. O-GlcNAcylation often occurs on serine (Ser) and threonine (Thr) residues of the specific substrate proteins via the addition of O-GlcNAc group by OGT. It has been known that O-GlcNAcylation is not only involved in many fundamental cellular processes, but also plays an important role in cancer development through various mechanisms. Recently, accumulating data reveal that O-GlcNAcylation at histones or non-histone proteins can lead to the start of the subsequent biological processes, suggesting that O-GlcNAcylation as 'protein code' or 'histone code' may provide recognition platforms or executive instructions for subsequent recruitment of proteins to carry out the specific functions. In this review, we summarize the interaction of O-GlcNAcylation and epigenetic changes, introduce recent research findings that link crosstalk between O-GlcNAcylation and epigenetic changes, and speculate on the potential coordination role of O-GlcNAcylation with epigenetic changes in intracellular biological processes.

  2. Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein.

    PubMed

    Piano, Valentina; Nenci, Simone; Magnani, Francesca; Aliverti, Alessandro; Mattevi, Andrea

    2016-12-02

    Although the precise functions of ether phospholipids are still poorly understood, significant alterations in their physiological levels are associated either to inherited disorders or to aggressive metastatic cancer. The essential precursor, alkyl-dihydroxyacetone phosphate (DHAP), for all ether phospholipids species is synthetized in two consecutive reactions performed by two enzymes sitting on the inner side of the peroxisomal membrane. Here, we report the characterization of the recombinant human DHAP acyl-transferase, which performs the first step in alkyl-DHAP synthesis. By exploring several expression systems and designing a number of constructs, we were able to purify the enzyme in its active form and we found that it is tightly bound to the membrane through the N-terminal residues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases.

    PubMed Central

    Alfenito, M R; Souer, E; Goodman, C D; Buell, R; Mol, J; Koes, R; Walbot, V

    1998-01-01

    Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1. PMID:9668133

  4. Glutathione-S-transferase P protects against endothelial dysfunction induced by exposure to tobacco smoke.

    PubMed

    Conklin, Daniel J; Haberzettl, Petra; Prough, Russell A; Bhatnagar, Aruni

    2009-05-01

    Exposure to tobacco smoke impairs endothelium-dependent arterial dilation. Reactive constituents of cigarette smoke are metabolized and detoxified by glutathione-S-transferases (GSTs). Although polymorphisms in GST genes are associated with the risk of cancer in smokers, the role of these enzymes in regulating the cardiovascular effects of smoking has not been studied. The P isoform of GST (GSTP), which catalyzes the conjugation of electrophilic molecules in cigarette smoke such as acrolein, was expressed in high abundance in the mouse lung and aorta. Exposure to tobacco smoke for 3 days (5 h/day) decreased total plasma protein. These changes were exaggerated in GSTP(-/-) mice. Aortic rings isolated from tobacco smoke-exposed GSTP(-/-) mice showed greater attenuation of ACh-evoked relaxation than those from GSTP(+/+) mice. The lung, plasma, and aorta of mice exposed to tobacco smoke or acrolein (for 5 h) accumulated more acrolein-adducted proteins than those tissues of mice exposed to air, indicating that exposure to tobacco smoke results in the systemic delivery of acrolein. Relative to GSTP(+/+) mice, modification of some proteins by acrolein was increased in the aorta of GSTP(-/-) mice. Aortic rings prepared from GSTP(-/-) mice that inhaled acrolein (1 ppm, 5 h/day for 3 days) or those exposed to acrolein in an organ bath showed diminished ACh-induced arterial relaxation more strongly than GSTP(+/+) mice. Acrolein-induced endothelial dysfunction was prevented by pretreatment of the aorta with N-acetylcysteine. These results indicate that GSTP protects against the endothelial dysfunction induced by tobacco smoke exposure and that this protection may be related to the detoxification of acrolein or other related cigarette smoke constituents.

  5. Rewiring the Glucose Transportation and Central Metabolic Pathways for Overproduction of N-Acetylglucosamine in Bacillus subtilis.

    PubMed

    Gu, Yang; Deng, Jieying; Liu, Yanfeng; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian; Liu, Long

    2017-10-01

    N-acetylglucosamine (GlcNAc) is an important amino sugar extensively used in the healthcare field. In a previous study, the recombinant Bacillus subtilis strain BSGN6-P xylA -glmS-pP43NMK-GNA1 (BN0-GNA1) had been constructed for microbial production of GlcNAc by pathway design and modular optimization. Here, the production of GlcNAc is further improved by rewiring both the glucose transportation and central metabolic pathways. First, the phosphotransferase system (PTS) is blocked by deletion of three genes, yyzE (encoding the PTS system transporter subunit IIA YyzE), ypqE (encoding the PTS system transporter subunit IIA YpqE), and ptsG (encoding the PTS system glucose-specific EIICBA component), resulting in 47.6% increase in the GlcNAc titer (from 6.5 ± 0.25 to 9.6 ± 0.16 g L -1 ) in shake flasks. Then, reinforcement of the expression of the glcP and glcK genes and optimization of glucose facilitator proteins are performed to promote glucose import and phosphorylation. Next, the competitive pathways for GlcNAc synthesis, namely glycolysis, peptidoglycan synthesis pathway, pentose phosphate pathway, and tricarboxylic acid cycle, are repressed by initiation codon-optimization strategies, and the GlcNAc titer in shake flasks is improved from 10.8 ± 0.25 to 13.2 ± 0.31 g L -1 . Finally, the GlcNAc titer is further increased to 42.1 ± 1.1 g L -1 in a 3-L fed-batch bioreactor, which is 1.72-fold that of the original strain, BN0-GNA1. This study shows considerably enhanced GlcNAc production, and the metabolic engineering strategy described here will be useful for engineering other prokaryotic microorganisms for the production of GlcNAc and related molecules. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Single and combined effects of aluminum (Al2O3) and zinc (ZnO) oxide nanoparticles in a freshwater fish, Carassius auratus.

    PubMed

    Benavides, María; Fernández-Lodeiro, Javier; Coelho, Pedro; Lodeiro, Carlos; Diniz, Mário S

    2016-12-01

    The increasing use of nanoparticles (NPs) worldwide has raised some concerns about their impact on the environment. The aim of the study was to assess the toxicity of metal oxide nanoparticles, singly or combined, in a freshwater fish (Carassius auratus). The fish were exposed for 7, 14, and 21 days to different concentrations of NPs (10 μg Al 2 O 3 .L -1 , 10 μg ZnO.L -1 , 10 μg Al 2 O 3 .L -1 plus 10 μg ZnO.L -1 , 100 μg Al 2 O 3 .L -1 , 100 μg ZnO.L -1 , and 100 μg Al 2 O 3 .L -1 plus 100 μg ZnO.L -1 ). At the end of each exposure period, antioxidant enzyme activity (catalase, glutathione-S-transferase, and superoxide dismutase), lipid peroxidation, and histopathology were assessed in the gills and livers of C. auratus. The results show an increase in catalase (CAT) and superoxide dismutase (SOD) activity in the gills and livers of fish, especially after 14 days of exposure to single and combined NPs, followed by a reduction at 21 days. An increase in glutathione S-transferase (GST) was observed in gills after 7 days for all tested NP concentrations (single and combined); while in livers, a significant increase was determined after 14 days of exposure to 100 μg.L -1 of both single ZnO and Al 2 O 3 NPs. Lipid peroxidation (LPO) significantly increased in gills after 7 days of exposure to 100 μg.L -1 Al 2 O 3 NPs (single or combined). In livers, LPO increased significantly after 7 days of exposure to all tested concentrations of both single ZnO and Al 2 O 3 (except for 10 μg Al 2 O 3 .L -1 ), and after 14 days of exposure to ZnO (10 and 100 μg.L -1 ) and Al 2 O 3 (100 μg.L -1 ) . The results from histological observations suggest that exposure to metal oxide NPs affected both livers and gills, presenting alterations such as gill hyperplasia and liver degeneration. However, the most pronounced effects were found in gills. In general, this study shows that the tested NPs, single or combined, are capable of causing sub-lethal effects

  7. Brain Immune Interactions as the Basis of Gulf War Illness: Gulf War Illness Consortium (GWIC)

    DTIC Science & Technology

    2015-10-01

    August to train all clinical staff and to ensure proper quality control measures are in place for the clinical studies. This has been followed up by...clinical and preclinical studies Training for researchers and clinical staff was completed at in-person meeting in Boston in August 2014 and continued to...Catechol-O- methyl transferase ( COMT ). COMT is associated with synaptic catecholamine neurotransmitters. COMT helps regulate cortical dopamine in the

  8. N-acetylglucosamine 6-Phosphate Deacetylase (nagA) Is Required for N-acetyl Glucosamine Assimilation in Gluconacetobacter xylinus

    PubMed Central

    Yadav, Vikas; Panilaitis, Bruce; Shi, Hai; Numuta, Keiji; Lee, Kyongbum; Kaplan, David L.

    2011-01-01

    Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tetr; named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species. PMID:21655093

  9. N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus.

    PubMed

    Yadav, Vikas; Panilaitis, Bruce; Shi, Hai; Numuta, Keiji; Lee, Kyongbum; Kaplan, David L

    2011-01-01

    Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.

  10. Glutathione Transferase as a Potential Marker for Gut Epithelial Injury versus the Protective Role of Breast Milk sIgA in Infants with Rota Virus Gastroenteritis

    PubMed Central

    Sherif, Lobna S.; Raouf, Randaa K. Abdel; Sayede, Rokaya M. El; Wakkadd, Amany S. El; Shoaib, Ashraf R.; Ali, Hanan M.; Refay, Amira S. El

    2015-01-01

    BACKGROUND: Secretory immunoglobulin A (SIgA) plays an important protective role in the recognition and clearance of enteric pathogens. AIM: This study was designed to assess if mucosal integrity “measured by secretory IgA (SIgA)” is a protective factor from more epithelial alteration “measured by glutathione transferase” in infants with Rota gastroenteritis and its relation to infants’ feeding pattern. PATIENTS AND METHODS: This study was conducted on 79 infants aged 6 months and less from those diagnosed as having gastroenteritis and admitted to Gastroenteritis Department in Abo El Rish Pediatric Hospital, Cairo University. Plasma glutathione s-transferases and Stool SIgA were measured using ELISA technique. Rota virus detection was done by Reverse transcriptase PCR. RESULTS: SIgA was found to be significantly positive in exclusive breast fed infants, Glutathione transferase was significantly more frequently positive in Rota positive cases than Rota negative cases by Reverse transcriptase PCR. A significant negative correlation between Glutathione transferase and Secretory IgA was found, (p < 0.05). CONCLUSION: Breast feeding should be encouraged and highly recommended in the first two years of life as it provides Secretory IgA to breast fed infants who in turn protect them against epithelial damage caused by Rota viral gastroenteritis. PMID:27275307

  11. An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

    PubMed

    Itzhaki, H; Maxson, J M; Woodson, W R

    1994-09-13

    The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene.

  12. SPINDLY, a Negative Regulator of Gibberellic Acid Signaling, Is Involved in the Plant Abiotic Stress Response1[W][OA

    PubMed Central

    Qin, Feng; Kodaira, Ken-Suke; Maruyama, Kyonoshin; Mizoi, Junya; Tran, Lam-Son Phan; Fujita, Yasunari; Morimoto, Kyoko; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2011-01-01

    The SPINDLY (SPY) gene was first identified as a negative regulator of plant gibberellic acid (GA) signaling because mutation of this gene phenocopies plants treated with an overdose of bioactive GA and results in insensitivity to a GA inhibitor during seed germination. The SPY gene encodes an O-linked N-acetylglucosamine transferase that can modify the target protein and modulate the protein activity in cells. In this study, we describe the strong salt and drought tolerance phenotypes of Arabidopsis (Arabidopsis thaliana) spy-1 and spy-3 mutants in addition to their GA-related phenotypes. SPY gene expression was found to be drought stress inducible and slightly responsive to salt stress. Transcriptome analysis of spy-3 revealed that many GA-responsive genes were up-regulated, which could explain the GA-overdosed phenotype of spy-3. Some stress-inducible genes were found to be up-regulated in spy-3, such as genes encoding late embryogenesis abundant proteins, Responsive to Dehydration20, and AREB1-like transcription factor, which may confer stress tolerance on spy-3. CKX3, a cytokinin (CK) catabolism gene, was up-regulated in spy-3; this up-regulation indicates that the mutant possesses reduced CK signaling, which is consistent with a positive role for SPY in CK signaling. Moreover, overexpression of SPY in transgenics (SPY overexpressing [SPY-OX]) impaired plant drought stress tolerance, opposite to the phenotype of spy. The expression levels of several genes, such as DREB1E/DDF1 and SNH1/WIN1, were decreased in SPY-OX but increased in spy-3. Taken together, these data indicate that SPY plays a negative role in plant abiotic stress tolerance, probably by integrating environmental stress signals via GA and CK cross talk. PMID:22013217

  13. Evolutionary conservation of regulated longevity assurance mechanisms

    PubMed Central

    McElwee, Joshua J; Schuster, Eugene; Blanc, Eric; Piper, Matthew D; Thomas, James H; Patel, Dhaval S; Selman, Colin; Withers, Dominic J; Thornton, Janet M; Partridge, Linda; Gems, David

    2007-01-01

    Background To what extent are the determinants of aging in animal species universal? Insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an evolutionarily conserved (public) regulator of longevity; yet it remains unclear whether the genes and biochemical processes through which IIS acts on aging are public or private (that is, lineage specific). To address this, we have applied a novel, multi-level cross-species comparative analysis to compare gene expression changes accompanying increased longevity in mutant nematodes, fruitflies and mice with reduced IIS. Results Surprisingly, there is little evolutionary conservation at the level of individual, orthologous genes or paralogous genes under IIS regulation. However, a number of gene categories are significantly enriched for genes whose expression changes in long-lived animals of all three species. Down-regulated categories include protein biosynthesis-associated genes. Up-regulated categories include sugar catabolism, energy generation, glutathione-S-transferases (GSTs) and several other categories linked to cellular detoxification (that is, phase 1 and phase 2 metabolism of xenobiotic and endobiotic toxins). Protein biosynthesis and GST activity have recently been linked to aging and longevity assurance, respectively. Conclusion These processes represent candidate, regulated mechanisms of longevity-control that are conserved across animal species. The longevity assurance mechanisms via which IIS acts appear to be lineage-specific at the gene level (private), but conserved at the process level (or semi-public). In the case of GSTs, and cellular detoxification generally, this suggests that the mechanisms of aging against which longevity assurance mechanisms act are, to some extent, lineage specific. PMID:17612391

  14. Transfer of Free Polymannose-type Oligosaccharides from the Cytosol to Lysosomes in Cultured Human Hepatocellular Carcinoma HEPG2 Cells

    PubMed Central

    Saint-Pol, Agnès; Bauvy, Chantal; Codogno, Patrice; Moore, Stuart E.H.

    1997-01-01

    Large, free polymannose oligosaccharides generated during glycoprotein biosynthesis rapidly appear in the cytosol of HepG2 cells where they undergo processing by a cytosolic endo H–like enzyme and a mannosidase to yield the linear isomer of Man5GlcNAc (Man[α1-2]Man[α1-2]Man[α1-3][Man α1-6]Man[β14]GlcNAc). Here we have examined the fate of these partially trimmed oligosaccharides in intact HepG2 cells. Subsequent to pulse–chase incubations with d-[2- 3H]mannose followed by permeabilization of cells with streptolysin O free oligosaccharides were isolated from the resulting cytosolic and membrane-bound compartments. Control pulse–chase experiments revealed that total cellular free oligosaccharides are lost from HepG2 cells with a half-life of 3–4 h. In contrast use of the vacuolar H+/ATPase inhibitor, concanamycin A, stabilized total cellular free oligosaccharides and enabled us to demonstrate a translocation of partially trimmed oligosaccharides from the cytosol into a membrane-bound compartment. This translocation process was unaffected by inhibitors of autophagy but inhibited if cells were treated with either 100 μM swainsonine, which provokes a cytosolic accumulation of large free oligosaccharides bearing 8-9 residues of mannose, or agents known to reduce cellular ATP levels which lead to the accumulation of the linear isomer of Man5GlcNAc in the cytosol. Subcellular fractionation studies on Percoll density gradients revealed that the cytosol-generated linear isomer of Man5GlcNAc is degraded in a membrane-bound compartment that cosediments with lysosomes. PMID:9008702

  15. A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

    PubMed Central

    Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

    2010-01-01

    Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control. PMID:20824165

  16. Bivalent Carbohydrate Binding Is Required for Biological Activity of Clitocybe nebularis Lectin (CNL), the N,N′-Diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc)-specific Lectin from Basidiomycete C. nebularis*

    PubMed Central

    Pohleven, Jure; Renko, Miha; Magister, Špela; Smith, David F.; Künzler, Markus; Štrukelj, Borut; Turk, Dušan; Kos, Janko; Sabotič, Jerica

    2012-01-01

    Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N′-diacetyllactosediamine (GalNAcβ1–4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its β-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency. PMID:22298779

  17. Identification of high-mannose and multiantennary complex-type N-linked glycans containing alpha-galactose epitopes from Nurse shark IgM heavy chain.

    PubMed

    Harvey, David J; Crispin, Max; Moffatt, Beryl E; Smith, Sylvia L; Sim, Robert B; Rudd, Pauline M; Dwek, Raymond A

    2009-11-01

    MALDI-TOF mass spectrometry, negative ion nano-electrospray MS/MS and exoglycosidase digestion were used to identify 36 N-linked glycans from 19S IgM heavy chain derived from the nurse shark (Ginglymostoma cirratum). The major glycan was the high-mannose compound, Man(6)GlcNAc(2) accompanied by small amounts of Man(5)GlcNAc(2), Man(7)GlcNAc(2) and Man(8)GlcNAc(2). Bi- and tri-antennary (isomer with a branched 3-antenna) complex-type glycans were also abundant, most contained a bisecting GlcNAc residue (beta1-->4-linked to the central mannose) and with varying numbers of alpha-galactose residues capping the antennae. Small amounts of monosialylated glycans were also found. This appears to be the first comprehensive study of glycosylation in this species of animal. The glycosylation pattern has implications for the mechanism of activation of the complement system by nurse shark IgM.

  18. Gal knockout and beyond.

    PubMed

    Zhong, R

    2007-01-01

    Recently, Galalpha1-3Galbeta1-4GlcNAc (Gal) knockout (k/o) pigs have been developed using genetic cloning technologies. This remarkable achievement has generated great enthusiasm in xenotransplantation studies. This review summarizes the current status of nonhuman primate experiments using Gal k/o pig organs. Briefly, when Gal k/o pig organs are transplanted into primates, hyperacute rejection does not occur. Although graft survival has been prolonged up to a few months in some cases, the overall results were not better than those using Gal-positive pig organs with human complement regulatory protein transgenes. Gal k/o pig kidneys rapidly developed rejection which was associated with increased anti-non-Gal antibodies. Although the precise mechanisms of Gal k/o pig organ rejection are not clear, it could result from incomplete deletion of Gal, up-regulation of new antigen (non-Gal antigen) and/or production of non-Gal antibodies. Future work in xenotransplantation should place emphasis on further modification of donors, such as combining human complement regulatory genes with Gal k/o, deleting non-Gal antigens and adding protective/surviving genes or a gene that inhibits coagulation. Induction of donor-specific T- and B-cell tolerance and promotion of accommodation are also warranted.

  19. Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

    PubMed

    Abunnaja, Maryam S; Kurogi, Katsuhisa; Mohammed, Yasir I; Sakakibara, Yoichi; Suiko, Masahito; Hassoun, Ezdihar A; Liu, Ming-Cheh

    2017-10-01

    Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined. © 2017 Wiley Periodicals, Inc.

  20. Cloning, Developmental, and Tissue-Specific Expression of Sucrose:Sucrose 1-Fructosyl Transferase from Taraxacum officinale. Fructan Localization in Roots1

    PubMed Central

    Van den Ende, Wim; Michiels, An; Van Wonterghem, Dominik; Vergauwen, Rudy; Van Laere, André

    2000-01-01

    Sucrose:sucrose 1-fructosyl transferase (1-SST) is the key enzyme initiating fructan synthesis in Asteraceae. Using reverse transcriptase-PCR, we isolated the cDNA for 1-SST from Taraxacum officinale. The cDNA-derived amino acid sequence showed very high homology to other Asteracean 1-SSTs (Cichorium intybus 86%, Cynara scolymus 82%, Helianthus tuberosus 80%), but homology to 1-SST from Allium cepa (46%) and Aspergillus foetidus (18%) was much lower. Fructan concentrations, 1-SST activities, 1-SST protein, and mRNA concentrations were compared in different organs during vegetative and generative development of T. officinale plants. Expression of 1-SST was abundant in young roots but very low in leaves. 1-SST was also expressed at the flowering stages in roots, stalks, and receptacles. A good correlation was found between northern and western blots showing transcriptional regulation of 1-SST. At the pre-flowering stage, 1-SST mRNA concentrations and 1-SST activities were higher in the root phloem than in the xylem, resulting in the higher fructan concentrations in the phloem. Fructan localization studies indicated that fructan is preferentially stored in phloem parenchyma cells in the vicinity of the secondary sieve tube elements. However, inulin-like crystals occasionally appeared in xylem vessels. PMID:10806226

  1. Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B.

    PubMed

    Borowiak, Maria; Fischer, Jennie; Hammerl, Jens A; Hendriksen, Rene S; Szabo, Istvan; Malorny, Burkhard

    2017-12-01

    Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L. A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing. In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011-13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed. Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

    PubMed Central

    Hahnvajanawong, Chariya; Chaiyagool, Jariya; Seubwai, Wunchana; Bhudhisawasdi, Vajarabhongsa; Namwat, Nisana; Khuntikeo, Narong; Sripa, Banchob; Pugkhem, Ake; Tassaneeyakul, Wichittra

    2012-01-01

    AIM: To determine whether expression of certain enzymes related to 5-fluorouracil (5-FU) metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma (CCA). METHODS: The histoculture drug response assay (HDRA) was performed using surgically resected CCA tissues. Tumor cell viability was determined morphologically with hematoxylin and eosin- and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues. The mRNA expression of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) was determined with real-time reverse transcriptase-polymerase chain reaction. The levels of gene expression and the sensitivity to 5-FU were evaluated. RESULTS: Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital, Khon Kaen University from 2007 to 2009. HDRA was used to determine the response of these CCA tissues to 5-FU. Based on the dose-response curve, 200 μg/mL 5-FU was selected as the test concentration. The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU. When the relationship between TP, OPRT, TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined, only OPRT mRNA expression was significantly correlated with the response to 5-FU. The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group (0.41 ± 0.25 vs 0.22 ± 0.12, P < 0.05). CONCLUSION: OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA. Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients. PMID:22912546

  3. Functional Expression of Enterobacterial O-Polysaccharide Biosynthesis Enzymes in Bacillus subtilis

    PubMed Central

    Schäffer, Christina; Wugeditsch, Thomas; Messner, Paul; Whitfield, Chris

    2002-01-01

    The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes wbaP (formerly rfbP), wecA (formerly rfe), and wbbO (formerly rfbF) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in Bacillus subtilis. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and N-acetylglucosaminyl 1-phosphate from UDP-N-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional wbbO gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In B. subtilis hosts, the genes were effectively transcribed under the vegII promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the tet resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in Escherichia coli and B. subtilis, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-α-d-14C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in B. subtilis varied between 20 and 75% of those measured in E. coli. PMID:12324313

  4. Reviewing Hit Discovery Literature for Difficult Targets: Glutathione Transferase Omega-1 as an Example.

    PubMed

    Xie, Yiyue; Dahlin, Jayme L; Oakley, Aaron J; Casarotto, Marco G; Board, Philip G; Baell, Jonathan B

    2018-05-10

    Early stage drug discovery reporting on relatively new or difficult targets is often associated with insufficient hit triage. Literature reviews of such targets seldom delve into the detail required to critically analyze the associated screening hits reported. Here we take the enzyme glutathione transferase omega-1 (GSTO1-1) as an example of a relatively difficult target and review the associated literature involving small-molecule inhibitors. As part of this process we deliberately pay closer-than-usual attention to assay interference and hit quality aspects. We believe this Perspective will be a useful guide for future development of GSTO1-1 inhibitors, as well serving as a template for future review formats of new or difficult targets.

  5. Gene Polymorphisms of Glutathione S-Transferase T1/M1 in Egyptian Children and Adolescents with Type 1 Diabetes Mellitus.

    PubMed

    Barseem, Naglaa; Elsamalehy, Mona

    2017-06-01

    Oxidative stress plays an important role in the pathogenesis of type 1 diabetes mellitus (T1DM). To evaluate the association of glutathione S-transferase mu 1 (GST M1) and glutathione S-transferase theta 1 (GST T1) polymorphisms with development of T1DM and disease-related risk factors. Measurement of fasting glucose, serum creatinine, lipid profile, and glycosylated hemoglobin (HbA1c), as well as evaluation of GST T1 and M1 genetic polymorphisms using polymerase chain reaction were done in 64 diabetic children and 41 controls. The diabetic group had significantly higher fasting glucose, HbA1c, and cholesterol levels. GST T1 null genotype was more frequent in the diabetic than the control group with 4.2-fold increased risk of T1DM (odds ratio=4.2; 95% confidence interval=1.6-11.5; p=0.03). Significant positive associations were found with lipid profile, HbA1c, and duration of illness but not with age, age at onset, and body mass index. Gene polymorphisms of the enzyme GST are associated with development of T1DM and disease-related risk factors.

  6. MicroR828 regulates lignin and H2O2 accumulation in sweet potato on wounding.

    PubMed

    Lin, Jeng-Shane; Lin, Chih-Ching; Lin, Hsin-Hung; Chen, Yu-Chi; Jeng, Shih-Tong

    2012-10-01

    MicroRNAs (miRNAs) are small noncoding RNAs which post-transcriptionally regulate gene expression by directing mRNA cleavage or translational inhibition. miRNAs play multiple roles in the growth, development and stress responses in plants. However, little is known of the wounding-responsive miRNAs and their regulation. Here, we investigated the expression patterns of microR828 (miR828) on wounding in sweet potato (Ipomoea batatas cv Tainung 57). The expression of miR828 was only detected in leaves, and was induced by wounding rather than by ethylene, hydrogen peroxide (H2O2), methyl jasmonate or nitric oxide (NO). Moreover, cyclic guanosine monophosphate (cGMP) was necessary for miR828 accumulation in leaves on wounding. Two miR828 target candidates, named IbMYB and IbTLD, were obtained by cDNA cloning, and their mRNA cleavage caused by miR828 was confirmed by cleavage site mapping, agro-infiltration and transgenics studies. The reduction in IbMYB and IbTLD expression coincided with the induction of miR828, demonstrating that IbMYB and IbTLD might be miR828 targets. Furthermore, transgenic sweet potato overexpressing miR828 precursor affected lignin and H2O2 contents. These results showed that cGMP could regulate wounding-responsive miR828, which repressed the expression of IbMYB and IbTLD. Subsequently, lignin and H2O2 were accumulated to participate in defense mechanisms. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

  7. Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis

    PubMed Central

    Zhang, Zhiyong; Zheng, Xixi; Yang, Jun; Messing, Joachim; Wu, Yongrui

    2016-01-01

    The maize endosperm-specific transcription factors opaque2 (O2) and prolamine-box binding factor (PBF) regulate storage protein zein genes. We show that they also control starch synthesis. The starch content in the PbfRNAi and o2 mutants was reduced by ∼5% and 11%, respectively, compared with normal genotypes. In the double-mutant PbfRNAi;o2, starch was decreased by 25%. Transcriptome analysis reveals that >1,000 genes were affected in each of the two mutants and in the double mutant; these genes were mainly enriched in sugar and protein metabolism. Pyruvate orthophosphate dikinase 1 and 2 (PPDKs) and starch synthase III (SSIII) are critical components in the starch biosynthetic enzyme complex. The expression of PPDK1, PPDK2, and SSIII and their protein levels are further reduced in the double mutants as compared with the single mutants. When the promoters of these genes were analyzed, we found a prolamine box and an O2 box that can be additively transactivated by PBF and O2. Starch synthase IIa (SSIIa, encoding another starch synthase for amylopectin) and starch branching enzyme 1 (SBEI, encoding one of the two main starch branching enzymes) are not directly regulated by PBF and O2, but their protein levels are significantly decreased in the o2 mutant and are further decreased in the double mutant, indicating that o2 and PbfRNAi may affect the levels of some other transcription factor(s) or mRNA regulatory factor(s) that in turn would affect the transcript and protein levels of SSIIa and SBEI. These findings show that three important traits—nutritional quality, calories, and yield—are linked through the same transcription factors. PMID:27621432

  8. Modelling (18)O2 and (16)O2 unidirectional fluxes in plants. IV: role of conductance and laws of its regulation in C3 plants.

    PubMed

    André, Marcel J

    2013-08-01

    Numerous studies focus on the measurement of conductances for CO2 transfer in plants and especially on their regulatory effects on photosynthesis. Measurement accuracy is strongly dependent on the model used and on the knowledge of the flow of photochemical energy generated by light in chloroplasts. The only accurate and precise method to quantify the linear electron flux (responsible for the production of reductive energy) is the direct measurement of O2 evolution, by (18)O2 labelling and mass spectrometry. The sharing of this energy between the carboxylation (P) and the oxygenation of photorespiration (PR) depends on the plant specificity factor (Sp) and on the corresponding atmospheric concentrations of CO2 and O2 (André, 2013). The concept of plant specificity factor simplifies the equations of the model. It gives a new expression of the effect of the conductance (g) between atmosphere and chloroplasts. Its quantitative effect on photosynthesis is easy to understand because it intervenes in the ratio of the plant specificity factor (Sp) to the specificity of Rubisco (Sr). Using this 'simple' model with the data of (18)O2 experiments, the calculation of conductance variations in response to CO2 and light was carried out. The good fitting of experimental data of O2 and CO2 exchanges confirms the validity of the simple model. The calculation of conductance variation during the increase of external CO2 concentration reveals a linear law of regulation between external and internal CO2 concentrations. During CO2 variations, the effects of g regulation tend to maintain a higher level of oxygenation (PR) in expense of a better carboxylation (P). Contrary to CO2, the variation of O2 creates a negative feedback effect compatible with a stabilization of atmospheric O2. The regulation of g amplifies this result. The effect of light in combination with CO2 is more complex. Below 800μmolquantam(-2)s(-1) the ratio PR/P is maintained unchangeable in expense of carboxylation

  9. Effects of miR-155 on proliferation and apoptosis by regulating FoxO3a/BIM in liver cancer cell line HCCLM3.

    PubMed

    Liao, W-W; Zhang, C; Liu, F-R; Wang, W-J

    2018-03-01

    MiR-155 has been shown to be up-regulated in hepatocellular carcinoma (HCC) patients. B-cell lymphoma-2 (Bcl-2) interacting mediator of cell death (BIM) regulates cell proliferation and apoptosis, as its down-regulation is involved in HCC onset. Transcriptional factor FoxO3a mediates BIM expression and is related to HCC pathogenesis. Bioinformatics analysis showed targeted regulation of FoxO3a by miR-155. This study aims to investigate whether miR-155 plays a role in mediating FoxO3a/BIM signal pathway and HCC occurrence. HCC patients were collected for tumor and adjacent tissues, in which microRNA-155 (miR-155) and FoxO3a expressions were examined. In vitro cultured HCCLM3, HepG2 and L-02 cells were tested for basal apoptotic rate by flow cytometry and were compared for miR-155 and FoxO3a expression. Dual-luciferase reporter gene assay demonstrated the targeted relationship between miR-155 and FoxO3a. HCCLM3 cells were treated with miR-155 inhibitor and/or FoxO3a-pMD18-T. Cell apoptosis and proliferation were examined by using flow cytometry and MTT assays, respectively. Western blot and spectrometry assay were employed to quantify the FoxO3a, BIM expressions, and caspase activity. Compared to adjacent tissues, HCC tissues had significantly higher miR-155 and significantly lower FoxO3a expression (p<0.05). HCCLM3 and HepG2 cells had significantly lower FoxO3a expression and basal apoptotic rate compared to L02 cells, whilst miR-155 level was significantly higher (p<0.05). MiR-155 targeted and inhibited 3'-UTR of FoxO3a, increasing BIM expression, caspase-3, and caspase-9 activities, and enhancing cell apoptosis and weakening proliferation. HCC tissues elevated the miR-155 and suppressed the FoxO3a expressions. MiR-155 targeted and inhibited FoxO3a expression to suppress the BIM, depress caspase-3 and caspase-9 activities, therefore inhibiting the HCC cell apoptosis and facilitating proliferation.

  10. O-GlcNAcylation Negatively Regulates Cardiomyogenic Fate in Adult Mouse Cardiac Mesenchymal Stromal Cells

    PubMed Central

    Zafir, Ayesha; Bradley, James A.; Long, Bethany W.; Muthusamy, Senthilkumar; Li, Qianhong; Hill, Bradford G.; Wysoczynski, Marcin; Prabhu, Sumanth D.; Bhatnagar, Aruni; Bolli, Roberto; Jones, Steven P.

    2015-01-01

    In both preclinical and clinical studies, cell transplantation of several cell types is used to promote repair of damaged organs and tissues. Nevertheless, despite the widespread use of such strategies, there remains little understanding of how the efficacy of cell therapy is regulated. We showed previously that augmentation of a unique, metabolically derived stress signal (i.e., O-GlcNAc) improves survival of cardiac mesenchymal stromal cells; however, it is not known whether enhancing O-GlcNAcylation affects lineage commitment or other aspects of cell competency. In this study, we assessed the role of O-GlcNAc in differentiation of cardiac mesenchymal stromal cells. Exposure of these cells to routine differentiation protocols in culture increased markers of the cardiomyogenic lineage such as Nkx2.5 and connexin 40, and augmented the abundance of transcripts associated with endothelial and fibroblast cell fates. Differentiation significantly decreased the abundance of O-GlcNAcylated proteins. To determine if O-GlcNAc is involved in stromal cell differentiation, O-GlcNAcylation was increased pharmacologically during the differentiation protocol. Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited. In addition, increasing O-GlcNAcylation further elevated smooth muscle actin expression. In addition to lineage commitment, we also evaluated proliferation and migration, and found that increasing O-GlcNAcylation did not significantly affect either; however, we found that O-GlcNAc transferase—the protein responsible for adding O-GlcNAc to proteins—is at least partially required for maintaining cellular proliferative and migratory capacities. We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function. O-GlcNAcylation and pathological conditions that may affect O-GlcNAc levels (such as diabetes) should be

  11. Redox regulation of mammalian sperm capacitation

    PubMed Central

    O’Flaherty, Cristian

    2015-01-01

    Capacitation is a series of morphological and metabolic changes necessary for the spermatozoon to achieve fertilizing ability. One of the earlier happenings during mammalian sperm capacitation is the production of reactive oxygen species (ROS) that will trigger and regulate a series of events including protein phosphorylation, in a time-dependent fashion. The identity of the sperm oxidase responsible for the production of ROS involved in capacitation is still elusive, and several candidates are discussed in this review. Interestingly, ROS-induced ROS formation has been described during human sperm capacitation. Redox signaling during capacitation is associated with changes in thiol groups of proteins located on the plasma membrane and subcellular compartments of the spermatozoon. Both, oxidation of thiols forming disulfide bridges and the increase on thiol content are necessary to regulate different sperm proteins associated with capacitation. Reducing equivalents such as NADH and NADPH are necessary to support capacitation in many species including humans. Lactate dehydrogenase, glucose-6-phospohate dehydrogenase, and isocitrate dehydrogenase are responsible in supplying NAD (P) H for sperm capacitation. Peroxiredoxins (PRDXs) are newly described enzymes with antioxidant properties that can protect mammalian spermatozoa; however, they are also candidates for assuring the regulation of redox signaling required for sperm capacitation. The dysregulation of PRDXs and of enzymes needed for their reactivation such as thioredoxin/thioredoxin reductase system and glutathione-S-transferases impairs sperm motility, capacitation, and promotes DNA damage in spermatozoa leading to male infertility. PMID:25926608

  12. Serum glutathione S-transferase Pi as predictor of the outcome and acute kidney injury in premature newborns.

    PubMed

    Stojanović, Vesna D; Barišić, Nenad A; Radovanović, Tanja D; Kovač, Nataša B; Djuran, Jelena D; Antić, Amira Peco E; Doronjski, Aleksandra D

    2018-07-01

    The incidence of acute kidney injury (AKI) among the neonates treated at the Neonatal Intensive Care Unit is high with high mortality rates. Glutathione S-transferase (GST) class Pi plays an important role in the protection of cells from cytotoxic and oncogenic agents. The aim of the study was to examine whether the levels of serum glutathione S-transferase Pi (GST Pi) determined after birth have any predictive value for the outcome and development of AKI in premature neonates. The prospective study included 36 premature neonates. The data about morbidity was gathered for all the neonates included in the study. The blood samples were taken in the first 6 h of life and GST Pi levels were measured. The mean values and standard deviations of GST Pi among the neonates who died and who survived were 1.904 ± 0.4535 vs 1.434 ± 0.444 ng/ml (p = 0.0128). Logistic regression revealed a statistically significant, positive correlation between GST Pi levels and death (p = 0.0180, OR7.5954; CI 1.4148-40.7748).The mean value of GST Pi levels in the neonates with AKI was higher than in neonates without AKI (p = 0.011). The conclusion of our study is that high levels of serum GST Pi in the first 6 h after birth are associated with an increased mortality and development of AKI in prematurely born neonates.

  13. p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

    PubMed Central

    Petrik, Deborah L; Karlen, Steven D; Cass, Cynthia L; Padmakshan, Dharshana; Lu, Fachuang; Liu, Sarah; Le Bris, Philippe; Antelme, Sébastien; Santoro, Nicholas; Wilkerson, Curtis G; Sibout, Richard; Lapierre, Catherine; Ralph, John; Sedbrook, John C

    2014-01-01

    Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta. PMID:24372757

  14. Unique N-Glycan Moieties of the 66-kDa Cell Wall Glycoprotein from the Red Microalga Porphyridium sp.

    PubMed Central

    Levy-Ontman, Oshrat; Arad, Shoshana (Malis); Harvey, David J.; Parsons, Thomas B.; Fairbanks, Antony; Tekoah, Yoram

    2011-01-01

    We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man8–9Xyl1–2Me3GlcNAc2. The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins. PMID:21515680

  15. Hydrothermal conversion of N-acetyl-d-glucosamine to 5-hydroxymethylfurfural using ionic liquid as a recycled catalyst in a water-dimethyl sulfoxide mixture.

    PubMed

    Zang, Hongjun; Yu, Songbai; Yu, Pengfei; Ding, Hongying; Du, Yannan; Yang, Yuchan; Zhang, Yiwen

    2017-04-10

    Here, N-acetyl-d-glucosamine (GlcNAc), the monomer composing the second most abundant biopolymer, chitin, was efficiently converted into 5-hydroxymethylfurfural (5-HMF) using ionic liquid (IL) catalysts in a water/dimethyl sulfoxide (DMSO) mixture solvent. Various reaction parameters, including reaction temperature and time, DMSO/water mass ratios and catalyst dosage were optimized. A series of ILs with different structures were analyzed to explore their impact on GlcNAc conversion. The substrate scope was expanded from GlcNAc to d-glucosamine, chitin, chitosan and monosaccharides, although 5-HMF yields obtained from polymers and other monosaccharides were generally lower than those from GlcNAc. Moreover, the IL N-methylimidazolium hydrogen sulfate ([Hmim][HSO 4 ]) exhibited the best catalyst performance (64.6% yield) when GlcNAc was dehydrated in a DMSO/water mixture at 180 °C for 6 h without the addition of extra catalysts. To summarize, these results could provide knowledge essential to the production of valuable chemicals that are derived from renewable marine resources and benefit biofuel-related applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein.

    PubMed

    Ohta, M; Hamako, J; Yamamoto, S; Hatta, H; Kim, M; Yamamoto, T; Oka, S; Mizuochi, T; Matsuura, F

    1991-10-01

    Asparagine-linked oligosaccharides present on hen egg-yolk immunoglobulin, termed IgY, were liberated from the protein by hydrazinolysis. After N-acetylation, the oligosaccharides were labelled with a UV-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-derivatized oligosaccharides were fractionated by anion exchange, normal phase and reversed phase HPLC, and their structures were determined by a combination of sugar composition analysis, methylation analysis, negative ion FAB-MS, 500 MHz 1H-NMR and sequential exoglycosidase digestions. IgY contained monoglucosylated oligomannose type oligosaccharides with structures of Glc alpha 1-3Man7-9-GlcNAc-GlcNAc, oligomannose type oligosaccharides with the size range of Man5-9GlcNAc-GlcNAc, and biantennary complex type oligosaccharides with core region structure of Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc. The glucosylated oligosaccharides, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, have not previously been reported in mature glycoproteins from any source.

  17. The 30 kDa protein co-purified with chick liver glutathione S-transferases is a carbonyl reductase.

    PubMed

    Tsai, S P; Wang, L Y; Yeh, H I; Tam, M F

    1996-02-08

    An unidentified 30 kDa protein was co-purified with chick liver glutathione S-transferases from S-hexylglutathione affinity column. The protein was isolated to apparent homogeneity with chromatofocusing. The molecular mass of the protein was determined to be 30 277 +/- 3 dalton by mass spectrometry. The protein was digested with Achromobacter proteinase I. Amino-acid sequence analyses of the resulting peptides show a high degree of identity with those of human carbonyl reductase. The protein is active with menadione as substrate. Thus, it is identified as chick liver carbonyl reductase.

  18. Forkhead Box O1 Regulates Macrophage Polarization Following Staphylococcus aureus Infection: Experimental Murine Data and Review of the Literature.

    PubMed

    Wang, Yu-Chen; Ma, Hong-Di; Yin, Xue-Ying; Wang, Yin-Hu; Liu, Qing-Zhi; Yang, Jing-Bo; Shi, Qing-Hua; Sun, Baolin; Gershwin, M Eric; Lian, Zhe-Xiong

    2016-12-01

    The functions of macrophages that lead to effective host responses are critical for protection against Staphylococcus aureus. Deep tissue-invading S. aureus initially countered by macrophages trigger macrophage accumulation and induce inflammatory responses through surface receptors, especially toll-like receptor 2 (TLR2). Here, we found that macrophages formed sporadic aggregates in the liver during infection. Within those aggregates, macrophages co-localized with T cells and were indispensable for their infiltration. In addition, we have focused on the mechanisms underlying the polarization of macrophages in Forkhead box transcription factor O1 (FoxO1) conditional knockout Lys Cre/+ FoxO1 fl/fl mice following S. aureus infection and report herein that macrophage M1-M2 polarization via TLR2 is intrinsically regulated by FoxO1. Indeed, for effective FoxO1 activity, stimulation of TLR2 is essential. However, following S. aureus challenge, there was a decrease in macrophage FoxO1, with increased phosphorylation of FoxO1 because of TLR2-mediated activation of PI3K/Akt and c-Raf/MEK/ERK pathway. Following infection in Lys Cre/+ FoxO1 fl/fl mice, mice became more susceptible to S. aureus with reduced macrophage aggregation in the liver and attenuated Th1 and Th17 responses. FoxO1 abrogation reduced M1 pro-inflammatory responses triggered by S. aureus and enhanced M2 polarization in macrophages. In contrast, overexpression of FoxO1 in macrophages increased pro-inflammatory mediators and functional surface molecule expression. In conclusion, macrophage FoxO1 is critical to promote M1 polarization and maintain a competent T cell immune response against S. aureus infection in the liver. FoxO1 regulates macrophage M1-M2 polarization downstream of TLR2 dynamically through phosphorylation.

  19. Quorum sensing transcriptional regulator QseA is essential for the expression of multiple virulence regulons of enterohemorrhagic Escherichia coli O157:H7

    USDA-ARS?s Scientific Manuscript database

    Introduction and Objectives: QseA is one of several transcriptional regulators that regulates the virulence gene expression in enterohemorrhagic Escherichia coli (EHEC) O157:H7 through quorum sensing. QseA has been shown to regulate the expression of the locus of enterocyte effacement (LEE), non-LEE...

  20. PDK-1/FoxO1 pathway in POMC neurons regulates Pomc expression and food intake.

    PubMed

    Iskandar, Kristy; Cao, Yongheng; Hayashi, Yoshitake; Nakata, Masanori; Takano, Eisuke; Yada, Toshihiko; Zhang, Changliang; Ogawa, Wataru; Oki, Miyo; Chua, Streamson; Itoh, Hiroshi; Noda, Tetsuo; Kasuga, Masato; Nakae, Jun

    2010-04-01

    Both insulin and leptin signaling converge on phosphatidylinositol 3-OH kinase [PI(3)K]/3-phosphoinositide-dependent protein kinase-1 (PDK-1)/protein kinase B (PKB, also known as Akt) in proopiomelanocortin (POMC) neurons. Forkhead box-containing protein-O1 (FoxO1) is inactivated in a PI(3)K-dependent manner. However, the interrelationship between PI(3)K/PDK-1/Akt and FoxO1, and the chronic effects of the overexpression of FoxO1 in POMC neurons on energy homeostasis has not been elucidated. To determine the extent to which PDK-1 and FoxO1 signaling in POMC neurons was responsible for energy homeostasis, we generated POMC neuron-specific Pdk1 knockout mice (POMCPdk1(-/-)) and mice selectively expressing a constitutively nuclear (CN)FoxO1 or transactivation-defective (Delta256)FoxO1 in POMC neurons (CNFoxO1(POMC) or Delta256FoxO1(POMC)). POMCPdk1(-/-) mice showed increased food intake and body weight accompanied by decreased expression of Pomc gene. The CNFoxO1(POMC) mice exhibited mild obesity and hyperphagia compared with POMCPdk1(-/-) mice. Although expression of the CNFoxO1 made POMCPdk1(-/-) mice more obese due to excessive suppression of Pomc gene, overexpression of Delta256FoxO1 in POMC neurons had no effects on metabolic phenotypes and Pomc expression levels of POMCPdk1(-/-) mice. These data suggest a requirement for PDK-1 and FoxO1 in transcriptional regulation of Pomc and food intake.